JP6656919B2 - 5-oxoprolinase, 5-oxoprolinase gene, and method for producing 5-oxoprolinase - Google Patents

Description

  The present invention relates to a 5-oxoprolinase, a 5-oxoprolinase gene, and a method for producing 5-oxoprolinase.

  In the production of soy sauce, yeast extract, or fermented seasoning, etc., L- released from protein raw materials such as soybean, wheat, gelatin, and casein by enzymatic degradation by endopeptidase and exopeptidase, or chemical degradation by acid, etc. Glutamic acid ("umami" component) is converted to L-pyroglutamic acid (no umami) by non-enzymatic reaction in the presence of heat and pressure, and as a result, umami is reduced. (Non-Patent Document 1). As a measure against this problem, an enzyme-decomposable seasoning in which 5-oxoprolinase is allowed to act on these foods has been proposed (Patent Document 1). The 5-oxopurinase disclosed in Patent Document 1 is derived from baker's yeast, and its amino acid sequence is not described.

5-oxoprolinase (5-oxoprolinase, 5-oxo-L-prolinase) is classified into EC 3.5.2.9 according to the classification standard of the Enzyme Committee of the International Union of Biochemistry, and includes pyroglutamase, pyroglutamase, and pyroglutamase. It is an enzyme also called glutamic acid hydrolase (pyroglutamic hydrolase, pyroglutamate hydrolase) and pyrrolidone carboxylic acid hydrolase (L-pyrrolidone carboxylate). 5-oxoprolinase is also referred to as L-pyroglutamic acid (L-pyroglutamic acid, 5-oxoproline, 5-oxoproline, 5-oxo-L-proline, (S) -5-oxopyrrolidine-2-carboxylic acid, and the like). ) was hydrolyzed in the presence of H _{2 O} and ATP, catalyzes the reaction to produce L- glutamic acid and phosphoric acid known as umami.

L- pyroglutamic acid ₊ 2H 2 O ₊ ATP → L- glutamic acid + ADP + phosphate as described above, 5-oxo-Pro Li proteinase, it is possible to generate an L- glutamic acid L- pyroglutamic acid, to act on the food or drink containing L- pyroglutamic acid Thereby, it is expected that the L-glutamic acid content in the food or drink is increased to improve umami.

  In addition, 5-oxoprolinase is expected to be used for purposes other than improving the umami of foods and drinks, for example, by utilizing the fact that 5-oxoprolinase acts specifically on the L-isomer, to prepare racemic pyroglutamic acid or glutamic acid. Separation of optical isomers from the body. Furthermore, for the purpose of quantifying L-pyroglutamic acid contained in foods, serum, urine and the like, 5-oxoprolinase can be used. As described above, 5-oxoprolinase is expected to have a wide variety of industrial uses.

  Until now, 5-oxoprolinase activity has been confirmed in several species, and extraction of enzymes and analysis of enzymological properties have been performed. For example, rats (liver (Non-patent document 2), kidney (Non-patent document 3)), genus Pseudomonas (Non-patent document 4), wheat germ (Non-patent document 5), genus Alcaligenes (Non-patent document) The 5-oxoprolinase of literature 6) is known. There is also an example in which 5-oxoprolinase protein is forcibly expressed in baker's yeast (Non-Patent Document 7).

  However, in spite of being an enzyme expected to be used in a wide variety of applications as described above, to date, 5-oxoprolinase, including the above-mentioned various known enzymes, has been mass-produced as an industrially usable product. However, there is no record of commercialization, and it is strongly desired to provide 5-oxoprolinase which can be suitably used for various uses.

JP-A-8-252075

Chemistry and Technology of Soy Sauce, edited by Tatsurorou Tochikura, p.181, Japan Brewing Association Scand. J. Clin. Lab. Invest. 32, 233-237, 1973 Proc. Nat. Acad. Sci USA 68, 2982-2985, 1971 Biochem. Biophys. Res. Commun. 56, 90-96, 1974 Plant Physiol. 62, 798-801, 1978 Agric. Biol. Chem. 52, 735-741, 1988 FEMS Yeast Res. 10, 394-401, 2010

  INDUSTRIAL APPLICABILITY The present invention is preferably used in various applications such as 5-oxoprolinase, for example, for improving the umami of foods and drinks, separating optical isomers from racemates, and quantifying L-pyroglutamic acid. An object of the present invention is to provide a 5-oxoprolinase that can be obtained.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and as a result, a novel 5-oxoprolinase which is remarkably different from various 5-oxoprolinases conventionally reported from microorganisms belonging to the genus Aspergillus. -Oxoprolinase was found and the gene encoding this enzyme protein was isolated. Further, a microorganism containing the 5-oxoprolinase gene or a microorganism having the ability to produce 5-oxoprolinase is cultured in a medium, and 5-oxoprolinase is collected from the obtained culture, and the enzyme is used in various manners. They have found that they have properties that can be suitably used in industrial applications, and have completed the present invention.

  That is, the present invention relates to the following.

(1) A protein having the following 5-oxoprolinase activity of (a) or (b).
(A) a protein having an amino acid sequence showing 80% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 and 2 and having 5-oxoprolinase activity; A protein having an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence represented by any of the above, and having 5-oxoprolinase activity (2) having the following enzymatic properties: And a protein having 5-oxoprolinase activity.
i) action: converts L-pyroglutamic acid to L-glutamic acid ii) molecular weight: the molecular weight of the polypeptide chain portion of the protein is about 140 kDa iii) optimal pH: pH 7.0 to pH 8.0 iv) stability pH: pH 6.0 to pH 9.0 v) Temperature stability: 80% or more activity remains after treatment at 37 ° C. for 15 minutes in a Tris-HCl buffer (pH 8.0) vi) Optimum temperature: Vii) ATP having a requirement for ATP, monovalent cation, and divalent cation in a Tris-HCl buffer (pH 8.0) at 25 ° C. to 45 ° C. (3) It is derived from a microorganism belonging to the genus Aspergillus. A) a protein having a 5-oxoprolinase activity;
(4) The protein having 5-oxoprolinase activity according to (3) above, which is derived from Aspergillus sojae.
(5) A 5-oxoprolinase gene encoding a protein having the following 5-oxoprolinase activity of (c) or (d).
(C) a protein having an amino acid sequence showing 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 and 2 and having 5-oxoprolinase activity; In the amino acid sequence represented by any one, one or several amino acids have an amino acid sequence in which addition, deletion, or substitution is performed, and have a 5-oxoprolinase activity. A) 5-oxoprolinase gene encoding a protein having 5-oxoprolinase activity having the DNA of (1).
(E) DNA having a base sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57
(F) hybridizing with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by any of SEQ ID NOs: 4, 5, 56 and 57 under stringent conditions, and 5-oxoprolinase DNA encoding an active protein
(7) A recombinant vector containing the 5-oxoprolinase gene according to (5) or (6).
(8) A transformant or transductant containing the recombinant vector according to (7).
(9) A method for producing 5-oxoprolinase, comprising culturing the transformant or transductant according to (8) above in a medium, and collecting 5-oxoprolinase from the resulting culture.
(10) The protein having 5-oxoprolinase activity according to any of the above (1) to (4), and the 5-oxoprolinase activity encoded by the gene according to the above (5) or (6). An umami ameliorating agent for food or drink, comprising the protein or the transformant or the transductant according to the above (8).
(11) A food or drink containing L-pyroglutamic acid is mixed with the protein contained in the umami improving agent according to (10), or the food or drink containing L-pyroglutamic acid is mixed with the umami improving agent according to (10). A method for producing umami food or drink, comprising fermenting using a transformant or a transductant contained in an agent.
(12) A method for separating L-pyroglutamic acid and D-pyroglutamic acid, comprising the following steps:
(i) The protein having 5-oxoprolinase activity according to any of the above (1) to (4) or the protein according to (5) or (6), in a solution containing L-pyroglutamic acid and D-pyroglutamic acid. Reacting a protein having 5-oxoprolinase activity encoded by the gene to hydrolyze L-pyroglutamic acid to L-glutamic acid, and
(ii) a step of separating L-glutamic acid and D-pyroglutamic acid produced in step (i),
The method comprising:

  This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2013-027844, which is a priority document of the present application.

  According to the present invention, it is possible to provide a novel 5-oxoprolinase that can be suitably used for the above-mentioned various uses. In one aspect of the invention, the 5-oxoprolinase of the invention is less susceptible to pH changes than known enzymes and can be used over a wide range of pH. Further, in one embodiment of the present invention, the 5-oxoprolinase of the present invention has high affinity for a substrate. In addition, Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii, which are examples of microorganisms derived from the 5-oxoprolinase of the present invention, are miso, soy sauce, sake, and sake. A microorganism that has been used for a long time in the manufacture of various brewed foods such as shochu and has a long eating experience. It is.

It is a figure which shows the optimal pH of the 5-oxoprolinase of this invention and the 5-oxoprolinase derived from yeast. It is a figure which shows the pH stability of 5-oxoprolinase of this invention and 5-oxoprolinase derived from yeast. It is a figure which shows the outline of the structure of the plasmid pAsAlpP for cassette production for Aspergillus genus expression. Examples of 5-oxoprolinase ortholog candidate genes include scaffold00004.806, scaffold00004.820, and scaffold00036.1302. It is a prediction figure of the exon-intron structure of the 5-oxoprolinase ortholog candidate gene of Aspergillus soya. The numbers in parentheses indicate the number of bases. It is a figure which shows the CBB staining image of the gel after SDS-PAGE of the cell extract which expressed the 5-oxoprolinase ortholog candidate protein derived from Aspergillus soya in Aspergillus soya and the protein purified by Ni-NTA Superflow column. . It is a figure which shows the relationship between the amount of addition of three kinds of 5-oxoprolinase ortholog candidate proteins derived from Aspergillus soya expressed by Aspergillus spp., And the L-glutamic acid concentration in the reaction solution after the reaction. It is a figure which shows the relationship between the kind of monovalent cation added to the reaction system, and relative activity. It is a figure which shows the relationship between the kind of divalent cation added to the reaction system, and relative activity. It is a figure which shows the optimal temperature of the 5-oxoprolinase of this invention and the 5-oxoprolinase derived from yeast. It is a figure which shows the temperature stability of the 5-oxoprolinase of this invention and the 5-oxoprolinase derived from yeast. It is a figure which shows the substrate specificity of the 5-oxoprolinase of this invention. It is a figure which shows the relationship between the addition amount of the 5-oxoprolinase ortholog candidate protein derived from Aspergillus oryzae expressed by the genus Aspergillus and the L-glutamic acid concentration in the reaction solution after the reaction. It is a figure which shows the relationship between the glutamic acid concentration in a reaction liquid, and the pyroglutamic acid concentration after the reaction of the tomato extract dilution liquid and the 5-oxoprolinase of this invention. (The values in parentheses indicate relative values when the amount of enzyme not added is 100.) It is a figure which shows the relationship between the glutamic acid concentration and the pyroglutamic acid concentration in the reaction liquid after the reaction of the concentrated soy sauce diluent and the 5-oxoprolinase of the present invention. (The values in parentheses indicate relative values when the amount of enzyme not added is 100.)

  Hereinafter, the present invention will be described in detail.

(Method for measuring 5-oxoprolinase activity)
The activity of the 5-oxoprolinase of the present invention (hereinafter referred to as “the present enzyme” in context) is determined by FEMS Yeast Res. (Vol. 10, pages 394 to 401, 2010), which can be measured by a modified method. In this method, the enzymatic activity is calculated by measuring L-glutamic acid generated by hydrolyzing an intramolecular amide bond of L-pyroglutamic acid. The amount of L-glutamic acid can be measured by a method using an enzyme acting on L-glutamic acid, such as L-glutamic acid oxidase, or by liquid chromatography. For example, the following measurement method can be used.

[Measurement method 1]
0.08 mL of a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid (manufactured by MP Biomedicals, manufacturing number 102781), 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0] and 0.08 mL 0.02 mL of an enzyme solution to be measured (prepared by diluting to an arbitrary concentration with a 50 mM Tris-HCl buffer (pH 8.0)) is mixed, and the enzyme reaction is performed at 37 ° C. for 1 hour. Next, the enzyme reaction solution is heated at 100 ° C. for 3 minutes to stop the enzyme reaction, and then the concentration of L-glutamic acid in the enzyme reaction solution is measured. If necessary, the reaction solution after the heating may be centrifuged (15,000 × g, 10 minutes) and the supernatant may be used for the measurement of L-glutamic acid. Other reagents described in this specification are manufactured by Wako Pure Chemical Industries, Nacalai Tesque, Sigma, Dojin Chemical Laboratory, Becton Dickinson, and Nippon Pharmaceutical Co., unless otherwise specified. And Takara Bio Co., Ltd., and reagents of which purity is distinguished use a special grade reagent.

For quantification of L-glutamic acid, "Yamasa L-glutamic acid detection kit II" (manufactured by Yamasa Shoyu Co., Ltd., containing L-glutamic acid oxidase) was used. 0.15 mL of the coloring reagent for L-glutamic acid detection was mixed with 0.13 mL of the coloring reagent for enzyme reaction, and 0.12 mL of the coloring reagent for detecting L-glutamic acid was mixed, and the absorbance at 600 nm after the reaction was measured at room temperature for 20 to 30 minutes. The L-glutamic acid concentration in the enzyme reaction solution is calculated (measured value) based on a calibration curve using an L-glutamic acid standard. In addition, a buffer solution is added in place of the substrate solution, and the same reaction is performed, and the concentration of L-glutamic acid in the reaction solution is calculated (blank value). From the obtained "blank value" and "measured value", the activity of 5-oxoprolinase can be calculated based on the following formula. The unit of the enzyme activity is defined as 1 unit (U) of the amount of the enzyme that produces 1 μmol of L-glutamic acid per minute under the above-mentioned conditions.

  [Glu] in the formula is the L-glutamic acid concentration (measured value) in the enzyme reaction solution, and [Glu blank] is the L of the reaction solution obtained by adding a 50 mM Tris-HCl buffer (pH 8.0) instead of the substrate solution. -Glutamic acid concentration (blank value), [enzyme] represents the concentration of 5-oxoprolinase in the reaction solution, and 60 represents the fraction of one hour.

(Enzymatic properties of 5-oxoprolinase of the present invention)
In one embodiment, the 5-oxoprolinase of the invention has the following enzymatic properties:

(1) Action: Hydrolyzes L-pyroglutamic acid having an intramolecular amide bond.

(Example of reaction:
L- pyroglutamic acid ₊ 2H 2 O ₊ ATP → L- glutamic acid + ADP + phosphate)
(2) Requirement: This enzyme exhibits ATP, monovalent cation, and divalent cation requirements.

(3) Optimum pH and stable pH range: When the activity of the present enzyme was measured at each pH by changing the pH of the buffer solution using the above-mentioned measurement method 1, the optimum pH of the present enzyme was pH 7.0 to pH 7.0. The pH is 8.0 (see FIG. 1). As shown in FIG. 1, the present enzyme can maintain 70% or more of the maximum activity in the above optimum pH range.

  When the residual activity of this enzyme solution after treatment at 4 ° C. for 16 hours in the range of pH 4.0 to 11.0 using the measuring method 1 was measured, the stable pH range of the enzyme was pH 6.0. 0 to 9.0 (see FIG. 2). As shown in FIG. 2, this enzyme can maintain 70% or more of the maximum activity when treated in the stable pH range. The enzyme is almost completely inactivated when treated under pH conditions of pH 5.0 or lower and pH 11.0 or higher.

(4) Optimum temperature range: The optimum temperature of the present enzyme is 37 ° C. when the activity at each temperature is measured by changing the temperature during the reaction using the measuring method 1 (see FIG. 9).

(5) Temperature stability: When the residual activity after treatment for 15 minutes at each temperature in a Tris-HCl buffer (pH 8.0) was measured using the above-mentioned measurement method 1, the enzyme was treated at 37 ° C. for 15 minutes. Retains 80% or more activity after treatment.

(6) Molecular weight: As determined by SDS polyacrylamide gel electrophoresis, the molecular weight of the present enzyme is about 140 kDa.

(7) Substrate specificity: The enzyme specifically hydrolyzes L-pyroglutamic acid to L-glutamic acid and does not hydrolyze D-pyroglutamic acid.

  In one embodiment, when the affinity of the 5-oxoprolinase of the present invention is expressed as a Km value for L-pyroglutamic acid, one of the orthologs of the present enzyme (As 4.806, described later) has a Km value. Is 93 μM, and the Km value of another ortholog of this enzyme (As4.820, described later) is 76 μM.

(Origin of the 5-oxoprolinase of the present invention)
Although the 5-oxoprolinase of the present invention is not limited by its origin as long as it has the enzymatic properties described herein, in one embodiment, the 5-oxoprolinase of the present invention comprises: Preferably, it is a protein derived from a microorganism, more preferably, a protein derived from a filamentous fungus, more preferably, a filamentous fungus belonging to the genus Aspergillus, such as Aspergillus oryzae, Aspergillus sojae. And proteins derived from Aspergillus tamari, Aspergillus niger, and the like.

  Aspergillus oryzae, Aspergillus soya, Aspergillus tamari and Aspergillus niger have been used in the production of brewed foods in Japan, such as miso, soy sauce, sake, and shochu, for a long time. It is a microorganism that is particularly important in industry because of its high reliability in use.

  Furthermore, the 5-oxoprolinase of the present invention also includes 5-oxoprolinase obtained by genetic recombination technology.

(Amino acid sequence of 5-oxoprolinase of the present invention)
In one embodiment, the 5-oxoprolinase of the present invention is a protein having the amino acid sequence shown in any of SEQ ID NOs: 1 and 2. For example, a protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 and 2 is obtained by cloning a 5-oxoprolinase gene derived from chromosomal DNA or cDNA of a filamentous fungus belonging to the genus Aspergillus. It can also be obtained by introducing into a suitable vector-host system for expression.

  Furthermore, the present invention has an amino acid sequence having 5-oxoprolinase activity and having one to several amino acids added, deleted or substituted in the amino acid sequence represented by any one of SEQ ID NOS: 1 and 2. Also encompasses proteins. The phrase "an amino acid sequence in which one or several (plural) amino acids are added, deleted or substituted" refers to any one of SEQ ID NOs: 1 and 2 as long as the desired 5-oxoprolinase activity is obtained. A sequence different from the amino acid sequence may be added to the represented amino acid sequence, or a part of the amino acid sequence may be deleted or substituted. Regarding the addition, deletion or substitution of amino acids, one or several amino acids means 1 to 10, for example 1 to 9, for example 1 to 8, for example 1 to 7, for example 1 to 6, for example 1 -5, for example 1-4, for example 1-3 amino acids. For example, even if the first methionine in the amino acid sequence represented by any of SEQ ID NOs: 1 and 2 is deleted, 5-oxoprolinase having the same enzymatic properties as the 5-oxoprolinase of the present invention. A protein having an amino acid sequence having such additions, deletions, substitutions, and the like is also included in the present invention as long as the protein exhibits the enzyme activity. In addition, the 5-oxoprolinase of the present invention has an amino acid sequence represented by any one of SEQ ID NOS: 1 and 2 in an amount of 80% or more, preferably 85% or more, more preferably 90%, and still more preferably 95% or more. More preferably, it includes a protein having an amino acid sequence showing 98% or more, more preferably 99% or more sequence identity.

(5-oxoprolinase gene of the present invention)
In one embodiment, the 5-oxoprolinase gene of the present invention is a 5-oxoprolinase gene encoding a protein having the above-mentioned enzymatic properties.

  In one embodiment, the 5-oxoprolinase gene of the present invention is a 5-oxoprolinase gene having a base sequence represented by any one of SEQ ID NOs: 56 and 57.

  Furthermore, the 5-oxoprolinase gene of the present invention is a 5-oxoprolinase gene which is introduced into a host, and after transcription of the gene, is spliced into a gene encoding a protein having the above-mentioned enzymatic properties. . Such a gene includes, for example, a 5-oxoprolinase gene having a base sequence represented by any of SEQ ID NOs: 4 and 5.

  Further, the 5-oxoprolinase gene of the present invention is complementary to a DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57, as long as it encodes a protein having 5-oxoprolinase activity. And a 5-oxoprolinase gene that encodes a protein that hybridizes with a DNA consisting of a specific base sequence under stringent conditions and has a 5-oxoprolinase activity.

  The "stringent conditions" are conditions under which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and the type of hybridization system to be used and the type, sequence and length of the probe are used. Depends on Such conditions can be determined by changing the temperature of hybridization, the temperature of washing, and the salt concentration. For example, when a signal of a non-specific hybrid is strongly detected, specificity can be increased by increasing the temperature of hybridization and washing and, if necessary, decreasing the salt concentration of washing. When no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, increasing the washing salt concentration. Such optimization can be easily performed by a researcher in this technical field.

  As specific examples of the stringent conditions, for example, a DNA probe is used as a probe, and hybridization is performed using 5 × SSC, 1.0% (w / v) nucleic acid hybridization blocking reagent (Boehringer Mannheim), Perform overnight (about 8 to 16 hours) using 0.1% (w / v) N-lauroyl sarcosine and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS. Do. The temperature at which hybridization and washing are performed is 65 ° C. or higher, preferably 68 ° C. or higher.

  In addition, the 5-oxoprolinase gene of the present invention is at least 80%, preferably at least 85%, more preferably at least 90%, the DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57. As described above, the 5-oxoprolinase gene, which more preferably has a sequence identity of 95% or more, more preferably 98% or more, and still more preferably 99% or more, and encodes a protein having a 5-oxoprolinase function, Include.

(Means for calculating sequence identity)
Examples of a program for calculating the sequence identity between two amino acid sequences or base sequences include, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 and Proc. Natl. Acad. USA 90: 5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can search a database for a sequence exhibiting high sequence identity to a given sequence by using, for example, the above-mentioned program. These are available, for example, on the website of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the United States National Center for Biotechnology Information.

  Each of the above methods is generally used to search a sequence showing sequence identity from a database. As a means for determining the sequence identity of an individual sequence, Genetyx network version 11.0.6 (Genetics) homology analysis can also be used. This method is based on the Lipman-Pearson method (Science 227: 1454-1441, 1985). When analyzing the sequence identity of a nucleotide sequence, a region encoding a protein (CDS or ORF) is used if possible.

(Cloning of 5-oxoprolinase gene by genetic engineering technique)
The gene encoding the 5-oxoprolinase of the present invention can be inserted into various suitable known vectors. Furthermore, this vector can be introduced into appropriate known hosts to produce a transformant or transductant into which a recombinant vector (recombinant DNA) containing the 5-oxoprolinase gene has been introduced. Methods for obtaining these genes, methods for obtaining gene sequence and amino acid sequence information, methods for manufacturing various vectors, and methods for preparing transformants are known to those skilled in the art, and examples thereof will be described later.

  For cloning the 5-oxoprolinase gene of the present invention, a generally used gene cloning method can be appropriately used. For example, chromosomal DNA or mRNA can be extracted from microbial cells or various cells capable of producing 5-oxoprolinase by a conventional method, for example, a method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). it can. Furthermore, cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared.

  In one embodiment, the 5-oxoprolinase gene can be obtained by cloning using a gene derived from chromosomal DNA or cDNA derived from a microorganism having the gene as a template. Examples of microorganisms derived from such genes include filamentous fungi, preferably filamentous fungi belonging to the genus Aspergillus, and more preferably Aspergillus sojae strain NBRC4239. More specifically, first, Aspergillus sojae NBRC4239 strain is cultured, and the obtained cells are frozen in liquid nitrogen, and then finely ground by physical grinding using a mortar or the like. The chromosomal DNA fraction is extracted from the cell fragments by a conventional method. For the extraction operation, a commercially available chromosomal DNA extraction kit can be used.

  Next, the DNA is amplified by performing a polymerase chain reaction (hereinafter referred to as “PCR”) using the chromosomal DNA as a template and synthetic primers complementary to the 5 ′ terminal sequence and the 3 ′ terminal sequence. As a primer, a primer having any sequence may be used as long as a DNA fragment containing the gene can be amplified. Examples thereof include primers represented by SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, and SEQ ID NOS: 11 and 12 designed with reference to the genome sequence of Aspergillus sojae. Using the prepared primer DNAs, a suitable PCR such as 5′RACE or 3′RACE is used to amplify the DNA containing the target gene fragment and ligate them to obtain a DNA containing the full-length target gene. Can be.

  In one embodiment, a 5-oxoprolinase gene can be constructed by using a chemical synthesis method in addition to obtaining it from a microorganism.

  Confirmation of a base sequence in an amplification product amplified by PCR or a chemically synthesized gene can be performed, for example, as follows. First, a DNA whose sequence is to be confirmed is inserted into an appropriate vector according to a conventional method to prepare a recombinant DNA. For cloning into a vector, a commercially available kit such as TA Cloning Kit (manufactured by Invitrogen), or pUC18 (manufactured by Takara Bio), pUC119 (manufactured by Takara Bio), pBR322 (manufactured by Takara Bio), pBluescript SK + ( Commercially available plasmid vector DNA such as Stratagene) and pYES2 / CT (Invitrogen) and commercially available bacteriophage vector DNA such as λEMBL3 (Stratagene) can be used. Using the recombinant DNA, a host cell, for example, Escherichia coli K-12 strain, preferably JM109 strain (manufactured by Takara Bio Inc.), DH5α strain (manufactured by Takara Bio Inc.) is transformed. . The recombinant DNA contained in the obtained transformant is purified using QIAGEN Plasmid Mini Kit (manufactured by QIAGEN) or the like.

  Then, the nucleotide sequence of each gene inserted into the recombinant DNA is determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983) or the like. As a specific sequence analyzer, for example, analysis can be performed using a Li-COR MODEL 4200L sequencer (manufactured by Aloka), a 370 DNA sequence system (manufactured by PerkinElmer), a CEQ2000XL DNA analysis system (manufactured by Beckman), or the like. Then, based on the determined base sequence, the amino acid sequence of the translated polypeptide, that is, the 5-oxoprolinase protein is determined.

(Construction of recombinant vector containing 5-oxoprolinase gene)
A recombinant vector (recombinant DNA) containing the 5-oxoprolinase gene of the present invention is constructed and transformed or transduced with the recombinant vector to obtain a recombinant microorganism capable of producing the enzyme. can do. The recombinant microorganism is also useful as one technique for efficiently producing the present enzyme.

  A recombinant vector containing a 5-oxoprolinase gene is constructed by combining a PCR amplification product containing a 5-oxoprolinase gene with various vectors in a form that allows expression of the 5-oxoprolinase gene. be able to. That is, it can be constructed, for example, by cutting out a DNA fragment containing the 5-oxoprolinase gene with an appropriate restriction enzyme and inserting it into a plasmid cut with an appropriate restriction enzyme. Alternatively, a DNA fragment containing the gene having a sequence homologous to the plasmid added to both ends and a DNA fragment derived from the plasmid amplified by inverse PCR are ligated using an In-Fusion Advantage PCR Cloning Kit (manufactured by Clontech). Can be obtained.

(Acquisition of transformant or transductant)
The method of transforming a microorganism using the recombinant vector obtained as described above varies depending on the microorganism used as a host and the type of the recombinant vector, but a known method can be appropriately used. For example, a technique of directly inserting into a host chromosome by using homologous recombination or a transposon, and a technique of introducing into a host by ligating on a vector such as a plasmid, cosmid, phage, virus, or artificial chromosome. And the like.

  In the method using homologous recombination, an upstream region of a recombination site on a chromosome, a sequence homologous to a downstream region, a suitable promoter and terminator, and a 5-oxoprolinase gene are ligated, and the Can be inserted. By forcibly expressing in a host under the control of its own high expression promoter, a transformant by self-cloning can be obtained. Examples of the high expression promoter include, for a microorganism belonging to a filamentous fungus, an α-amylase gene (amy) promoter region, a TEF1 promoter region as a translation elongation factor, an alkaline protease gene (alp) promoter region, and the like.

  In the method using a vector, the cloned 5-oxoprolinase gene can be converted into a plasmid, cosmid, phage, virus, chromosomal integration type, artificial chromosome, or other vector used for transformation of prokaryotic or eukaryotic cells by a conventional method. And the host corresponding to each vector can be transformed or transduced by a conventional method.

  As such a suitable vector-host system, any one that can produce 5-oxoprolinase in the host can be used, and the filamentous fungus expression vector pST14 (Mol. Gen. Genet. 218) can be used. , 99-104, 1989) and filamentous fungi, yeast expression vectors [YEpFLAG-1 (manufactured by SIGMA), pYES2 (manufactured by Invitrogen), pYD1 (manufactured by Invitrogen), pAUR123 (manufactured by Takara Bio) and yeast Saccharomyces cerevisiae system, Escherichia coli expression vector [pBluescriptII (Stratagene), pUC (Takara Bio) pET (Invitrogen), pRSET (Invitrogen), pTE (S System such as ratagene Co., Ltd.)] and E. coli Escherichia coli (Escherichia coli), and the like.

  In addition, the above-described vector may contain a marker gene for enabling selection of transformed cells. Examples of the marker gene include a gene that complements the auxotrophy of the host, such as URA3, pyrG, and niaD, and a resistance gene to drugs such as ampicillin, kanamycin, and oligomycin. In addition, the recombinant vector desirably contains a promoter or other control sequences (eg, an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) capable of expressing the gene of the present invention in a host cell. Examples of the promoter include a suitable inducible promoter and a constitutive promoter, and specific examples include a GAL1 promoter, an alp promoter, an amyB promoter, and a lac promoter. However, the function required for the expression of the 5-oxoprolinase gene is not necessarily required when the DNA fragment containing the inserted 5-oxoprolinase gene has that function. In the case of performing transformation by a co-transformation method, it is not always necessary to have a marker gene on the vector DNA. Tags for purification can also be attached. For example, by connecting a linker sequence appropriately upstream or downstream of the 5-oxoprolinase gene and connecting a histidine-encoding base sequence of 6 or more codons, purification using a nickel column can be enabled.

(Transformant or transductant host)
As the host cell, any organism can be used as long as it can produce the present enzyme by transformation or transduction with a recombinant vector containing the 5-oxoprolinase gene. Alternatively, eukaryotes, for example, E. coli or yeast, as well as other bacteria, fungi, actinomycetes, and other microorganisms or animal cells can be used, but are not limited thereto.

  Examples of lower eukaryotic host cells include filamentous fungi and yeast. Microorganisms classified as filamentous fungi include, for example, Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, and Mucor belonging to the genus Tricorderma. Filamentous fungi. Specific preferred microorganisms which belong to these genera and which produce 5-oxoprolinase used in the present invention include Aspergillus sojae, Aspergillus oryzae, Aspergillus oryzae, and Aspergillus oryzae. Nidulans (Aspergillus nidulans), Aspergillus niger (Aspergillus niger), Penicillium chrysogenum (Penicillium chrysogenum) and the like can be mentioned. More specifically, Aspergillus sojae NBRC4239 strain can be mentioned. As a method for transforming a filamentous fungus, a known method, for example, a method using polyethylene glycol and calcium chloride after protoplasting (Mol. Gen. Genet. 218, 99-104, 1989) can be used.

  Examples of microorganisms classified as yeast include yeast belonging to the genus Zygosaccharomyces, the genus Saccharomyces, the genus Pichia, the genus Candida, and the like. Specific preferred microorganisms which belong to these genera and which produce 5-oxoprolinase used in the present invention include Zygosaccharomyces rouxii, Saccharomyces cerevisiae, and Saccharomyces. -Pombe (Saccharomyces pombe), Pichia pastoris (Pichia pastoris), Candida antarctica (Candida antarctica), etc. are mentioned. More specifically, Zygosaccharomyces rouxii NBRC1876 strain can be mentioned. As a method for transforming the yeast, known methods, for example, a method using lithium acetate (Meth. Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003)) 481) -484) can be preferably used, but the present invention is not limited thereto. Transformation may be performed using any arbitrary method including a spheroplast method and a glass bead method.

  Examples of higher eukaryotic host cells include insect cells and mammalian cells. More specifically, silkworm cells (Sf9 cells), Chinese hamster ovarian cells (Chinese hamster ovarian cells: CHO cells) (both are distributed from the ATCC) and the like can be mentioned. As a method for transforming a mammalian cell, a calcium phosphate method, an electroporation method, a lipofection method, a microinjection method, a virus vector method, and the like can be used. You just have to make the switch.

  Examples of prokaryotic host cells include microorganisms belonging to the genus Escherichia, for example, Escherichia coli K-12 strain, preferably BL21 (DE3) strain, JM109 strain, and DH5α strain (all of which are Takara Bio Inc.). And the like). They can be transformed or transduced to obtain host cells (transformants) into which the DNA has been introduced. As a method for transferring a recombinant vector to such a host cell, for example, a method for transferring a recombinant DNA in the presence of calcium ions or an electroporation method can be used. Further, commercially available competent cells (for example, ECOS Competent Escherichia coli BL21 (DE3); manufactured by Nippon Gene Co., Ltd.) may be used.

(Production of 5-oxoprolinase of the present invention)
The 5-oxoprolinase of the present invention can be produced using various known enzyme production methods. For example, a 5-oxoprolinase-producing microorganism is cultured in a medium to produce a desired 5-oxoprolinase, and the enzyme can be collected from the culture or inside the cultured cells. Also, the present invention encompasses the 5-oxoprolinase gene of the present invention or a recombinant vector containing the 5-oxoprolinase gene, cultivates a microorganism having the ability to produce 5-oxoprolinase, and extracts 5-oxoprolinase from the culture. The enzyme can be collected.

  As the microorganism used for producing the present enzyme, any microorganism may be used, regardless of the presence or absence of genetic recombination, as long as it has the ability to produce the present enzyme. Microorganisms such as filamentous fungi and actinomycetes or animal cells can be used. Specific examples include Escherichia coli belonging to the genus Escherichia, yeast belonging to the genus Saccharomyces, filamentous fungi belonging to the genus Aspergillus, and mammalian cells. Specifically, for example, Aspergillus oryzae RIB40 (ATCC 42149) strain, Aspergillus sojae NBRC4239 strain and the like can be mentioned. It is also possible to use a mutant of the strain. As a method for obtaining a mutant of the strain, for example, a method of causing a mutation in the original strain using a radioisotope, ultraviolet light, nitrosoguanidine, or the like can be used.

  As a matter of course, the microorganism used for production of the present enzyme may have the ability to produce the present enzyme and also have the ability to produce another conventional 5-oxoprolinase. For example, when the enzyme is produced by recombination, a case may be assumed in which a microorganism used as a host also produces another 5-oxoprolinase of the host itself, which is originally possessed by the microorganism. In addition, a microorganism having both the ability to produce both types of 5-oxoprolinase containing the present enzyme and a microorganism having the ability to produce certain 5-oxoprolinase are originally added to a plurality of kinds of microorganisms containing the present enzyme. It is also envisioned that a gene encoding 5-oxoprolinase may be introduced to have the ability to produce multiple types of 5-oxoprolinase.

  In the case where 5-oxoprolinase is produced using a microorganism having the ability to produce a plurality of types of 5-oxoprolinase, culturing is performed under various culture conditions to obtain 5-oxoprolinase. It is also possible to produce the enzyme by arbitrarily changing the production ratio of -oxoprolinase, or to obtain only the desired 5-oxoprolinase using any known separation and purification technique, or Alternatively, a crude 5-oxoprolinase containing the present enzyme can be obtained without going through a special separation / purification step.

  The microorganism is cultured in a medium, and the present enzyme is collected from the resulting culture. As a culture method, a usual solid culture method or liquid culture method can be adopted.

  As the medium, a normal medium for culturing microorganisms, that is, any of a synthetic medium and a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients at an appropriate ratio.

  The carbon source may be any assimilable carbon compound, for example, glucose, maltose, starch hydrolyzate, glycerin, fructose, molasses and the like. The nitrogen source may be any available nitrogen compound, such as yeast extract, tryptone, peptone, soyton, meat extract, corn steep liquor, soy flour, soybean or wheat bran leaching solution, amino acids, ammonium sulfate, Ammonium nitrate and the like. Examples of the inorganic substance include various salts such as sodium chloride, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium carbonate, sodium acetate, calcium chloride and the like. If necessary, vitamins and antifoaming agents may be added. In addition, if there is a nutrient source or a component which can improve the production amount of 5-oxoprolinase of the present invention by adding it, it may be used alone or in combination. In the case of a transformed microorganism, a promoter-inducing substance such as galactose or isopropyl-1-thio-β-galactoside (IPTG) is added as necessary to produce 5-oxoprolinase. These medium components can be used alone or in appropriate combination.

  Culture conditions vary depending on the microorganism to be cultured. For example, the initial pH of the medium is adjusted to pH 5 to 10, the culture temperature is set to 20 to 40 ° C., the culture time is set to 10 to 25 hours, or 1 to 7 days. It is performed by culturing, shaking culturing, static culturing, or the like.

For example, 2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (w / v) KH as an example of a medium and culture conditions for culturing a microorganism of the genus Aspergillus. ₂ PO ₄ , 0.05% (w / v) MgSO ₄ .7H ₂ O, 0.1% (w / v) casamino acid, weakly acidic without pH adjustment at 30 ° C. and 120 rpm Shaking culture for 2 to 4 days is mentioned. As an example of a culture medium and culture conditions for culturing yeast, a 2% (w / v) bactopeptone, 1% (w / v) bactoeast extract, 2% (w / v) glucose medium was used. , 30 ° C., 200 rpm for 24 hours with shaking. For example, the cultivation of Escherichia coli is performed at a culture temperature of 10 to 42 ° C., preferably at a culture temperature of about 25 ° C. for 4 to 24 hours, more preferably at a culture temperature of about 25 ° C. for 4 to 8 hours. What is necessary is just to implement by culture | cultivation, static culture, etc. In culturing mammalian cells and insect cells, select a medium in which various sugars, amino acids, inorganic salts, vitamins, serum, growth hormone, etc. are mixed according to each cell, and stand at 37 ° C. for 1 to 7 days. Culture or shaking culture is mentioned. If necessary, the cells can be cultured in an incubator supplied with 5% carbon dioxide. Alternatively, a commercially available culture medium (manufactured by Nissui Pharmaceutical Co., Invitrogen Co., Ltd., Sigma-Aldrich Co., etc.) may be used.

  After the culture of the enzyme-producing microorganism, 5-oxoprolinase can be collected from the culture using conventional enzyme collecting means. For example, when 5-oxoprolinase is released outside the cells, the cells may be separated by filtration or centrifugation, and the culture solution containing 5-oxoprolinase may be recovered as a crude enzyme solution. it can.

  Alternatively, when 5-oxoprolinase is present in the cells, the cells are separated from the culture by, for example, filtration, centrifugation, or the like, and the 5-oxoprolinase is collected from the cells. Is preferred. For example, an ultrasonic crusher, a French press, a dyno mill, a method of destroying cells using a disrupting means such as a mortar, a method of lysing bacterial cell walls using a cell wall lysing enzyme such as lysozyme, Triton X-100, etc. A method of extracting an enzyme from cells using a surfactant or the like may be employed alone or in combination, and then insoluble substances may be removed by filtration or centrifugation to obtain a crude enzyme solution.

  In order to further purify 5-oxoprolinase from the obtained crude enzyme solution, an ordinary protein purification method can be used. Specifically, for example, ammonium sulfate precipitation, organic solvent precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography (for example, a nickel column, etc.), electrophoresis, etc. And these can be used alone or in appropriate combination.

(Advantage in the application of the 5-oxoprolinase of the present invention to various uses)
Since the 5-oxoprolinase of the present invention works well at pH 7.0 to 8.0, it can be suitably used for applications and use steps in a wide pH range including a neutral range. In addition, since the stable pH range of the 5-oxoprolinase of the present invention is wide, even when the enzyme is used as a quantitative reagent, a food processing aid, or the like, it is hardly affected by changes in the pH environment. Has advantages.

  In addition, the 5-oxoprolinase of the present invention has an optimum temperature range in a medium temperature range. Therefore, when the 5-oxoprolinase of the present invention is used as a reagent or in food processing, etc., a predetermined action and effect can be exerted without excessive heating, and can be incorporated into food or a reagent. When the reaction is carried out, it can be expected that thermal denaturation of various components in foods and reagents can be suppressed to a small extent.

  As described above, this enzyme works well without heating more than necessary, but has a certain heat resistance, and can coexist with the heating process used in food processing etc. Therefore, it is also suitable for practical use.

  As described above, the 5-oxoprolinase of the present invention has a certain heat resistance and is rapidly sterilized under heat sterilization conditions widely used in the production of ordinary seasonings, for example, under conditions such as a maximum temperature of 95 ° C. Therefore, there is no need to incorporate a special heating step for inactivating the enzyme during the production process.

  Furthermore, the present enzyme has a small Km value with respect to L-pyroglutamic acid, can catalyze the reaction more efficiently, and can be expected to shorten the reaction time and reduce the amount of enzyme used.

  As mentioned above, there has been no report that 5-oxoprolinase has been put to practical use for foods or reagents, but even when compared with the properties of known enzymes reported in some documents, the present invention 5-oxoprolinase is excellent in the above-mentioned points, and has an advantage in application to various uses.

(A device for the production of the 5-oxoprolinase of the present invention for application in various uses)
When producing the 5-oxoprolinase of the present invention for applications in various applications, various production steps devised by those skilled in the art with the intention of achieving better effects in subsequent steps. Can also be adopted.

  For example, powdering at a specific pH, immobilization to a sensor, etc., heating at a high temperature, etc., applications where removal or addition of a specific component is preferred, or formulation of this enzyme, If it is necessary to coexist with another enzyme or reagent component that is known to be suitable for a specific range of pH and temperature conditions that are different from the characteristics, if necessary, individually set the conditions in the upstream manufacturing process It can also be optimized.

(Production of food and drink using 5-oxoprolinase of the present invention)
By processing various foods and beverages using the 5-oxoprolinase of the present invention obtained as described above, umami-based foods and beverages containing higher concentrations of L-glutamic acid and umami were improved. Food and drink can be manufactured. Specifically, the food and drink containing L-pyroglutamic acid are brought into contact with or mixed with the present enzyme, or the food and drink containing L-pyroglutamic acid are fermented with a microorganism expressing the present enzyme to thereby provide the food and drink. By hydrolyzing L-pyroglutamic acid in the product and converting it into L-glutamic acid which is important as a taste component, it is possible to obtain a food or drink having an unprecedented excellent taste power. In the specification of the present application, increasing the concentration of L-glutamic acid and / or decreasing the concentration of L-pyroglutamic acid contained in food and drink is referred to as improving the umami of the food and drink. L-glutamic acid is converted to L-pyroglutamic acid by a non-enzymatic reaction in the presence of heat or pressure. Therefore, the present enzyme can also be used to maintain the umami of foods and drinks by hydrolyzing L-glutamic acid in foods and drinks that have been converted to L-pyroglutamic acid again into L-glutamic acid. Such maintenance of umami is also included in the improvement of umami referred to herein.

  That is, the present enzyme can be used as an umami improving agent for converting L-pyroglutamic acid without umami to L-glutamic acid, and the microorganism expressing the enzyme can be used as a microorganism for producing fermented foods rich in umami. The microorganism is preferably a microorganism that highly expresses the present enzyme or a microorganism that has high activity of the present enzyme.

  Foods and drinks to which the present enzyme can be applied are not particularly limited as long as they contain L-glutamic acid or L-pyroglutamic acid, and include, for example, vegetables such as tomato and Chinese cabbage, grains such as flour, mushrooms such as mushrooms and shiitake, and soybeans. And various fermented foods such as seaweeds such as kelp, seaweed, soy sauce, fish sauce, miso, bonito flakes, shaved flakes, and various processed foods including canned foods, retort foods, instant foods, and frozen foods.

  In one embodiment, the umami improving agent of the present invention comprises the enzyme in any suitable form. For example, the enzyme may be in the form of a dissolved state (including a diluted state and a concentrated state), a gel, a solid powder, a granule and the like. In another embodiment, the umami improving agent of the present invention contains a transformant or a transductant (microorganism) expressing the present enzyme. In this case, the microorganism expressing the present enzyme can be in any form such as a culture solution, a culture, a suspension, a solid powder, or a granule. The umami improving agent of the present invention may optionally contain other components such as excipients and stabilizers.

  The amount of 5-oxoprolinase and the details of the enzyme treatment conditions suitable for the production of foods and drinks with umami and improved umami taste depend on the food and drink to be processed and the concentration of L-glutamic acid required. May be set as appropriate. The progress of the enzymatic reaction of the present enzyme can be verified, and the evaluation of umami improvement after fermentation can be performed by measuring the concentration of L-glutamic acid in foods and drinks using known methods such as enzymatic methods or chromatography, A known sensory evaluation may be performed. In general, the threshold value of L-glutamic acid concentration at which humans feel umami is said to be about 0.03%, but is influenced by co-existing components, and the effect is considered to be synergistic. Since this enzyme works well at pH 7.0 to 8.0, it can be used in a wide pH range including a neutral range. In general, many foods have low pH, but the present enzyme can be widely used for processing such foods and drinks.

  When using this enzyme in food or drink, or when fermenting food or drink using microorganisms that produce this enzyme, it is necessary to obtain the enzyme from microorganisms that can be safely used in food or drink, It is also important to ferment foods and drinks using available microorganisms. Aspergillus oryzae and Aspergillus sojae, which are examples of microorganisms derived from this enzyme, have high enzyme productivity and high reliability for long-term use and high reliability of Aspergillus oryzae. And has long been used in the manufacture of brewed foods in Japan, such as miso, soy sauce, and sake. Such microorganisms can be suitably used in applications of processing food and drink.

(Production of L-pyroglutamic acid measuring reagent using 5-oxoprolinase of the present invention)
The 5-oxoprolinase of the present invention can also be used for measuring L-pyroglutamic acid. For example, a sample whose L-pyroglutamic acid content is to be measured is subjected to ultrafiltration to remove a protein component, and a solution appropriately diluted with a 50 mM Tris-HCl buffer (pH 8.0) is used as a measurement solution. Amicon Ultra-0.5 mL 10K (manufactured by Millipore) or the like can be used for ultrafiltration. The solution for measurement is reacted with the present enzyme in the presence of ATP, a monovalent cation and a divalent cation to cause a reaction from L-pyroglutamic acid to L-glutamic acid. For example, the concentration of L-pyroglutamic acid in a sample can be calculated by measuring the concentration by the method of measurement method 1. During the reaction, the enzyme concentration and the reaction time can be appropriately set. Further, the measuring reagent may contain any components such as preservatives, buffers, stabilizers, and other components not directly involved in the reaction of the present enzyme.

(Resolution of racemic body using 5-oxoprolinase of the present invention)
This enzyme uses L-pyroglutamic acid as a substrate, and D-pyroglutamic acid does not become a substrate. D-glutamic acid and D-pyroglutamic acid are important starting materials in the production of drugs, food additives, and pesticides, but their production requires organic synthesis and resolution of the racemic form. More expensive than racemic. The present enzyme hydrolyzes L-pyroglutamic acid as an impurity to obtain high-purity D-pyroglutamic acid, and hydrolyzes the obtained D-pyroglutamic acid by any method to convert D-glutamic acid. Obtainable.

  For example, L-pyroglutamic acid is specifically converted to L-glutamic acid by reacting a mixture of L-pyroglutamic acid and D-pyroglutamic acid with the present enzyme in a reaction solution according to assay method 1. As a method for separating L-glutamic acid and D-pyroglutamic acid, an ordinary compound separation method can be used. Specifically, for example, reverse phase chromatography, ion exchange chromatography, crystallization, and the like can be used. A method including such a series of steps is referred to herein as a method for resolving racemic pyroglutamic acid. In addition, modification such as derivatization can be appropriately performed in resolution of the racemate.

  Confirmation that L-pyroglutamic acid was converted to L-glutamic acid is performed by detecting the remaining amount of L-pyroglutamic acid, or measuring DL-pyroglutamic acid, DL-glutamic acid, and L-glutamic acid. Can be performed. For example, DL-pyroglutamic acid can be separated by liquid chromatography, after derivatization with bromothymol blue, the degree of ultraviolet absorption can be detected, and it can be calculated from a calibration curve using a standard. As shown in FIG. 1, it can be measured by an enzymatic method using L-glutamic acid oxidase. The L-pyroglutamic acid converted to L-glutamic acid by the present enzyme in the DL-pyroglutamic acid mixture is the concentration of L-glutamic acid generated after the reaction, or the concentration of DL-pyroglutamic acid before the reaction and the concentration of DL-pyroglutamic acid before the reaction. It can be calculated from the difference in pyroglutamic acid concentration.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by those examples.

(A. Acquisition of 5-oxoprolinase ortholog candidate protein from microorganism belonging to genus Aspergillus)
1. Search for 5-oxoprolinase ortholog candidate genes in Aspergillus soya Rat 5-oxoprolinase (NCBI Reference Sequence: NP_446356. 1) and its yeast ortholog, OXP1 / YKL215c (NCBI Reference Sequence: 70N1). Using sequence data, we attempted to search for genes with relatively high sequence identity from Aspergillus species belonging to filamentous fungi. The Blast program (tblastn) was used for the search. As a microorganism belonging to the genus Aspergillus DNA data, Aspergillus sojae (Aspergillus sojae) NBRC4239 shares genome sequence of (DDBJ / EMBL / GenBank DNA databases, Accession numbers for the 65 scaffold sequences; DF093557-DF093585, DNA RESEARCH 18,165-176, 2011) was used.

  As a result of the search, three genes (scaffold00004.806, scaffold00004.820, and scaffold00036.13022) shown in SEQ ID NOs: 4 to 6 were found as sequence regions having relatively high gene sequence identity.

  When sequence identity at the amino acid level was compared using genetic information processing software Genetyx network version 11.0.6 (Genetics), the scaffold00004.806, scaffold00004.820, and scaffold00036.16.102 were compared with those of rat 5- The sequence identities with oxoprolinase were 51.5%, 55.2% and 48.6%, respectively, and the sequence identities with the yeast ortholog OXP1 / YKL215c were 51.1%, 54.1% and 46, respectively. 0.9%.

  The sequence information of these three regions was used as a clue for searching for a 5-oxoprolinase ortholog candidate molecule in the genus Aspergillus, and the following operation was carried out.

2. Genomic DNA extraction from Aspergillus sojae Aspergillus soya NBRC4239 strain in a baffled Erlenmeyer flask in PD medium (2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (W / v) KH ₂ PO ₄ , 0.05% (w / v) MgSO ₄ .7H ₂ O, 0.1% (w / v) casamino acid) is shake-cultured at 30 ° C. for 1 to 2 days. The cells were collected by filtration. The collected cells were frozen in liquid nitrogen and ground using a mortar and pestle to obtain fine powdered cell fragments.

  About 300 μL of Nuclei Lysis Solution (promega) warmed to 65 ° C. was added with about 1 spatula of the powdered bacterial cells, vortexed, and incubated at 65 ° C. for 15 minutes. Next, RNase (manufactured by Nippon Gene Co., Ltd.) was added to a concentration of 50 μg / mL and incubated at 37 ° C. for 15 minutes. Thereafter, 125 μL of Protein Precipitation Solution (manufactured by Promega) was added, vortexed vigorously for 20 seconds, and centrifuged at 14,000 × g for 5 minutes. The obtained supernatant was mixed with 300 μL of isopropanol, and centrifuged at 11,000 × g for 3 minutes, and the precipitate obtained was rinsed with 70% ethanol to collect genomic DNA derived from Aspergillus soya NBRC4239 strain. TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) was added to the genomic DNA, and the mixture was incubated at 50 ° C. for 5 minutes to dissolve, thereby preparing a genomic DNA solution having a desired concentration.

3. Cloning of Ortholog Candidate Genes of 5-Oxoprolinase in Aspergillus soya Using the genomic DNA obtained above as a template, three candidate 5-oxoprolinase ortholog genes of Aspergillus soya (scafffold00004.806, scaffold00004.820, and scaffold00036.1302) was attempted to be amplified by PCR. Specifically, primers of SEQ ID NOs: 7 and 8 are used for scaffold00004.806, SEQ ID NOs: 9 and 10 are used for scaffold00004.820, and primers of SEQ ID NOS: 11 and 12 are used for scaffold00036.13022, and Prime Star Max (Takara) is used as a reaction reagent. The extension reaction was carried out using a Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit. The DNA fragments (approximately 4000 base pairs) considered to be the DNA fragments of the three types of 5-oxoprolinase ortholog candidate genes amplified by the above-mentioned PCR were separated in a 1% agarose gel, respectively, and QIAquick Gel Extraction Kit (QIAGEN) Was used for purification. In addition, pAsN, a plasmid vector derived from pUC19, was used as a vector for cloning. pAsN contains the Aspergillus soya-derived nitrate reductase gene (including the promoter region and the terminator region), the Aspergillus soya-derived tef1 gene promoter region (Ptef1, upstream 748 bp, SEQ ID NO: 15), and Aspergillus at the LacZ locus of pUC19. A vector prepared by introducing a terminator region (Talp, downstream 800 bp, SEQ ID NO: 16) of a soya-derived alkaline protease gene. Using pAsN as a template and the primers for inverse PCR of SEQ ID NOs: 13 and 14, the PCR was used to amplify the sequence of a plasmid containing Talp on the 5 'side and Ptefl on the 3' side as a DNA fragment. Was separated in a 1% agarose gel and purified using QIAquick Gel Extraction Kit.

  Then, the purified DNA fragments considered to be the DNA fragments of the three 5-oxoprolinase ortholog candidate genes were ligated to the DNA fragment derived from the plasmid pAsN using In-Fusion Advantage PCR Cloning Kit (manufactured by Clontech). did. As a result, a recombinant plasmid was obtained in which the DNA fragment considered to be the DNA fragment of the 5-oxoprolinase ortholog candidate gene amplified by PCR was connected between the Ptef1 region and the Talp region of the plasmid pAsN.

  After transforming Escherichia coli JM109 strain by a conventional method using these recombinant plasmids, each of the recombinant plasmids was extracted from the transformant, and each DNA sequence inserted into the plasmid was analyzed. As a result of the analysis, the DNA fragments considered to be the DNA fragments of the 5-oxoprolinase ortholog candidate gene were the sequences of scaffold00004.806, scaffold00004.820, and scaffold00036.10.302, which are present in the genomic DNA of Aspergillus soya NBRC4239, respectively. Was confirmed to match.

  In accordance with scaffold00004.806, scaffold00004.820, and scaffold00036.13022, which are the names of the gene sequences found in the database search, pAsN-4.806 pre mRNA and pAsN-4.820 pre mRNA were respectively associated with the plasmids. , PAsN-36.1302 pre mRNA.

4. Preparation of Expression Vector for Aspergillus spp.To introduce the ortholog candidate gene of 5-oxoprolinase obtained by the above method into genomic DNA of Aspergillus soya NBRC4239 strain, a plasmid for preparing a DNA fragment for transformation was prepared as follows. It was produced as follows. As a gene recombination site on the genomic DNA of Aspergillus soya NBRC4239 strain, an alkaline protease locus confirmed to have no effect on the growth of Aspergillus spp. Was selected.

  First, each 5-oxoprolinase ortholog candidate gene in the three types of recombinant plasmids (pAsN-4.806, pAsN-4.820, pAsN-36.1302) obtained by the above-described method is located upstream thereof. Amplification was performed by PCR using the primers of SEQ ID NOS: 17 and 18, together with the Ptefl region and the Talp region located downstream. Ptefl is a promoter for expressing a 5-oxoprolinase ortholog candidate gene, and Talp is a sequence complementary to the downstream region of the homologous recombination site of the gene.

  Further, as a sequence complementary to the upstream region of the homologous recombination site of the gene, a promoter region of an alkaline protease gene (Palp, 1515 bp upstream, 15% bp, 15% bp) using Aspergillus soja NBRC4239 strain genomic DNA as a template and primers of SEQ ID NOs: 19 and 20 as primers. SEQ ID NO: 21) was amplified by PCR. In addition, as a gene for complementing the auxotrophy of the host, which is used to confirm that a transformant was obtained in the transformation operation described below, Aspergillus soya NBRC4239 strain genomic DNA was used as a template, and SEQ ID NO: 22 was used. Using the primers No. 23 and No. 23, the peripheral region of the gene that complements the uridine requirement (pyrG, 1838 bp including 407 bp upstream, 896 bp coding and 535 bp downstream, SEQ ID NO: 24) was amplified by PCR.

  Furthermore, using the plasmid pUC19 (manufactured by Takara Bio Inc.) as a template and the primers for inverse PCR of SEQ ID NOS: 25 and 26, the 166th to 393rd bases of the plasmid pUC19 (the base numbers correspond to the restriction map attached to the reagent) (Including MCS, origin of replication, ampicillin resistance gene) was amplified by PCR. The extension reaction was performed using Prime Star Max (manufactured by Takara Bio Inc.) as a reaction reagent according to the attached protocol.

  Each DNA fragment amplified by the above PCR was separated in a 1% agarose gel, and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). When the DNA fragments are ligated to each other in the following operation, about 15 bp of the same sequence as an adjacent DNA fragment is added to each of the primers, and the primers are added using the In-Fusion Advantage PCR Cloning Kit (Clontech). , Can be connected. That is, a DNA fragment derived from pUC19 and a DNA fragment of Palp, a DNA fragment of Palp and a DNA fragment of pyrG, a DNA fragment of pyrG and a DNA fragment of Ptef1, a DNA fragment of Ptef1 and a 5-oxoprolinase ortholog candidate gene (scafffold00004.806) , Scaffold00004.820 or scaffold00036.1302), the DNA fragment of the 5-oxoprolinase ortholog candidate gene and the DNA fragment of Talp, and the DNA fragment of Talp and the DNA fragment derived from pUC19 can be ligated. As a result, as shown in FIG. 3, Palp, pyrG, Ptef1, 5-oxoprolinase ortholog candidate genes (scaffold00004.806, scaffold0000.820, or scaffold00036.1302) are located upstream of the multicloning site (MCS) of pUC19. And Talp sequences were sequentially ligated to obtain three types of recombinant plasmids.

  After transformation of Escherichia coli JM109 strain by a conventional method using the recombinant plasmid, the recombinant plasmid was extracted from the transformant, and each DNA sequence inserted into the plasmid was analyzed. The sequence of the enzyme ortholog candidate gene (scaffold00004.806, scaffold00004.820, or scaffold00036.13022) matches scaffold00004.806, scaffold00004.820, and scaf.fol with the sequences of scaffold00004.806 and scaffold00004.806 present in the genomic DNA of Aspergillus soya NBRC4239 strain. It was confirmed.

  Corresponding to the names of the derived gene sequences scaffold00004.806, scaffold00004.820, and scaffold00036.13022, the respective plasmids were respectively pAsAlpP-4.806 pre mRNA, pAsAlpP-4.820 pre mRNA and pAsAlp-36. 1302 pre mRNA.

5. Acquisition of CDS sequence of 5-oxoprolinase ortholog candidate gene and addition of His tag sequence in Aspergillus soya The above-mentioned three exon-intron structures of 5-oxoprolinase ortholog candidate genes were predicted. It was also thought that there were two exons and one intron (see FIG. 4). Specifically, out of 3,986 bases out of 3,986 bases of scaffold00004.806 (ortholog candidate gene 1, SEQ ID NO: 4), base position 2524 out of 3,899 bases of scaffold0000.820 (ortholog candidate gene 2, SEQ ID NO: 5) From No. 2579 to No. 3579 of the 3917 bases of scaffold00036.13022 (ortholog candidate gene 3, SEQ ID NO: 6) were predicted to be introns, respectively.

  In order to obtain a sequence excluding introns (hereinafter referred to as CDS), a region predicted to be an exon was amplified by PCR, and exon 1 and exon 2 were ligated. That is, using the plasmid pAsAlpP-4.806 pre mRNA prepared above as a template and the primers of SEQ ID NOS: 17 and 27, the exon 1 region of scaffold00004.806 to which Ptef1 was linked on the upstream side was compared with SEQ ID NO: 30 and The exon 2 region of scaffold00004.806 was amplified by PCR using 31 primers. Using the pAsAlpP-4.820 pre mRNA as a template and the primers of SEQ ID NOs: 17 and 28, the exon 1 region of scaffold00004.820 to which Ptefl was linked on the upstream side was used, and the primers of SEQ ID NOs: 32 and 33 were used. The exon 2 region of scaffold00004.820 was amplified by PCR. Using the pAsAlpP-36.1302 pre mRNA as a template and the primers of SEQ ID NOS: 17 and 29, the exon 1 region of scaffold 00036.1302 with Ptefl linked upstream was used, and the primers of SEQ ID NOs: 34 and 35 were used. The exon 2 region of scaffold00036.1302 was amplified by PCR. Further, using pAsAlpP-4.820 pre mRNA as a template, the sequence of a plasmid containing Talp on the 5 ′ side and Palp and pyrG on the 3 ′ side was amplified by PCR using primers for inverse PCR of SEQ ID NOS: 13 and 23. A DNA fragment was obtained. An extension reaction was performed using Prime Star Max as a reaction reagent according to the attached protocol.

  The DNA fragment amplified by the PCR was separated in a 1% agarose gel, purified by QIAquick Gel Extraction Kit, and the exon 1 region in which Ptef1 was linked to the upstream side for each of the three types of 5-oxoprolinase ortholog candidate genes. And the DNA fragment of the exon 2 region were ligated to a DNA fragment derived from a plasmid using In-Fusion Advantage PCR Cloning Kit, whereby the intron predicted sequences of ortholog candidate genes of three types of 5-oxoprolinase were ligated. Thus, a plasmid incorporating the sequence excluding the above was obtained.

  The recombinant plasmids were associated with pAsAlpP-4.806 CDS, pAsAlpP-4.820 CDS, corresponding to the names of gene sequences scaffold00004.806, scaffold00004.820, and scaffold00036.102302 found by database search. And pAsAlpP-36.1302 CDS.

  Next, in order to simplify the purification process of the expressed protein, a plasmid for expressing a His-tag fusion protein was constructed. Using the three types of plasmids pAsAlpP-4.806 CDS, pAsAlpP-4.820 CDS, and pAsAlpP-36.1302 CDS prepared above as templates, SEQ ID NOS: 36 and 31, SEQ ID NOs: 37 and 33, SEQ ID NOs: 38 and 38, respectively. Using 35 primers, a 24-base sequence encoding eight histidine sequences was added to the 5 ′ side of the 5-oxoprolinase ortholog candidate gene (scaffold00004.806, scaffold00004.820, and scaffold00036.1302) CDS. Three types of DNA fragments were amplified by PCR.

  Further, using pAsAlpP-4.806 pre mRNA as a template, the sequence of the plasmid containing Talp on the 5 ′ side and Palp, pyrG and Ptef1 on the 3 ′ side using the primers for inverse PCR of SEQ ID NOs: 13 and 14 was subjected to PCR. Amplified by An extension reaction was performed using Prime Star Max as a reaction reagent according to the attached protocol.

  Each of the DNA fragments amplified by the above PCR was separated in a 1% agarose gel, purified using a QIAquick Gel Extraction Kit, and 8 histidines were added to the 5 'side of the CDS of the three candidate 5-oxoprolinase ortholog candidate genes. A DNA fragment derived from a plasmid containing Talp on the 5 'side and Palp, pyrG and Ptef1 on the 3' side using In-Fusion Advantage PCR Cloning Kit, each of the DNA fragments to which the 24-base sequence encoding the above sequence was added. Oxoprolinase ortholog candidate gene in which a 24-base sequence encoding a sequence of eight palidines, Palp, pyrG, Ptef1, was added upstream of the multicloning site (MCS) of pUC19. (Scaffold00004.806, scaffold00004.820, or scaffold00036.1302) CDS, and the sequence of Talp acquires the three recombinant plasmids coupled in order.

  After transforming Escherichia coli JM109 strain by a conventional method using the recombinant plasmid, the recombinant plasmid was extracted from the transformant, and each DNA sequence inserted in the plasmid was analyzed. As a result of the analysis, the CDS of a 5-oxoprolinase ortholog candidate gene to which a 24-base sequence encoding a sequence of Palp, pyrG, Ptefl, and eight histidines was added, and the respective sequences of Talp were the genome of Aspergillus soya NBRC4239 strain. It was confirmed that the sequence matched the sequence on the DNA.

  As described above, the His tag sequence is added to the three 5-oxoprolinase ortholog candidate genes (scaffold00004.806, scaffold00004.820, and scaffold00036.13022), and it is predicted that the gene is an intron. An operation to remove the sequence to be performed was performed.

  Plasmids containing each of the three genes subjected to the above-described operations were subjected to pAsAlpP-His-4.806 CDS and pAsAlpP- corresponding to the names of the derived gene sequences scaffold00004.806, scaffold00004.820, and scaffold00036.1302. His-4.820 CDS and pAsAlpP-His-36.1302 CDS.

6. Acquisition of 5-oxoprolinase ortholog candidate gene transformant of Aspergillus soya NBRC4239 strain Using pAsAlpP-His-4.806 CDS prepared as described above as a template, primers of SEQ ID NOS: 39 and 40, and , Ptef1, and a DNA fragment to which the CDS region of 5-oxoprolinase ortholog candidate gene scaffold00004.806 added with a His tag sequence and a Talp were ligated, using pAsAlpP-His-4.820 CDS as a template, SEQ ID NO: 41 and SEQ ID NO: 41. Using 40 primers, the CDS region of 5-oxoprolinase ortholog candidate gene scaffold0000.820 to which Palp, pyrG, Ptefl, and His tag sequences were added, and Talp were ligated. Using the primers of SEQ ID NOs: 41 and 42, a 5-oxoprolinase ortholog candidate gene scafffold00036.13022 to which a Palp, pyrG, Ptefl, and His tag sequence were added using the pAsAlpP-His-36.1302 CDS as a template and the CDS as a template. And the DNA fragment to which the Talp was linked were amplified by PCR, respectively. The elongation reaction was carried out using KOD Dash Neo (manufactured by TOYOBO) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit.

  The above three amplified DNA fragments (for each of the 5-oxoprolinase ortholog candidate genes, the CDS region of the 5-oxoprolinase ortholog candidate gene added with a Palp, pyrG, Ptefl, and His tag sequence, and Talp were ligated). The DNA fragment) was dissolved in TE after ethanol precipitation to prepare a DNA solution of a desired concentration, and a pyrG-disrupted strain (48 bp upstream of the pyrG gene, 896 bp coding region, and 896 bp downstream of the pyrG gene) was prepared from Aspergillus soya NBRC4239 by the following procedure. 240 bp deletion strain).

First, Aspergillus soya NBRC4239 strain (conidia of a pyrG-disrupted strain derived from Aspergillus soya NBRC4239 strain cultured on a Maltz plate, about 1/10 in a 9 cm petri dish) to be used for transformation was suspended in a 0.01% Tween80 solution. PD medium supplemented with uridine to a concentration of 10 mM (2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (w / v) KH ₂ PO ₄ , 0.05% ( (w / v) 150 mL of MgSO ₄ .7H ₂ O, 0.1% (w / v casamino acid) and incubating with shaking for about 18 hours. After the culture, the cells were collected by filtration, and a DNA fragment amplified by PCR for transformation (Palp, pyrG, Ptef1, and a His tag sequence were added to each of the three 5-oxoprolinase ortholog candidate genes) Transformation was performed by the protoplast-PEG method using a TE solution (about 10 μg of DNA / 20 μL) of the CDS region of the 5-oxoprolinase ortholog candidate gene and the DNA fragment to which Talp was linked.

  In each of the obtained transformants, a sequence in which the CDS region of a 5-oxoprolinase ortholog candidate gene in which pyrG, Ptefl, and His tag sequences are added to the alkaline protease locus of NBRC4239 strain is introduced. . These strains can be grown in a uridine-free medium by introducing pyrG, a gene that complements uridine auxotrophy, so that strains into which the target gene has been introduced can be selected.

  Furthermore, it was confirmed that the transformant was a strain into which the gene was introduced at the target locus by colony PCR. That is, the conidia of the transformant scraped with a toothpick are suspended in TE and used as a template for PCR, and the primers of SEQ ID NOs: 43 and 44 are used to convert the alkaline protease locus from the 237th base upstream to the 277th base downstream. The sandwiched region was amplified by PCR. KOD FX (manufactured by TOYOBO) was used as a reaction reagent, and after a heat treatment at 94 ° C. for 5 minutes, an extension reaction was performed according to the attached protocol. As a result, in the transformant strain, pyrG, Ptef1, and His tag sequences were added in place of the alkaline protease gene (alp, 1389 bp, SEQ ID NO: 45) at the alkaline protease locus of the genomic DNA of Aspergillus soya NBRC4239 strain. -A homologous recombination at the alkaline protease locus was detected in the transformant by detecting a signal of about 7 kbp indicating the presence of DNA (about 6.6 kbp) to which the CDS region of the oxoprolinase ortholog candidate gene was linked. I confirmed that it was done.

  Using the above-mentioned method, obtaining transformants (genes of Aspergillus genus transformed with 5-oxoprolinase ortholog candidate genes) derived from the three candidate genes of 5-oxoprolinase ortholog derived from Aspergillus soya. Was completed. The transformants of the genus Aspergillus by the respective 5-oxoprolinase ortholog candidate genes correspond to NBRC4239-His-4.806, respectively, corresponding to the names of the derived genes, scaffold00004.806, scaffold0000.820, and scaffold00036.1302. The strains were named CDS strain, NBRC4239-His-4.820 CDS strain, and NBRC4239-His-36.1302 CDS strain.

7. Acquisition of 5-oxoprolinase ortholog candidate protein derived from Aspergillus soya 5-oxoprolinase candidate introduced into the genomic DNA of a transformant of the genus Aspergillus by the 5-oxoprolinase ortholog candidate gene obtained as described above. A 24-base sequence encoding a sequence of eight histidines was added upstream of the gene. By culturing a transformant of the genus Aspergillus, a tag of eight histidines was fused to the N-terminus. An oxoprolinase ortholog candidate protein can be expressed.

The N-terminal His tag-fused 5-oxoprolinase ortholog candidate protein can be purified by a conventional method using a Ni column. That is, transformants of the genus Aspergillus (NBRC4239-His-4.806 CDS strain, NBRC4239-His-4.820 CDS strain, and NBRC4239-His-36.1302 CDS strain) by the 5-oxoprolinase ortholog candidate gene. The conidia were transferred to a PD medium (2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (w / v) KH ₂ PO ₄ , 0.05% (w / v) MgSO ₄・ Inoculate in 7H ₂ O, 0.1% (w / v casamino acid) at 1 × 10 ⁵ cells / mL, culture at 30 ° C. and 120 rpm for 2 days, and collect cells by filtration did.

  The collected bacterial cells were frozen in liquid nitrogen, and ground using a mortar and pestle to obtain fine powdery bacterial cell fragments, which were extracted with 1/10 to 1/20 of the culture solution containing 2 mM PMSF. The suspension was suspended in a buffer (50 mM Tris, 300 mM NaCl, 20% (w / v) glycerol, 0.5 mM L-pyroglutamic acid, pH 8.0). The centrifuged supernatant of the obtained suspension was filtered through a 0.2 μm filter to obtain a cell extract (crude enzyme solution).

  Thereafter, a part of the bacterial cell extract was applied to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind the His-tag fused 5-oxoprolinase ortholog candidate protein, and washed with an extraction buffer containing 20 mM imidazole. A fraction containing a 5-oxoprolinase ortholog candidate protein eluted with an extraction buffer containing 250 mM imidazole was obtained. If necessary, the fraction containing the 5-oxoprolinase ortholog candidate protein was further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The fraction containing the 5-oxoprolinase ortholog candidate protein was dialyzed against an extraction buffer to obtain a purified enzyme solution.

  By the above-mentioned enzyme purification operation, 100 to 300 μg of 5-oxoprolinase ortholog candidate protein was obtained per 100 mL of culture. Hereinafter, each of the 5-oxoprolinase ortholog candidate proteins is associated with the name of the derived gene (scaffold00004.806, scaffold00004.820, and scaffold00036.13022), as 4.806, As4.8820, and As36.1302. Notation.

8. Confirmation of Purification Degree of Each 5-Oxoprolinase Ortholog Candidate Protein by SDS-PAGE Cell Extract from the Transformant Containing As4.806, As4.820, and As36.1302 (5-oxoprolinase Ortholog Candidate) The protein crude enzyme solution) and a part of the purified protein (purified enzyme solution) were subjected to SDS-polyacrylamide gel electrophoresis. The electrophoresis was performed according to the method of Laemmli, to which a Tris SDSβME sample treatment solution (manufactured by Cosmo Bio) was added, and a heat treatment was performed at 100 ° C. for 5 minutes. Electrophoresis was performed using Tris-Glycine SDS Running buffer (manufactured by Invitrogen). The result of performing CBB staining of the gel after the completion of the electrophoresis is shown in FIG. This confirmed that the 5-oxoprolinase ortholog candidate proteins (As4.806, As4.820, As36.1302) were purified with high purity. The molecular weight was about 140 kDa (portion marked with a black triangle), which was consistent with the size calculated from the sequence of the gene disclosed in the present invention.

  The amount of each protein was subjected to electrophoresis with 500 μg of wet bacteria in a cell extract of an enzyme derived from Aspergillus soya, and 1 μg of a purified protein solution of an enzyme derived from Aspergillus soya. For reference, for the yeast ortholog OXP1 / YKL215c, which is a known 5-oxoprolinase, a cell extract and a purified protein solution are prepared in the same manner as described below, and 100 μL of the cell extract and 1 μg of the purified protein are electrophoresed. Was served.

(B. Acquisition of yeast-derived 5-oxoprolinase)
As a reference enzyme for comparing various properties of the 5-oxoprolinase of the present invention with a known enzyme, yeast-derived OXP1 / reported as an ortholog protein of 5-oxoprolinase derived from a microorganism as in the present invention. YKL215c was prepared. In addition, about acquisition of the said 5-oxoprolinase from yeast, FEMS Yeast Res. (Vol. 10, pages 394 to 401, 2010) and the like, and various operations were performed.

1. Genomic DNA Extraction from Saccharomyces cerevisiae Saccharomyces cerevisiae strain INVsc1 (manufactured by Invitrogen) was converted to YPD medium (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w) / V) D-glucose) overnight, followed by shaking culture, followed by centrifugation at 13,000 xg for 1 minute to collect the cells. After the collected cells were washed with sterile milli-Q water, 300 μL of Nuclei Lysis Solution (manufactured by Promega) and glass beads for multi-beads shocker (manufactured by Yasui Kikai) having a diameter of 0.5 mm and a diameter of 0.5 mm were added, and a bead shocker ( (Yasui Kikai Co., Ltd.) (2000 rpm, crushed for 60 seconds, allowed to stand for 30 seconds, and repeated 8 times). Then, after incubating at 65 ° C. for 15 minutes, RNase (Nippon Gene) was added at 50 μg / mL, and the mixture was incubated at 37 ° C. for 15 minutes. 125 μL of Protein Precipitation Solution (Promega) was added, vortexed vigorously for 10 seconds, and centrifuged at 17,000 × g for 3 minutes.

  The obtained supernatant was mixed with 300 μL of isopropanol, and the precipitate obtained by centrifugation at 17,000 × g for 10 minutes was rinsed with 70% ethanol to collect yeast-derived genomic DNA. An appropriate amount of TE was added to the collected genomic DNA, and the mixture was incubated at 50 ° C. for 5 minutes to dissolve, thereby preparing a genomic DNA solution having a desired concentration.

2. Cloning of Yeast OXP1 / YKL215c and Preparation of Yeast Transformant Using genomic DNA of Saccharomyces cerevisiae strain INVsc1 as a template, primers shown in SEQ ID NOs: 46 and 47, and a recognition sequence of histidine and a restriction enzyme SacI on the 5 'side A coding region (3861 bp, SEQ ID NO: 48) of OXP1 / YKL215c, to which a 24-base sequence encoding eight sequences and a recognition sequence for a restriction enzyme XhoI was added to the 3 ′ side, was amplified by PCR. An extension reaction was performed using Prime Star Max (manufactured by Takara Bio Inc.) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit.

  The DNA fragment considered to be OXP1 / YKL215c to which a 24-base sequence encoding a restriction enzyme recognition sequence and eight histidine sequences amplified by the PCR was added, separated in a 1% agarose gel, and then separated in a QIAquick Gel Extraction Kit. (QIAGEN), cut with restriction enzymes SacI (Takara Bio Inc.) and XhoI (BioLabs), separated in 1% agarose gel, and again using QIA quick Gel Extraction Kit. Purified. The purified DNA fragment was ligated to the SacI-XhoI site of a yeast expression plasmid pYES2 / CT (Invitrogen) using ligation high ver2 (TOYOBO) to prepare a yeast transformant. A recombinant plasmid pYES2-His-YKL215c was constructed.

  Next, Escherichia coli JM109 strain was transformed by a conventional method using the above-mentioned recombinant plasmid pYES2-His-YKL215c, and the above-mentioned recombinant plasmid pYES2-His-YKL215c was extracted from the transformant and inserted into the plasmid. DNA sequence analysis was performed. As a result of the analysis, it was confirmed that the DNA fragment considered to be OXP1 / YKL215c to which the 24-base sequence encoding the eight histidine sequences had been added was identical to the sequence of Saccharomyces cerevisiae OXP1 / YKL215c.

  Then, S. cerevisiae was expressed using the plasmid pYES2-His-YKL215c. c. The Saccharomyces cerevisiae strain INVsc1 was transformed using the EasyComp ™ Transformation kit (manufactured by Invitrogen) according to the attached manual, and the expression of URA3, a uracil synthesis gene encoded by pYES2 / CT, resulted in a medium without uracil. A yeast transformant using OXP1 / YKL215c was selected on the basis of the fact that uracil is not required by being able to grow.

3. Obtaining Yeast OXP1 / YKL215c Protein A 24-base sequence encoding a sequence of eight histidines is located upstream of the OXP1 / YKL215c gene in the plasmid introduced into the yeast transformant with OXP1 / YKL215c obtained as described above. By culturing the yeast transformant in the presence of galactose, expression of the OXP1 / YKL215c protein in which eight histidine tags are fused to the N-terminal can be induced. The N-terminal His tag-fused OXP1 / YKL215c protein can be purified by a conventional method using a Ni column.

  That is, the above-mentioned yeast transformant by OXP1 / YKL215c was transformed into a liquid medium for preculture (0.67% (w / v) amino acid-free yeast nitrogen base (manufactured by Becton Dickinson), 0.192% (w / V) A uracil-free yeast synthetic dropout medium additive (Sigma), 2.0% (w / v raffinose) was cultured with shaking at 30 ° C for 24 hours. Then, a liquid medium for main culture (0.67% (w / v) amino acid-free yeast nitrogen base, 0.192% (w / v) uracil-free yeast synthetic dropout medium supplement, 2.5% (W / v) D-galactose, 0.75% (w / v) raffinose), diluted 10-fold, cultured at 30 ° C. for 12 to 18 hours, and centrifuged at 500 × g for 1 to 5 minutes. Was recovered. The collected cells were extracted with an extraction buffer (50 mM Tris, 300 mM NaCl, 20% (w / v) glycerol, 0.5 mM L-pyroglutamic acid, pH 8.0) containing 1/10 to 1/20 the volume of the culture solution and containing 2 mM PMSF. 0) and crushed using a multi-beads shocker. Alternatively, the collected cells are frozen in liquid nitrogen, ground using a mortar and a pestle to form fine powdery cell pieces, and 1/10 to 1/25 of the 2 mM PMSF in the culture solution is added. It was suspended in the extraction buffer containing.

  The centrifugal supernatants of the two kinds of disrupted cell suspensions were filtered with a 0.2 μm filter to obtain a cell extract (crude enzyme solution). Thereafter, the bacterial cell extract is applied to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind the His tag-fused OXP1 / YKL215c protein, washed with an extraction buffer containing 20 mM imidazole, and eluted with an extraction buffer containing 250 mM. OXP1 / YKL215c protein-containing fraction was obtained.

  If necessary, the OXP1 / YKL215c protein-containing fraction was further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The OXP1 / YKL215c protein-containing fraction was dialyzed against an extraction buffer to obtain a purified enzyme solution. By the above-mentioned enzyme purification, 55 to 200 μg of OXP1 / YKL215c protein (hereinafter referred to as “YKL215c”) was obtained per 100 ml of culture.

4. Confirmation of Purification Degree of OXP1 / YKL215c by SDS-PAGE SDS-polyacrylamide gel was obtained by extracting a cell extract (crude enzyme solution) derived from a yeast transformant by OXP1 / YKL215c and a part of the purified protein (purified enzyme solution). It was subjected to electrophoresis. The electrophoresis was performed according to the method of Laemmli, to which a Tris SDSβME sample treatment solution (manufactured by Cosmo Bio) was added, and a heat treatment was performed at 100 ° C. for 5 minutes. Electrophoresis was performed using Tris-Glycine SDS Running buffer (manufactured by Invitrogen). The result of performing CBB staining of the gel after the completion of the electrophoresis is shown in FIG. This confirmed that YKL215c was purified with high purity. The molecular weight was about 140 kDa, which was consistent with the size calculated from the amino acid sequence of YKL215c (1286 amino acids, SEQ ID NO: 55).

  The amount of each protein was equivalent to 100 μL of the culture solution in the cell extract, and 1 μg of the protein in the purified protein solution was subjected to electrophoresis.

(Confirmation of the activity of a candidate protein of 5-oxoprolinase ortholog derived from Aspergillus soja expressed in C. Aspergillus)
Detection of L-glutamic acid producing activity by reaction of 1.5-oxoprolinase ortholog candidate protein with L-pyroglutamic acid 7. 5-oxoprolinase ortholog candidate proteins (As4.806, As4.820, and As36.1302) derived from Aspergillus soya obtained by the above method, and B. 3. 5-oxoprolinase activity was confirmed by using the yeast-derived 5-oxoprolinase yeast ortholog protein YKL215c obtained according to the following procedure.

  0.08 mL of an L-pyroglutamic acid solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, pH 8.0] and the above-mentioned 5-oxoprolyl diluted to 200 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) 0.02 mL of the solution of the candidate protein for the ortholog of the enzyme (As4.806, As4.820, and As36.1302) was mixed, and the enzyme reaction was performed at 37 ° C. for 1 hour. For reference, 0.08 mL of L-pyroglutamic acid solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, pH 8.0] and YKL215c solution 0 diluted to 200 μg / mL with 50 mM Tris-HCl buffer (pH 8.0) .02 mL was mixed and an enzyme reaction was carried out at 37 ° C. for 1 hour. After heating the reaction solution at 100 ° C. for 3 minutes to stop the enzyme reaction, the concentration of L-glutamic acid in the enzyme reaction solution was measured.

  For quantification of L-glutamic acid, "Yamasa L-glutamic acid detection kit II" (manufactured by Yamasa Shoyu Co., Ltd.) was used, and 0.02 mL of the enzyme reaction mixture was mixed with 0.15 mL of the L-glutamic acid detection coloring reagent of the kit. Then, the absorbance at 600 nm after the reaction at room temperature for 20 to 30 minutes was measured. The L-glutamic acid concentration in the enzyme reaction solution was calculated using a calibration curve based on an L-glutamic acid standard, and the presence or absence of enzyme activity was determined from the L-glutamic acid concentration in the reaction solution.

  As a result, when any of the 5-oxoprolinase ortholog candidate proteins (As4.806, As4.820, and As36.1302) derived from Aspergillus soya, and the 5-oxoprolinase yeast ortholog protein YKL215c were used in the reaction, The L-glutamic acid in the reaction solution was below the detection limit (5 μM). That is, most of L-pyroglutamic acid (0.5 mM) added to the reaction solution was not converted into L-glutamic acid, and 5-oxoprolinase activity could not be detected.

2. Detection of reaction and activity between 5-oxoprolinase ortholog candidate protein and L-pyroglutamic acid in the presence of ATP and metal ions In response to the above results, the present inventors converted L-pyroglutamic acid to L-glutamic acid. It was expected that there might be a reaction mechanism involving phosphorylation in the conversion of. An ATP-requiring enzyme may require the presence of a metal ion for its catalytic activity. It was also considered that the reason why conversion to L-glutamic acid could not be detected by the reaction of the candidate protein for 5-oxoprolinase ortholog with L-pyroglutamic acid was a lack of cofactor in the reaction system. Then, next, the reaction between the 5-oxoprolinase ortholog candidate protein and L-pyroglutamic acid as a substrate was performed in the presence of ATP and metal ions.

  Specifically, the above-described A.I. 7. 5-oxoprolinase ortholog candidate proteins (As4.806, As4.820, and As36.1302) derived from Aspergillus soya obtained by the above method, and B. 3. 5-oxoprolinase activity was confirmed by using the yeast-derived 5-oxoprolinase yeast ortholog protein YKL215c obtained according to the following procedure. In addition, regarding the activity measurement of the enzyme, FEMS Yeast Res. (Vol. 10, pp. 394-401, 2010). The enzymatic activity can be calculated by measuring enzymatically the degree of increase in L-glutamic acid generated by hydrolyzing the intramolecular amide bond of L-pyroglutamic acid.

  0.08 mL of a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid (manufactured by MP Biomedicals), 6.25 mM ATP (manufactured by ORIENTAL YEAST), 12.5 mM MgCl2, 187.5 mM KCl, pH 8.0], 0.02 mL of each 5-oxoprolinase ortholog candidate protein (As4.806, As4.820, and As36.1302) solution (diluted to 200 μg / mL using a 50 mM Tris-HCl buffer (pH 8.0)) Were mixed, and an enzyme reaction was carried out at 37 ° C. for 1 hour. Since L-pyroglutamic acid shows strong acidity, it was dissolved in a buffer solution, adjusted to a pH at the time of use, and then mixed with another reagent to prepare a substrate solution. Specifically, 10 mmol of L-pyroglutamic acid and 10 mmol of Tris were dissolved in about 80 mL of water, adjusted to pH 8 with hydrochloric acid, and then adjusted to 100 mL. Similarly, also in the following Examples, the reagent in which L-pyroglutamic acid was dissolved in various buffers was used after dissolving L-pyroglutamic acid in various buffers and adjusted to the pH at the time of use. .

  In addition, the reagents described in the present specification are manufactured by Wako Pure Chemical Industries, Nacalai Tesque, Sigma, Dojindo Laboratories, Becton Dickinson, and Nippon Pharmaceutical Co., unless otherwise specified. And reagents manufactured by Takara Bio Inc., and those having a distinction in purity used reagents of a special grade.

For reference, 0.08 mL of a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0] and a YKL215c solution (50 mM Tris- 0.02 mL of HCl buffer (diluted to 200 μg / mL with pH 8.0) was mixed, and an enzyme reaction was performed at 37 ° C. for 1 hour.

  After the reaction, each enzyme reaction solution was heated at 100 ° C. for 3 minutes to stop the enzyme reaction, and then the concentration of L-glutamic acid in the enzyme reaction solution was measured.

For quantification of L-glutamic acid, "Yamasa L-glutamic acid detection kit II" (manufactured by Yamasa Shoyu Co., Ltd.) was used, and 0.15 mL of the L-glutamic acid detection coloring reagent of the kit was added to 0.02 mL of the enzyme reaction solution. After mixing, the absorbance at 600 nm after the reaction at room temperature for 20 to 30 minutes was measured (measured value). Further, instead of the substrate solution, 0.08 mL of 50 mM Tris-HCl buffer (pH 8.0) was used, and the buffer and each of the 5-oxoprolinase ortholog candidate proteins (As4.806, As4.820, As4.820, And As36.1302), or a yeast ortholog protein YKL215c solution (diluted to 200 μg / mL using a 50 mM Tris-HCl buffer (pH 8.0)), and reacted at 37 ° C. for 1 hour. The L-glutamic acid concentration in the solution was measured (blank value). Based on the obtained "blank value" and "measured value", the activity of 5-oxoprolinase was calculated based on the following formula. The unit of the enzyme activity was defined as 1 unit (U) of the amount of the enzyme producing 1 μmol of L-glutamic acid per minute under the above-mentioned conditions.

  [Glu] in the formula is the L-glutamic acid concentration (measured value) in the enzyme reaction solution, and [Glu blank] is the L of the reaction solution obtained by adding a 50 mM Tris-HCl buffer (pH 8.0) instead of the substrate solution. -Glutamic acid concentration (blank value), [enzyme] represents the concentration of 5-oxoprolinase in the reaction solution, and 60 represents the fraction of one hour.

Table 1 shows the results of examining the activity of 5-oxoprolinase calculated for each Aspergillus soya-derived 5-oxoprolinase ortholog candidate protein and yeast-derived ortholog protein YKL215c based on the above-described activity measurement method.

  The above C.I. 1. As shown in Table 1, the L-glutamic acid concentration in the reaction solution was lower than the detection limit in the reaction of each purified protein with the Tris-HCl buffer, but as shown in Table 1, 5 L-glutamic acid derived from Aspergillus soya When a purified protein of -oxoprolinase candidate protein As4.806 and As4.820 was reacted with a substrate solution, the concentration of L-glutamic acid in the reaction solution was reduced in the same manner as the known 5-oxoprolinase YKL215c. It increased to 100 μM or more.

  From this, YKL215c, As4.806, and As4.820 are reactions that convert L-pyroglutamic acid to L-glutamic acid in the presence of ATP, potassium ions that are monovalent cations, and magnesium ions that are divalent cations. , Ie, 5-oxoprolinase activity.

  On the other hand, in Aspergillus soya-derived 5-oxoprolinase candidate protein As36.1302, by reaction with L-pyroglutamic acid even in the presence of ATP, potassium ion which is a monovalent cation, and magnesium ion which is a divalent cation. The L-glutamic acid concentration in the reaction solution did not increase, and no 5-oxoprolinase activity could be detected.

  The reason that the 5-oxoprolinase activity of the 5-oxoprolinase ortholog candidate protein As36.1302 was not detected was also expected to be that the enzyme titer was low and the amount of enzyme was insufficient. The amount of enzyme was further increased, and the presence or absence of activity was similarly confirmed.

A solution of the above-mentioned 5-oxoprolinase ortholog candidate protein (As36.1302) from Aspergillus soya diluted to 100, 200, 500, and 1000 μg / mL with 50 mM Tris-HCl buffer (pH 8.0), Tris-HCl buffer The above-mentioned 5-oxoprolinase ortholog (As4.806, As4.820) solution prepared from Aspergillus soya diluted to 100, 200, 500, and 1000 μg / mL with the liquid (pH 8.0) was prepared, and each solution was 0.02 mL. Was mixed with 0.08 mL of a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0], and the enzyme was incubated at 37 ° C. for 1 hour. The reaction was performed. As described above, C.I. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzyme reaction was measured, and the presence or absence of the activity of the enzyme was examined. The relationship between the amount of protein added and the concentration of L-glutamic acid in each of the 5-oxoprolinase orthologs and 5-oxoprolinase ortholog candidate proteins derived from Aspergillus soya was as shown in FIG.

  As shown in FIG. 6, in As4.806 (plotted with white triangles in the figure) and As4.820 (plotted with white circles in the figure), the L-glutamic acid concentration increased depending on the added protein concentration. .

  On the other hand, in the reaction system to which the 5-oxoprolinase ortholog candidate protein As36.1302 (plotted with a white square in the figure) was added, even if the amount of addition was increased to 200 μg / mL, L-glutamic acid in the reaction solution was not added. The concentration did not increase and no 5-oxoprolinase activity could be detected.

  Thus, of the three types of proteins obtained from the three candidate genes having the same degree of sequence identity by the above-described series of techniques, As4.806 and As4.820 were Aspergillus soya of 5-oxoprolinase. It turned out to be an ortholog.

  The amino acid sequences encoded by the open reading frames predicted from the sequences of SEQ ID NOs: 56, 57 and 58, which correspond to the amino acid sequences of As4.806, As4.820 and As36.1302, are shown in SEQ ID NOs: 1, 2 and 3. Shown in

(Enzymatic properties of 5-oxoprolinase from Aspergillus)
The properties of the enzymes of As4.806 and As4.820, which are 5-oxoprolinases of the present invention whose activity was confirmed in Example 1, were examined as follows. In addition, various properties of the enzyme were similarly examined for YKL215c derived from yeast, which is a known 5-oxoprolinase, as a reference enzyme. The results are as shown in Table 2.

In addition, each 5-oxoprolinase was prepared as described in Example 1 unless otherwise specified. 7. And 5-oxoprolinase derived from Aspergillus soya (As4.806 and As4.820) obtained by the method described in Example 1. 3. The yeast-derived 5-oxoprolinase YKL215c obtained by the method described in (1) was used.

(1) ATP Requirement 2. Showed that As4.806 and As4.820 had 5-oxoprolinase activity in the presence of ATP, potassium ions that are monovalent cations, and magnesium ions that are divalent cations. Therefore, it was investigated whether ATP is a cofactor necessary for catalyzing the reaction from L-pyroglutamic acid to L-glutamic acid.

Specifically, C.I. 2. ATP-free substrate was added to a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0] according to the method described. Using a solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0], the 5-oxoprolinase derived from Aspergillus soya (As 4.806 and As 4.820). ) The reaction with the solution was performed. The concentration of L-glutamic acid in the reaction solution was measured, and the C.C. 2. Based on the formula described, the activity of 5-oxoprolinase was calculated. The unit of the enzyme activity was defined as 1 unit (U) of the amount of the enzyme producing 1 μmol of L-glutamic acid per minute under the conditions of this test.

  For each Aspergillus soya-derived 5-oxoprolinase, a reaction with L-pyroglutamic acid was performed in the presence or absence of ATP, and the activity of 5-oxoprolinase calculated based on the above-described activity measurement method was shown in Table 3. Shown in As a result, 5-oxoprolinase activity was detected in the presence of ATP, but no 5-oxoprolinase activity was detected in both As4.806 and As4.820 in the absence of ATP.

This indicates that the 5-oxoprolinases As4.806 and As4.820 of the present invention are ATP-requiring 5-oxoprolinases.

(2) Monovalent cation, divalent cation requirement C.I. 2. In the present invention, As4.806 and As4.820, which are 5-oxoprolinases of the present invention, exhibit 5-oxoprolinase activity in the presence of ATP, potassium ions that are monovalent cations, and magnesium ions that are divalent cations. Was found to have. Therefore, it was investigated whether monovalent cations and divalent cations are cofactors necessary for catalyzing the reaction from L-pyroglutamic acid to L-glutamic acid.

Specifically, first, a substrate solution [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0], a substrate solution not containing potassium ions [ 50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , pH 8.0], and a substrate solution containing no magnesium ion [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, pH 8.0] was prepared. Next, the above-mentioned 5-oxoprolinase (As4.806 and As4.820) solution derived from Aspergillus soya diluted to 200 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) was prepared. The three kinds of substrate solutions (0.08 mL) and the two kinds of enzyme solutions (0.02 mL) were mixed, and an enzyme reaction was performed at 37 ° C. for 1 hour. C. of Example 1. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzyme reaction was measured, and the activity of 5-oxoprolinase was calculated.

  For each Aspergillus soya-derived 5-oxoprolinase, a reaction with L-pyroglutamic acid was performed in the presence or absence of ATP, and the activity of 5-oxoprolinase calculated based on the above-described activity measurement method was determined. It was shown to. As a result, 5-oxoprolinase activity was detected in the presence of both potassium ions and magnesium ions, but in the absence of potassium ions or magnesium ions, both As4.806 and As4.820 were detected. , 5-oxoprolinase activity was not detected.

From this, it was found that the 5-oxoprolinases As4.806 and As4.820 of the present invention were 5-oxoprolinases requiring monovalent cations and divalent cations.

  Next, the types of monovalent cations and divalent cations capable of catalyzing the reaction from L-pyroglutamic acid to L-glutamic acid by As4.806 and As4.820 were examined.

In the examination of the type of monovalent cation, a substrate solution containing no monovalent cation [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgSO ₄ , pH 8.0], a substrate solution containing KCl [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgSO ₄ , 187.5 mM KCl, pH 8.0], substrate solution containing NH ₄ Cl [50 mM Tris, 0.625 mM L-pyroglutamic acid Glutamic acid, 6.25 mM ATP, 12.5 mM MgSO ₄ , 187.5 mM NH ₄ Cl, pH 8.0], substrate solution containing CsCl [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgSO ₄ , 187.5 mM CsCl, pH 8 .0] substrate solution [50 mM Tris containing LiCl, 0.625 mM L-pyroglutamic _{acid, 6.25mM ATP, 12.5mM MgSO 4,} 187.5mM LiCl, pH8.0 ], substrate solution containing NaCl [50 mM Tris , 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgSO ₄ , 187.5 mM NaCl, pH 8.0], respectively. The above-mentioned 5-oxoprolinase (As4.806 and As4.820) solution derived from Aspergillus soya diluted to 300 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) was prepared, and 0.02 mL of each was prepared. Six types of substrate solutions were mixed with 0.08 mL, and an enzyme reaction was performed at 37 ° C. for 1 hour. For reference, 0.02 mL of the yeast-derived 5-oxoprolinase YKL215c solution diluted to 50 μg / mL with a 50 mM Tris (pH 8.0) buffer and 0.08 mL of the six types of substrate solutions were mixed, and 37 The enzyme reaction was carried out at a temperature of 1 hour. C. of Example 1. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzyme reaction was measured, and the presence or absence of enzyme activity was examined.

  As a result, as shown in FIG. 7, the 5-oxoprolinase activity of As4.806 (black hatched bar in the figure) and As4.820 (black bar in the figure) is detected by the addition. A plurality of monovalent cations were confirmed. The activity was detected when an ammonium ion, a cesium ion, or the like was added in addition to the potassium ion. In particular, a relatively high activity was detected by adding ammonium ions. The activity of the reference enzyme YKL215c (white bars in the figure) was detected when ammonium ions and cesium ions were added. By examining the concentration of each cation, it is considered that a monovalent cation capable of catalyzing the reaction of 5-oxoprolinase from L-pyroglutamic acid to L-glutamic acid can be selected other than potassium ion.

In the examination of the type of divalent cation, a substrate solution containing no divalent cation [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, pH 8.0], and a substrate solution containing MgSO ₄ [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, 12.5 mM MgSO ₄ , pH 8.0], substrate solution containing CoSO ₄ [50 mM Tris, 0.625 mM L-pyroglutamic acid , 6.25 mM ATP, 187.5 mM KCl, 12.5 mM CoSO ₄ , pH 8.0], a substrate solution containing MnSO ₄ [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, 12.5 mM MnSO ₄ , pH 8.0 ], A substrate solution containing Ca (CH ₃ COOH) ₂ [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, 12.5 mM Ca (CH ₃ COOH) ₂ , pH 8.0] , A substrate solution containing FeSO ₄ [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, 12.5 mM FeSO ₄ , pH 8.0], a substrate solution containing NiSO ₄ [50 mM Tris, 0.625 mM L-pyroglutamic acid, 6.25mM ATP, 187.5mM KCl, 12.5mM NiSO 4, pH8.0 ], substrate solution [50 mM Tris containing CuSO _4, 0.625 mM L-pyroglutamic acid, 6.25 mM ATP, 187.5 mM KCl, 12.5 m CuSO _4, pH 8.0], a substrate solution [50 mM Tris containing ZnSO _4, 0.625 mM L-pyroglutamic acid, 6.25mM ATP, 187.5mM KCl, 12.5mM ZnSO 4, pH8.0 ] was prepared respectively . The above-mentioned 5-oxoprolinase (As4.806 and As4.820) solution derived from Aspergillus soya diluted to 300 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) was prepared, and 0.02 mL of each was prepared. Nine kinds of substrate solutions were mixed with 0.08 mL, and an enzyme reaction was performed at 37 ° C. for 1 hour.

  For reference, 0.02 mL of the yeast-derived 5-oxoprolinase YKL215c solution diluted to 300 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) was mixed with 0.08 mL of the nine types of substrate solutions. At 37 ° C. for 1 hour. After heating the reaction solution at 100 ° C. for 3 minutes to stop the enzyme reaction, 5 μL of 1 M HCl was added to the reaction solution, mixed, and then subjected to ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). As a result, a deproteinized fraction was prepared. The deproteinized fraction was subjected to ion exchange chromatography, and the separated amino acids were detected by a post-labeling method using ninhydrin. The concentration of L-glutamic acid in the solution after the above-mentioned enzyme reaction was calculated using a calibration curve based on an L-glutamic acid standard, and the presence or absence of the activity of the enzyme was examined.

  As a result, as shown in FIG. 8, the 5-oxoprolinase activity of As4.806 (black hatched bar on white background) and As4.820 (black bar in FIG. 8) is detected by the addition. Several divalent cations were confirmed. Relatively high activity was detected when manganese ions or iron ions were added in addition to magnesium ions. The activity of the reference enzyme YKL215c (white bar in the figure) was detected when cobalt ion, manganese ion, iron ion or nickel ion was added. By examining the concentration of each cation, it is considered that divalent cations that can catalyze the reaction of 5-oxoprolinase from L-pyroglutamic acid to L-glutamic acid can be selected in addition to magnesium ions.

(3) Optimum pH
The optimal pH of As4.806 and As4.820, which are 5-oxoprolinases of the present invention, was examined. The results are shown in FIG.

Specifically, a 55.6 mM Tris-HCl buffer (pH 8.0, 9.0, plotted with a black circle in the figure), a 55.6 mM citrate-NaOH buffer (pH 4.0, pH 5.0, pH 6) 5.0, plotted with black squares in the figure), 55.6 mM phosphate-NaOH buffer (pH 6.0, pH 7.0, pH 8.0, plotted with open triangles in the figure), 55.6 mM HEPES-NaOH Buffer solution (pH 7.0, pH 8.0, plotted with x mark in the figure), 55.6 mM glycine-NaOH buffer solution (pH 9.0, H9.5, pH 10.0, pH 10.5, open oblique square in the figure) , 55.6 mM CHES-NaOH buffer (pH 9.0, pH 10.0, plotted with a white square in the figure), 55.6 mM CAPS-NaOH buffer (pH 10.0, pH 1.0, FIG Central America mark the plot) at pH adjusting substrate solution (0.556mM L- pyroglutamic acid, 5.56 mm _ATP, 11.1 mM MgCl 2, including 166.7MM KCl) and 0.09 mL, 50 mM 0.01 mL of the above-mentioned 5-oxoprolinase ortholog candidate protein (As4.806, As4.820) solution diluted to 400 μg / mL with a Tris-HCl buffer (pH 8.0) is mixed, and the mixture is mixed at 37 ° C. for 1 hour. An enzymatic reaction was performed.

  As a reference, 0.09 mL of the substrate solution prepared with the above buffers having different pHs and the yeast-derived 5-oxoprolinase YKL215c diluted to 600 μg / mL with the 50 mM Tris-HCl buffer (pH 8.0) buffer were used. 0.01 mL of the solution was mixed, and an enzyme reaction was performed at 37 ° C. for 1 hour. C. of Example 1. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzymatic reaction was measured, and the measured value at the pH where the L-glutamic acid concentration was the highest was set at 100, and the relative activities were compared.

  As a result, the above-mentioned 5-oxoprolinase As4.806 and As4.820 derived from Aspergillus soya of the present invention showed the highest activity at pH 7.0 or pH 8.0, and reached pH 7.0 to 8.0. It had a suitable pH. When viewed individually, the relative activity of As4.806 was the highest at pH 7.0, and the surrounding area showed 70% or more of the maximum relative activity value in the range of pH 7.0 to 9.0. Therefore, it was considered that the composition can be suitably used in this range.

  Further, the relative activity of As4.820 was the highest at pH 8.0, and as a surrounding area, 70% or more of the maximum relative activity value was shown in the pH range of 7.0 to 9.0. It was considered that this range could be suitably used.

  On the other hand, the relative activity of the known yeast-derived 5-oxoprolinase YKL215c was highest at pH 10.0, and at pH 9.5 or lower and at pH 10.5 or higher, the activity was 70% or less of the maximum relative activity value. It became. Furthermore, in the range of pH 7.0-8.0 in which Asoxolus soya-derived 5-oxoprolinase As4.806 and As4.820 can be suitably used, the activity was only 40% or less of the maximum relative activity value. From the above, it was considered that the 5-oxoprolinase derived from Aspergillus soya of the present invention is more suitable for practical use in the neutral region than the known enzymes.

(4) pH Stability The pH stability of As4.806 and As4.820, which are 5-oxoprolinases of the present invention, was examined in the range of pH 4.0 to 11.0. The results are shown in FIG.

Specifically, a 100 mM Tris-HCl buffer (pH 8.0, 9.0, plotted with a black circle in the figure), a 100 mM citrate-NaOH buffer (pH 4.0, pH 5.0, pH 6.0, FIG. 100 mM phosphoric acid-NaOH buffer (pH 6.0, pH 7.0, pH 8.0, plotted with open triangles in the figure), 100 mM HEPES-NaOH buffer (pH 7.0, pH 8) 0.0, plotted with x marks in the figure), 100 mM glycine-NaOH buffer (pH 9.0, H9.5, pH 10.0, pH 10.5, plotted with open diagonal square marks in the figure), 100 mM CHES-NaOH buffer Solution (pH 9.0, pH 10.0, plotted with white squares in the figure), 100 mM CAPS-NaOH buffer (pH 10.0, pH 11.0, FIG. 0.015 mL of a solution of 5-oxoprolinase (As4.806 and As4.820) from Aspergillus soya diluted to 600 μg / mL with 50 mM Tris-HCl buffer (pH 8.0). 015 mL of each was mixed and stored at 4 ° C. for 16 hours. 0.01 mL of a solution of As4.806 and As4.820 stored at 4 ° C. and a substrate solution [50 mM Tris, 0.556 mM L-pyroglutamic acid, 5.56 mM ATP, 11.1 mM MgCl ₂ , 166.7 mM KCl, pH 8 .0], and enzymatic reaction was carried out at 37 ° C for 1 hour.

For reference, 0.015 mL of the above-mentioned various 100 mM buffers having different pHs and 0.015 mL of the above-mentioned 5-oxoprolinase YKL215c solution derived from yeast diluted to 200 μg / mL with a 50 mM Tris-HCl buffer (pH 8.0) are used. Were mixed and stored at 4 ° C. for 16 hours. 0.01 mL of a solution storing the YKL215c at 4 ° C. and 0.09 mL of a substrate solution [50 mM CHES, 0.556 mM L-pyroglutamic acid, 5.56 mM ATP, 11.1 mM MgCl ₂ , 166.7 mM KCl, pH 10.0] Were mixed and an enzyme reaction was carried out at 37 ° C. for 1 hour. The enzyme-reacted solution was heated at 100 ° C. for 3 minutes to stop the enzyme reaction, and then centrifuged (15,000 × g, 10 minutes) to obtain a supernatant as a sample for measuring L-glutamic acid. The sample for measuring L-glutamic acid was prepared according to Example 1. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzymatic reaction was measured, and the measured value at the pH where the L-glutamic acid concentration was the highest was set at 100, and the relative activities were compared.

  As a result, the above-mentioned 5-oxoprolinase As4.806 and As4.820 derived from Aspergillus soya of the present invention had a maximum relative activity of 70 at pH 6.0 to 9.0 after treatment at 4 ° C. for 16 hours. %, The stable pH range of Aspergillus soya-derived 5-oxoprolinase was considered to be pH 6.0 to 9.0. In addition, it was found that when stored at pH 5.0 or lower and pH 11.0 or higher, it was almost completely inactivated.

  In addition, since yeast-derived 5-oxoprolinase YKL215c, which is a reference enzyme, exhibited a residual activity of 70% or more of the maximum relative activity value in the pH range of 9.0 to 10.0, yeast-derived 5-oxoprolinase was used. Was considered to be pH 9.0 to 10.0.

  As described above, the stable pH range of the 5-oxoprolinase derived from Aspergillus soya of the present invention ranges from a weakly acidic region to a weakly alkaline region, and is in a wider pH range including a neutral to acidic range than the known enzyme YKL215c. Was found to be highly stable.

(5) Optimum temperature In the determination of the optimum temperature, the C.I. 2. Was incubated at 4, 15, 25, 37, 45, 55, and 65 ° C. for 1 hour, and the 5-oxoprolinase activity detection reaction solution described in 2. The L-glutamic acid concentration was measured according to the method described in (1). The relative activity was compared with the measured value at the temperature where the L-glutamic acid concentration was the highest as 100.

  As shown in FIG. 9, the 5-oxoprolinase As 4.806 derived from Aspergillus soya of the present invention (plotted with open triangles in the figure) and As4.820 (plotted with open squares in the figure) at 37 ° C. Since the maximum relative activity was shown in the vicinity, and 80% or less of the maximum relative activity was shown at 25 ° C. or less and at 45 ° C. or more, the optimal temperature of As4.806 and As4.820 was considered to be around 37 ° C. .

  The yeast-derived 5-oxoprolinase YKL215c (plotted with x in the figure) showed the maximum relative activity at around 45 ° C and 90% of the maximum relative activity at around 37 ° C. At 25 ° C or lower and 55 ° C or higher, 80% or less of the maximum relative activity was shown. Therefore, it was considered that the optimum temperature of YKL215c was around 45 ° C.

(6) Temperature stability In the study of temperature stability, 0.03 mL of the 5-oxoprolinase (As4.806 or As4.820) solution of the present invention diluted with 50 mM Tris-HCl buffer (pH 8.0) was used. After incubation at 4, 37, 45, 50, 55, 60, 65, 70 ° C. for 15 minutes, the substrate solution [50 mM Tris, 0.714 mM L-pyroglutamic acid, 7.14 mM ATP, 14.2 mM MgCl ₂ , 214. 3 mM KCl, pH 8.0] 0.07 mL, and an enzyme reaction was carried out at 37 ° C. for 1 hour. C. of Example 1. 2. According to the method described, the concentration of L-glutamic acid in the solution after the enzyme reaction was measured, and the relative activity was compared with the measured value in the reaction solution of the enzyme stored at 4 ° C. without heat treatment as 100.

  As shown in FIG. 10, the above-mentioned 5-oxoprolinase As 4.806 derived from Aspergillus soya (plotted with white triangles) in the present invention has a residual activity of 80% or more after heat treatment at 37 ° C. for 15 minutes. And was found to be stable up to about 37 ° C.

  The 5-oxoprolinase As4.820 derived from Aspergillus soya of the present invention (plotted with white squares in the figure) has a residual activity of 80% or more after heat treatment at 45 ° C and is stable up to about 45 ° C. It turned out to be. In addition, 5-oxoprolinase YKL215c derived from yeast (plotted with x in the figure) had a residual activity of 80% or more after heat treatment at 45 ° C, and was found to be stable up to about 45 ° C.

  As described above, in As4.806 and As4.820, no decrease in the activity was detected at the optimal reaction temperature of 37 ° C., indicating that the enzyme reaction can be stably performed.

(7) Km value for L-pyroglutamic acid In order to examine the affinity of this enzyme for L-pyroglutamic acid as a substrate, the C.I. 2. In the described activity measurement method, the activity was measured by changing the concentration of L-pyroglutamic acid as a substrate, and the Michaelis constant (Km) was calculated using Hanes-Woolf Plot.

  As a result, the Km value of 5-oxoprolinase As4.806 of the present invention with respect to L-pyroglutamic acid was 93 μM, and the Km value of As4.820 with respect to L-pyroglutamic acid was 76 μM.

  In contrast, the Km value of yeast-derived 5-oxoprolinase YKL215c for L-pyroglutamic acid was 513 μM.

  That is, the 5-oxoprolinase of the present invention has a Km value several to six times lower than that of the known yeast 5-oxoprolinase, indicating that the enzyme is a high-affinity enzyme. It is expected that the reaction can proceed.

(8) Substrate specificity While As4.806 and As4.820, which are 5-oxoprolinases of the present invention, specifically react with and hydrolyze L-pyroglutamic acid, D-pyroglutamic acid is a substrate. Not sure. In the determination of substrate specificity, the presence or absence of 5-oxoprolinase activity was determined based on the production of glutamate and the decrease in pyroglutamic acid.

Specifically, C.I. 2. According to the method described, the substrate solution [50 mM Tris, 2.5 mM L-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0] and L-pyroglutamic acid were added to D- A substrate solution [50 mM Tris, 2.5 mM D-pyroglutamic acid (manufactured by MP Biomedicals, production number 156461), 6.25 mM ATP, 12.5 mM MgCl ₂ , 187.5 mM KCl, pH 8.0] changed to pyroglutamic acid was used. Prepared. Next, in Example 5-2. 7.68 units / 1200 μg using Aspergillus soya-derived 5-oxoprolinase As4.806-solid solution (to be described later, 50 mM Tris-HCl buffer (pH 8.0)) produced to wheat bran obtained according to the method described. / Diluted to / mL), and 2. of Example 3. Using a yeast-expressed Aspergillus soya-derived 5-oxoprolinase SC-As4.820 solution obtained according to the method described above (to 12.75 units / 1500 μg / mL using a 50 mM Tris-HCl buffer (pH 8.0) described below). Dilution) was prepared. The two kinds of substrate solutions (0.28 mL) and the two kinds of enzyme solutions (0.07 mL) were mixed, and an enzyme reaction was performed at 37 ° C. for 4 hours.

  For reference, 0.28 mL of each of the two substrate solutions and 0.07 mL of 10 mM Tris-HCl buffer (pH 8.0) containing no enzyme were mixed, and an enzyme reaction was performed at 37 ° C. for 4 hours. Was.

  After the reaction, 360 μL of 0.022 M HCl was added to 0.04 mL of the reaction solution, mixed, and subjected to ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) to prepare a protein-free fraction. The deproteinized fraction was subjected to ion exchange chromatography, and the amino acids after separation were detected by a post-labeling method using ninhydrin. The concentration of glutamic acid in the solution after the enzyme reaction was calculated using a calibration curve with a glutamic acid standard, and the presence or absence of enzyme activity was examined.

  Subsequently, a protein-free fraction was prepared by ultrafiltration of the reaction solution using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The deproteinized fraction was subjected to ion exchange chromatography, and the separated organic acid was detected by a post-labeling method using bromothymol blue. The concentration of pyroglutamic acid in the solution after the enzymatic reaction was calculated using a calibration curve based on a pyroglutamic acid sample, and the presence or absence of a decrease in the concentration of pyroglutamic acid was examined. The results are shown in FIG.

  As shown in FIG. 11, when L-pyroglutamic acid is reacted with As4.806-solid, or when L-pyroglutamic acid is reacted with SC-As4.820, the glutamic acid concentration in the reaction solution increases ( The upper row in the figure, black bars) and the concentration of pyroglutamic acid decreased (lower row, black bars in the figure). On the other hand, when D-pyroglutamic acid was allowed to react with each of the 5-oxoprolinases, the glutamic acid concentration hardly changed (the upper part in the figure, black hatched bars on a white background), and no decrease in pyroglutamic acid was observed. (Lower bar in the figure, black hatched bar on white background).

  This indicated that 5-oxoprolinase As4.806 and As4.820 use L-pyroglutamic acid as a substrate and do not use D-pyroglutamic acid as a substrate.

  As described above, the 5-oxoprolinase of the present invention provides different uses and usage scenes because the optimum pH is different from that of known 5-oxoprolinase, and particularly, the optimum pH and pH biased toward alkaline. Known enzymes having stability are particularly excellent when used under weakly acidic to neutral to slightly alkaline conditions, which have been difficult to improve the effect. For example, as an example of use under such pH conditions, it is possible to envisage, for example, during the actual use process of various food processing, and in addition to this, a new use scene that could not be suitably used with known enzymes It is expected to provide.

  In addition, when the 5-oxoprolinase of the present invention is used for applications such as reagents or food processing, the 5-oxoprolinase is unlikely to be affected by changes in the pH of the test environment, and is considered to be easy to handle and easy to use. Therefore, it can be said that it is superior to the known 5-oxoprolinase. Since such properties broaden versatility, it can be suitably used even in applications and use steps that are difficult to use with known 5-oxoprolinase, and variations in the practical use of 5-oxoprolinase It can be said that it contributes to.

  Furthermore, the 5-oxoprolinase of the present invention has a small Km value for L-pyroglutamic acid, can catalyze the reaction more efficiently, and has the advantages that a reduction in reaction time and a reduction in the amount of enzyme used can be expected.

(Production of 5-oxoprolinase from Aspergillus spp. By yeast)
C. of Example 1. 2. It was confirmed that the Aspergillus soya-derived 5-oxoprolinases As4.806 and As4.820 of the present invention obtained in the above can be produced using a yeast which is a heterologous host as a host. Aspergillus soya-derived 5-oxoprolinase ortholog candidate As36.1302, which could not be confirmed, was confirmed to obtain an active protein by using yeast as a host.

1. Preparation of yeast expression plasmid for 5-oxoprolinase gene derived from Aspergillus soya and a candidate gene for 5-oxoprolinase ortholog and acquisition of yeast transformant 5. The plasmid pAsAlpP-4. Into which the sequence excluding the intron predicted sequence of the 5-oxoprolinase ortholog genes (scaffold00004.806, scaffold00004.820) and the 5-oxoprolinase ortholog candidate gene (scafffold00036.13022) obtained in the above step was introduced. From 806 CDS, pAsAlpP-4.820 CDS, pAsAlpP-36.1302 CDS, a DNA fragment of each gene amplified by PCR was introduced into pYES2 / CT (manufactured by Invitrogen) to obtain a yeast expression plasmid.

  Specifically, using the pAsAlpP-4.806 CDS as a template and the primers shown in SEQ ID NOS: 49 and 50, a 24 base sequence encoding a recognition sequence of the restriction enzyme EcoRI and a sequence of 8 histidines was placed on the 5 'side. A DNA fragment of the CDS of scaffold00004.806 to which the recognition sequence of the restriction enzyme NotI was added to the sequence 3 ′ side was amplified by PCR. Using pAsAlpP-4.820 CDS as a template and the primers shown in SEQ ID NOs: 51 and 52, a 24 base sequence encoding the recognition sequence of the restriction enzyme KpnI and 8 histidine sequences on the 5 ′ side, 3 ′ side A DNA fragment of CDS of scaffold00004.820 to which a recognition sequence of a restriction enzyme NotI was added was amplified by PCR. Using pAsAlpP-36.1302 CDS as a template and the primers shown in SEQ ID NOS: 53 and 54, a 24 base sequence encoding the recognition sequence of the restriction enzyme EcoRI and 8 histidine sequences on the 5 'side, and the 3' side Then, a DNA fragment of CDS of scaffold00036.13022 added with a recognition sequence of a restriction enzyme NotI was amplified by PCR.

  An extension reaction was performed using Prime Star Max (manufactured by Takara Bio Inc.) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit.

  The three types of DNA fragments amplified by the PCR were separated in a 1% agarose gel, purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN), and subjected to restriction enzymes EcoRI (manufactured by NEB) and KpnI-HF (manufactured by NEB). After cutting with BioLabs) and NotI (manufactured by Takara Bio Inc.), they were separated in a 1% agarose gel, and purified again using QIA quick Gel Extraction Kit. The purified DNA fragment was ligated to the EcoRI-NotI site and the KpnI-NotI site of pYES2 / CT (Invitrogen) using ligation high ver2 (manufactured by TOYOBO), and to the pYES2 / CT multicloning site. A 5-oxoprolinase ortholog gene and a 5-oxoprolinase ortholog candidate gene (scafffold00004.806, scaffold00004.820, or scaffold00036.13.102) to which a 24-base sequence encoding 8 histidine sequences has been added to the 5 ′ side. Recombinant plasmids for the production of three types of yeast transformants incorporating CDS were obtained.

  After transforming Escherichia coli JM109 strain by a conventional method using the recombinant plasmid, the recombinant plasmid was extracted from the transformant, and the DNA sequence inserted into the plasmid was analyzed. As a result of the analysis, the CDS sequence of the 5-oxoprolinase ortholog candidate gene to which a 24-base sequence encoding a sequence of eight histidines was added on the 5 ′ side was identical to the sequence on the genomic DNA of Aspergillus soya NBRC4239 strain. Make sure you do. Plasmids containing the three types of genes that had undergone the above operations were respectively subjected to pYES2-His-4.806 and pYES2-His corresponding to the names of the derived gene sequences, scaffold00004.806, scaffold00004.820, and scaffold00036.1302. -4.820 and pYES2-His-36.1302.

  Using the three plasmids pYES2-His-4.806 CDS, pYES2-His-4.820 CDS, and pYES2-His-36.1302 CDS, S. c. Saccharomyces cerevisiae strain INVsc1 was transformed using EasyComp ™ Transformation kit (manufactured by Invitrogen) according to the attached manual, and the expression of URA3, a uracil synthesis gene encoded by pYES2 / CT, resulted in a medium without uracil. A yeast transformant was selected using a 5-oxoprolinase gene derived from Aspergillus soya and a candidate gene for a 5-oxoprolinase ortholog, using as an index that uracil was not required for growth. The yeast transformants with the three types of 5-oxoprolinase genes derived from the three Aspergillus soya and 5-oxoprolinase ortholog candidate genes correspond to the names of the derived genes, scaffold00004.806, scaffold00004.820, and scaffold00036.1302. The strains were named INVsc1-His-4.806 CDS strain, INVsc1-His-4.820 CDS strain, and INVsc1-His-36.1302 CDS strain, respectively.

2. Acquisition of yeast-expressed Aspergillus soya-derived 5-oxoprolinase and ortholog candidate protein Yeast transformant INVsc1- by Aspergillus soya-derived 5-oxoprolinase gene and 5-oxoprolinase ortholog candidate gene obtained as described above 5-oxoprolinase gene and 5-oxoprolinase ortholog candidate in plasmids introduced into His-4.806 CDS strain, INVsc1-His-4.820 CDS strain, and INVsc1-His-36.1302 CDS strain A 24-base sequence encoding a sequence of 8 histidines is added upstream of the gene. By culturing a yeast transformant in the presence of galactose, a tag of 8 histidines is fused to the N-terminal. 5-oxopro Linase and 5-oxoprolinase ortholog candidate protein can be induced to be expressed, and can be purified by a conventional method using a Ni column. That is, the aforementioned INVsc1-His-4.806 CDS strain, INVsc1-His-4.820 CDS strain, and INVsc1-His-36.1302 CDS strain were transformed into a preculture liquid medium (0.67% (w / v)). ) Amino acid-free yeast nitrogen base (manufactured by Becton Dickinson), 0.192% (w / v) Additive for uracil-free yeast synthetic dropout medium (manufactured by Sigma), 2.0% (w / v) ) In Raffinose), shaking culture was performed at 30 ° C for 24 hours. Then, a liquid medium for main culture (0.67% (w / v) amino acid-free yeast nitrogen base, 0.192% (w / v) uracil-free yeast synthetic dropout medium supplement, 2.5% (W / v) D-galactose, 0.75% (w / v) raffinose) diluted 10-fold, cultured at 30 ° C for 12 to 18 hours, and centrifuged at 1,500 xg for 1 to 5 minutes. The cells were collected.

  The collected cells were extracted with an extraction buffer (50 mM Tris, 300 mM NaCl, 20% (w / v) glycerol, 0.1 mM DTT, 0.5 mM L) containing 1/10 to 1/20 the volume of the culture solution and 2 mM PMSF. -Pyroglutamic acid, pH 8.0), added with glass beads (manufactured by Yasui Kikai Co., Ltd.) having a diameter of 0.5 mm for a multi-bead shocker, and crushed using a multi-bead shocker (manufactured by Yasui Kikai) (2000 rpm). , Crushed for 60 seconds, and allowed to stand for 30 seconds, and this was repeated eight times). The centrifugal supernatant of the disrupted cell suspension was filtered through a 0.2 μm filter to obtain a cell extract.

  Thereafter, the bacterial cell extract is applied to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind His-tagged 5-oxoprolinase and His-tagged 5-oxoprolinase ortholog candidate protein, and to extract containing 20 mM imidazole. After washing with a buffer, a His-tagged protein-containing fraction eluted with an extraction buffer containing 250 mM was obtained.

  The three types of His-tag fusion protein-containing fractions were further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) and dialyzed against the extraction buffer to obtain a purified enzyme solution. Hereinafter, the respective His tag-fused 5-oxoprolinase and His-tag fused 5-oxoprolinase ortholog candidate proteins are represented by the names of the genes derived from them (scaffold00004.806, scaffold00004.820, and scaffold00036.13.102). It is described as SC-As 4.806, SC-As 4.820, SC-As 36.1302. As a result of the above enzyme purification, 10 to 20 μg of SC-As 4.806, 75 to 400 μg of SC-As 4.820, and 10 to 20 μg of SC-As36.1302 were obtained per 100 ml of culture.

3. Confirmation of activity of yeast-expressed 5-oxoprolinase derived from Aspergillus soya. Obtained from Aspergillus soya 5-oxoprolinase proteins (SC-As4.806 and SC-As4.820) and 5-oxoprolinase ortholog candidate protein (SC-As36.1302) expressed in yeast. B. of Example 1. 3. Of the yeast-derived 5-oxoprolinase YKL215c obtained by C. 2. The 5-oxoprolinase activity was measured according to the method described in (1). Table 5 shows the results.

  As shown in Table 5, Aspergillus soya-derived 5-oxoprolinase SC-As4.806, SC-As4.820, and yeast-derived 5-oxoprolinase YKL215c expressed in yeast were reacted with the substrate solution. , 5-oxoprolinase activity was detected. From this fact, 5-oxoprolinase As4.806 and As4.820 derived from Aspergillus soya exhibit 5-oxoprolinase activity even when expressed in yeast, and have activity even when expressed heterologously. It was confirmed that it was possible.

  In the 5-oxoprolinase ortholog candidate protein SC-As36.1302 derived from Aspergillus soya expressed in yeast, the L-glutamic acid concentration did not increase due to the reaction with the substrate solution, and 5-oxoprolinase activity was detected. Did not. That is, it was confirmed that the 5-oxoprolinase ortholog candidate protein As36.1302 derived from Aspergillus soya did not show 5-oxoprolinase activity even when expressed in yeast.

(Production of 5-oxoprolinase from Aspergillus sp. By Escherichia coli)
It has been reported that yeast-derived 5-oxoprolinase YKL215c could not be expressed in Escherichia coli (FEMS Yeast Res. 10, 394-401, 2010). However, it is unclear about the aspergillus-derived pyroglutamic acid ring-opening enzyme, and if expression and large-scale purification of the enzyme retaining its activity in Escherichia coli becomes possible, the enzyme used for performance evaluation can be easily obtained. C. of Example 1. 2. It was confirmed that the 5-oxoprolinases As4.806 and As4.820 derived from Aspergillus soya of the present invention obtained in the above can be produced using Escherichia coli which is a heterologous host as a host.

1. Preparation of Escherichia coli Expression Plasmid for 5-oxoprolinase Gene Derived from Aspergillus soya and Acquisition of Escherichia coli Transformant 5. The plasmid pAsAlpP-His-4.806 CDS into which the CDS of the 5-oxoprolinase ortholog genes (scaffold00004.806 and scaffold00004.820) to which a 24-base sequence encoding the sequence of 8 histidines was added, which was obtained in step 2, was added. A plasmid for Escherichia coli expression was obtained from pAsAlpP-His-4.820 CDS by introducing a DNA fragment of each gene amplified by PCR into pET-22b (+) (Novagen).

  Specifically, using pAsAlpP-His-4.806 CDS as a template and the primers shown in SEQ ID NOS: 59 and 60, a scaffold 000004 in which a 24-base sequence encoding a sequence of eight histidines was added to the 5 'side. The DNA fragment of the CDS of .806 was sequenced from pAsAlpP-His-4.820 CDS using the primers shown in SEQ ID NOs: 61 and 62 as a 24-base sequence encoding 8 histidine sequences on the 5 'side. The DNA fragment of CDS of scaffold00004.820 to which was added was amplified by PCR.

  Furthermore, using plasmid pET-22b (+) as a template and the primers for inverse PCR of SEQ ID NOs: 63 and 64, the sequence excluding the region flanked by restriction sites NdeI and NotI of plasmid pET-22b (+). Was amplified by PCR. The extension reaction was performed using Prime Star Max (manufactured by Takara Bio Inc.) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) according to the protocol attached to the reaction reagent kit.

  Each DNA fragment amplified by the above-mentioned PCR was separated in a 1% agarose gel, and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Restriction of each DNA fragment obtained by adding a 24-base sequence encoding eight histidine sequences to the 5 ′ side of the CDS of the two types of 5-oxoprolinase ortholog candidate genes using the In-Fusion Advantage PCR Cloning Kit By ligating to the plasmid pET-22b (+) excluding the region flanked by the enzyme sites NdeI and NotI, the region between the NdeI and NotI of the multicloning site (MCS) of the plasmid pET-22b (+) was Preparation of two types of Escherichia coli transformants in which the CDS of a 5-oxoprolinase ortholog gene (scaffold00004.806 or scaffold00004.820) to which a 24-base sequence encoding 8 histidine sequences was added on the 5 ′ side was incorporated. To obtain a recombinant plasmid.

  After transforming Escherichia coli JM109 strain by a conventional method using the recombinant plasmid, the recombinant plasmid was extracted from the transformant, and the DNA sequence inserted into the plasmid was analyzed. As a result of the analysis, the CDS sequence of the 5-oxoprolinase ortholog candidate gene to which a 24-base sequence encoding a sequence of eight histidines was added on the 5 ′ side was identical to the sequence on the genomic DNA of Aspergillus soya NBRC4239 strain. Make sure you do. Plasmids containing the two types of genes subjected to the above operations were respectively associated with pET-22b (+)-His-4.806 CDS corresponding to the names of the derived gene sequences scaffold00004.806 and scaffold00004.820. It was named pET-22b (+)-His-4.820 CDS.

  Using the two types of plasmids, pET-22b (+)-His-4.806 CDS and pET-22b (+)-His-4.820 CDS, Escherichia coli ECOS ™ competent Escherichia coli BL21 (DE3) strain (Manufactured by Nippon Gene Co., Ltd.) according to a standard method, and the expression of ampicillin resistance gene encoded by pET-22b (+) was used as an index to enable growth on an ampicillin-supplemented medium. A transformant of Escherichia coli with the Nase gene was selected. The Escherichia coli transformant with each Aspergillus soya-derived 5-oxoprolinase gene corresponds to the BL21-His-4.806 CDS strain corresponding to scaffold00004.806 and scaffold00004.820, respectively. It was named BL21-His-4.820 CDS strain.

2. Acquisition of 5-oxoprolinase protein derived from Escherichia coli-expressing Aspergillus genus Escherichia coli transformants BL21-His-4.806 CDS strain and BL21-His using the 5-oxoprolinase gene derived from Aspergillus soya obtained as described above. -4.820 A 24-base sequence encoding a sequence of eight histidines was added upstream of the 5-oxoprolinase gene and the 5-oxoprolinase ortholog candidate gene in the plasmid introduced into the CDS strain. By culturing the Escherichia coli transformant in the presence of IPTG, it is possible to induce the expression of a 5-oxoprolinase protein in which eight histidine tags are fused to the N-terminal side. And can be purified. That is, the BL21-His-4.806 CDS strain and the BL21-His-4.820 CDS strain were transformed into an LB liquid medium (1.0% (w / v) polypeptone (Becton) containing 50 μg / mL ampicillin. Dickinson), 0.5% (w / v) yeast extract (Becton Dickinson), 0.5% (w / v) NaCl (Wako Pure Chemical Industries) at 30 ° C. overnight, The culture was shake-cultured, diluted 100-fold with an LB liquid medium containing 50 μg / mL ampicillin, shake-cultured for 3.0 hours, added with IPTG to a concentration of 0.1 mM, and further added at 30 ° C. for 2 hours. The cells were cultured with shaking for 0.5 to 3.0 hours, and the cells were collected by centrifugation at 5,000 xg for 3 minutes.

  The collected cells were frozen in liquid nitrogen, ground using a mortar and a pestle to form fine powdery cell pieces, and then extracted with an extraction buffer (50 mM Tris) containing 1/12 volume of the culture solution containing 2 mM PMSF. , 300 mM NaCl, 20% (w / v) glycerol, 0.5 mM L-pyroglutamic acid, pH 8.0) and sonicated to reduce the viscosity of the suspension. The centrifugal supernatant of the disrupted cell suspension was filtered through a 0.2 μm filter to obtain a cell extract.

  Thereafter, the cell extract is applied to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind the His-tagged 5-oxoprolinase, washed with an extraction buffer containing 20 mM imidazole, and eluted with an extraction buffer containing 250 mM. Thus, a fraction containing a His-tag fusion protein was obtained.

  The two types of His-tag fusion protein-containing fractions were further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) and then dialyzed against an extraction buffer to obtain a purified enzyme solution. Hereinafter, each of the His tag-fused 5-oxoprolinases is referred to as EC-As 4.806 and EC-As 4.820, corresponding to the names of the derived genes (scaffold00004.806 and scaffold00004.820). As a result of the above enzyme purification, 55 μg of protein was obtained for EC-As 4.806 and 63 μg of EC-As 4.820 was obtained per 100 ml of culture.

3. Confirmation of the activity of 5-oxoprolinase derived from Escherichia coli genus Aspergillus sp. The 5-oxoprolinase protein (EC-As 4.806 and EC-As 4.820) derived from Aspergillus soya and expressed in Escherichia coli obtained by the method described in Example 1 was used. 2. The 5-oxoprolinase activity was measured according to the method described in (1). Table 6 shows the results.

  As shown in Table 6, 5-oxoprolinase EC-As 4.806 and EC-As4.820 derived from Aspergillus soya expressed in Escherichia coli detected 5-oxoprolinase activity by reaction with the substrate solution. Was done. From this fact, 5-oxoprolinase As 4.806 and As4.820 derived from Aspergillus soya exhibit 5-oxoprolinase activity when expressed in Escherichia coli and have activity even when heterologously expressed. It was confirmed that expression was possible. It is also clear that sugar chains are unnecessary in order to have the function of pyroglutamic acid ring-opening enzyme derived from Aspergillus, and if it is possible to express and mass-purify the enzyme retaining its activity in Escherichia coli, the enzyme used for performance evaluation Can be easily obtained.

  As described above, the 5-oxoprolinase of the present invention can be produced without limiting the host to filamentous fungi such as Aspergillus. The fact that heterologous expression is possible is advantageous when selecting a host for the purpose of increasing the expression level, or when confirming at the protein level when the enzyme is improved, and contributes to the practical significance of the present invention.

(Production of Aspergillus-derived 5-oxoprolinase and 5-oxoprolinase ortholog candidate protein expressed in Aspergillus in solid culture)
In the manufacture of various brewed foods such as miso, soy sauce, sake, and shochu, koji is produced by solid-culture of koji mold. Generally, it is known that solid culture and liquid culture show different expression patterns, and it is considered that the expression level and specific activity of the enzyme may be different from those in liquid culture.

  Therefore, the A.I. 6. By culturing the transformant of Aspergillus soya NBRC4239 strain of the present invention obtained by the above-mentioned 5-oxoprolinase As4.806 derived from Aspergillus soya and the transformant obtained by As4.820 in solid culture using wheat bran. To confirm that a protein having an activity can be produced. 2. In Aspergillus soya-derived 5-oxoprolinase ortholog candidate protein As36.1302, the activity of which could not be confirmed, a solid culture using wheat bran was performed to confirm that an active protein was obtained. Was.

1. Acquisition of 5-oxoprolinase and 5-oxoprolinase candidate protein from Aspergillus soya from a wheat bran culture 6. Aspergillus genus transformant NBRC4239-His-4.806 CDS strain, NBRC4239-His-4.820 CDS strain and 5-oxoprolinase ortholog candidate gene by Aspergillus soya-derived 5-oxoprolinase gene obtained in Up to the 5-oxoprolinase gene and the 5-oxoprolinase ortholog candidate gene introduced into the genomic DNA of the Aspergillus transformant NBRC4239-His-36.1302 CDS strain, eight histidine sequences are coded. And a 5-oxoprolinase and a 5-oxoprolinase ortholog candidate protein in which eight histidine tags are fused to the N-terminal side by culturing an Aspergillus transformant. Can be present induction, it can be purified using a Ni column by a conventional method.

First, 8 mL of water was added to 10 g of wheat bran, and 5 g of each was placed in a 150 mL Erlenmeyer flask and autoclaved to prepare an 80% sprinkled wheat bran solid medium. Next, the A.I. 6. 5 × 10 ⁶ conidia of the NBRC4239-His-4.806 CDS strain, the NBRC4239-His-4.820 CDS strain, and the NBRC4239-His-36.1302 CDS strain obtained in the above were used for the aforementioned 80% watered wheat bran. The cells were cultured in a solid medium and cultured at 30 ° C. When the hypha was growing, it was maintained and cultured again. A culture cultured for 42 hours showing hyphal elongation and showing almost no conidia was collected and frozen with liquid nitrogen.

  1 g of the collected wheat bran culture was ground in liquid nitrogen using a mortar and pestle, and the fine powdered bacterial cells obtained were extracted with 10 mL of an extraction buffer containing 2 mM PMSF (50 mM Tris, 300 mM NaCl, 20 mM NaCl). % (W / v) glycerol, 0.5 mM L-pyroglutamic acid, pH 8.0). The centrifuged supernatant of the obtained suspension was filtered with a 0.2 μm filter to obtain a wheat bran culture extract.

  Thereafter, this wheat bran culture extract was subjected to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind His tag-fused 5-oxoprolinase and His tag-fused 5-oxoprolinase ortholog candidate protein, and imidazole 20 mM was obtained. After washing with an extraction buffer containing 250 mM, a fraction containing a His-tag fusion protein eluted with an extraction buffer containing 250 mM was obtained.

  The three types of His-tag fusion protein-containing fractions were further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) and dialyzed against the extraction buffer to obtain a purified enzyme solution. Hereinafter, each of the His-tag fusion 5-oxoprolinase protein and the His-tag fusion 5-oxoprolinase ortholog candidate protein is referred to as the name of the derived gene (scaffold00004.806, scaffold00004.820, and scaffold00036.16.102). , As4.806-solid, As4.820-solid, and As36.1302-solid. By the above enzyme purification, 43 μg of As4.806-solid, 57 μg of As4.820-solid, and 47 μg of As36.1302-solid were obtained per 100 ml of culture.

2. Example 1 of Embodiment 5 Of the activity of 5-oxoprolinase and 5-oxoprolinase candidate protein derived from Aspergillus soya purified from a wheat bran culture obtained by the method described in 1 of Example 5 above. 5-oxoprolinase (As4.806-solid and As4.820-solid) and 5-oxoprolinase ortholog candidate protein (As36.1302-solid) derived from Aspergillus soya purified from a wheat bran culture obtained by About C. of Example 1. 2. The 5-oxoprolinase activity was measured according to the method described in (1). Table 7 shows the results.

  As shown in Table 7, the 5-oxoprolinase proteins As4.806-solid and As4.820-solid derived from Aspergillus soya purified from the wheat bran culture increased the glutamic acid concentration by reaction with the substrate solution. From this fact, 5-oxoprolinase As 4.806 and As4.820 derived from Aspergillus soya exhibit 5-oxoprolinase activity even when expressed up to wheat bran, and can be produced with the activity. Was confirmed.

  In addition, in Aspergillus soya-derived 5-oxoprolinase ortholog candidate protein As36.1302-solid expressed up to wheat bran, the L-glutamic acid concentration did not increase due to the reaction with the substrate solution, and the 5-oxoprolinase activity was detected. Was not done. That is, it was confirmed that the 5-oxoprolinase ortholog candidate protein As36.1302 derived from Aspergillus soya did not exhibit 5-oxoprolinase activity even when expressed up to wheat bran.

  Furthermore, it has been shown that 5-oxoprolinase As 4.806-solid derived from Aspergillus soya, which was produced up to wheat bran, can be expressed while maintaining an activity several times or more higher than that in liquid culture. It was suggested that glutamic acid could be produced.

(Acquisition of 5-oxoprolinase ortholog candidate protein from Aspergillus oryzae)
1. Search for 5-oxoprolinase ortholog candidate genes in Aspergillus oryzae Using sequence data of the protein of Aspergillus soya 5-oxoprolinase As4.820, a gene having relatively high sequence identity with them was obtained from Aspergillus oryzae. Tried to explore. The Blast program (tblastn) was used for the search. As the DNA data of Aspergillus oryzae, the genomic sequence of Aspergillus oryzae RIB40 strain (Database of National Institute of Technology and Evaluation: DOGAN, Nature 438 (7071), 1157-1161, 2005) was used. .

  As a result of the search, a gene AO09010300443 (3843 bp) represented by SEQ ID NO: 65 was found as a sequence region having relatively high gene sequence identity. The amino acid sequence encoded by the open reading frame predicted from the DNA sequence of AO09010300443 is shown in SEQ ID NO: 66.

  When sequence identity at the amino acid level was compared using genetic information processing software Genetyx network version 11.0.6 (Genetics), the sequence identity between AO09010300443 and Aspergillus soya 5-oxoprolinase As4.820 was obtained. Its sex was 99.0%, suggesting that it was a 5-oxoprolinase ortholog of Aspergillus oryzae. The sequence identity between AO09010300443 and rat 5-oxoprolinase was 55.0%, and the sequence identity with yeast-derived 5-oxoprolinase, OXP / YKL215c, was 54.1%. This is the same as C.I. 2. Showed 51.5% sequence identity between Aspergillus soya 5-oxoprolinase As4.820 and rat 5-oxoprolinase, which were shown to have 5-oxoprolinase activity, and As4.820 and yeast ortholog. The fact that the sequence identity with 5-oxoprolinase was as low as 51.1% suggested that AO09010300443 is a 5-oxoprolinase ortholog of Aspergillus oryzae.

  Therefore, an experiment was conducted to confirm the presence or absence of 5-oxoprolinase activity of the protein encoded by AO09010300443 using the DNA sequence information of AO09010300443.

2. Extraction of Genomic DNA from Aspergillus oryzae Aspergillus oryzae RIB40 strain in a baffled Erlenmeyer flask in PD medium (2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (W / v) KH ₂ PO ₄ , 0.05% (w / v) MgSO ₄ .7H ₂ O, 0.1% (w / v) casamino acid) is shake-cultured at 30 ° C. for 1 to 2 days. The cells were collected by filtration. The collected cells were frozen in liquid nitrogen and ground using a mortar and pestle to obtain fine powdered cell fragments.

  About 300 μL of Nuclei Lysis Solution (promega) warmed to 65 ° C. was added with about 1 spatula of the powdered bacterial cells, vortexed, and incubated at 65 ° C. for 15 minutes. Next, RNase (manufactured by Nippon Gene Co., Ltd.) was added to a concentration of 50 μg / mL and incubated at 37 ° C. for 15 minutes. Thereafter, 125 μL of Protein Precipitation Solution (manufactured by Promega) was added, vortexed vigorously for 20 seconds, and centrifuged at 14,000 × g for 5 minutes. The resulting supernatant was mixed with 300 μL of isopropanol, and centrifuged at 11,000 × g for 3 minutes, and the precipitate obtained was rinsed with 70% ethanol to collect genomic DNA derived from Aspergillus oryzae RIB40 strain. TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) was added to the genomic DNA, and the mixture was incubated at 50 ° C. for 5 minutes to dissolve, thereby preparing a genomic DNA solution having a desired concentration.

3. Cloning of candidate gene for 5-oxoprolinase ortholog in Aspergillus oryzae and preparation of expression vector for Aspergillus spp. As in the 5-oxoprolinase ortholog gene, it was thought that there were two exons and one intron. Specifically, out of the 3899 bases shown in SEQ ID NO: 67, positions 2524 to 2579 were predicted to be introns, and it was predicted that splicing would generate the CDS of SEQ ID NO: 65. In this experiment, the sequence of pre mRNA represented by SEQ ID NO: 67 was used for the next operation without removing the predicted intron sequence.

  Experiment 6-2. 5-oxoprolinase ortholog candidate of Aspergillus oryzae having added thereto a 24-base sequence encoding 8 histidine sequences on the 5 ′ side using the genomic DNA obtained by the above as a template and primers of SEQ ID NOs: 68 and 69 The sequence of pre mRNA of gene AO09010300443 was amplified by PCR. An extension reaction was performed using Prime Star Max (manufactured by Takara Bio Inc.) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit. A DNA fragment considered as a pre-mRNA of AO09010300443 added with a 24-base sequence encoding a sequence of eight histidines amplified by the above PCR was separated in a 1% agarose gel, and QIAquick Gel Extraction Kit (manufactured by QIAGEN) Purified using In addition, as a cloning vector, A. 4. The recombinant plasmid pAsAlpP-4.806 pre mRNA (prepared to the upstream of the multicloning site (MCS) of pUC19, Palp, pyrG, Ptef1, pre-mRNA of 5-oxoprolinase ortholog candidate gene scafffold00004.806, and Talp The sequence on the plasmid side containing Talp on the 5 'side and Palp, pyrG and Ptef1 on the 3' side using the primers for inverse PCR of SEQ ID NOs. Amplified by PCR. Next, the DNA fragment was separated in a 1% agarose gel and purified using QIAquick Gel Extraction Kit.

  The two types of DNA fragments obtained as described above were ligated using In-Fusion Advantage PCR Cloning Kit (manufactured by Clontech), and a 24-base sequence encoding a sequence of eight histidines was added to the 5 ′ side. A DNA fragment considered to be a DNA fragment of the 5-oxoprolinase ortholog candidate gene AO09010300443 pre mRNA of Aspergillus oryzae was ligated between the Ptef1 region and the Talp region of the plasmid pAsAlpP, and a recombinant plasmid for producing a koji mold transformant was obtained. Obtained. The obtained recombinant plasmid was named pAsAlpP-AO090103000443 pre mRNA corresponding to AO090103000443 which is the name of the gene from which it was derived.

  After transforming Escherichia coli JM109 strain by a conventional method using this recombinant plasmid, the recombinant plasmid was extracted from the transformant, and each DNA sequence inserted into the plasmid was analyzed. As a result of the analysis, the DNA fragment considered to be the DNA fragment of the 5-oxoprolinase ortholog candidate gene AO090103000443 pre mRNA is identical to the sequence of AO090103000443 (SEQ ID NO: 67) present in the genomic DNA of Aspergillus oryzae RIB40 strain. It was confirmed.

4. Acquisition of Transformant of Aspergillus soya NBRC4239 Strain from Aspergillus oryzae 5-oxoprolinase Ortholog Candidate Gene Using pAsAlpP-AO090103000443 pre mRNA as a template and primers of SEQ ID NOs: 41 and 40, , PyrG, Ptef1, and a 5-oxoprolinase ortholog candidate gene AO09010300443 pre mRNA to which a His tag sequence was added, and a DNA fragment to which Talp were linked were amplified by PCR. The elongation reaction was carried out using KOD Dash Neo (manufactured by TOYOBO) as a reaction reagent and Mastercycler gradient (manufactured by Eppendorf) as an apparatus according to the protocol attached to the reaction reagent kit.

  The DNA fragment (Palp, pyrG, Ptefl, a 5-oxoprolinase ortholog candidate gene AO090103000443 pre mRNA to which a His tag sequence is added, and a DNA fragment to which Talp is ligated) and a DNA fragment to which Talp is ligated are dissolved in TE after ethanol precipitation. Then, a DNA solution having a desired concentration was prepared and used for transformation of a pyrG-disrupted strain (48 bp upstream, 896 bp coding region and 240 bp downstream deletion of the pyrG gene) derived from Aspergillus soya NBRC4239 by the following procedure.

First, Aspergillus soya NBRC4239 strain (conidia of a pyrG-disrupted strain derived from Aspergillus soya NBRC4239 strain cultured on a Maltz plate, about 1/10 in a 9 cm petri dish) to be used for transformation was suspended in a 0.01% Tween80 solution. PD medium supplemented with uridine to a concentration of 10 mM (2% (w / v) dextrin, 1% (w / v) polypeptone, 0.5% (w / v) KH ₂ PO ₄ , 0.05% ( (w / v) 150 mL of MgSO ₄ .7H ₂ O, 0.1% (w / v casamino acid) and incubating with shaking for about 18 hours. After the culture, the cells were collected by filtration, and a DNA fragment (Palp, pyrG, Ptefl, a 5-oxoprolinase ortholog candidate gene A0090103000443 pre mRNA to which a His tag sequence was added) amplified by PCR for transformation, and Transformation was performed by a protoplast-PEG method using a TE solution (about 10 μg of DNA / 20 μL) of a Talp-linked DNA fragment.

  In each of the obtained transformants, a sequence in which a 5-oxoprolinase ortholog candidate gene AO090103000443 pre mRNA in which pyrG, Ptefl and a His tag sequence are added to the alkaline protease locus of NBRC4239 strain is introduced. . These strains can be grown in a uridine-free medium by introducing pyrG, a gene that complements uridine auxotrophy, so that strains into which the target gene has been introduced can be selected.

  Furthermore, it was confirmed that the transformant was a strain into which the gene was introduced at the target locus by colony PCR. That is, the conidia of the transformant scraped with a toothpick are suspended in TE and used as a template for PCR, and the primers of SEQ ID NOs: 43 and 44 are used to convert the alkaline protease locus from the 237th base upstream to the 277th base downstream. The sandwiched region was amplified by PCR. KOD FX (manufactured by TOYOBO) was used as a reaction reagent, and after a heat treatment at 94 ° C. for 5 minutes, an extension reaction was performed according to the attached protocol. As a result, in the transformant strain, pyrG, Ptefl, and His tag sequences were added in place of the alkaline protease gene (alp, 1389 bp, SEQ ID NO: 45) at the alkaline protease locus of the genomic DNA of Aspergillus soya NBRC4239 strain. Oxoprolinase ortholog candidate gene AO09010300443 pre A homologous recombination at the alkaline protease locus was detected in the transformant by detecting a signal of about 7 kbp indicating the presence of DNA (about 6.6 kbp) linked to pre mRNA. I confirmed that it was done.

  Obtaining a transformant derived from a candidate gene for a 5-oxoprolinase ortholog derived from Aspergillus oryzae (a transformant of the genus Aspergillus using a candidate gene for 5-oxoprolinase ortholog of Aspergillus oryzae) by the above method. Was completed. The transformant was named NBRC4239-His-AO090103000443 pre mRNA strain corresponding to the name of the derived gene AO09010300443.

5. Acquisition of 5-oxoprolinase ortholog candidate protein derived from Aspergillus oryzae 5-oxoprolinase ortholog candidate gene of Aspergillus oryzae obtained as described above was introduced into the genomic DNA of a transformant of the genus Aspergillus by the 5-oxoprolinase ortholog candidate gene. Up to the oxoprolinase candidate gene, a 24-base sequence coding for 8 histidine sequences is added. By culturing a transformant of Aspergillus, 8 histidine tags are fused to the N-terminal. The obtained 5-oxoprolinase candidate protein can be expressed.

The N-terminal His tag-fused 5-oxoprolinase candidate protein can be purified by a conventional method using a Ni column. That is, the conidia of a transformant of the genus Aspergillus (NBRC4239-His-AO090103000443 pre mRNA strain) by a candidate gene for 5-oxoprolinase ortholog of Aspergillus oryzae were transferred to a PD medium (2% (w / v) dextrin, 1% (w / v) _{polypeptone, 0.5% (w / v)} KH 2 PO 4, in _{0.05% (w / v) MgSO} 4 · 7H 2 O, 0.1% (w / v) casamino acids) The cells were inoculated at 1 × 10 ⁵ cells / mL, cultured at 30 ° C. and 120 rpm for 2 days, and the cells were collected by filtration.

  The collected bacterial cells were frozen in liquid nitrogen, and ground using a mortar and pestle to obtain fine powdery bacterial cell fragments. An extraction buffer (50 mM Tris) containing 1/13 of the culture solution containing 2 mM PMSF was used. , 300 mM NaCl, 20% (w / v) glycerol, 0.5 mM L-pyroglutamic acid, pH 8.0). The centrifuged supernatant of the obtained suspension was filtered through a 0.2 μm filter to obtain a cell extract (crude enzyme solution).

  Thereafter, a part of the bacterial cell extract was applied to a Ni-NTA Superflow column (manufactured by QIAGEN) to bind the His-tag fused 5-oxoprolinase ortholog candidate protein, and washed with an extraction buffer containing 20 mM imidazole. A fraction containing a 5-oxoprolinase ortholog candidate protein eluted with an extraction buffer containing 250 mM imidazole was obtained. If necessary, the fraction containing the 5-oxoprolinase ortholog candidate protein was further concentrated using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The fraction containing the 5-oxoprolinase ortholog candidate protein was dialyzed against an extraction buffer to obtain a purified enzyme solution.

  By the above-mentioned enzyme purification operation, 283 μg of 5-oxoprolinase ortholog candidate protein was obtained per 100 mL of culture.

6. Confirmation of activity of 5-oxoprolinase candidate protein derived from Aspergillus oryzae expressed in Aspergillus spp. Of the 5-oxoprolinase candidate protein derived from Aspergillus oryzae obtained by the method described in Example 1. 2. The 5-oxoprolinase activity was measured according to the method described in (1). The relationship between the amount of protein added and the L-glutamic acid concentration was as shown in FIG.

  As shown in FIG. 12, the concentration of L-glutamic acid increased in a concentration-dependent manner of the added AO09010300443 protein, indicating that it was a 5-oxoprolinase ortholog of Aspergillus oryzae.

This indicated that 5-oxoprolinase derived from Aspergillus sp. Is not limited to Aspergillus soya, and that highly homologous molecules are widely present in Aspergillus sp.

  It is considered that the use of the 5-oxoprolinase of the present invention for the processing of food and drink can be used for improving the umami of food and drink. In other words, in a food or drink containing a large amount of pyroglutamic acid, when pyroglutamic acid is hydrolyzed with 5-oxoprolinase, glutamate, which is important as a taste component, can be liberated in large quantities, and has an unprecedented superior taste power. It is expected that food and drink can be obtained.

  Therefore, the effect of increasing the glutamic acid concentration by treating tomato extract and soy sauce, which are foods having high pyroglutamic acid content, with 5-oxoprolinase derived from Aspergillus sp. According to the following procedure was examined.

(A. Increase in the amount of glutamic acid in tomato extract by 5-oxoprolinase derived from Aspergillus)
The tomato extract (manufactured by Lyco Red, Brix 60%) was diluted with Milli-Q water and adjusted to pH 8 with 1M KOH. The 5-oxoprolinase used in the reaction was prepared according to 2. The yeast-expressed 5-oxoprolinase SC-As4.820 derived from Aspergillus soya obtained according to the method described in Example 1. 2. The activity of 5-oxoprolinase was measured according to the method described, and used for a reaction solution with tomato extract.

  Specifically, 0.16 mL of a tomato extract diluted solution [8-fold diluted mato extract, 6.25 mM ATP, pH 8.0] and 0.16 mL of Example 3 were used. 0.04 mL of a 5-oxoprolinase ortholog protein SC-As 4.820 solution (diluted to 10.6 unit / 1200 μg / mL using a 10 mM Tris-HCl buffer (pH 8.0)) obtained according to the method described in Each was mixed and subjected to an enzyme reaction at 37 ° C. for 4 hours.

  As a reference, 0.16 mL of a tomato extract diluent [8-fold diluted mato extract, 6.25 mM ATP, pH 8.0] and 10 mM Tris-HCl buffer (instead of the 5-oxoprolinase ortholog protein solution) were used. pH 8.0), bovine serum albumin (hereinafter referred to as BSA) solution (diluted to 1200 μg / mL using 10 mM Tris-HCl buffer (pH 8.0)), or A. 7. 0.04 mL of an As36.1302 solution (diluted to 1200 μg / mL using a 10 mM Tris-HCl buffer (pH 8.0)) obtained according to the method described above was mixed, and the enzyme reaction was carried out at 37 ° C. for 4 hours. Was done.

  After the reaction, 0.4 mL of Milli-Q water was added to the above reaction solution to prepare a three-fold dilution. The diluted solution was subjected to ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) to prepare a protein-free fraction. The deproteinized fraction was subjected to ion exchange chromatography, and the separated organic acid was detected by a post-labeling method using bromothymol blue. The concentration of pyroglutamic acid in the solution after the enzymatic reaction was calculated using a calibration curve based on a pyroglutamic acid sample, and the presence or absence of a decrease in the concentration of pyroglutamic acid was examined. Subsequently, 0.28 mL of 0.03 M HCl was added to and mixed with 0.12 mL of the diluted solution, and a protein-free fraction was prepared by ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The deproteinized fraction was subjected to ion exchange chromatography, and the amino acids after separation were detected by a post-labeling method using ninhydrin. The concentration of glutamic acid in the solution after the enzyme reaction was calculated using a calibration curve with a glutamic acid standard, and the presence or absence of enzyme activity was examined.

  As a result, as shown in FIG. 13, by adding SC-As4.820 to the diluted tomato extract, the glutamic acid concentration in the reaction solution increased (white bar in the figure), and the pyroglutamic acid concentration decreased (FIG. 13). Black diagonal bar on a white background).

  On the other hand, instead of SC-As4.820, 10 mM Tris-HCl buffer (pH 8.0), BSA solution, and C.I. 2. As shown in the figure, even if an As36.1302 solution that did not show 5-oxoprolinase activity in a 50 mM Tris-HCl buffer (pH 8.0) was added, the glutamate concentration in the reaction solution did not increase, and pyroglutamic acid was not increased. There was almost no change in the concentration, and no 5-oxoprolinase activity could be detected.

  Accordingly, it was shown that by treating the tomato extract, which is a food having a high pyroglutamic acid content, with 5-oxoprolinase derived from Aspergillus soya by the above-described series of techniques, there is an effect of increasing the glutamic acid concentration.

(Increase in amount of glutamic acid in soy sauce by 5-oxoprolinase derived from B. Aspergillus)
Dark soy sauce (Kikkoman, specialty round soy soy sauce) was diluted with Milli-Q water and adjusted to pH 8 with 1M KOH. The 5-oxoprolinase used in the reaction was prepared according to 2. The yeast-expressed 5-oxoprolinase SC-As4.820 derived from Aspergillus soya obtained according to the method described in Example 1. 2. The activity of 5-oxoprolinase was measured according to the method described, and used in a reaction solution with concentrated soy sauce.

  Specifically, 0.16 mL of concentrated soy sauce diluent [three-fold diluted concentrated soy sauce, 6.25 mM ATP, pH 8.0] and 0.16 mL of Example 3 were used. 0.04 mL of a 5-oxoprolinase ortholog protein SC-As 4.820 solution (diluted to 4.43 units / 600 μg / mL using a 10 mM Tris-HCl buffer (pH 8.0)) obtained according to the method described in the following. Each was mixed and subjected to an enzyme reaction at 37 ° C. for 4 hours.

  As a reference, 0.16 mL of a concentrated soy sauce diluent [three-fold diluted concentrated soy sauce, 6.25 mM ATP, pH 8.0] and 10 mM Tris-HCl buffer instead of the 5-oxoprolinase ortholog protein solution were used. (PH 8.0), BSA solution (diluted to 1200 μg / mL using 10 mM Tris-HCl buffer (pH 8.0)), or A. 7. 0.04 mL of an As36.1302 solution (diluted to 600 μg / mL using a 10 mM Tris-HCl buffer (pH 8.0)) obtained according to the method described above was mixed, and the enzyme reaction was carried out at 37 ° C. for 4 hours. went.

  After the reaction, 0.4 mL of Milli-Q water was added to the above reaction solution to prepare a three-fold dilution. The diluted solution was subjected to ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore) to prepare a protein-free fraction. The deproteinized fraction was subjected to ion exchange chromatography, and the separated organic acid was detected by a post-labeling method using bromothymol blue. The concentration of pyroglutamic acid in the solution after the enzymatic reaction was calculated using a calibration curve based on a pyroglutamic acid sample, and the presence or absence of a decrease in the concentration of pyroglutamic acid was examined. Subsequently, 0.28 mL of 0.03 M HCl was added to and mixed with 0.12 mL of the diluent, and a protein-free fraction was prepared by ultrafiltration using Amicon Ultra-0.5 mL 10K (manufactured by Millipore). The deproteinized fraction was subjected to ion exchange chromatography, and the amino acids after separation were detected by a post-labeling method using ninhydrin. The concentration of glutamic acid in the solution after the enzyme reaction was calculated using a calibration curve with a glutamic acid standard, and the presence or absence of enzyme activity was examined.

  As a result, as shown in FIG. 14, by adding SC-As4.820 to the diluted dark soy sauce, the glutamic acid concentration in the reaction solution was increased (white bar in the diagram), and the pyroglutamic acid concentration was decreased (in the diagram) Black diagonal bar on white background).

  On the other hand, instead of SC-As4.820, 10 mM Tris-HCl buffer (pH 8.0), BSA solution, and C.I. 2. As shown in the above, using As36.102 solution (10 mM Tris-HCl buffer (pH 8.0)) that did not show 5-oxoprolinase activity in 50 mM Tris-HCl buffer (pH 8.0), the concentration was increased to 1200 μg / mL. (Dilution) was added, the glutamate concentration in the reaction solution did not increase, and 5-oxoprolinase activity could not be detected.

  Thus, it was shown that by treating the soy sauce, which is a food having a high pyroglutamic acid content, with 5-oxoprolinase derived from Aspergillus sp. By the above-described series of techniques, there is an effect of increasing the glutamic acid concentration.

  From the above results, in a food or drink containing a large amount of pyroglutamic acid, pyroglutamic acid can be hydrolyzed to important glutamic acid as a taste component by the 5-oxoprolinase of the present invention, which is an unprecedented excellent presentation. It is expected that a food or drink having a taste can be obtained.

(Resolution of racemic body using 5-oxoprolinase of the present invention)
Racemates have the same chemical and physical properties and are difficult to distinguish under normal conditions. A method of forming and separating diastereomers using an optical resolving agent, a method of crystallization, and a method of using a column for optical resolution are used. As shown in Example 2, this enzyme uses L-pyroglutamic acid as a substrate, and D-pyroglutamic acid does not become a substrate. By utilizing this property, racemic pyroglutamic acid can be optically resolved.

  As an example, the following procedure can be used. Specifically, C.I. 2. According to the method described, a mixture of L-pyroglutamic acid and D-pyroglutamic acid [50 mM Tris, 2.5 mM L-pyroglutamic acid, 2.5 mM D-pyroglutamic acid, 6.25 mM ATP, 12.5 mM MgCl2, 187 0.5 mM KCl, pH 8.0], and 2. A yeast-expressed Aspergillus soya-derived 5-oxoprolinase SC-As4.820 solution (diluted using a 50 mM Tris-HCl buffer (pH 8.0) described below) obtained according to the method described above was prepared. The two kinds of substrate solutions (0.4 mL) and the two kinds of enzyme solutions (0.1 mL) were mixed, and an enzyme reaction was performed at 37 ° C. for 4 hours.

  After the reaction, the reaction solution was filtered through a PVDF filter having a pore size of 0.45 μm to prepare a protein-free fraction, which was applied to a column for reverse phase chromatography, CAPCELL PAK C18 MGIII (Shiseido, 20 × 250 mm). After injecting the reaction solution, eluent A (0.1% trichloroacetic acid aqueous solution) and eluent B (0.1% trichloroacetic acid / 80% acetonitrile solution) are used as eluents, and the ratio of eluent B is gradually increased. As a result, an eluted fraction was obtained. When the ultraviolet absorption at 210 nm of the eluate was measured, two peaks were detected. A plurality of fractions corresponding to each peak were mixed and concentrated by an evaporator according to a standard method. For the concentrated fraction, the concentrations of glutamic acid and pyroglutamic acid were measured according to the method described in Example 2, (8). Further, C.I. 2. The L-glutamic acid concentration was measured according to the method described in (1).

  As a result, glutamic acid (concentration almost the same as that of L-glutamic acid) was detected in the fraction containing the peak having an early retention time, and pyroglutamic acid was not detected. Thus, it was confirmed that L-glutamic acid could be collected. In the fraction containing the peak with a slow retention time, glutamic acid was not detected and pyroglutamic acid was detected. In addition, since L-pyroglutamic acid was below the detection limit when detected, the fraction containing a peak with a slow retention time did not contain L-pyroglutamic acid, indicating that D-pyroglutamic acid was obtained with high purity. confirmed. It is known that L-pyroglutamic acid can be obtained again by treating the L-glutamic acid solution under high temperature and high pressure under acidic conditions. As described above, it was shown that by using this enzyme, it is possible to resolve L-pyroglutamic acid and D-pyroglutamic acid from a racemic pyroglutamic acid.

  Depending on the type of column used, pyroglutamic acid may be contained in a fraction containing a peak having a fast retention time, and L-glutamic acid may be contained in a fraction containing a peak having a late retention time. In this case, the peak position is previously determined using a standard substance (pyroglutamic acid or L-glutamic acid), and L-pyroglutamic acid and D-pyroglutamic acid are separated from the racemic pyroglutamic acid by the same procedure as described above. Can be separated.

  When using this enzyme in food or drink, or when fermenting food or drink using microorganisms that produce this enzyme, it is necessary to obtain the enzyme from microorganisms that can be safely used in food or drink, It is also important to ferment foods and drinks using available microorganisms. The 5-oxoprolinase gene of the present invention is a gene isolated from an Aspergillus bacterium that has experienced eating. In the use of food and drink for processing, the use of a 5-oxoprolinase gene or protein derived from a genus Aspergillus leads to safe and secure food and is considered to be extremely useful in industry.

  All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (5)

A protein having the following 5-oxoprolinase activity of (a) or (b) ,
(A) a protein having an amino acid sequence showing 98% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having 5-oxoprolinase activity, or 5-oxo having the amino acid sequence of SEQ ID NO: 2 A protein having prolinase activity,
(B) a protein having an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 and 2, and which has 5-oxoprolinase activity
An umami improver for food and drink, including A protein derived from Aspergillus sojae having the following enzymological properties and having 5-oxoprolinase activity :
i) action: converts L-pyroglutamic acid to L-glutamic acid ii) molecular weight: the molecular weight of the polypeptide chain portion of the protein is 140 kDa or about 140 kDa as measured by SDS polyacrylamide gel electrophoresis iii) ) Optimum pH: pH 7.0-pH 8.0 iv) Stable pH: pH 6.0-pH 9.0 v) Temperature stability: 15 at 37 ° C. in Tris-HCl buffer (pH 8.0) Vi) Optimum temperature: 25 ° C to 45 ° C in Tris-HCl buffer (pH 8.0) vii) ATP, monovalent cation, and divalent cation requirement Show
An umami improver for food and drink, including A 5-oxoprolinase gene encoding a protein having 5-oxoprolinase activity, having the following DNA of (e) or (f) :
(E) DNA having a base sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57
(F) hybridizing with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57 under stringent conditions, and 5-oxoprolinase DNA encoding an active protein
A umami ameliorating agent for food and drink, comprising a protein having 5-oxoprolinase activity encoded by: (E) DNA having a base sequence represented by any of SEQ ID NOs: 4, 5, 56 and 57, or
(F) hybridizing with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 4, 5, 56 and 57 under stringent conditions, and 5-oxoprolinase DNA encoding an active protein
An umami improving agent for food or drink, comprising a transformant or a transductant containing a recombinant vector containing: The food or drink containing L-pyroglutamic acid and the protein contained in the umami improving agent according to claim 1, 2 or 3 , or the food or drink containing L-pyroglutamic acid according to claim 4 or the umami according to claim 4. A method for producing a food or drink having an umami taste, comprising fermenting using a transformant or a transductant contained in an improving agent.

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