JPH07106149B2 - Aminopeptidase and use thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel aminopeptidase and a method for producing the same, a microorganism producing the enzyme, a method for producing a D-amino acid using the enzyme or the microorganism, and The present invention relates to a method for producing a D-amino acid N-substituted amide using the oxygen or the microorganism. D-alanine alkylamide is useful as a raw material for synthesizing artificial sweeteners (Japanese Patent Publication No. 9320/1986).

[Conventional technology]

Aminopeptidase is an exopeptidase that sequentially releases amino acids from the N-terminus of a peptide (EC
3.4.11.) "Enzyme Handbook", p.531-536, Asakura Shoten 1982. ) Aminopeptidase normally acts on peptides consisting of L-amino acids in an L stereospecific manner to release L-amino acids. Among them, aminopeptidases that act non-stereospecifically on peptides having N-terminals of D-amino acids are slightly known, but their reaction rate is generally L
-Much slower than the reaction rate for peptides consisting of amino acids.

Robinson et al. (Journal of Biological Chemistry, 202,
1 (1953)), Hops (Archives of Biochemistry and
Biophysics, 114,567 (1966)), Minamiura et al. (Journa
l of Fermentation Technology, 33,653 (1969), Plascot et al. (Journal of Biochemistry, 75,185 (197
4)) shows that aminopeptidases derived from various organisms are L-
It acts not only on peptides consisting of amino acids, but also on D-
Although it is reported that it slightly acts on peptides having N-terminal amino acids, these are not aminopeptidases that specifically act only on peptides consisting of D-amino acids.

Jerry et al. (Journal of Basic Microbiology, 5,299
(1986)) is Brevibacterium.
It has been reported that an acylamide amide hydrolase (EC3.5.1.4) produced by a genus bacterium also acts on D-alanine amide, but it is not a D stereospecific hydrolysis.
Also, this enzyme is not an aminopeptidase.

JP 57-13000, JP 59-159789, JP 60-3644
6, JP-A-62-55097 and JP-A-62-253397 describe a method for enzymatically hydrolyzing DL-amino acid amide or L-amino acid amide to a corresponding L-amino acid by various microorganisms. However, it is not described which enzyme is involved from the viewpoint of enzyme chemistry. Further, no D stereospecific hydrolysis of D-amino acid amide-containing products is described.

Japanese Patent Application Laid-Open No. 60-184392 discloses Achromobacte.
r), a method for enzymatically hydrolyzing a D-amino acid amide to a corresponding D-amino acid by a bacterial cell of the genus Alcaligenes, and Kurthia, but from an enzymatic chemical point of view. , No description of what enzyme is involved. Further, the described hydrolysis reaction uses D-amino acid amide as a raw material, and D-stereospecific hydrolysis of a D-amino acid amide-containing product has not been confirmed.

Japanese Patent Laid-Open No. 61-96989 discloses Rhodococcus erythropolis (Rho
dococcus etythropolis) to enzymatically hydrolyze D-alanine amide, D-valine amide, D-aminobutyric acid amide, D-leucine amide, D-serine amide, or D-threonine amide to the corresponding D-amino acid. Although claimed and described in the Examples are methods of enzymatically hydrolyzing D-alanine amide, D-valine amide and D-leucine amide to the corresponding D-amino acids, from an enzymatic chemistry point of view any No mention is made of whether the enzyme is involved. Further, no D stereospecific hydrolysis of D-amino acid amide-containing products is described.

JP-A-61-274690 discloses Pseudomonas fluorescens, Rhodococcus erythropolis, and D-methionine amide, D-glutamine amide by Serratia mercescens cells.
D-threonine amide, D-leucine amide, D-phenylalanine amide, D-tyrosine amide, and D-
A method for hydrolyzing valinamide to the corresponding D-amino acid is described, but it is not described which enzyme is involved from the enzymatic chemistry point of view.
Further, the described hydrolysis reaction uses D-amino acid amide as a raw material, and D stereospecific hydrolysis of a mixture of D-amino acid amide and L-amino acid amide is not described at all.

Japanese Patent Publication No. 61-68 describes a method for producing a D-amino acid peptidase derived from actinomycetes belonging to the genus Streptomyces which acts on an oligopeptide containing a D-amino acid.
This enzyme is a carboxypeptidase-like enzyme that acts on the C-terminus of peptides, and is not an aminopeptidase specific to D-amino acid derivatives.

Therefore, no aminopeptidase specific to the D-amino acid derivative has been known so far, and using this enzyme, D-amino acid
A method for synthesizing a D-amino acid by subjecting an amino acid amide-containing material as a raw material to D stereospecific hydrolysis is not known at all.

A method for synthesizing D-amino acids using an enzyme or a microorganism is a method of using D stereospecific amino acid acylase derived from a normal bacterium belonging to the genus Streptomyces to produce N-acetyl DL.
-Amino acid is optically resolved to give D-phenylglycine or D-
Methods for synthesizing valine (JP-B-53-36035 and JP-A-63-39598, respectively), D stereospecific hydrolysis of hydantoin compounds by Pseudomonas bacteria (JP-B-56-1911), α-keto acid As a raw material, a method for synthesizing D-amino acids excluding D-alanine using D-amino acid transaminase and an amino group donor regeneration system (JP-A-62-205790) and the like can be mentioned. However, a method of synthesizing a D-amino acid amide using a D-amino acid amide-containing material as a raw material and using an enzyme specific to the D-amino acid amide is not known at all.

By the way, peptide synthesis is mainly carried out by a chemical synthesis method, but there are problems that a protective group is required, racemization and side reactions easily occur. On the other hand, in recent years, a technique has been developed in which an amide bond hydrolase such as peptidase or protease is used as a catalyst to synthesize a peptide under reverse reaction conditions. The enzyme-based peptide bond synthesis reaction is an excellent method because the reaction conditions are mild and the above-mentioned problems of the chemical synthesis method can be avoided.

The peptide synthesis method by the enzyme method has so far been trypsin,
Endopeptidases such as α-chymotrypsin, pepsin, papain, thermolysin, subtilisin and pronase, and carboxypeptidase which is an exopeptidase have been used. Important methods of enzymatic peptide synthesis include the reverse reaction of peptide bond hydrolysis and the aminolysis method of amino acid alkyl esters.In both synthetic methods, the amino group of the amino acid derivative that serves as the acid moiety of the substrate is Everything had to be protected. On the other hand, studies using aminopeptidases, which are exopeptidases that sequentially release amino acids from the N-terminus of peptides, have not been known so far.

D-amino acids are rare amino acids in nature. Natural proteins are composed of L-amino acids, and therefore, the method of using these hydrolases in a peptide bond synthesis reaction is L-amino acid.
-Developed as a method of synthesizing peptides consisting of amino acids. A synthetic reaction of a peptide containing a D-amino acid using a known protein hydrolase has been reported. For example,
Morihara et al. (Journal of Biochemistry, 84, 1277 (197
8)) shows a method for the catalytic synthesis of α-chymotrypsin of ZL-phenylalanyl-D-leucinamide. Since then, Stova and Petkov (FEBS Letters, 1
83,103 (1985), West and Wong (Journal of O)
rganic Chimstry, 51,2728 (1986)), Barbus and Wong (Journal of Chemical Society, Chemical Communi
ncations, 1987,533), Margoline and Kryvanov (Journ
al of American Chemical Society, 109, 3802 (198
7)), Matos et al. (Biotechnology Letters 9,233 (198
7)) describes a method for synthesizing peptides containing D-amino acids using α-chymotrypsin or lipase as a contact. However, the synthetic methods using these endopeptidases all use a compound in which the amino group of the amino acid derivative of the acid moiety is protected as a substrate, and the synthesis using a compound in which the amino group is free has not been described at all. Absent. Also, in these syntheses, the D-amino acid is included in the amino moiety, which is different from the present invention.

Margoline et al., (Journal of American Chemical Socie
ry, 109,7885 (1987)), a synthesis using N-formyl D-alanine 2-chloroethyl ester as an acid moiety and D-alanine amide or the like as an amine moiety with subtilisin as a catalyst is reported.

However, the enzymatic synthesis methods of D-amino acid amides reported in these reports all require a protecting group at the acid moiety, which is different from the present invention. Also, DL in the acid part
-A method for synthesizing a D-amino acid amide by stereoselective aminolysis using an amino acid derivative is not known.

[Problems to be Solved by the Invention]

Therefore, the present invention provides an aminopeptidase specific to a D-amino acid derivative which has never been known to exist, a novel method for producing the enzyme, and D-using the enzyme.
(EN) It is intended to provide a novel method for producing an amino acid, and a novel method for producing a D-amino acid amide which stereoselectively converts an amino acid derivative of an acid moiety into a D-amino acid amide using the enzyme.

[Means for Solving the Problems]

The present inventors extensively screened a strain having an aminopeptidase activity acting specifically on a D-amino acid derivative in order to develop a novel microorganism producing the enzyme and a novel method for producing the enzyme. However, it has been found that many bacteria produce a novel D-amino acid aminopeptidase.

Therefore, the present invention provides an aminopeptidase characterized by acting specifically on a D-amino acid derivative; culturing a bacterium that produces an aminopeptidase, and collecting the enzyme from the culture. Production method: A method for producing a D-amino acid, which comprises reacting the enzyme or a substance containing the enzyme with a D-amino acid derivative to produce a D-amino acid, and collecting the D-amino acid;
And reacting a D-amino acid derivative with an amine in the presence of the oxygen or the content of the enzyme to form a D-amino acid N-
The present invention provides a method for producing a D-amino acid N-substituted amide, which comprises producing a substituted amide and collecting this.

[Specific explanation]

(1) Microorganisms The microorganisms used in the present invention may be any microorganisms capable of producing an aminopeptidase specific to D-amino acids, and such microorganisms may be selected from preservative bacteria. It can be separated from the natural world.

Such microorganisms include Achromobacter cycolastes (Achromobacter cyc) which was found in preserved strains.
cloclastes) IAM 1013, Achromobacter delicatulus IAM 1433, Flavobacterium
esteroaromaticum) IFO 3751, flavobacterium
F. suaverolens IFO 3752, Bacillus cereus IFO 3001, Bacillus
B. sphaericus IFO 3341, Bacillus sphaericus IFO 3527, Bacillus thiamnolyticus IAM 1034, Bacillus stearothermophilus IFO 12550,
Micrococcus roseus IFO
3768, Micrococcus sp. SCRC 4
14, Corynebacter Spedonium (Corynebacter
ium spedonicum) IFO 3306, Pseudomonas aeruginosa IFO 3080, Pseudomonas aeruginosa IFO 12653 (P.putida), Protaminobacter ruber IFO 3708,
Mycobacterium smegmatis
egmatis) IFO 3082, Streptomyces griseolus IFO 3403, and Sptreptomyces fulvissimu
s) IFO 13482 can be mentioned. These preserved bacteria can be freely obtained from IFO or IAM under the above-mentioned deposit numbers.

As a microorganism belonging to Cellulomonas, Cellulomonas sp.S.
CRC 631 (Kyoto University Faculty of Agriculture, AKU-676, Yamada et al., Ag
ricultural and Biological Chemistry, 46 , 2325, 1982). Cellulomonas sp.SCRC
631 remarks to Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology 9
Deposited as 918 (FERM P-9918). As a microorganism belonging to Micrococcus, Micrococcus sp.SCRC 41
4 (Kyoto University Faculty of Agriculture AKU-510, Ogata et al., Agricult
ural and Biological Chemistry, 30 , 176, 1966). Micrococcus sp.SCRC 41
4 Microbiology Research Institute of Microbiology Research Institute, Institute of Microbial Technology, AIST 991
Deposited as No. 7 (FFRM P-9917).

Examples of microorganisms belonging to Achromobacter include, for example, a new strain Achromobacter sp. SCRC isolated by the present inventor.
C1-38, Achromobacter sp, SCRC C1-16, Achromobacter sp.SCRC C1-17 can be mentioned. The mycological properties of these strains are very similar, and Achromobacter sp. SCRC C1-38 as a representative strain of these strains was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microbiology Research Institute No. 9916 (FERM). P
−9916) has been deposited. Examples of the microorganism belonging to Arthrobacter include the new strain Arthrobacter sp. SCRC C2-9 and Arthrobacter sp. SCRC N1-31 isolated according to the present invention. The chrysanthemological properties of these strains are very similar, and Arthrobacter sp.SCRC N1-31 as a representative strain of these strains is in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microbiology Research Institute No. 9915 ( FERM P−99
15) has been deposited.

The source of isolation of these strains is Sagami City, Kanagawa Prefecture.

The above-mentioned novel strain has mycological properties as shown in Table 1.

Based on the above mycological properties, Chester and Cooper (Jorn
al of Clinical Microbiology, 9,425 (1979)) and M
anual of Clinical Microbiology 4th ed., P 330, (19
85), the SCRC C1-38, SCRC C1-16,
The SCRC C1-17 strain was identified as follows. That is,
Gram-negative, no spore formation, bacilli, motility, aerobic, oxidase positive, and acid formation from glucose. From these properties, it is clear that the bacterium belongs to the genus Achromobacter. Meanwhile, Bergey's Manu
al of Systematic Bacteriology 1st ed., Vol.2., p 126
SCRC C2-9 and SCRC N1-31 were identified as follows according to the 6 classification criteria. That is, gram positive, coryneform type, non-motile, aerobic, catalase positive, and mycolic acid negative, which contains lysine in peptide glycans. From these properties, it is clear that the bacteria belong to the genus Arthrobacter.

It is also possible to obtain mutations in these strains to obtain strains with higher productivity. Further, the gene involved in the production of aminopeptidase existing in the cells of these strains is excised, and this is inserted into an appropriate vector, for example, a plasmid, and using this vector, an appropriate host, for example, Escherichia coli (Escherchia coli) It is also possible to artificially create the aminopeptidase-producing strain of the present invention by transforming a heterologous host such as or yeast or a homologous host such as Achromobacter or Arthrobacter.

(2) Method for producing enzyme When the above-mentioned microorganism is cultured to produce the aminopeptidase of the present invention, any basal nutrient medium may be used as long as the microorganism of the present invention can grow therein. . This medium contains one or more kinds of nitrogen sources such as ammonium sulfate, yeast extract, peptone, and meat extract. If necessary, glucose, starch, glycerin, etc. can be added to this medium as a carbon source.
It is preferable to add inorganic salts such as dipotassium phosphate, sodium chloride and magnesium sulfate to this medium. It is also preferable to add a small amount of D-amino acid amide that can serve as an enzyme inducer. The additive of D-amino acid amide varies depending on the composition of the basal medium and the nature of the strain to be cultured, but is about 0.01 to 5%.

The culture may use either a solid medium or a liquid medium, but in order to obtain a large amount of the target enzyme, a liquid medium is used,
It is preferable to culture under aerobic conditions by shaking culture, aeration / agitation culture and the like. The culture temperature may be any temperature within the temperature range in which the bacteria grow and aminopeptidase is produced, but it is preferably 25 to 45 ° C. pH is 5
11, preferably in the range of 6-10. The culture time may be selected so that the enzyme activity is expressed, but is preferably 6 to 72 hours.

Next, the aminopeptidase of the present invention is adopted from the obtained culture, and a conventional enzyme purification method can be used as a purification method. The crude enzyme is obtained by centrifugation or the like, and further treated with protamine sulfate or streptomycin sulfate, salt folding, organic solvent precipitation, adsorption chromatography, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. Then, a uniform crystalline enzyme preparation can be isolated by a known method such as crystallization by adding a salt such as ammonium sulfate or polyethylene glycol.

The use form of aminopeptidase used in this method is not particularly limited. For example, it is needless to say that the purified enzyme can be used in a culture medium containing cells, live bacterial cells, air-dried bacterial cells dehydrated by acetone or the like, crushed bacterial cells, various stages. It is possible to use a partially purified product that has been purified to. Further, it is possible to use those obtained by immobilizing these enzymes or enzyme-containing substances with polyacrylamide, photocrosslinkable resin, polyurethane resin, kappa carrageenan, sodium alginate, ion exchange resin, semipermeable membrane, polymeric enzyme modifier, etc. I can.

(3) Method for measuring titer In the present invention, titer was measured by the following method. Tris-HCl (pH8.0) 50 μmol, D-alanine amide 5 μmo
l, and mix the appropriate amount of sample to 0.5 ml,
After reacting at 30 ° C. for 10 minutes, the reaction was stopped by immersing in boiling water for 3 minutes, and the produced D-alanine was quantified by the following method. That is, 0.5 ml of the above reaction solution,
Phenol 10.6 μmol, 4-aminoantipyrine 0.79 μm
ol and 5 units of peroxidase to make 1.5 ml,
After incubating at 5 ° C for 5 minutes, 0.144 units of D-amino acid oxidase was added to make 1.6 ml, and the mixture was shaken at 37 ° C for 60 minutes. Soak this in boiling water for 3 minutes to stop the reaction,
The absorbance at 500 nm was measured, and the amount of D-alanine in the reaction solution was determined from the calibration curve. 1 μmol D- per minute
The amount of enzyme that produces alanine was defined as 1 unit.

The aminopeptidase activity using D-alanine-p-nitroanilide as a substrate was measured as follows. That is, D
-Alanine-p-nitroanilide 10 µmol, phosphate buffer (pH 7.0) 100 µmol and 1 ml of reaction solution containing enzyme were kept at 30 ° C for 10 minutes, and then the absorbance at 405 nm was measured to determine the absorption of p-nitroaniline. D in the reaction solution from the coefficient
-The amount of alanine was determined. On the other hand, the enzyme activity for L-alanine amide was measured as follows. That is, 5 μmol of L-alanine amide and 50 μmo of tris-hydrochloric acid (pH8.0)
After 0.5 ml of the reaction solution containing 1 and the enzyme was kept at 30 ° C. for 10 minutes, it was immersed in boiling water for 3 minutes to stop the reaction, and the L-alanine produced was quantified by the following method. That is, glycine-KCl-KOH (pH 10.4) 100 μmol, NAD ⁺ 2.5
1 ml of the reaction solution containing μmol, the above reaction solution, and 0.5 unit of L-alanine dehydrogenase was kept at 30 ° C. for 5 minutes, and the increase in absorbance at 340 nm derived from NADH produced was measured by a spectrophotometer to obtain a calibration curve. The amount of L-alanine in the reaction solution was calculated from the above. One unit was the amount of enzyme that produced 1 μmol of L-alanine per minute.

(4) Properties of enzyme As an example of the aminopeptidase of the present invention, SCRC C1-3
The aminopeptidase produced by 8 has the following properties.

(1) Action: Catalyze the reaction represented by the following formula.

D-amino acid amide + H ₂ O → D-amino acid + NH ₃ (2) Substrate specificity: This enzyme uses D-alanine amide as the best substrate. An amide of D-alanine and ammonia having an N-terminal which is free, an amide of D-alanine and various alkylamines, an ester of D-alanine, and a peptide serve as substrates. An example of this is shown in Table 2 below.

(3) Optimum pH: The optimum pH is around pH 8.5.

(4) pH stability: pH 7.0 when the residual activity was measured after incubation at 30 ° C for 1 hour in buffer solution (0.05M) of each pH.
Stable around ~ 10.0.

(5) Optimum temperature: maximum activity around 45 ° C.

(6) Temperature stability: When the residual activity was measured after treatment in 0.1 M phosphate buffer (pH 8.0) at each temperature for 10 minutes, 80% activity remained at 45 ° C.

(7) Absorption spectrum: It has a maximum absorption at 281 nm.

(8) Effects of metal ions and inhibitors: The activity is inhibited by metal ions such as silver and mercury and SH inhibitors such as RCMB.

(9) Isoelectric point: Approximately 4.2 when measured by focal electrophoresis using an ampholine.

(10) Molecular weight: High performance liquid chromatography (TSKG30
00SW) will be calculated as about 122,000.

(11) Molecular weight of subunit: Calculated to be about 59,000 by SDS-polyacrylamide gel electrophoresis.

(12) Uniformity: high performance liquid chromatography (TSK Ph
enyl-5PW) gives a single peak as shown in FIG. 5A. In addition, SDS-polyacrylamide gel electrophoresis (1
0.0%, pH 7.2) gives a single band as shown in FIG.

(5) Production of D-amino acid The present invention also provides a method for producing a D-amino acid. In this method, an aminopeptidase or a substance containing it is used to convert a derivative of D-amino acid into a D-amino acid, and the D-amino acid is collected.

The aminopeptidase of the present invention has the following reaction: Can be catalyzed, and this reaction can be used to produce a D-amino acid from a D-amino acid derivative.

The method of the present invention can be most effectively used for the production of D-alanine, but the production of D-2-aminobutyric acid, D-valine, D-norvaline, D-phenylglycine, D-homophenylalanine, etc. Can also be applied for.

As a D-amino acid derivative as a substrate, a D-amino acid amide, N, or N-, depending on the type of X in the reaction formula (I),
-Substituted D-amino acid amide, D-amino acid ester, N
A peptide having a D-amino acid at the terminal can be used. Representative examples of the substituent of the N-substituted D-amino acid include a lower alkyl group such as a methyl group, an ethyl group, a propyl group and the like, and thus a D-amino acid N-lower alkylamide such as a D-amino acid as a substrate. −
N-methylamide, -N-ethylamide, -N-propylamide and the like can be used. Furthermore, derivatives having a group containing an aromatic group as the N-substituent and various amides shown in Table 2 can be used.
As the substrate ester, for example, those in which the α-carboxyl group of D-amino acid is esterified with a lower alkanol, for example, methyl ester, ethyl ester, propyl ester and the like can be used. Substrate peptides include D-amino acid-Gly, D-amino acid-D-Ala-D
Various peptides shown in Table 2 such as -Ala, D-amino acid-L-Ala-L-Ala and the like can be used.

However, it is preferable to use an inexpensive substrate for industrially producing D-amino acids, and for this purpose, N
It is preferred to use unsubstituted D-amino acid amides, such as D-alanine amide, D-2-butyric acid amide and the like. The D-amino acid amide used in the present invention is, for example, α-aminonitrile synthesized by synthesizing each D-amino acid methyl ester according to a known method and then reacting with ammonia gas, or by the Strecker method. Can be hydrated chemically or enzymatically. In addition, D-amino acid amide, which is a by-product during the optical resolution of DL-amino acid amide with an enzyme, can also be used.

As a raw material, not only the above D-amino acid derivative but also D
It is also possible to use mixtures of amino acid derivatives and D-amino acid derivatives with L-amino acid derivatives. When this mixture is used, according to the method of the present invention, the D-amino acid derivative is stereospecifically converted into D-amino acid, and D, L
-A stereoisomerically pure D-amino acid can be easily produced from a mixture of amino acid derivatives.

In the method of the present invention, aminopeptidase or its inclusion is used as a reaction catalyst. Here, the aminopeptidase means an enzyme that acts on the substrate as described above in a D stereoisomeric manner to produce a D-amino acid, and includes aminopeptidase, peptidase, protease, esterase,
It includes those commonly referred to as acidase, amylase and the like. Examples of such an enzyme include the enzymes produced by microorganisms as described above. However, in addition to the purely isolated enzyme, any material having the enzymatic activity of aminopeptidase can be used in the method of the present invention. As such an enzyme active material, for example, the broth of the above-mentioned microorganisms, that is, a mixture of cultured cells and medium, separated cultured cells, culture supernatant,
A treated product of bacterial cells or the like can be used. As the treated bacterial cells, dried bacterial cells, dried bacterial cells treated with a solvent such as acetone or ethanol, crushed bacterial cells, any step of the enzyme purification process described in the item (2) of the method for producing the enzyme And the partially purified product obtained in. It is also possible to use an immobilized enzyme product obtained by immobilizing the above-mentioned bacterial cells or a treated product of various bacterial cells by a conventional enzyme immobilization method. The immobilization carrier is
Polyacrylamide, a photocrosslinkable resin, a polyurethane resin, kappa carrageenan, sodium alginate, an ion exchange resin, a polymeric enzyme modifier, or a semipermeable membrane can be used.

In industrial practice, it is advantageous to use live cells, immobilized cells and the like.

The amount of aminopeptidase in the reaction solution depends on the substrate such as D-
The concentration varies depending on the concentration of the amino acid amide and is not particularly limited, but usually 1 to 100,000 units is convenient.

The amount of the D-amino acid derivative as a raw material, for example, amide, added is
The amount of the enzyme varies depending on the concentration of the enzyme in the reaction solution and the like, and is not particularly limited as long as it does not inhibit the reaction, but 1 to 500 g / l is convenient. When it is used at low concentration, it can be used in the form of free base, but when it is used at relatively high concentration, for example, it is used in the form of hydrochloride, tosylate, etc. from the viewpoint of pH adjustment. Is preferred. In the batch type reaction, the D-amino acid derivative or its inclusion or its salt may be added all at once at the start of the reaction, or may be added in a plurality of divided portions as the reaction progresses, or continuously.

As the reaction medium, water, acetone, acenitrile, DMS
An aqueous solution having a buffering effect containing O, DMF and the like can be used. As the buffer, for example, Tris-HCl buffer, phosphate buffer, imidazole-HCl buffer, HEPES-
NaOH buffer, TRICINE-NaOH buffer, sodium carbonate-
Sodium hydrogen carbonate buffer, boric acid-NaOH buffer and the like can be used. It is also possible to use an organic solvent which is immiscible with water, such as a ketone, an ether, a hydrocarbon, an aromatic olefin, a halogenated hydrocarbon, an organic acid ester, an alcohol or a nitrile. For example, methyl butyl ketone, isopropyl ether, petroleum ether, hexane,
Heptane, cyclohexane, carbon tetrachloride, chloroform, methylene dichloride, trichloroethane, benzene, toluene, xylene, ethyl acetate, butyryl acetate, butanol, hexanol, octanol and the like can be used together with water. Further, a mixture of these organic solvents can be used, or a two-layer system with an organic solvent saturated with water, an aqueous solution having a buffering action, micelles, reverse micelles or emulsions can be used for the reaction.

The reaction pH is pH 5 to 11, preferably pH 6 to 10.

The reaction temperature can be considered in the same manner as the reaction pH,
It is usually 20 to 60 ° C, preferably 25 to 50 ° C.

Although the reaction time is not particularly limited, the reaction is maintained until the substrate D-amino acid amide-containing material is converted into D-amino acid in a sufficient yield depending on the substrate concentration of the reaction mixture, the enzyme titer and the like.

The D-amino acid produced can be arbitrarily purified and collected by a conventional method. For example, after completion of the reaction, trichloroacetic acid is added to precipitate the protein, and the cells are filtered together with the bacterial cells (if present), purified from the filtrate with an ion exchange resin or the like, and crystallized.

(6) Method for producing D-amino acid N-substituted amide The present invention further provides a method for producing a D-amino acid N-substituted amide. In this method, a derivative of a D-amino acid is reacted with an N-substituted amine in the presence of aminopeptidase or a substance containing the aminopeptidase to produce a D-amino acid N-substituted amide, which is collected.

The aminopeptidase of the present invention has the following reversible reaction: Can be catalyzed and this reaction can be used to produce D-amino acid N-substituted amides.

This method can be used most effectively for the preparation of D-alanine N-substituted amides, but the D-amino acid moiety is eg D-2-aminobutyric acid, D-valine, D-norvaline, D- It can also be used for the preparation of D-amino acid N-substituted amides such as phenylglycine.

As the substrate D-amino acid derivative, the D-amino acid derivative described in the method (5) for producing D-amino acid can be used, and from the viewpoint of reactivity, an ester of D-amino acid, such as methyl ester or ethyl. Esters, propyl esters and the like are preferable. As the gaseous raw material, the D-amino acid derivative as described above can be used, or a mixture of these D-amino acid derivative and L-amino acid derivative can be used. When using a D, L-mixture, the enzyme acts in a D stereospecific manner
-Amino acid N-substituted amides are selectively produced.

As the other substrate, an amine, an amine having an amino group bonded to a primary carbon, secondary carbon or tertiary carbon is used. Examples of the N-substituent include an alkyl group, a cycloalkyl group, a cycloalkyl-alkyl group, an aryl group, an aralkyl group and a heterocyclic group. The alkyl group includes, for example, a linear or branched alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a 3-pentyl group, and the like, and has 1 carbon atom.
It can be up to 20 alkyl groups. Examples of the cycloalkyl group include cyclopentyl group and cyclohexyl group. Examples of the cycloalkyl-alkyl group include dicyclopropylmethyl group, fentyl group,
Examples thereof include t-butylcyclopropylmethyl group. In addition, examples of the aryl group include a phenyl group, and examples of the aralkyl group include a benzyl group. Examples of the heterocyclic group include a tetrahydrothiophen-3-yne group, a thietan-3-yl group, and a tetramethyl-1,1-dioxoethane-3-yl group. These groups may be further substituted with other substituents.

As the enzyme or its components used in this method, various forms described in the method for producing D-amino acid (5) can be used.

As the reaction medium, various media described in the method for producing D-amino acid (5) can be used. D-
In the amino acid N-substituted amide synthesis reaction, the amino acid ester of the substrate and the D-amino acid N-substituted amide of the product are
Since it is possible to undergo enzymatic or non-enzymatic hydrolysis, it is better to minimize the presence of water, and the reaction in an organic solvent is particularly desirable.

The addition amount of the D-amino acid derivative and the amine, which are the substrates, is
The amount of the enzyme varies depending on the concentration of the enzyme in the reaction solution and the like, and is not particularly limited as long as it does not inhibit the reaction, but 1 to 500 g / l is convenient. When it is used at a low concentration, it can be used in the form of a free base, but when it is used at a relatively high concentration, for example, it is used in the form of a hydrochloride or tosylate as a pH adjusting agent. It is preferable from the viewpoint. In the batch type reaction, the substrate may be added all at once at the start of the reaction, or may be decomposed multiple times as the reaction progresses or may be added continuously.

The reaction pH is pH 5 to 11, preferably pH 6 to 10.

The reaction temperature can be considered in the same manner as the reaction pH,
It is usually 20 to 60 ° C, preferably 25 to 50 ° C.

The reaction time is not particularly limited, but the reaction is maintained until the substrate amino acid amide or amino acid ester is converted to the D-amino acid N-substituted amide with a sufficient yield depending on the substrate concentration of the reaction mixture, the enzyme titer, etc. To do.

The D-amino acid N-substituted amide thus produced can be optionally purified and collected by a conventional method. For example, after completion of the reaction, the bacterial cells and the immobilized enzyme agent (if present) are filtered, and the D-amino acid N-substituted amide contained in the filtrate is purified by solvent extraction, ion exchange resin, or the like, Crystallize.

Next, the present invention will be described more specifically by way of examples.

Example 1. Purification of aminopeptidase from Achromobacter sp. SCRC C1-38 Glucose 0.1%, tryptone 0.5%, yeast extract 0.5
%, K ₂ HPO ₄ 0.1% and adjusted to pH 7.0, 10 liters of the medium were sterilized by heating at 120 ° C. for 15 minutes. This was inoculated with Achromobacter sp. SCRC C1-38 (Microtechnology Research Institute, Microbiology No. 9916), and cultured by shaking at 30 ° C. for about 18 hours to obtain a bacterial cell having a wet weight of 90 g. After washing the cells with physiological saline, a phosphate buffer solution (pH: 0.1 mM EDTA and 5 mM 2-mercaptoethanol) was added.
7.0) Suspended in 300 ml and sonicated at 9 KHz for about 20 minutes (total about 2.5 hours) to disrupt the cells. Crushed cells are 14,
It was removed by centrifugation at 000 × g for 20 minutes to obtain an elementary extract containing aminopeptidase. To this cell-free extract, 3.8 g of protamine sulfate was added, and after stirring for 30 minutes, 14,000 xg,
The precipitate was removed by centrifugation for 20 minutes. Solid ammonium sulfate was added to the supernatant to make it 30% saturated with ammonium sulfate. After stirring for 30 minutes, the formed precipitate was removed by centrifugation at 14,000 xg for 20 minutes. Solid ammonium sulfate was added to this supernatant to make 90% ammonium sulfate saturation. 14,000 ×
The enzyme-active precipitate obtained by centrifugation at 20 g for 20 minutes was dissolved in a small amount of 0.01 M phosphate buffer (pH 7.0), and 0.01 M containing EDTA and 5 mM 2-mercaptoethanol was added. It was dialyzed against a phosphate buffer (pH 7.0). The enzyme solution was previously equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol.
-Pass through a Toyopearl 650M column, add 0.1 mM EDTA, 5
Contains mM 2-mercaptoethanol and 0.1 M NaCl
Elution was performed with 0.1 M phosphate buffer (pH 7.0). The active fractions were collected, 0.1 mM was dialyzed with 0.01 M phosphate buffer (pH 7.0) containing EDTA and 5 mM 2-mercaptoethanol, and then passed through a hydroxyapatite column equilibrated with the same buffer in advance to obtain 0.1 mM. The enzyme was eluted with a linear concentration gradient of 0.01 M to 0.5 M phosphate buffer (pH 7.0) containing EDTA and 5 mM 2-mercaptoethanol. Collect the active fractions, add ammonium sulfate to achieve 30% saturation, and then
0.1 mM EDTA, 5 mM 2-mercaptoethanol, and 30
Pass it through a column of Butyl Toyopearl equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 100% saturated ammonium sulfate to obtain 0.1 mM E
The enzyme was eluted with a linear gradient of 0.01 M acid buffer (pH 7.0) containing 30% to 0% saturated ammonium sulfate containing DTA and 5 mM 2-mercaptoethanol. Collect active fractions, 0.1 mM
0.01M with EDTA and 5mM 2-mercaptoethanol
After dialysis with phosphate buffer (pH 7.0), concentrate to approx.
M EDTA and 5 mM 2-mercaptoethanol and 0.1 MN
Gel filtration chromatography was performed on Sephadex G-200 equilibrated with 0.05 M phosphate buffer (pH 7.0) containing aCl. Thus, about 4,40 aminopeptidases
Purified 0 times. The specific activity in this purification process is shown in Table 3.

This enzyme gave a single peak by Pheny-5PW column chromatography (Fig. 1) and proved to be homogeneous on SDS-polyacrylamide gel electrophoresis (Fig. 2).

Example 2. Partial purification of aminopeptidase from Achromobacter sp. SCRS C1-38 Glucol 0.1%, tryptone 0.5%, yeast extract 0.5
%, K ₂ HPO ₄ 0.1%, and adjusted to pH 7.0, 20 liter of medium was sterilized by heating at 120 ° C. for 15 minutes. This was inoculated with Achromobacter sp. SCRC C1-38 (Microtechnology Research Institute, Microbial Co. No. 9916) and shake-cultured at 30 ° C for about 18 hours to obtain a microbial cell having a wet weight of about 186 g. After washing the cells with physiological saline, 0.1 mM EDTA and
Phosphate buffer containing 5 mM 2-mercaptoethanol (pH 7.
0) Suspended in 300 ml and sonicated at 9 KHz for about 20 minutes (total about 5 hours) to crush the cells. Crushed cells are 14,0
It was removed by centrifugation at 00 × g for 20 minutes to obtain an elementary extract containing aminopeptidase. Add 7.6 g of protamine sulfate to this cell-free extract, stir for 30 minutes, and then add 14,000 × g, 20
The precipitate was removed by centrifugation for 1 minute. Solid ammonium sulfate was added to this supernatant to make it 30% saturated with ammonium sulfate.
After stirring for 30 minutes, the purified precipitate was removed by centrifugation at 14,000 xg for 20 minutes. Solid ammonium sulfate was added to this supernatant to make 90% ammonium sulfate saturation. 20 at 14,000 xg
The precipitate with enzyme activity obtained by centrifugation for 10 minutes was dissolved in a small amount of 0.01M phosphate buffer (pH 7.0)
0.0 with M EDTAH and 5 mM 2-mercaptoethanol
It was dialyzed against 1M phosphate buffer (pH 7.0). This enzyme solution was preliminarily equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol, and passed through a column of DEAE-Toyopearl 650M to prepare 0.1 mM EDTA and 5 mM of 2 mM.
-Mercaptoethanol and 0.01M with 0.1M NaCl
Elution was performed with a phosphate buffer (pH 7.0). Collect active categories, 3
After adding ammonium sulfate to achieve 0% saturation, 0.1 mM in advance
EDTA, 5 mM 2-mercaptoethanol, and a column of Butyl Toyopearl equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 30% saturated ammonium sulfate, 0.1 mM EDTA and 5 mM 2- 30% to 0% including mercaptoethanol
The enzyme was eluted with a saturated concentration of ammonium sulfate of 0.01 M in a linear concentration gradient of 0.01 M phosphate buffer (pH 7.0).

The active fractions were collected and dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. In this way, the aminopeptidase was partially purified about 1,500 times in a yield of about 100%. Table 4 shows specific activity and recovery rate in this purification process.

Example 3. Synthesis of D-alanine from DL-alanine amide by partially purified enzyme of Achromobacter sp. SCRC C1-38. DL-alanine amide hydrochloride 934 mg (0.0075 mol) was dissolved in 0.2 M phosphate buffer (pH 7.0) 75 ml and dialyzed with 0.01 M phosphate buffer (pH 7.0) 210 units of aminopeptidase (Example 2) Partially purified specific activity of 30 units / mg of enzyme) was added and the mixture was incubated at 37 ° C for 1 hour. D formed in the reaction solution
- alanine Amberlite IRA-400 a (Cl ^-) adsorbed on a column, washed with water, and eluted with 1N hydrochloric acid. This solution was concentrated under reduced pressure and adsorbed on a Dowex 50W x 8 (H ⁺ ) column,
After washing with water, it was eluted with 1N aqueous ammonia. Concentrated under reduced pressure,
313 mg (46.9%) of D-alani was obtained. The obtained D-alanine was recrystallized from water-methanol-isopropyl alcohol-ether and the spectral data was compared with that of commercially available D-alanine.

Specific irradiance [α] = -14.15 ° (c = 6.6, 1N HCl) (standard ▲ [α] ²⁰ _D ▼ = -14 to -15 ° (c = 6,1H HCl)).

mp 289 ~ 291 ℃ (standard sample 291 ~ 293 ℃).

MS, m / e 44 (21%), 57 (22%), 75 (29%), 90 (100
%, M ⁺ ).

Elemental analysis value Calculated value Actual value C 40.44 40.24 H 7.92 8.12 N 15.72 15.55 Glucose 0.1%, Tryptone 0.5%, Yeast extract 0.5
%, K ₂ HPO ₄ 0.1%, pH 7.0 adjusted to 200 ml
After heat sterilization at 120 ° C. for 15 minutes, Flavobacterium suaverolens IFO3752 was inoculated and cultured for 20 hours.
After washing the cells with physiological saline, the cells were disrupted in the same manner as in Example 1, and the disrupted cells were removed by centrifugation. The supernatant was dialyzed against 0.01 M phosphate buffer overnight to obtain a cell-free extract.

DL-alanine amide hydrochloride 2.49 mg (20 μmol), phosphate buffer (pH 7.0) 100 μmol, the above cell-free extract 200 μl,
A reaction solution containing 10 μmol of D-cycloserine in 1 ml at 30 ° C.
Allowed to react for 10 minutes. Quantification of D-alanine contained in the reaction solution by boiling using D-amine oxidase
It contained 0.46 μmol D-alanine. On the other hand, when L-alanine contained in the reaction solution was quantified using L-amino acid dehydrogenase, it contained 0.048 μmol of L-alanine.

Example 4. Synthesis of D-alanine-3-aminopentanamide in water using a partially producing enzyme from Achromobacter sp. SCRC C1-38. 0.1 mmol D-alanine methyl ester hydrochloride, 0.5 mmol 3-aminopentane, 13.2 units aminopeptidase (partially purified specific activity 30 units / mg enzyme in Example 2)
The reaction liquid containing 1 mL of water in 1 ml of water was kept warm at 30 ° C. The amino acid amide produced in the reaction solution was subjected to paper chromatography by a conventional method, color was developed by spraying ninhydrin, spots were extracted, and then quantified from a calibration curve prepared separately as a chemically synthesized amino acid amide standard sample. As a result, when the reaction time was 7.5 minutes, the yield was 78%.
Then, D-alanine-3-aminopentanamide was formed.

Example 5. Synthesis of D-alanine-3-aminopentanamide in water using immobilized enzyme from Achromobacter sp. SCRC C1-38. Fukui and Tanaka (Advance in Biochemical Engineering / Biotechno
logy 29,1 (1984)) photocrosslinkable resin ENTG
It was fixed in the form of a film with -3800 and cut into smaller pieces. D-alanine methyl ester hydrochloride 0.1 mmol, 3-
0.5 g of immobilized enzyme containing 0.5 mmol of aminopentane and 0.94 unit of aminopeptidase (enzyme with partial activity of 30 units / mg partially purified in Example 2) was added to make 1 ml of reaction solution, and the temperature was 30 ° C.
I kept it warm. After 120 minutes from the reaction, quantification according to the method of Example 4 revealed that D-alanine-3-aminopentanamide was formed in the reaction solution in a yield of 24%.

Similarly, using the enzyme immobilized with the photo-crosslinkable resin ENTP-2000, 180 minutes after the reaction, D-alanine-3-aminopentanamide was synthesized with a yield of 33%.

Similarly, when the enzyme (1.6 units) immobilized with urethane resin PU-6 was reacted for 60 minutes with the above reaction composition,
D-alanine-3-aminopentanamide could be synthesized with a yield of 29%.

Example 6. Synthesis of D-alanine-3-aminopentanamide in organic solvent using immobilized enzyme from Achromobacter sp. SCRC C1-38. The enzyme derived from Achromobacter sp. SCRC C1-38 was added to Fukui and Tanaka (Advance in Biochemical Engineering / Biotech
nology 29,1 (1984)) urethane resin PU
It was fixed at -6 and further finely cut. D-alanine methyl ester hydrochloride 0.1 mmol, 3-aminopentane 0.5
0.2 g of the immobilized enzyme containing 1 mmol of aminopeptidase (1.6 units) (partially purified specific activity of 30 units / mg of enzyme in Example 2) was added to butyl acetate, benzene, trichloroethane, toluene, and isopropyl ether in a water-saturated solution. One kind was added to 1 ml and kept at 30 ° C. Reaction 60
After minutes, D-alanine-3-aminopentanamide was synthesized in yields of 100,100,90,62,22%, respectively.

Example 7. Synthesis of D-alanine-3-aminopentanamide in organic solvent using immobilized enzyme from Achromobacter sp. SCRC C1-38. The enzyme derived from Achromobacter sp. SCRC C1-38 was immobilized with urethane resin PU-6 and further cut into smaller pieces. Any one of D-alanine methyl ester hydrochloride, DL-alanine methyl ester hydrochloride, and L-alanine methyl ester hydrochloride 0.1 mmol, 3-aminopentane 0.5 mmol, aminopeptidase 1.6 units (partially purified in Example 2) The enzyme was added together with 0.2 g of the immobilized enzyme containing 30 units / mg of specific activity) to 1 ml of a saturated solution of butyl acetate in water and kept at 30 ° C.
After 120 minutes in the reaction solution using D-alanine methyl ester hydrochloride as a substrate, D-alanine-3-aminopentanamide was produced in a yield of 95%.

In the reaction solution using DL-alanine methyl ester hydrochloride as a substrate, D-alanine-3-aminopentanamide was produced in a yield of 50% after 12 hours. In the reaction solution using L-alanine methyl ester hydrochloride as a substrate, even after 12 hours, no D
-Alanine-3-aminopentanamide was not formed.

10 ml of water-saturated butyl acetate containing an enzyme derived from Achromobacter sp. SCRC C1-38 immobilized with PU-6 urethane prepolymer and containing 0.1 M DL-alanine methyl ester hydrochloride and 0.5 M 3-aminopentane. 2.0 g was added and shaken at 30 ° C. for 6 hours. The reaction solution was concentrated under reduced pressure, and 4N HCl / ethyl acetate was added to give a hydrochloride. After drying over anhydrous magnesium sulfate and concentrating, the condensation product was purified to a single product, 87.9 mg (yield
45.2%, theoretical yield 90.4%).

The product was further converted to B with (Boc) ₂ O in the presence of triethylamine.
oc, and silica gel column chromatography (Hexane
/ Bac-D-Ala-NH purified with ethyl acetate = 1/1) Was obtained.

The obtained Boc-modified D-alanine-3-aminopentanamide was in good agreement with that of the authentic sample in the spectral data.

Specific rotation ▲ [α] ²⁰ _D ▼ ＝ ＋ 50.56 ° (c = 1.25, CHC
l ₃ ) (Standard ▲ [α] ²⁰ _D ▼ + 50.90 ° (c = 1.21, CHC
l ₃ ).

Example 8. Synthesis of D-alanine-3-aminopentanamide in an organic solvent using immobilized cells derived from Achromobacter sp. SCRC C1-38. Achromobacter sp.SCRC C1-38 live cells 200 mg (0.042
Unit) photocrosslinkable resin ENTG-3800 immobilized product, D-alanine methyl ester hydrochloride 0.1 mmol, 3-aminopentane
1 ml reaction mixture consisting of water-saturated butyl acetate containing 0.5 mmol
Was kept at 30 ° C, and after 240 minutes, the yield was 38% and D
-Alanine-3-aminopentanamide could be synthesized.

Example 9. D-alanine-3-in an organic solvent using acetone-dried cells derived from Achromobacter sp. SCRC C1-38.
Synthesis of aminopentanamide Achromobacter sp. SCRC C1-38 acetone dried cells 10
0 mg (4 units), D-alanine methyl ester hydrochloride 0.1
1 ml of a water-saturated butyl acetate solution containing 0.5 mmol and 3-aminopentane 0.5 mmol was kept warm at 30 ° C.
After 40 minutes, D-alanine-3-aminopentanamide could be synthesized with a yield of 5%.

Example 10. Synthesis of various D-alanine N-substituted-amides in organic solvents using immobilized enzyme from Achromobacter sp. SCRC C1-38. Enzyme from Achromobacter sp. SCRC C1-3 (Example 2
The partially purified enzyme having a specific activity of 30 units / mg) was immobilized with urethane resin PU-6 and further cut into fine pieces. D
-Alanine methyl ester hydrochloride 0.1 mmol, 0.5 mmol of n-butylamine or benzylamine, 0.2 g of immobilized enzyme containing 1.6 units of amine peptidase was added to 1 ml of butyl acetate aqueous solution, and the mixture was added at 30 ° C for 8 hours. Kept warm. The yields of D-alanine n-butylamide and D-alanine benzylamide were 70 and 14%, respectively. The yield was calculated as a control with separately synthesized D-alanine alkylamide. That is, a sample was dansylated by a conventional method, paper chromatography was performed by a descending method using hexane as a solvent, a spot emitting fluorescence was cut out, extracted with methanol, and a calibration curve was prepared from the absorbance at 205 nm and calculated.

Example 11. Synthesis of D-alanine-3-aminopentanamide in organic solvent using immobilized enzyme from Achromobacter sp. SCRC C1-38. The enzyme derived from Achromobacter sp. SCRC C1-38 was immobilized with urethane resin PU-6 and further cut into smaller pieces. D-alanine amide hydrochloride, DL-alanine amide hydrochloride, L-
0.1 mmol of any of alanine amide hydrochloride, 0.5 mmol of 3-aminopentane, 1.6 units of aminopeptidase (enzyme having a specific activity of 30 units / cm partially purified in Example 2)
Saturated butyl acetate solution (1 ml) with immobilized enzyme (0.2 g)
It was added to the inside and kept warm at 30 ° C. After 480 minutes in the reaction solution using D-alanine amide hydrochloride as a substrate, D-alanine-3-aminopentanamide was produced at a yield of 97%. DL-
In the reaction solution using alanine amide hydrochloride as a substrate, D-alanine-3-aminopentanamide was produced in a yield of 48% after 600 minutes. In the reaction solution using L-alanine amide hydrochloride as a substrate, D-alanine-3-aminopentanamide was not produced even after 24 hours.

Example 12 Glycerol 0.1%, Tryptone 0.5%, Yeast extract 0.5
%, K ₂ HPO ₄ 0.1% D-alanine amide hydrochloride, p
100 ml of medium prepared in H7.0, after heat sterilization for 15 minutes,
Various strains were inoculated and shake cultured at 30 ° C. for about 16 hours.
After washing the cells with physiological saline, the cells were suspended in 0.1 M phosphate buffer (pH 7.0) and sonicated at 9 KHz for 5 minutes. The disrupted bacterial cells were removed by centrifugation to obtain an elementary extract. This elementary extract was dialyzed against 0.01M phosphate buffer (pH 7.0) at 4 ° C overnight. The aminopeptidase activity with respect to D-alanine-p-nitroanilide and L-alanine amide in the enzyme solution thus obtained was measured by the method described in "[Specific Description] (3) Measurement of titer". . The results are shown in Table 5.

Reference Example 1. Synthesis of artificial sweetener (I) Synthesis of β-benzyloxy-N-benzyloxycarbonyl-L-aspartyl-D-alanine-3-aminopentanamide D-alanine-3-aminopentanamide hydrochloride 85 mg (0.568 mmol) synthesized by an enzymatic reaction )as well as 203 mg (0.57 mmol) of (β-benzyloxy-N-loxycarbonyl-L-aspartic acid) in an argon atmosphere DMF
It was dissolved in 10 ml. Under water cooling, 77 mg (0.57 mmol) of 1-hydroxybenzotriazole and 57.6 mg (0.57 mmol) of N-methylmorpholine were added to the reaction mixture. Then, the reaction solution was cooled to -20 ° C, and 118 mg of dicyclohexylcarbodiimide
5 ml of a DMF solution (0.57 mmol) was added dropwise. The temperature was gradually raised to room temperature, and after stirring overnight, the produced dicyclohexylurea was filtered off. The reaction solution was concentrated under reduced pressure, methylene chloride was added, and the generated crystals were separated by filtration. 5 layers of methylene chloride
% Sodium hydrogen carbonate, water, 2N hydrochloric acid, water, and then saturated brine, dried over anhydrous magnesium sulfate, and decolorized with activated carbon. After distilling off the solvent, methylene chloride-
Recrystallize with hexane to give 244 mg (86.4 mg) of colorless crystals.
%) (Ii) Synthesis of L-aspartyl-D-alanine-3-aminopentanamide Under an argon atmosphere, 2 protected dipeptides 252 mg (0.497 mmo
l) is dissolved in methanol (15 ml) and 10% Pd-C (10 m
g) was added and the atmosphere was replaced with hydrogen. After stirring overnight at room temperature, the product was filtered through Celite, concentrated under reduced pressure, and recrystallized from water-methanol to give 133 mg (99.8%) of dippetide as a colorless solid.
Was obtained as a single item.

Specific rotation ▲ [α] ²⁰ _D ▼ + 26.92 ° (c = 1.30 CH ₃ OH).

mp.221-223 ° C.

MS, m / e 29 (26%), 44 (100%), 57 (24%), 88 (46
%), 159 (26%), 274 (5%, M ⁺ ) Elemental analysis value Calculated value Actual value C 52.73 53.16 H 8.48 8.40 N 15.37 14.99

[Brief description of drawings]

FIG. 1 shows the elution profile of the purified enzyme of the present invention by Phenyl-5PW column chromatography, showing that the enzyme of the present invention is homogeneous. FIG. 2 is a sketch of the results of SDS-polyacrylamide gel electrophoresis of the purified enzyme of the present invention and shows that the enzyme of the present invention is homogeneous.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 9/48 C12R 1:07) (C12N 9/48 C12R 1: 265) (C12N 9/48 (C12R 1:38) (C12N 9/48 C12R 1:32) (C12N 9/48 C12R 1:06) (C12N 9/48 C12R 1: 465) (C12N 9/48 C12R 1:01)

Claims (3)

[Claims] 1. The following properties: (1) Action: Catalyze the reaction represented by the following formula: D-amino acid amide + H ₂ O → D-amino acid + NH ₃ (2) Substrate specificity: D-alanine amide is good A substrate is an amide of D-alanine and ammonia, which is a free N-terminal, an amide of D-alanine and various alkylamines, an ester of D-alanine, etc .: (3) Optimum pH: pH 8. The optimum value is around 5: (4) pH stability: pH 7.0 to 1 when the residual activity after incubation at 30 ° C for 1 hour in buffer solution (0.05M) of each pH is measured.
There is around 0.0: (5) Optimum temperature: maximum activity around 45 ° C:
And (6) Molecular weight: high performance liquid chromatography (TSKG3000
Calculated as about 122,000 by SW): Aminopeptidase having: 2. A genus of Achromobacter,
Corynebacterium genus, Flavobacterium genus, Bacillus genus
us), Micrococcus genus, Cellulomonas genus, Pseudomonas
s) genus, Protaminobacter genus, Mycobacterium genus, Arthrobacter genus, or Streptomyces (St
D-amino acid amide, which may be N-substituted in the culture, bacterial cells, or treated product of bacterial cells of the genus reptomyces)
A D-amino acid is stereospecifically produced by acting on a peptide having a D-amino acid at the terminus, a D-amino acid ester, a salt thereof, or a mixture thereof with a corresponding L-amino acid derivative. And a method for producing a D-amino acid. 3. A genus Achromobacter,
Corynebacterium (Flavobacterium), Bacillus (Bacill)
us), Micrococcus genus, Cellulomonas genus, Pseudomonas
s) genus, Protaminobacter genus, Mycobacterium genus, Arthrobacter genus, or Streptomyces (St
D-amino acid amide that may be N-substituted in the presence of a culture, bacterial cell, or treated product of bacterial cells of the genus reptomyces, a peptide having a D-amino acid at the N-terminus, or a D-amino acid ester Or a salt thereof, or a mixture of these with a corresponding L-amino acid derivative, and an amine or a salt thereof to stereospecifically produce a D-amino acid N-substituted amide or a salt thereof. And a method for producing a D-amino acid N-substituted amide.