JP4884733B2 - D-Lactate dehydrogenase - Google Patents

Description

  The present invention relates to a D-lactate dehydrogenase protein of Lactobacillus bacteria, a nucleic acid encoding the protein, and a method for producing the protein.

  Lactate dehydrogenase has attracted attention as a conjugate enzyme for measuring alanine aminotransferase activity, particularly in clinical examination. Natural optical isomers of D-form and L-form are known for lactic acid, D-lactate dehydrogenase acts only on D-form lactic acid, and L-lactate dehydrogenase acts only on L-form. .

  The conventionally known D-lactate dehydrogenase (EC 1.1.1.28) is more α-hydroxyglutarate in the reagent for measuring alanine aminotransferase activity than L-lactate dehydrogenase (EC 1.1.1.27). It is excellent in that there is almost no blank reaction that is considered to be derived from the activity of α-hydroxyglutarate dehydrogenase. However, conventionally known D-lactate dehydrogenase has low stability in solution, and long-term storage in solution is difficult when used as a clinical test reagent. Furthermore, since the Km value for pyruvic acid is high, a high concentration range could not be measured.

From the above, D-lactate dehydrogenase having excellent long-term storage stability in a solution and preferably having a low Km value for pyruvate has been desired.
Japanese Patent No. 2739473 Japanese Patent No. 3555950 JP 09-000247 A JP 10-084946 A Japanese Patent Laid-Open No. 10-084952 JP2002-136293 Microbiological Reviews, Vol.44, p.106-139 (1980), Garvie EI. Methods in Enzymology, Vol.89, p.35-40 (1982), Brandt RB. Appl. Environ. Microbiol. 37: 379-382 (1979) Appl. Environ. Microbiol. 65 (9), 4002-4007 (1999) Proc. Natl. Acad. Sci. USA 102 (11), 3906-3912 (2005) Eur. J. Biochem. 208 (3), 799-805 (1992) J. Biol. Chem. 267 (12), 8499-8513 (1992) FEBS Lett. 290 (1-2), 61-64 (1991) J. Biol. Chem. 266 (19), 12588-12594 (1991) Submitted (05-DEC-2003) Microbiology, College of Life Science, Nankai University, 94 Weijin Road, Tianjin, Tianjin 300071, PRC Appl. Environ. Microbiol. 70 (10), 6290-6295 (2004) Clin. Chem. Acta, 237: 43-58,1995

  An object of the present invention is to provide a nucleic acid encoding a mutant Lactobacillus bacterium D-lactate dehydrogenase protein. Here, in the mutant D-lactate dehydrogenase protein, one or more amino acid residues are substituted with another amino acid residue in the natural amino acid sequence, and the storage stability in a solution is high. It is an improved protein.

  In one embodiment of the present invention, the mutant D-lactate dehydrogenase protein is selected from the group consisting of amino acid residues corresponding to the ninth, 156th, 195th, 275th, 286th and 340th positions of SEQ ID NO: 1. One or more selected amino acid residues are replaced with amino acid residues other than natural amino acid residues.

In the present invention, preferably, in the mutant D-lactate dehydrogenase protein, the amino acid residue glycine corresponding to position 195 of SEQ ID NO: 1 is substituted with cysteine.
Another object of the present invention is to provide an expression vector containing the nucleic acid of the present invention, and a transformant transformed with the expression vector.

  Another object of the present invention is to provide a method for producing a mutant Lactobacillus bacterium D-lactate dehydrogenase protein. The production method of the present invention includes transforming a host cell with an expression vector containing the nucleic acid of the present invention, and culturing the transformed cell under conditions that allow expression of the nucleic acid.

Another object of the present invention is to provide a mutant Lactobacillus bacterium D-lactate dehydrogenase protein produced by the method of the present invention.
The present invention is also a mutant Lactobacillus D-lactate dehydrogenase protein, wherein one or more amino acid residues are replaced with another amino acid residue in the native amino acid sequence, and An object of the present invention is to provide a D-lactate dehydrogenase protein of the bacterium belonging to the genus Lactobacillus, which has improved storage stability in a solution. The D-lactate dehydrogenase protein of the present invention preferably exhibits the following pH stability and / or thermal stability: pH stability: 5.5 to 8.5 (25 ° C., 7 days)
Thermal stability: <37-55 ° C. (pH 9.0, 15 minutes).

Another object of the present invention is to provide a kit for measuring alanine aminotransferase activity, comprising the mutant D-lactate dehydrogenase protein of the present invention and instructions for use.
Another object of the present invention is to provide a nucleic acid encoding a natural D-lactate dehydrogenase protein. The nucleic acid of the present invention comprises
below:
(A) a nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(C) a nucleic acid capable of hybridizing under stringent conditions to the nucleic acid of (a) or (b) and encoding a protein having D-lactate dehydrogenase activity; and (d) the amino acid of SEQ ID NO: 1 A protein comprising an amino acid sequence at least 80% identical to the sequence and selected from the group consisting of nucleic acids encoding proteins having D-lactate dehydrogenase activity.

The nucleic acid of the present invention is preferably Lactobacillus sp. It encodes a D-lactate dehydrogenase protein derived from NBRC14511.
Another object of the present invention is to provide an expression vector containing the nucleic acid of the present invention and a transformant transformed with the expression vector of the present invention.

The present invention further includes the following:
(A) a nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(C) a nucleic acid capable of hybridizing under stringent conditions to the nucleic acid of (a) or (b) and encoding a protein having D-lactate dehydrogenase activity; and (d) the amino acid of SEQ ID NO: 1 A natural D-lactate dehydrogenase encoded by a nucleic acid selected from the group consisting of a nucleic acid encoding a protein having an amino acid sequence at least 80% identical to the sequence and having D-lactate dehydrogenase activity The object is to provide a protein.

The present inventor has come up with the present invention as a result of diligent efforts and research to solve the above problems.
Specifically, first, as a result of selecting and comparing 10 known lactic acid bacteria, Lactobacillus sp. Was selected as a bacterium candidate for obtaining an enzyme having a lower Km value for pyruvate than D-lactate dehydrogenase. NBRC14511 (National Institute of Technology and Evaluation (NITE) strain registration number, available from the same corporation) (Suzuki, K., Benno, Y., Mitsuoka, T., Takebe, S., Kobashi, K. & Hase , J. (1979). Appl. Environ. Microbiol. 37: 379-382).

  A gene (nucleic acid) encoding a novel D-lactate dehydrogenase protein was isolated from the bacterium (SEQ ID NO: 2). Furthermore, using the nucleic acid, D-lactate dehydrogenase protein (SEQ ID NO: 1) was expressed by genetic engineering to confirm the activity, and the present invention was conceived. Lactobacillus sp. The novel D-lactate dehydrogenase protein derived from NBRC14511 has the advantage that the Km value for pyruvate is lower than that of known proteins.

  Furthermore, the present inventors continued this research in order to improve the storage stability in the solution of D-lactate dehydrogenase. And, from the natural amino acid sequence of D-lactate dehydrogenase, one or two or more amino acids are randomly substituted with another amino acid, thereby improving mutant D-lactate dehydrogenation in solution. The present invention was conceived by successfully obtaining a protein having enzyme activity.

In one aspect, the present invention provides a nucleic acid encoding a mutant Lactobacillus bacterium D-lactate dehydrogenase protein, wherein the nucleic acid encodes a mutant Lactobacillus bacterium D-lactate dehydrogenase protein, In the mutant D-lactate dehydrogenase protein, one or more amino acid residues are replaced with another amino acid residue in the natural amino acid sequence, and the storage stability in solution is improved. And the nucleic acid is a protein.

The “D-lactate dehydrogenase protein” is an enzyme protein having catalytic activity for removing hydrogen from D-lactic acid, and generally promotes the following reaction reversibly.
Reaction: D-lactic acid + NAD ⁺ ⇔ pyruvic acid + NADH + H ⁺
Moreover, although it is not necessarily limited, it has the following biochemical properties.

Molecular weight: about 43,000-45,000 (preferably about 44,000)
(Per SDS-PAGE subunit)
Km value: pyruvate about 0.14-about 0.30 mM
NADH about 0.10 to about 0.15 mM
Optimum pH: about 6.5 to about 7.5
The method for measuring the enzyme activity of “D-lactate dehydrogenase protein” is not limited and can be examined using a known method. Although not limited, for example, at a measurement temperature of 25 ° C. and a measurement wavelength of 340 nm, 1 μmol of NAD ⁺ produced per minute under the reaction conditions is defined as 1 unit, and the enzyme activity is defined as D-lactate dehydrogenase activity. It may be defined. Thereby, the amount of NAD ⁺ produced by the reverse reaction of the above reaction formula is measured.

  As a reaction solution for activity measurement, for example, a potassium phosphate buffer containing pyruvic acid and NADH as substrates can be used. The composition of the buffer is not particularly limited, and the following composition was employed in the examples.

Buffer composition for LDH activity measurement Final concentration potassium phosphate buffer 0.1M
(PH 7.0)
Pyruvate 2mM
NADH 0.22mM
Alternatively, the enzyme activity may be determined by measuring the amount of D-lactic acid produced by a reverse reaction in the same manner as NAD ⁺ . Alternatively, pyruvic acid or NADH by a positive reaction may be measured. Those skilled in the art can appropriately adopt known measurement methods.

  “Lactobacillus bacteria” are also called lactobacilli and resistant lactobacilli, and the species is not particularly limited. For example, the following D-lactate dehydrogenase proteins derived from Lactobacillus species are known.

1) Lactobacillus johnsonii
ACCESSION AF071558
AUTHORS Lapierre, L., Germond, JE, Ott, A., Delley, M. and Mollet, B.
TITLE D-Lactate dehydrogenase gene (ldhD) inactivation and resulting metabolic effects in the Lactobacillus johnsonii strains La1 and N312
JOURNAL Appl. Environ. Microbiol. 65 (9), 4002-4007 (1999)
PUBMED 10473408
2) Lactobacillus acidophilus NCFM
ACCESSION CP000033
AUTHORS Altermann, E., Russell, WM, Azcarate-Peril, MA, Barrangou, R., Buck, BL, McAuliffe, O., Souther, N., Dobson, A., Duong, T., Callanan, M., Lick, S., Hamrick, A., Cano, R. And Klaenhammer, TR
TITLE Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM
JOURNAL Proc. Natl. Acad. Sci. USA 102 (11), 3906-3912 (2005)
PUBMED 15671160
3) L.helveticus
ACCESSION X66723
AUTHORS Kochhar, S., Hottinger, H., Chuard, N., Taylor, PG, Atkinson, T., Scawen, MD and Nicholls, DJ
TITLE Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli
JOURNAL Eur. J. Biochem. 208 (3), 799-805 (1992)
PUBMED 1396685
4) Lactobacillus delbrueckii (Lactobacillus delbrueckii-2)
ACCESSION M85224
AUTHORS Kochhar, S., Hunziker, PE, Leong-Morgenthaler, P. and Hottinger, H.
TITLE Primary structure, physicochemical properties, and chemical modification of NAD (+)-dependent D-lactate dehydrogenase.Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site
JOURNAL J. Biol. Chem. 267 (12), 8499-8513 (1992)
PUBMED 1569100
5) L. delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii)
ACCESSION X60220
AUTHORS Bernard, N., Ferain, T., Garmyn, D., Hols, P. and Delcour, J.
TITLE Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp.bulgaricus by complementation in Escherichia coli
JOURNAL FEBS Lett. 290 (1-2), 61-64 (1991)
PUBMED 1915894
6) Lactobacillus plantarum
ACCESSION D90339
AUTHORS Taguchi, H. And Ohta, T.
TITLE D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family.Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum
JOURNAL J. Biol. Chem. 266 (19), 12588-12594 (1991)
PUBMED 1840590
7) Lactobacillus sp. MD-1
ACCESSION AY496011
REFERENCE 1 (bases 1 to 996)
AUTHORS Li, J., Liu, R., Liang, F. And Zhang, X.
TITLE Cloning and sequence analysis of D-Lactate dehydrogenase gene in Lactobacillus sp. MD-1
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 996)
AUTHORS Li, J., Liu, R., Liang, F. And Zhang, X.
TITLE Direct Submission
JOURNAL Submitted (05-DEC-2003) Microbiology, College of Life Science, Nankai University, 94 Weijin Road, Tianjin, Tianjin 300071, PRC
8) Lactobacillus sp. MONT4
ACCESSION AY301012
AUTHORS Weekes, J. and Yuksel, GU
TITLE Molecular Characterization of Two Lactate Dehydrogenase Genes with a Novel Structural Organization on the Genome of Lactobacillus sp. Strain MONT4
JOURNAL Appl. Environ. Microbiol. 70 (10), 6290-6295 (2004)
PUBMED 15466577
In the present invention, “nucleic acid” includes synthetic DNA and synthetic RNA in addition to DNA, RNA and cDNA. Furthermore, vectors containing these are also within the scope of the “nucleic acid” of the present invention. Unless specifically stated, either double-stranded or single-stranded embodiments are included. In the case of a double strand, when a base sequence is referred to in this specification, it is intended to include its complementary base sequence. In one preferred embodiment, the nucleic acid is substantially free of contaminating endogenous components. The nucleic acid molecule is preferably in a substantially pure form at least once and can be obtained using standard biochemical methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring, NY). (Such as those outlined in Harbor (1989)) from DNA or RNA that has been isolated in amounts or concentrations that allow the identification, manipulation, and recovery of its component nucleotide sequences.

  The present invention relates to a nucleic acid encoding a mutant Lactobacillus bacterium D-lactate dehydrogenase protein. Wherein the mutant D-lactate dehydrogenase protein is: 1) one or more amino acid residues are replaced with another amino acid residue in the natural amino acid sequence, and 2) a solution The storage stability in is improved.

  As described above, a natural (wild-type) Lactobacillus bacterium D-lactate dehydrogenase protein is known, and its amino acid sequence and base sequence are also known. The present invention conserves D-lactate dehydrogenase protein in solution by replacing one or more amino acid residues with another amino acid residue different from the natural amino acid residue in the natural amino acid sequence. Based on success in improving stability.

  Examples described later in this specification use random mutagenesis to create mutants in which mutations are randomly introduced into the amino acid sequence of the D-lactate dehydrogenase protein of Lactobacillus bacteria, Alternatively, pH stability was measured, and those having higher residual activity than natural proteins were selected as preferred mutants of the present invention. In this way, more mutant D-lactate dehydrogenase proteins are prepared using random mutagenesis, and the storage stability in the solution is improved, that is, the thermal stability and / or the pH stability is improved. It is possible to obtain further mutants by repeatedly selecting those that are improved. In Example 4, a mutant having improved storage stability (solution stability) in a solution compared to the wild type under conditions of 40 ° C. and 15 minutes was selected. By changing the screening conditions, it is possible to further obtain mutants by relaxing the temperature conditions to be selected or increasing the storage time. Those skilled in the art can ascertain the preferred embodiments of the site and number at which the amino acid of the present invention is substituted according to the disclosure of the present specification.

Alternatively, when a mutant is produced by targeting a specific site in the sequence, known site-directed mutagenesis can be employed.
In the present invention, the “thermal stability” of the D-lactate dehydrogenase protein is not limited, but for example, as described in Example 6, the treatment is performed at pH 9.0 for 15 minutes. This means that the temperature at which the remaining activity of 75% is maintained is higher than that of the wild type D-lactate dehydrogenase protein. Specifically, as shown in Table 1, Lactobacillus sp. The wild type D-lactate dehydrogenase protein derived from NBRC14511 had a temperature (thermal stability) at which the remaining activity of 75% was maintained at 30 ° C. On the other hand, all of the mutants of the present invention were stable from 37 ° C to 55 ° C.

  In the present invention, it is not limited that “thermal stability” of D-lactate dehydrogenase protein has been improved. For example, as described in Example 6, the protein was stored at 25 ° C. for 7 days. In this case, it means that the pH range in which the remaining activity of 90% is maintained is wider than that of the wild type D-lactate dehydrogenase protein. Specifically, as shown in Table 1, Lactobacillus sp. In the wild-type D-lactate dehydrogenase protein derived from NBRC14511, the range in which the residual activity was maintained at 90% (pH stability) was pH 5.5-8.0. In contrast, all of the mutants of the present invention were stable at a higher pH of at least pH 5.5-8.5. More preferably, it was stable at pH 5.5-9.5, and more preferably at pH 5.5-10.0.

  In addition to “thermal stability” and / or “pH stability”, “storage stability in solution” includes a long period of time under normal conditions (for example, optimum pH such as 25 ° C. and pH 7.0). This includes stability during storage.

  1 and 2 show Lactobacillus sp. Prepared in Examples as an embodiment of the present invention. The thermostability and pH stability of a mutant D-lactate dehydrogenase derived from NBRC14511 are shown. The variant “G195C” is a variant in which the 195th glycine in SEQ ID NO: 1 is substituted with cysteine. The mutant “E9G / E275K / I286V” is a mutant in which the ninth glutamic acid is replaced by glycine, the 275th glutamic acid is replaced by lysine, and the 286th isoleucine is replaced by valine in SEQ ID NO: 1. Mutant “I286V” is a mutant in which 286th isoleucine is replaced with valine, and “K156R / N340K” has 156th lysine replaced with arginine and 340th asparagine replaced with lysine. It is a mutant. All the mutants have improved “thermostability” and / or “pH stability” as compared to wild-type D-lactate dehydrogenase derived from the same species.

  Therefore, in the present invention, preferably, the non-limiting D-lactate dehydrogenase protein corresponds to the ninth, 156th, 195th, 275th, 286th and 340th positions of SEQ ID NO: 1. One or more amino acid residues selected from the group consisting of amino acid residues are substituted with amino acid residues different from natural amino acid residues. SEQ ID NO: 1 is Lactobacillus. sp. It is the amino acid sequence of the natural type D-lactate dehydrogenase protein of (NBRC14511). The protein is encoded by a nucleic acid having the base sequence of SEQ ID NO: 2. “The amino acid residues corresponding to the 9th, 156th, 195th, 275th, 286th and 340th positions of SEQ ID NO: 1” preferably mean these amino acid residues in SEQ ID NO: 1. However, the present invention is not limited to this. When the amino acid sequence of a natural D-lactate dehydrogenase protein derived from another bacterium of another species of Lactobacillus is aligned with the amino acid sequence of SEQ ID NO: 1, Similarly, the mutant protein of the present invention can be obtained by substituting an amino acid residue present at a position corresponding to a residue with an amino acid residue different from the natural amino acid residue.

  For example, FIG. 3 shows Lactobacillus. sp. The amino acid sequence of (NBRC14511) natural D-lactate dehydrogenase protein and the D-lactate dehydrogenase protein derived from the known genus Lactobacillus, which was referred to when preparing a probe for isolating the gene encoding the protein These five amino acid sequences are aligned so as to maximize homology (alignment). As shown in FIG. 3, for example, Lactobacillus. sp. It can be seen that the 195th amino acid residue of SEQ ID NO: 1 of (NBRC14511) corresponds to the 167th or 168th position in the D-lactate dehydrogenase protein derived from other species, and both are glycine.

  FIG. 4 further shows that Lactobacillus. acidophilus, Lactobacillus. sp. MD-1 and Lactobacillus. sp. The amino acid sequences of D-lactate dehydrogenase proteins derived from three species derived from the genus Lactobacillus of MONT4 are aligned and arranged. For the convenience of alignment so that the homology is maximized, for example, the 195th amino acid residue glycine of SEQ ID NO: 1 is represented as the 197th amino acid residue in FIG.

Preferably, said mutant D-lactate dehydrogenase protein satisfies one or more of any of the following conditions:
1) the amino acid residue corresponding to position 9 of SEQ ID NO: 1 is substituted with glycine;
2) the amino acid residue corresponding to position 156 of SEQ ID NO: 1 is substituted with arginine;
3) the amino acid residue corresponding to position 195 of SEQ ID NO: 1 is substituted with cysteine;
4) the amino acid residue corresponding to position 275 of SEQ ID NO: 1 is substituted with lysine;
5) The amino acid residue corresponding to position 286 of SEQ ID NO: 1 is substituted with valine; and 6) The amino acid residue corresponding to position 340 of SEQ ID NO: 1 is replaced with lysine. Residues are not limited to the above. Those skilled in the art can appropriately employ substitutions of amino acid residues other than those described above based on the disclosure of the present specification.

  In the amino acid residues corresponding to the 9th, 156th, 195th, 275th, 286th and 340th positions of SEQ ID NO: 1, the substitution may be performed at one position or may occur at any plural positions (up to 6 positions). Good. Examples of such a preferable replacement location are the 195th single, the 286th single, the 9th / 275th / 286th combination, or the 156th / 340th combination.

  Preferably, it is a variant in which the amino acid residue corresponding to position 195 of SEQ ID NO: 1 is substituted alone. More preferably, the amino acid residue corresponding to position 195 of SEQ ID NO: 1 is substituted with cysteine. Most preferably, the amino acid residue glycine corresponding to position 195 of SEQ ID NO: 1 is substituted with cysteine.

Natural D-Lactate Dehydrogenase Underlying the Variant of the Invention In one aspect, the present invention relates to a “natural” Lactobacillus bacterial D-lactate dehydrogenase protein, wherein one or more natural amino acid sequences are present. By substituting one amino acid residue with another amino acid residue, a mutant protein having improved storage stability in the solution is provided. In the present specification, the “natural” D-lactate dehydrogenase protein is not only one that has actually been confirmed to exist naturally in Lactobacillus bacteria, but also the amino acid sequence and activity of the protein that has been confirmed. Also included are homologous proteins that differ in some amino acid sequences that are not involved in, but do not change the solution stability of the enzyme.

  Although not necessarily limited, in the present invention, preferably, the Lactobacillus bacterium is Lactobacillus sp. NBRC14511, the natural amino acid sequence of the D-lactate dehydrogenase protein has the amino acid sequence set forth in SEQ ID NO: 1. That is, preferably, the mutant of the present invention is a mutant from the natural amino acid sequence shown in SEQ ID NO: 1. The natural amino acid sequence of D-lactate dehydrogenase protein is preferably encoded by the base sequence of SEQ ID NO: 2. That is, preferably, the nucleic acid encoding the mutant of the present invention is a mutant from the natural base sequence shown in SEQ ID NO: 2.

  One or more codons may encode the same amino acid, which is called the degeneracy of the genetic code. For this reason, a DNA sequence that does not completely match SEQ ID NO: 2 may encode a protein having the same amino acid sequence as SEQ ID NO: 1. Such degenerate DNA sequences may result from silent mutations (eg, occurring during PCR amplification) or may be the product of intentional mutagenesis of native sequences. Since the degenerate DNA sequence encodes the amino acid sequence of SEQ ID NO: 1 in the same manner as natural SEQ ID NO: 2, a nucleic acid encoding the mutant D-lactate dehydrogenase protein (variant) of the present invention is prepared. Can be the basis of

  In addition to SEQ ID NO: 1, for example, the amino acid sequences of known D-lactate dehydrogenase proteins of known Lactobacillus bacteria described in FIGS. 3 and 4 can also serve as a basis for producing the mutant of the present invention. However, the present invention is not limited to these, and any homologue that does not change the solution stability of the enzyme, even if the amino acid sequence of one or more amino acid sequences is deleted, added or substituted in the natural sequence, is not limited. The “natural amino acid sequence” can be the basis for the production of the mutant of the present invention. Such “amino acid mutation” is 1 to plural, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5.

  These homologues are at least about 70%, preferably about 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more with the natural amino acid sequence, eg, SEQ ID NO: 1. Have identity.

  The percent amino acid identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by Needleman, S .; B. And Wunsch, C.I. D. (J. Mol. Biol., 48: 443-453, 1970) and determined by comparing sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). May be. Preferred default parameters for the GAP program include: (1) Henikoff, S and Henikoff, J. et al. G. (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992), scoring matrix, blossum 62; (2) 12 gap weights; (3) 4 gap length weights; And (4) no penalty for end gaps.

  Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). The program can be used on the Internet from the website of National Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ). Various conditions (parameters) for homology search by the BLAST program are described in detail on the same site, and some settings can be appropriately changed, but the search is usually performed using default values.

  A nucleic acid encoding a homologue that forms the basis of the production of the mutant of the present invention is also a nucleic acid having a natural base sequence, for example, a stringent condition that is moderately or highly stringent under conditions stringent to SEQ ID NO: 2, for example. And a nucleic acid encoding a homologous protein that can hybridize under mild conditions and exhibits solution-stable D-lactate dehydrogenase activity.

“Stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA, pH 8.0, about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C. , Other similar hybridization solutions such as Stark's solution), and the use of washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Preferably moderately stringent conditions include hybridization conditions of about 50 ° C. and 6 × SSC. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, More preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg hybridization conditions as described above, and approximately 68 ° C., 0.2 × SSC, 0.1% Defined with SDS washing. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete.

In order to achieve the desired degree of stringency by applying the basic principles known to those skilled in the art and governing the hybridization reaction and duplex stability, as further described below, the wash temperature It should be understood that the wash salt concentration can be adjusted as needed (see, eg, Sambrook et al., 2001). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the length of the hybrid can be determined by juxtaposing the nucleic acid sequences and identifying the region or regions with optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be the length of less than 50 base pairs, not should be 5-25 less ℃ than hybrid melting temperature (T _m), T m is determined by the following equation Is done. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (A + T base number) +4 (G + C base number). For hybrids longer than 18 base pairs, T _m = 81.5 ° C. + 16.6 (log ₁₀ [Na ⁺ ]) + 41 (molar fraction [G + C]) − 0.63 (% formamide) −500 / n, where N is the number of bases in the hybrid, and [Na ⁺ ] is the sodium ion concentration in the hybridization buffer (1 × SSC [Na ⁺ ] = 0.165 M). Preferably, each such hybridizing nucleic acid is at least 8 nucleotides (or more preferably at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 1% of the length of the nucleic acid to which it hybridizes (more preferably at least 25%, or at least 50%, or at least 70%, and most preferably at least 80) At least 50% (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least with the nucleic acid to which it hybridizes 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%). Here, sequence identity, as described in more detail above, by comparing the sequences of hybridizing nucleic acids aligned in parallel to maximize identity with overlapping portions while minimizing sequence gaps. It is determined.

  The percent nucleic acid identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing sequence information using a computer program. A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux et al., 1984, Nucl. Acids Res. 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters of the “GAP” program are: (1) GCG implementation of a unitary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and Schwartz and Dayhoff supervised “Atlas of Polypeptide Sequence and Structure” National Biomedical Research Foundation, pages 353-358, 1979, Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986 weighted amino acid comparison matrix; or other comparable comparison matrix; (2) 30 penalties for each gap of amino acids and one additional penalty for each symbol in each gap; or each of the nucleotide sequences Includes 50 penalties for gaps and an additional 3 penalties for each symbol in each gap; (3) no penalty to end gaps; and (4) no maximum penalty for long gaps. Other sequence comparison programs used by those skilled in the art include, for example, the National Medical Library website: http: // www. ncbi. nlm. nih. gov / blast / bl2seq / bls. The BLASTN program available for use by html, version 2.2.7, or the UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 can be found at the following Internet site: http: // blast. Wustl. It is described in edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) Segments of query sequences with low compositional complexity (Woughton and Federhen SEG program (Computers and Chemistry, 1993); Woton and Federhen, 1996, “Analysis of compositionally biased regions in sequence data bases” or 71: 66. , Segments consisting of short-periodic internal repeats (Cl including a filter for masking verie and States (determined by the XNU program of Computers and Chemistry, 1993), and (B) a threshold of statistical significance for reporting a fit to the database sequence, or According to the statistical model of E-score (Karlin and Altschul, 1990), the expected probability of a match that is simply found by chance; if the statistical significance due to a match is greater than the E-score threshold, this fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e 30,1e-40,1e-50,1e-75, or a 1e-100.

  Similarly, the natural nucleic acid of the present invention differs from a known natural base sequence, for example, SEQ ID NO: 2, due to deletion, insertion or substitution of one or more bases, but is solution stable. A nucleic acid encoding a homologous protein exhibiting D-lactate dehydrogenase activity. As long as the “solution stability” of the enzyme is maintained, the number of bases to be deleted, inserted or substituted is not particularly limited, but is preferably 1 to 500, more preferably 1 to 200, most preferably 1. From 100 to 100.

  Certain amino acids may be substituted, for example, with residues having similar physicochemical properties. Examples of such conservative substitutions are those that replace one aliphatic residue with each other, eg Ile, Val, Leu, or Ala; one such as between Lys and Arg, Glu and Asp, or between Gln and Asn. Substitution from one polar residue to another; or substitution of another aromatic residue, such as substituting one another for Phe, Trp, or Tyr. Other conservative substitutions are well known, for example, substitution of entire regions with similar hydrophobic properties. A person skilled in the art can make a desired deletion, insertion or substitution by a well-known genetic engineering technique using, for example, site-directed mutagenesis described in Sambrook et al. (2001) (described above) and the like. Is possible.

Expression vector containing the nucleic acid of the present invention, transformant, and method for producing the mutant protein of the present invention The present invention further comprises an expression vector containing the nucleic acid of the present invention, and a transformant transformed with the expression vector. I will provide a. The transformant is preferably E. coli.

  The present invention also provides a mutant Lactobacillus bacterium comprising transforming a host cell with an expression vector comprising the nucleic acid of the present invention and culturing the transformed cell under conditions that allow expression of the nucleic acid. A method for producing a D-lactate dehydrogenase protein is provided.

  A method for producing the mutant Lactobacillus bacterium D-lactate dehydrogenase protein of the present invention is described below. Expression, isolation and purification of the proteins and fragments of the present invention are not limited and may be accomplished by any suitable technique.

  In addition to the nucleic acid of the present invention, the expression vector of the present invention can generally be contained in a vector containing a selectable marker and an origin of replication for propagation in a host. The vector further includes appropriate transcriptional or translational control sequences, such as those derived from mammalian, microbial, viral, or insect genes, optionally linked to the nucleic acids of the invention.

  Examples of regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that regulate transcription and translation initiation and termination. A nucleotide sequence is operably linked when a regulatory sequence is functionally related to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence regulates transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the host cell and a selection gene that identifies the transformant are generally incorporated into the expression vector. As the selection marker, a commonly used marker can be used by a conventional method. Examples include tetracycline, ampicillin, or antibiotic resistance genes such as kanamycin or neomycin, hygromycin or spectinomycin.

  Suitable host cells for nucleic acids encoding the mutant Lactobacillus D-lactate dehydrogenase proteins of the present invention include prokaryotes, yeast, and higher order eukaryotic cells, each of which is described below. To discuss.

  The protein of the invention to be expressed in such host cells can also be a fusion protein comprising a region derived from a heterologous protein. For example, such regions can be included to allow protein secretion, improved stability, enhanced purification, targeting, or oligomerization. For example, a nucleic acid sequence encoding an appropriate signal peptide can be incorporated into an expression vector. A nucleic acid sequence encoding a signal peptide (secretory leader) can be fused in-frame to the nucleic acid of the invention so that it is translated as a fusion protein comprising the signal peptide. A signal peptide that functions in the intended host cell promotes extracellular secretion of the packed protein. It is possible to exchange the natural signal sequence with a heterologous signal peptide.

  Suitable host cells for expression of the proteins of the invention include prokaryotes, yeast, and higher order eukaryotic cells. Prokaryotic hosts suitable for use in the expression of these proteins include Pseudomonas, along with bacteria of the genus Escherichia (eg JM109, DH5α, HB101, etc.), Bacillus, and Salmonella. Members of the genera (Pseudomonas), Streptomyces, and Staphylococcus are included. For expression in prokaryotic cells, eg, in E. coli, the polynucleotide molecule encoding the protein of the invention preferably includes an N-terminal methionine residue that facilitates expression of the recombinant protein. The N-terminal Met can optionally be cleaved from the expressed protein.

  Expression vectors for use with cellular hosts generally comprise one or more phenotypically selectable markers. Such genes, for example, encode proteins that confer antibiotic resistance or proteins that provide autotrophic requirements. A wide range of such vectors are readily available from commercial sources. Examples include the pGEM vector (Promega), the pSPORT vector, and the pPROEX vector (InVitrogen, Life Technologies, Carlsbad, Calif.), The Bluescript vector (Stratagene), and the pQE vector (Qiagen).

  In Examples described later, pTRP-ldhD was used as an expression vector in E. coli. The construction of the pTRP vector is described, for example, in Uchida, K., Kondoh, K. and Matsuo, Y. et al. (Clin. Chem. Acta, 237: 43-58, 1995).

  The proteins of the invention can also be expressed in yeast host cells from genera, including Saccharomyces, Pichia, and Kluberomyces. Preferred yeast hosts are S. cerevisiae. S. cerevisiae and P. cerevisiae. P. pastoris. Yeast vectors will often contain an origin of replication sequence derived from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. . Vectors that can replicate in both yeast and E. coli (referred to as shuttle vectors) can also be used. In addition to the features described above for yeast vectors, shuttle vectors will also include sequences for replication and selection in E. coli. Direct secretion of a protein expressed in a yeast host can be achieved by including a nucleotide sequence encoding a yeast alpha factor leader sequence at the 5 'end of the nucleotide sequence encoding the protein of the invention. A vector is simply a gene DNA in a vector such as a plasmid that can be prepared by ligating a desired gene to a recombination vector available in the industry (eg, plasmid DNA) by a conventional method. As a method for incorporating the fragment, for example, Sambrook, J. et al. , And Russell, D.C. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. (New York: Cold Spring Harbor Laboratory Press) and the like. For convenience, a commercially available ligation kit (for example, manufactured by Takara Shuzo Co., Ltd.) can also be used.

  Methods for introducing a vector into a host cell are generally described in Sambrook, J. et al. (2001) (described above), a calcium phosphate method or a calcium chloride / rubidium chloride method, an electroporation method, an electroinjection method, a method using chemical treatment such as PEG, a method using a gene gun, and the like.

  Insect host cell culture systems can also be used to express the proteins of the invention. The proteins of the invention are preferably expressed using a baculovirus expression system, as described, for example, in the review of Luckow and Summers ((1988), BioTechnology 6:47).

  It is also possible to express the protein of the present invention in a mammalian host cell. Non-limiting examples of suitable mammalian host cell lines include monkey kidney cell COS-7 strain (Gluzman et al. (1981), Cell 23: 175-182), Chinese hamster ovary (CHO) cells (Puck et al. (1958). , PNAS USA 60: 1275-1281), CV-1 (Fischer et al. (1970), Int. J. Cancer 5: 21-27) and human cervical cancer cells (HELA) (ATCC CCL 2).

The selection of an expression vector suitable for expression of the protein of the invention will depend on the particular mammalian host cell being used. Examples of suitable expression vectors include pcDNA3.1 / Hygro ⁺ (Invitrogen), pDC409, and pSVL. Expression vectors for use in mammalian host cells can include transcriptional and translational regulatory sequences derived from the viral genome. Commonly used promoter and enhancer sequences that can be used to express proteins include, but are not limited to, human cytomegalovirus (CMV), adenovirus 2, polyomavirus, and simian virus. 40 (SV40) origin is included. Methods for constructing mammalian expression vectors are disclosed, for example, in Okayama and Berg ((1982) Mol. Cell. Biol. 2: 161-170), Cosman et al. ((1986) Mol. Immunol. 23: 935-941). The

  Known to facilitate insertion into specific vectors, modification of nucleic acid molecules (eg, by modifying restriction sites), ease of use in specific expression systems or hosts (eg, using preferred host codons), etc. And is intended for use in the present invention. Genetic engineering methods for producing proteins include expression of nucleic acid molecules in cell-free expression systems, in cellular hosts, in tissues, and in animal models, according to known methods.

  The protein of the present invention can be prepared by culturing a transformant (transformed host cell) under culture conditions suitable for recombinant protein expression. The resulting expressed protein can then be purified from such cultures (ie media or cell extracts) using known purification methods such as selective precipitation with various salts, gel filtration and ion exchange chromatography. It is. Protein purification also includes an affinity column containing an agent that will bind to the protein; one or more column steps on an affinity resin such as concanavalin A-agarose, Cibacrom Blue 3GA Sepharose®; phenyl ether, butyl ether Or one or more steps involving hydrophobic interaction chromatography using a resin such as propyl ether; or immunoaffinity chromatography using antibodies that specifically bind to one or more epitopes of the protein of the invention It is possible. Alternatively, the proteins of the invention can also be expressed in a form that will facilitate purification. For example, it can be expressed as a fusion protein, that is, it can be fused with maltose binding protein (MBP), glutathione-S-transferase (GST) and the like. Proteins can also be purified by tagging with epitopes and subsequently using specific antibodies directed against such epitopes. One such epitope (“Flag®”) is commercially available from Kodak (New Haven, Conn.).

  Finally, using one or more RP-HPLC steps using hydrophobic reverse phase high performance liquid chromatography (RP-HPLC) media, eg silica gel with pendant methyl or other aliphatic groups, the protein can be further It is possible to purify. It is also possible to provide a substantially homogeneous isolated recombinant protein using some or all of the various purification steps described above. A protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein”; such an isolated protein of the present invention binds to a protein of the present invention. Isolated antibodies, fragments, variants, binding partners and the like.

  The proteins of the invention can also be produced by known conventional chemical synthesis. Methods of constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The amino acid sequence of the protein constructed by synthesis shares primary, secondary or tertiary structure and / or conformational properties with the protein of the present invention produced by genetic engineering, including enzyme activity, solution stability, It may have the same biological properties as the protein of the present invention.

Mutant Lactobacillus D-Lactate Dehydrogenase Protein The present invention further provides a mutant Lactobacillus bacterium D-lactate dehydrogenase protein. The mutant Lactobacillus bacterium D-lactate dehydrogenase protein of the present invention has one or more amino acid residues substituted in the natural amino acid sequence with another amino acid residue, and is stored in solution. It is characterized by improved stability.

The protein of the present invention is preferably produced by the production method using the nucleic acid of the present invention described above.
The mutant D-lactate dehydrogenase protein of the bacterium belonging to the genus Lactobacillus of the present invention is as described in detail in the section "Nucleic acid encoding the D-lactate dehydrogenase protein of the mutated Lactobacillus bacterium".

  The protein of the present invention is preferably one or more amino acids selected from the group consisting of amino acid residues corresponding to the 9th, 156th, 195th, 275th, 286th and 340th positions of SEQ ID NO: 1 The residue is substituted with an amino acid residue different from the natural amino acid residue.

The mutant protein of the present invention has improved storage stability in solution than the natural protein. Preferably, the following pH stability and / or thermal stability are satisfied.
pH stability: 5.5-8.5 (25 ° C., 7 days)
Thermal stability: <37-55 ° C. (pH 9.0, 15 minutes).

More preferably, the pH stability is 5.5 to 9.0, still more preferably the pH stability is 5.5 to 9.5, and even more preferably the pH stability is 5.5 to 10.0.
Moreover, although it is not necessarily limited, it has the following biochemical properties.

Molecular weight: about 43,000-45,000 (preferably about 44,000)
(Per SDS-PAGE subunit)
Km value: pyruvate about 0.14-about 0.30 mM
NADH about 0.10 to about 0.15 mM
Optimum pH: about 6.5 to about 7.5
These molecular weights, Km values, and optimum pH are substantially the same as the wild-type Lactobacillus bacteria D-lactate dehydrogenase protein, and are common.

Kit for measuring alanine aminotransferase activity The D-lactate dehydrogenase (D-LDH) of the present invention can function as a conjugate enzyme of alanine aminotransferase (ALT). The specific reaction is as follows.

L-alanine + 2-oxoglutaric acid
→ pyruvic acid + L-glutamic acid
ALT
Pyruvic acid + NADH + H ⁺
→ D-lactic acid + NAD ⁺
D-LDH

The activity of alanine aminotransferase can be measured using the D-lactate dehydrogenase of the present invention. Therefore, the present invention provides a kit for measuring alanine aminotransferase activity. The kit of the present invention includes a mutant D-lactate dehydrogenase protein and instructions for use. Further, if desired, Tris buffer, L-alanine, 2-oxoglutarate, NADH and the like may be contained.

Natural D-lactate dehydrogenase protein, nucleic acid, etc. In the present invention, Lactobacillus sp. Nucleic acid encoding a novel D-lactate dehydrogenase protein was isolated from NBRC14511. Lactobacillus sp. Isolated in the present invention. The nucleic acid derived from NBRC14511 has the base sequence set forth in SEQ ID NO: 1 and encodes the amino acid sequence set forth in SEQ ID NO: 2.

  Lactobacillus sp. Of the present invention. NBRC14511-derived D-lactate dehydrogenase has the closest known D-lactate dehydrogenase protein from Lactobacillus johnsonii with an amino acid sequence of 72.6% and a nucleic acid sequence of 67.3%. .

Therefore, in another aspect, the present invention provides a nucleic acid encoding a natural D-lactate dehydrogenase protein. The nucleic acid of the present invention comprises
below:
(A) a nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(C) a nucleic acid capable of hybridizing under stringent conditions to the nucleic acid of (a) or (b) and encoding a protein having D-lactate dehydrogenase activity; and (d) the amino acid of SEQ ID NO: 1 A protein comprising an amino acid sequence at least 80% identical to the sequence and selected from the group consisting of nucleic acids encoding proteins having D-lactate dehydrogenase activity.

The definition of “under stringent conditions” is the same as that described in the section of “Natural D-lactate dehydrogenase as the basis of the mutant of the present invention”.
The definition of “identity” is also the same as that described in the section “Natural D-Lactate Dehydrogenase as the basis of the mutant of the present invention”. More preferably, the native protein of the present invention comprises an amino acid sequence that is at least 85%, more preferably 90%, even more preferably 95% identical to the amino acid sequence of SEQ ID NO: 1. The nucleic acid encoding the natural protein of the present invention preferably comprises a base sequence that is at least 70%, more preferably 80%, and even more preferably 90% identical to the nucleic acid of SEQ ID NO: 2.

The nucleic acid of the present invention is preferably Lactobacillus sp. It encodes a D-lactate dehydrogenase protein derived from NBRC14511.
More preferably, the nucleic acid of the present invention is either (a) or (b) below.

(A) a nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO: 1; or (b) a nucleic acid comprising the base sequence of SEQ ID NO: 2.
Another object of the present invention is to provide an expression vector containing the nucleic acid of the present invention and a transformant transformed with the expression vector of the present invention. The definitions of “expression vector” and “transformant” are the same as those described in the section “Method for producing expression vector containing the nucleic acid of the present invention, transformant, and mutant protein of the present invention”.

The present invention further provides a natural D-lactate dehydrogenase protein. The proteins of the present invention are:
(A) a nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(C) a nucleic acid capable of hybridizing under stringent conditions to the nucleic acid of (a) or (b) and encoding a protein having D-lactate dehydrogenase activity; and (d) the amino acid of SEQ ID NO: 1 A protein comprising an amino acid sequence at least 80% identical to the sequence and encoded by a nucleic acid selected from the group consisting of nucleic acids encoding proteins having D-lactate dehydrogenase activity.

The protein of the invention preferably comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 1.
The protein of the invention preferably has the advantage that the Km value for pyruvate is lower than that known. Without limitation, the Km value for pyruvic acid is preferably 0.20 mM or less, more preferably 0.18 mM or less.

  The present invention also provides a method for producing the novel D-lactate dehydrogenase protein of the present invention. The production method of the present invention includes transforming a host cell with an expression vector containing the nucleic acid of the present invention, and culturing the transformed cell under conditions that allow expression of the nucleic acid.

The present invention further provides a kit for measuring alanine aminotransferase activity, comprising the novel D-lactate dehydrogenase protein of the present invention and instructions for use.
Abbreviation of base In the description and sequence listing in this specification, symbols in the base sequence mean the following bases.

a: adenine g: guanine c: cytosine t: thymine u: uracil r: guanine or adenine y: thymine / uracil or cytosine m: adenine or cytosine k: guanine or thymine / uracil s: guanine or cytosine w: adenine or thymine / Uracil b: guanine or cytosine or thymine / uracil d: adenine or guanine or thymine / uracil h: adenine or cytosine or thymine / uracil v: adenine or guanine or cytosine n: adenine or guanine or cytosine or thymine / uracil, unknown, or Other bases

In the present invention, a novel D-lactate dehydrogenase protein and a nucleic acid encoding the protein are provided.
In another embodiment of the present invention, a mutant D-lactate dehydrogenase protein having improved stability in an alkaline solution by random mutagenesis can be obtained, and the nucleotide sequence of the gene encoding the protein has been clarified. . Expression vectors into which these genes had been introduced were introduced into E. coli to obtain transformants. Further, by culturing the obtained transformant in a large amount, the present protein can be produced in a large amount, stably and easily.

  Moreover, since the mutant D-lactate dehydrase has high stability in solution, it can be stored for a long period of time, and is particularly useful as a conjugate reagent for measuring alanine aminotransferase activity.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 Lactobacillus sp. Cloning of NBRC14511-derived D-lactate dehydrogenase gene 1) Lactobacillus sp. NBRC14511 was statically cultured at 37 ° C. for 15 hours in MRS medium (manufactured by Difco) and collected.

  2) Current Protocols in Molecular Biology 2.4.1 John Wiley & Sons Inc. , (1987-2000), genomic DNA was prepared from the cells.

  3) Degenerate PCR was performed using the following primers designed based on the base sequences of five known Lactobacillus genus D-LDH genes. The obtained DNA fragment of about 700 bp was subjected to DNA sequence analysis, and a DNA probe was prepared using ECL Direct Labeling Module (manufactured by Amersham Biosciences).

Sense primer (SEQ ID NO: 3):
5'-ggctcgagcgtacacgtgtggtgtgayaacwtbgayrtks-3 '
Antisense primer (SEQ ID NO: 4):
5'-ccgatcccagtgtagaargcvvgtrtgtggwgtwaycarwac-3 '
4) Lactobacillus sp. Genomic DNA prepared from NBRC14511 was treated with restriction enzymes PstI and HindIII (manufactured by Takara Bio Inc.), then subjected to agarose electrophoresis, and Southern hybridization was performed using the prepared DNA probe. As a result of using ECL Detection Reagents (manufactured by Amersham Biosciences), a positive band was detected at a position of about 4 kb.

5) A DNA fragment in the vicinity of about 4 kb was collected by GFX ^™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences), and the restriction enzyme P was used in advance using LIGAFAST ^™ RAPID DNA LIGATION SYSTEM (Promega) III Introduced into pBluescript II KS ⁺ (2.9 kb) vector treated with Takara Bio Inc., transformed with E. coli DH5α strain, LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 1 .4% agar, 50 μg / ml ampicillin (pH 7.4)) and cultured at 30 ° C. for 18 hours.

  6) From the grown colonies, transformants containing the D-lactate dehydrogenase gene were obtained by colony hybridization using the DNA probe used in Southern hybridization.

  7) A plasmid was extracted from the obtained transformant, and the D-lactate dehydrogenase gene sequence and amino acid sequence were determined by a DNA sequencer (3100 Genetic Analyzer, manufactured by Applied Biosystems).

Example 2 Construction of D-Lactate Dehydrogenase Gene Expression System 1) Using the obtained plasmid containing D-lactate dehydrogenase gene as a template, 5 ′ end side (sense primer) and 3 ′ end side (antisense) PCR was performed using the following primers provided with EcoRI and BamHI restriction enzyme sites in the primer), and a DNA fragment of about 1.1 kb was confirmed by agarose electrophoresis. The PCR reaction composition was (in 50 μl) with 1 U KOD plus polymerase, 10 pg template DNA, 30 μM sense primer, 30 μM antisense primer, 0.2 mM dNTP Mix, 1 mM MgSO ₄ , 1 × buffer. The PCR reaction was repeated 30 cycles of 94 ° C. for 30 seconds (denaturation), 60 ° C. for 30 seconds (annealing), and 68 ° C. for 2 minutes (extension).

Sense primer (SEQ ID NO: 5):
5'-ggaagctttgaattcatggcagaaacagagtgtatatccag-3 '
Antisense primer (SEQ ID NO: 6):
5'-ccgatccttaaccaactttaccaccgtgtc-3 '
2) The obtained PCR product was treated with restriction enzymes EcoRI and BamHI (manufactured by Takara Bio Inc.), and after agarose electrophoresis, recovered by GFX ^™ PCR DNA and Gel Band Purification Kit (manufactured by Amersham Biosciences). The D-lactate dehydrogenase gene fragment was introduced into the expression vector pTRP (2.9 kb) previously treated with restriction enzymes EcoRI and BamHI (Takara Bio) using LIGAFAST ^™ RAPID DNA LIGATION SYSTEM (manufactured by Promega). did.

Using the obtained plasmid as a template, QuikChange ^™ Site-Directed Mutagenesis Kits (manufactured by STRATAGENE) was used to destroy the EcoRI restriction enzyme site in the D-lactate dehydrogenase gene, and the expression plasmid pTRP-ldhD Got. Next, E. coli JM109 strain was transformed to obtain E. coli recombinant pTRP-ldhD / JM109.

Sense primer (SEQ ID NO: 7):
5'-catgctgctattcaaaacctcgcatttacgcccaaaaccgtc-3 '
Antisense primer (SEQ ID NO: 8):
5'-gaccttgggtttggcgtaaaatgcgagctgtttgaatagcagcatg-3 '
Example 3 Purification method 1) E. coli recombinant pTRP-ldhD / JM109 was cultured in Super broth (3.5% polypeptone, 2% yeast extract, 0.5% NaCl pH7.5) at 37 ° C. for 9 hours and collected for 9 hours. Fungus.

  2) 5.8 g of the obtained wet cells were suspended in 30 ml of extraction buffer (20 mM potassium phosphate buffer pH 7.5, 5 mM 2-mercaptoethanol, 0.5 mM EDTA), and subjected to ultrasonic disruption (ASTRASON manufactured by MISONIX). ULTRASONIC PROCESSOTR XL) and centrifugation (18,000 × g, 15 minutes, 4 ° C. Model J2-21 Centrifuge manufactured by BECKMAN) to prepare a crude extract. The expression level of D-lactate dehydrogenase in the obtained crude extract was about 800 U / mg protein.

The LDH activity was measured at a measurement temperature of 25 ° C. and a measurement wavelength of 340 nm with the reaction liquid composition described in the following table. In addition, 1 μmol of NAD ⁺ produced per minute under these reaction conditions was defined as 1 unit, and the enzyme activity was defined as D-lactate dehydrogenase activity.

Buffer composition for LDH activity measurement Final concentration potassium phosphate buffer 0.1M
(PH 7.0)
Pyruvate 2mM
NADH 0.22mM
3) The cell-free extract was added to DEAE-Cellulofine A-500 (manufactured by Seikagaku Corporation) equilibrated with buffer A (10 mM potassium phosphate buffer pH 7.5, 5 mM 2-mercaptoethanol, 0.5 mM EDTA). Then, after washing with buffer A, the protein of interest was eluted with a linear gradient of 0-0.3 M NaCl.

  4) Ammonium sulfate was added to the active fraction to a final concentration of 1.0 M, and the mixture was stirred for 1 hour, and then buffer B (10 mM K-phosphate buffer pH 7.5, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 1 The solution was passed through Phenyl-Sepharose CL 4B (manufactured by Amersham Biosciences) equilibrated with (0.0M ammonium sulfate), washed with buffer B, and the target protein was eluted with a linear gradient of 1.0-0M ammonium sulfate. The obtained active fraction was dialyzed against buffer A, and a purified preparation of recombinant protein having a specific activity of 1,500 U / mg protein was obtained. As a result of staining with CBBR-250 after SDS-PAGE, this purified sample showed an approximately single band of about 44,000.

Example 4 Random mutagenesis and screening 1) pTRP-ldhD was treated with restriction enzymes EcoRI and BamHI (Takara Bio), and D-lactate dehydrogenase gene was converted into GFX ^™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences) And introduced into a pBluescript II KS ⁺ (2.9 kb) vector previously treated with restriction enzymes EcoRI and BamHI (manufactured by Takara Bio Inc.) using LIGAFAST ^™ RAPID DNA LIGATION SYSTEM (manufactured by Promega). The plasmid pBluescript-ldhD was obtained.

2) The obtained plasmid was subjected to error-prone PCR using Diversity PCR random mutation Kit (Clontech). PCR reaction composition (per 50 μl): 1 × TITANIUM Taq polymerase, 1 ng pBluescript-ldhD, 0.2 μM sense primer, 0.2 μM antisense primer, 1 × Diversify dNTP Mix, 0.04 mM dGTP, 0.16 mM dGTP, 0.16 mM MnSO ₄ This was performed in 1 × TITANIUM Taq buffer. The PCR reaction was 94 ° C. for 30 seconds (denaturation) and 68 ° C. for 2 minutes (elongation) for 25 cycles, and a 1.1-kb PCR product corresponding to the D-lactate dehydrogenase gene was confirmed by agarose electrophoresis.

Sense primer (SEQ ID NO: 9):
5'-gtaaaacgaggggccagtgagcg-3 '
Antisense primer (SEQ ID NO: 10):
5'-cactaaaaggagaaaagactgggag-3 '
3) The amplified DNA fragment was treated with restriction enzymes EcoRI and BamHI (manufactured by Takara Bio Inc.) and purified by GFX ^™ PCR DNA and Gel Band Purification Kit (manufactured by Amersham Biosciences). The entire amount of the obtained DNA fragment was introduced into an expression vector pTRP (2.9 kb) previously treated with restriction enzymes EcoRI and BamHI (manufactured by Takara Bio Inc.) using LIGAFAST ^™ RAPID DNA LIGATION SYSTEM (manufactured by Promega). The JM109 strain was transformed to prepare a mutant enzyme library.

  4) Inoculate 96-well deep well (Super broth (3.5% polypeptone, 2% yeast extract, 0.5% NaCl pH 7.5) 0.6 ml / well) from the mutant enzyme library at 37 ° C, After 17 hours of shaking culture, the cells were collected by centrifugation. The obtained microbial cells were suspended in an extraction buffer (same as that during purification), then crushed and centrifuged to obtain a crude extract.

  5) Each crude extract was diluted with buffer C (0.1 M Tris-HCl buffer pH 9.0, 0.1 mg / ml BSA), heat-treated at 40 ° C. for 15 minutes, LDH activity was measured, and residual activity was measured. Calculated.

  6) As a result, 4 clones with improved stability over the wild type were selected. As a result of DNA sequence analysis of the resulting 4 clones, a transformant (G195C) in which the 195th glycine was replaced with cysteine, the 9th glutamic acid was replaced with glycine, the 275th glutamic acid was replaced with lysine, and the 286th isoleucine Transformed with valine (E9G / E275K / I286V), 286th isoleucine substituted with valine (I286V), 156th lysine replaced with arginine, 340th asparagine replaced with lysine It was confirmed that it was a transformant (K156R / N340K).

7) Purification method is the same as wild type.
5 Preparation and Screening of 20 Amino Acid Substitutes 1) Using pTRP-ldhD as a template and using the following primers, QuikChange ^™ Site-Directed Mutagenesis Kits (manufactured by STRATAGENE), mutated the 195th glycine, then E. coli The JM109 strain was transformed to produce 20 types of substitutes substituted with other amino acids.

Sense primer (SEQ ID NO: 11):
5'-gcaatattggaannktttcggcgctaaggttaattgcatatgaccc-3 '
Antisense primer (SEQ ID NO: 12):
5'-ccttagcgccgaamnnttccataattttcatataacacgtcc-3 '
2) 20 kinds of amino acid substitutions were inoculated into 96-well deep wells (Super broth (3.5% polypeptone, 2% yeast extract, 0.5% NaCl pH 7.5) 0.6 ml / well), 37 The cells were collected by centrifugation at 17 ° C. for 17 hours. The obtained microbial cells were suspended in an extraction buffer (same as that during purification), then crushed and centrifuged to obtain a crude extract.

  3) Each crude extract was diluted with buffer C (0.1 M Tris-HCl buffer pH 9.0, 0.1 mg / ml BSA), heat-treated at 55 ° C. for 60 minutes, LDH activity was measured, and residual activity was measured. Calculated.

4) As a result, the transformant in which the 195th glycine was replaced with cysteine was the most stable.
Example 6 Physicochemical Properties of Mutant D-Lactate Dehydrogenase Protein of the Present Invention The physicochemical properties obtained in the examples are summarized in Table 1 below.

１）熱安定性に関して
本発明の酵素を１００ｍＭ トリス−塩酸バッファー ｐＨ９．０（０．１ｍｇ／ｍｌ ＢＳＡを含む）中で２５−７０℃、１５分間熱処理後ＬＤＨ活性を測定し、残存活性を算出した。結果は図１・表１に示す通りで、野生型は３０℃まで安定であるのに対し、変異型は３７−５５℃まで安定であった。

1) Thermal stability The enzyme of the present invention was measured for LDH activity after heat treatment at 25-70 ° C. for 15 minutes in 100 mM Tris-HCl buffer pH 9.0 (including 0.1 mg / ml BSA) to calculate the residual activity. did. The results are shown in FIG. 1 and Table 1. The wild type was stable up to 30 ° C., whereas the mutant type was stable up to 37-55 ° C.

2) Regarding pH stability The enzyme of the present invention was added to 100 mM potassium phosphate buffer pH 5.5-7.5, 100 mM Tris-hydrochloric acid buffer pH 7.5-9.0, 100 mM glycine-sodium hydroxide buffer pH 9.0-10.0. LDH activity was measured after storage at 25 ° C. for 7 days in 0.1% (w / v) Tween 80, and the residual activity was calculated.

The results are shown in FIG. 2 and Table 1. The wild type was stable up to pH 5.5-8.0, whereas the mutant type was stable up to at least about pH 5.5-8.5.
3) Regarding the Km value, they were 0.14-0.29 mM (pyruvic acid) and 0.10-0.13 mM (NADH).

4) About optimum pH It was about pH 6.5-7.5.
5) Regarding molecular weight As a result of conducting SDS-PAGE, the molecular weight was about 44,000.

FIG. 1 shows the thermal stability of D-lactate dehydrogenase protein. The residual activity was calculated by measuring LDH activity after heat treatment at 25-70 ° C. for 15 minutes in 100 mM Tris-HCl buffer pH 9.0 (containing 0.1 mg / ml BSA). The black circle / solid line represents “G195C”, the black diamond / dotted line represents “E9G / E275K / I286V”, the black triangle / solid line represents “I286V”, and the black square / dotted line represents “K156R / N340K”. White circles and solid lines indicate control “wild type”. FIG. 2 shows the pH stability of D-lactate dehydrogenase protein. LDH activity was measured after storage for 7 days at 25 ° C. in 100 mM Tris-HCl buffer pH 7.5-9.0 (including 0.1% (w / v) Tween 80), and the residual activity was calculated. The black circle / solid line represents “G195C”, the black diamond / dotted line represents “E9G / E275K / I286V”, the black triangle / solid line represents “I286V”, and the black square / dotted line represents “K156R / N340K”. The white triangle and solid line indicate the “wild type” of the control. Lactobacillus. sp. The amino acid sequence of (NBRC14511) natural D-lactate dehydrogenase protein and the D-lactate dehydrogenase protein derived from the known genus Lactobacillus, which was referred to when preparing a probe for isolating the gene encoding the protein Are arranged so that the homology is maximized. Lactobacillus. sp. The amino acid sequence of the natural type D-lactate dehydrogenase protein of (NBRC14511) and the eight kinds of amino acid sequences of the known D-lactate dehydrogenase proteins derived from the genus Lactobacillus were arranged so as to maximize the homology. Is. Lactobacillus. sp. The amino acid sequence of the natural type D-lactate dehydrogenase protein of (NBRC14511) and the eight kinds of amino acid sequences of the known D-lactate dehydrogenase proteins derived from the genus Lactobacillus were arranged so as to maximize the homology. Is.

Claims (6)

A nucleic acid encoding a mutant Lactobacillus bacterium D-lactate dehydrogenase protein having improved storage stability in solution,
Here, the mutant D-lactate dehydrogenase protein satisfies any of the following conditions :
1) The 195th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine;
2) The ninth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine, the 275th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with lysine, and 286 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1 has been substituted with valine; or
3) The 156th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine, and the 340th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with lysine.
Said nucleic acid. An expression vector comprising the nucleic acid according to claim 1 . A transformant transformed with the expression vector of claim 2 . A mutant Lactobacillus bacterium D comprising transforming a host cell with an expression vector comprising the nucleic acid of claim 1 and culturing the transformed cell under conditions that allow the expression of the nucleic acid. -Method for producing lactate dehydrogenase protein. A mutant D-lactate dehydrogenase protein of the genus Lactobacillus having improved storage stability in solution, which satisfies any of the following conditions :
1) The 195th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine;
2) The ninth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine, the 275th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with lysine, and The amino acid residue at position 286 of SEQ ID NO: 1 is substituted with valine; or 3) the amino acid residue at position 156 of amino acid sequence set forth in SEQ ID NO: 1 is replaced with arginine; , 340th amino acid residue of the amino acid sequence of SEQ ID NO: 1 is substituted with lysine,
A D-lactate dehydrogenase protein of the bacterium belonging to the genus Lactobacillus. A kit for measuring alanine aminotransferase activity, comprising the mutant D-lactate dehydrogenase protein according to claim 5 and instructions for use.

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