WO2007069509A1

WO2007069509A1 - Novel hydantoinase and process for producing n-carbamoyl d-amino acid

Info

Publication number: WO2007069509A1
Application number: PCT/JP2006/324337
Authority: WO
Inventors: Makoto Ueda; Masakatsu Nishihachijo; Hirokazu Nanba
Original assignee: Kaneka Corporation
Priority date: 2005-12-12
Filing date: 2006-12-06
Publication date: 2007-06-21
Also published as: JP5159319B2; JPWO2007069509A1

Abstract

A novel hydantoinase is provided. Further, processes for producing the hydantoinase and N-carbamoyl D-amino acid using the same are provided. The invention provides hydantoinase derived from an Aneurinibacillus sp. strain, KNK491C, a gene encoding the hydantoinase, a recombinant plasmid containing the gene, and a transformant into which the hydantoinase gene has been introduced. Further, the process for producing hydantoinase in which the Aneurinibacillus sp. strain, KNK491C or the transformant is cultured and the hydantoinase is collected is provided. Further, the process for producing N-carbamoyl D-amino acid by allowing the enzyme to act on racemic 5-substituted hydantoin is provided.

Description

Specification

Novel Hydantoinase and N-Strength Lumamoyl _ D _ Method for Producing Amino Acid Technical Field

[0001] The present invention relates to an enzyme (hereinafter referred to as hydantoinase) which hydrolyzes a novel 5-substituted hydantoin produced by microorganisms into D-selective hydrolyzate and converts it into the corresponding N-strength rubamoyl D-amino acid. Is a microorganism having an ability to produce hydantoinase! /, And relates to a method for producing N-strength rubamoyl-D-amino acid using a transformant.

Background art

[0002] An optically active N-strength D-amino acid is a compound that can be easily converted into the corresponding optically active D-amino acid, and the optically active D-amino acid is useful as a synthetic intermediate for pharmaceuticals and the like. A compound. As a method for producing N-strength rubamoyl-D-amino acid, the enzyme produced by microorganisms is allowed to act on 5-substituted hydantoin to be selectively hydrolyzed to the corresponding N-strength rubamoyl-D-amino acid. A method of converting to is generally known (Patent Documents 1, 2, and 3).

[0003] Further, as a technique for producing a transformant having hydantoinase activity by introducing a gene encoding hydantoinase, an example using a hydantoinase gene derived from a thermophilic bacterium (Patent Document 4), Examples using hydantoinase genes derived from microorganisms belonging to the genus Gropacteria (Patent Document 5), examples using hydantoinase genes derived from microorganisms belonging to the genus Bacillus, Agrobacterium, and Pseudomonas (patent documents) 6) etc. are known.

Patent Document 1: JP-A-53-91189

Patent Document 2: JP-A 53-44690

Patent Document 3: JP-A 53-133688

Patent Document 4: Japanese Patent Laid-Open No. 62-87089

Patent Document 5: International Publication No. WO94Z00577

Patent Document 6: International Publication No. WOWO96Z20275 Disclosure of the invention

Problems to be solved by the invention

[0004] The hydantoinase produced by the above-mentioned microorganisms or transformants described as the background art generally has a higher degree of substitution at the 5-position of the substrate hydantoin (or is bulky and has a side chain). However, for example, for a hydantoin with a small substituent at the 5-position such as 5-methylhydantoin, the stereoselectivity is not strict and the optical purity is high. —It may be difficult to obtain strong rubermoyl-D-amino acids.

[0005] One of the objects of the present invention is to provide a novel hydantoinase exhibiting high stereoselectivity regardless of the size of the substituent of the 5-substituted hydantoin substrate. Another object of the present invention is to clarify the amino acid sequence of the hydantoinase, the DNA sequence of the gene, a microorganism or transformant having the ability to produce the enzyme, and a method for producing the hydantoinase using them Is to provide. Furthermore, one of the objects of the present invention is to provide an efficient method for producing N-strength rubamoyl-D-amino acid using the hydantoinase.

Means for solving the problem

[0006] In view of the above-mentioned problems, the present inventors have made an Aneurinibacillus sp.

) KNK491C strain (FERM BP-10735) Strength While active against various 5-substituted hydantoins, it is highly stereocable to substrates such as hydantoins with a very small 5-position substituent such as 5-methylhydantoin. It was found to produce hydantoinase that exhibits selectivity.

[0007] Furthermore, the hydantoinase gene was isolated and expressed in a host microorganism, and a highly active transformant was bred. By allowing the microorganism or transformant producing hydantoinase obtained in the present invention to act on 5-substituted hydantoin, it becomes possible to efficiently produce N-carbamoyl-D-amino acid, thereby completing the present invention. It came to do.

[0008] The present invention has one or more of the following features.

1) One feature of the present invention is the following polypeptide (a), (b) or (c): (a) a polypeptide having the amino acid sequence ability shown in SEQ ID NO: 1 in the Sequence Listing;

(b) a polypeptide having one or several amino acid substitutions, insertions, deletions and Zs or additions to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and having hydantoinase activity;

(c) A polypeptide comprising an amino acid sequence having 73% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and having hydantoinase activity.

5) One feature of the present invention is the following DNA (d), (e), (f) or (g):

(d) DNA having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing,

(e) DNA encoding a polypeptide having a base sequence in which one or several bases are substituted, inserted, deleted and Z or added in the base sequence shown in SEQ ID NO: 2 in the sequence listing and having hydantoinase activity ,

(f) a DNA encoding a polypeptide comprising a base sequence having 69% or more homology with the base sequence shown in SEQ ID NO: 2 in the sequence listing, and having a hydantoinase activity;

(g) A DNA that hybridizes under stringent conditions with a DNA complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing and encodes a polypeptide having hydantoinase activity.

6) One feature of the present invention is a recombinant plasmid containing the DNA.

7) One feature of the present invention is a transformant obtained by transforming a host microorganism with the recombinant plasmid.

8) One feature of the present invention is a microorganism having the ability to produce the polypeptide and belonging to the genus Aneurinibacillus.

9) One feature of the present invention is a method for producing hydantoinase, comprising culturing a microorganism capable of producing the polypeptide, accumulating the polypeptide in the culture, and collecting the polypeptide. is there.

11) One feature of the present invention is that the above-mentioned hydantoinase (polypeptide having hydantoinase activity), a transformant, or a microorganism is allowed to act on 5-substituted hydantoin to produce N-carbamoyl D-amino acids (for example, N-strength rubamoyl). D—a—amino acid). The invention's effect

[0009] The present invention has the above-mentioned constitutional power and can efficiently produce a novel hydantoinase. In addition, by using the microorganism or transformant that produces the hydantoinase, N-force rubermoyl-D-amino acids with high optical purity can be efficiently produced from various hydantoins including those with a small substituent at the 5-position. can do.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail based on embodiments. The scope of the present invention is not limited by the embodiments and examples.

1.Measurement of hydantoinase activity

In embodiments, the measurement of hydantoinase activity of a polypeptide is generated in a reaction.

N-strength rubamoyl amino acid can be quantified by the color development method using p-dimethylaminobenzaldehyde shown below.

[0011] Substrate solution 4mL consisting of 500mL DL-5-methylhydantoin, 0.1M M manganese sulfate, 0.1M sodium carbonate Z sodium bicarbonate buffer (PH8.7) After mixing at 45 ° C for 10 minutes, stop the reaction by adding 2 mL of 2N hydrochloric acid. After adding lmL of 10% (wZv) p-dimethylaminobenzaldehyde Z35% hydrochloric acid solution to this reaction solution and mixing, the absorbance at 440 nm was measured, and the N-- produced in the reaction solution was measured based on a calibration curve prepared in advance. Quantify the amount of force rubyamolanin.

2.Microorganism

The polypeptide of the embodiment can acquire microbial power having hydantoinase activity. The microorganism is not particularly limited as long as it is a microorganism that produces the same polypeptide, but examples include microorganisms belonging to the genus Aneurinibacillus, and among them, Aneurinibacillus sp. Is preferable. More preferred is the Aneurinibacillus sp. K NK491C strain newly isolated from soil by the present inventors. The Aneurini bacillus sp. KNK491C strain obtained in the present invention was assigned the accession number FERM BP-1073 5 on November 20, 2006. Deposited at the Deposit Center (Dai 305-85 66 Tsukuba Sakaihigashi 1-1-1 Tsukuba, Ibaraki Prefecture). The following shows the mycological properties of the Aneurinibacillus sp. KNK491C strain.

2- 1. Form

1) Aspergillus having a diameter of 0.8 / ζ πι, length of 2.0 to 3. O / z m

2) Gram staining: indefinite

3) Mobility: Yes

4) Sporulation: yes

2- 2. Culture characteristics

1) Colony morphology in Nutrientagar (Oxoid) medium: pale yellow, irregular, central ridge, fringe, smooth surface, opaque

2) Growth temperature: 20 ° C viable, 30 ° C viable, 45 ° C viable, 55 ° C viable

3) Growth under anaerobic conditions: None

2- 3. Physiological tests

1) Catalase activity: +

2) Oxidase activity: +

3) Acid / gas production from glucose: Z—

4) OZF test (oxidation Z fermentation)

5) Oxidation test

Glicero Norre: + Erythritol:

D arabinose:

Larabinose:

Ribose: +

D Xylose:

L-xylose:

Adonitor:

β-methylolone D xylose: one

Galactose: Fructose: +

Mannose: one

Sonorebose: One

Rhamnose: One

Zunoreshito Nore: One

Inositol: One

Manetor: One

Sorbitol: One

a-Methyl-D-Mannose: One a-Methyl-D-Gnore Course: One

N-Acetyldarcosamine: One Amygdalin: One

Albumin: one

Esclin: One

Salicin: one

Cellobiose: One

Maltose: one

Lactose: one

Meribi talent: One

White sugar: —

Tore / Rose Loose: One

Inulin: One

Meletitose: One

Rafuinos: One

Starch: —

Glycogen: One

Xylitol: One

Gentiobiose: One D Alla North:

D lyxose:

D Tagatose:

D Fucos:

L course:

D arabitonore:

L arabitol:

Darconate:

2—Ketogluconic acid:

5—Ketogluconic acid: —

6) β-galactosidase activity:

7) Arginine dihydrolase activity:

8) Lysine decarboxylase activity:

9) Ortinin decarboxylase activity:

10) Usability of citrate:

11) H 2 S production:

2

12) urease activity:

13) Tributophane deaminase activity:

14) Indole production:

15) Casein production (VP): +

16) Gelatinase activity:

17) Nitrate reduction: +

18) Casein hydrolysis:

19) Utilization

L sodium aspartate: +

D Fructose: +

Sodium fumarate: +

D—Sodium Dalconate: + D—Darcosamine: One

Sodium glutamate: +

DL Sodium glycerate: —

Glicero Norre: +

DL Sodium lactate: +

Ratatoose:

D Sodium malate: +

Manoletos:

Putrescine: +

L Sorbose: One

Sucrose:

L Sodium tartrate: —

D Trueno Sucrose:

2-4. 16S rDNA nucleotide sequence analysis

The 16SrDNA base sequence of the Aneurinibacillus sp. KNK491C strain was determined, and homology search was performed against the international base sequence database (GeneBank, DDBJ, EMBL) using BLAST as search software. As a result, the bacterium having the highest homology was the Aneurinibacillus migulanus strain DSM2895, and the homology rate was 99.9%.

[0013] Note that the microorganism that produces the polypeptide of the embodiment may be a wild strain of the above-described microorganism, or may be a mutant strain with improved mutation. Mutant strains are obtained by methods well known to those skilled in the art, such as UV irradiation, treatment with drugs such as N-methyl Ν 'Nitro-N N-trosoguanidine (NTG), ethyl methanesulfonate (E MS), etc. be able to.

[0014] The medium for culturing the microorganism producing the polypeptide of the present invention is not particularly limited as long as the microorganism can grow. For example, as a carbon source, carbohydrates such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid, and esters thereof, oils such as rapeseed oil and soybean oil, and nitrogen sources Ammonium sulfate, sodium nitrate, peptone, casamino acid, corn steep liquor, Ingredients such as sesame, yeast extract, inorganic salts such as magnesium sulfate, sodium salt sodium, calcium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, etc. Ordinary liquid media can be used.

[0015] Furthermore, substances that enhance the production of hydantoinase, for example, pyrimidine metabolites such as uracil, metal salts such as manganese salt of manganese salt and manganese sulfate, etc. may be added in a small amount. The concentration of these hydantoinase production-enhancing substances in the medium is preferably 0.001 to 10% by weight, more preferably 0.01 to 1% by weight.

[0016] The culture is usually carried out aerobically, and the culture temperature is preferably 10 ° C or higher and 60 ° C or lower, more preferably 20 ° C or higher and 50 ° C or lower. The culture pH is preferably 3 or more and 11 or less, more preferably PH 5 or more and 9 or less, and the culture time can be 1 day or more and 5 days or less. In addition, either a batch or continuous culture method may be used.

[0017] After completion of the culture, the culture fluid force is clarified by centrifugation or the like, and the microbial cells are disrupted by means such as ultrasonic disruption to obtain a crude enzyme solution. The polypeptide of the present invention can be obtained by purifying the crude enzyme solution by a salting-out method, a column chromatography method or the like.

3.Amino acid sequence

The polypeptide of the present invention may be a natural enzyme obtained from a microorganism as described above, or a recombinant enzyme produced using a gene recombination technique. Examples of the natural enzyme include the polypeptide represented by SEQ ID NO: 1 in the sequence listing.

[0018] Further, the polypeptide according to the embodiment of the present invention is an amino acid in which one or several amino acids are substituted, inserted, deleted and Z or added in the amino acid sequence shown in SEQ ID NO: 1 in Sequence Listing. It may be a polypeptide comprising a sequence and having hydantoinase activity, or 73% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% with the amino acid sequence shown in SEQ ID NO: 1. It may be a polypeptide having an amino acid sequence having homology of at least%, more preferably at least 99%, and having hydantoinase activity. `` Several amino acids '' is not limited as long as hydantoinase activity is not lost, but is preferably 20 amino acids or less, more preferably 15 amino acids or less, even more preferably 10 amino acids or less, most preferably No more than 5, 4, 3, or 2. The “homology” in the embodiment uses a method well known to those skilled in the art, sequence analysis software, and the like. Can be obtained. Here, as an example, homology search of genetic information processing software GENETY X Ver. 7Z network version (manufactured by Genetics) was used.

[0019] The "polypeptide having hydantoinase activity" means that the activity can be detected under the activity measurement conditions described in the above item "1. Measurement of hydantoinase activity". It is preferable to have an activity of 10% or more when the polypeptide having the amino acid sequence ability shown in No. 1 is used, more preferably 40% or more, 60% or more, more preferably 80% or more. It refers to a polypeptide that exhibits activity.

4. DNA

“DNA” as an embodiment of the present invention will be described. The DNA of the present invention may be DNA encoding the above polypeptide! It may be DNA represented by SEQ ID NO: 2 in the Sequence Listing, and 1 or several bases are substituted, inserted, deleted and Z or added in the base sequence represented by SEQ ID NO: 2 in the Sequence Listing. It may be a DNA encoding a polypeptide having a unique base sequence and having a hydantoinase activity! /. The term “several bases” means that the number is not limited as long as the polypeptide encoded by DNA does not lose hydantoinase activity, but it is preferably 50 bases or less, more preferably 30 bases or less, and even more. Preferably no more than 20 amino acids, most preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0020] Further, 69% or more, preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, more preferably 99% with the base sequence represented by SEQ ID NO: 2. It may be DNA that encodes a polypeptide having the above-mentioned homology and having hydantoinase activity! / ヽ.

[0021] "DNA" as an embodiment of the present invention is hybridized under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, and has a hydantoinase activity. It can be DNA encoding a polypeptide! /. DNA that also has a base sequence ability complementary to the base sequence shown in SEQ ID NO: 2 and DNA that hybridizes under stringent conditions are defined as the “combination-one” hybridization method, the plaque “hybridization method”, or the Southern detection method. When the hybridization method is performed, a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 specifically forms a hybrid. Say DNA. The stringent conditions include, for example, 75 mM trisodium citrate, 750 mM sodium chloride, 0.5% sodium dodecyl sulfate, 0.1% ushi serum albumin, 0.1% polyvinyl pyrrolidone, and 0.1% trisodium citrate, 150 mM sodium chloride, and 0.1% after performing a noble hybridization at 65 ° C in an aqueous solution that also has compositional power of l% Ficoll 400 (Amersham Biosciences) This refers to the conditions under which cleaning is performed at 60 ° C. using an aqueous solution composed of 1% sodium dodecyl sulfate. Preferably, after carrying out the hybridization under the above conditions, an aqueous solution having a composition of 15 mM trisodium citrate, 150 mM sodium chloride, and 0.1% sodium dodecyl sulfate is used at 65 ° C. More preferably, after hybridization is performed under the above conditions, 1.5 mM trisodium citrate, 15 mM sodium chloride salt, and 0.1% sodium dodecyl sulfate are added. Cleaning is performed at 65 ° C using an aqueous solution that also has compositional power.

[0022] The DNA (hydantoinase gene) of the present invention can be obtained from a microorganism having hydantoinase activity as described above. In order to obtain the target DNA, for example, the following method can be used.

[0023] First, chromosomal DNA is isolated from a microorganism having hydantoinase activity. Chromosomal DNA is obtained from cultured cells using a method such as Marmur method (see J. Mol. Biol., 3,208 (1961)). Next, after partially digesting the chromosomal DNA with an appropriate restriction enzyme such as Sau3AI, various chromosomal DNA fragments are ligated with pUCl 8 cleaved with an appropriate vector plasmid such as the restriction enzyme BamHI using T4 DNA ligase. Can be obtained as a single gene library. In order to select the plasmid containing the target hydantoinase gene from this gene library, there is a method of detecting the protein produced by the transformant as an indicator of enzyme activity or the like (see Example 1 described later), or Is a method for purifying the target hydantoinase, analyzing its N-terminal or internal amino acid sequence, synthesizing the corresponding DNA primer, and detecting the presence of the gene by using a hybrid or PCR method. Etc.

[0024] The nucleotide sequence of the chromosomal DNA fragment inserted into the selected plasmid is analyzed to determine the open reading frame (ORF) present in the fragment. Known from within this ORF By selecting one having a high homology with the base sequence of hydantoinase, the full-length base sequence of the target hydantoinase gene can be obtained.

5.Transformant 'vector

By using the DNA obtained by the above method or a recombinant plasmid obtained by incorporating the DNA into a vector, a host microorganism can be transformed to obtain a transformant.

[0025] As the host and vector, the host-vector system described in "Recombinant DNA Experiment Guidelines" (Science and Technology Agency, Research and Development Bureau, Life Science Division, revised on March 22, 1996) can be used. For example, the hosts include the genus Escherichia, the genus Pseudomonas, the genus Flavobacterium, the genus Bacillus, the Serratia, the genus Corynebacterium, the Brevi The genus Brevibacterium, the genus Agrobacterium, the genus Acetobacter, the genus Gluconobacter, the genus Lactobacillus, the genus Streptococcus or the Streptococcus It is possible to use microorganisms belonging to the genus (Streptomyces).

[0026] As the vector, a plasmid, phage or derivative thereof derived from a microorganism capable of autonomous replication in the above host can be used. Among them, as a host microorganism, for example, Escherichia coli i whose bacteriological properties are well known to those skilled in the art and provided for transformation, and a vector that can autonomously replicate in the microorganism as a vector. It is preferable to use it. Examples of such vectors include pUC18 (Takara Bio Inc.), pUC19 (Takara Bio Inc.), pBR322 (Takara Bio Inc.), PACYC184 (stock Based on the description in the specification of International Publication No. WO94Z03613, pSTV28 (Takara Bio Inc.), PSTV29 (Takara Bio Inc.), pSClOl (Funakoshi Inc.), pT7Blue (Takara Bio Inc.) Therefore, pUCNT that can be produced by those skilled in the art, or derivatives thereof can be mentioned. These derivatives are modified promoters, terminators, enhancers, SD sequences, replication start sites (or i), and other regulatory genes for the purpose of increasing enzyme production and plasmid stability. Stuff, drug resistance, clawing This refers to a modified site of the restriction enzyme site.

[0027] As an example of a transformant, a recombinant plasmid pALHIOl in which DNA obtained as described above from Aneurinibacillus sp. KN K491C strain is incorporated into pUCNT is used. Escherichia coli) HB101 can be transformed to obtain a transformant Escherichia coli HB101 (pALHIOl). The bacteriological properties of Escherichia coli HBlOl are described in “BIOCHEMICAL S FOR LIFE SCIENCE” (Toyobo Co., Ltd., 1993, pages 116-119) and various other known literatures. Is well known. Escherichia coli HB101 (pALHIOl) has the same mycological properties as Escherichia coli HB101, except that it can produce specific enzymes by genetic recombination.

[0028] The transformant Escherichia coli HB101 (pALHIOl) obtained as an embodiment of the present invention was incorporated as an independent administrative agency, National Institute of Advanced Industrial Science and Technology, under the accession number FERM P-20712 on November 15, 2005. It has been deposited at the Research Center for Patent Biological Deposits (Dai 305-8566, 1-1-1 Tsukuba Satohi, Ibaraki Prefecture).

[0029] It should be noted that the recombinant DNA technology used in the present invention is well known in the art, for example,

Molecular loning 2nd Edition (Cold bpnng Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).

6. Culture of transformants

By culturing the above-mentioned transformant capable of producing the hydantoinase of the present invention, the enzyme can be produced in large quantities, and can be used for the production of N-strength Lubamoyl D-amino acid.

[0030] Culture of microorganisms may be performed using a normal medium. The medium used for the culture may be a normal medium containing nutrients such as a carbon source, a nitrogen source and inorganic salts. Addition of organic micronutrients such as vitamins and amino acids to this often gives favorable results. As the carbon source, carbohydrates such as glucose or sucrose, organic acids such as acetic acid, alcohols and the like are appropriately used. Nitrogen sources include ammonium salt, a Water, ammonia gas, urea, yeast extract, peptone, corn steep liquor, etc. are used. Examples of inorganic salts include phosphates, manganese salts, magnesium salts, potassium salts, sodium salts, calcium salts, iron salts, sulfates, and chlorine.

[0031] The culture temperature is preferably 25 ° C or higher and 40 ° C or lower, more preferably 25 ° C or higher and 37 ° C or lower. The culture pH is preferably pH 4 or more and 9 or less, more preferably pH 5 or more and 8 or less. In addition, either a batch or continuous culture method may be used.

[0032] If necessary, a treatment for inducing an enzyme such as addition of isopropyl 1- 1 13-D-galatatoside (IPTG), ratatose or the like can be performed.

7. N—Strengthening Rubamoyl D Amino Acid Production Method

An efficient method for producing N-carbamoyl-D amino acids by using the hydantoinase obtained in the embodiment will be described. N-force rubermoyl-D-amino acids can be obtained by allowing hydantoinase to act on 5-substituted hydantoins and hydrolyzing them stereoselectively. 5-Substituted hydantoins may be racemic or optically active, such as L or D. The stereoselective hydrolysis reaction with hydantoinase and the chemical racemization of the substrate proceed at the same time, so that any force when using a 5-substituted hydantoin of racemic, L-, or D-form as a substrate can be used. It can be converted to the corresponding N-strength rubermoyl D-amino acid.

[0033] The following general formula (1) which is a substrate of this reaction

[0034] [Chemical 1]

[0035] The 5-substituted hydantoin represented by the formula is not particularly limited as long as it is hydrolyzed by the present enzyme. Examples of the substituent represented by R include the number of carbons optionally having a substituent. Examples include an alkyl group having 1 to 20 carbon atoms, an aralkyl group having 7 to 20 carbon atoms which may have a substituent, or an aryl group having 6 to 20 carbon atoms which may have a substituent. Can do. The alkyl group having 1 to 20 carbon atoms which may have a substituent in R is not particularly limited, and examples thereof include a methyl group, an isopropyl group, an isobutyl group, a 1 methylpropyl group, a strong rubamoylmethyl group, 2-force rubamoylethyl, hydroxymethyl, 1-hydroxysityl, mercaptomethyl, 2-methylthioethyl, (1 mercapto-1-methyl) ethyl, 4-aminobutyl, 3-gua-dinopropyl, 4 (5) -Imidazolemethyl group, ethyl group, n-propyl group, nbutyl group, chloromethyl group, methoxymethyl group, 2 hydroxyethyl group, 3 aminopropyl group, 2 cyanoethyl group, 3 cyanopropyl group, 4 ( Examples thereof include a benzoylamino) butyl group and a 2-methoxycarbo-ruethyl group.

[0036] The aralkyl group having 7 to 20 carbon atoms which may have a substituent is not particularly limited, and examples thereof include a benzyl group, an indolylmethyl group, a 4-hydroxybenzyl group, a 2 fluorobenzyl group, 3 Examples thereof include a fluorinated benzyl group, a 4 fluorinated benzyl group, and a 3, 4-methylenedibenzyl group. It may have a substituent! Examples of the reel group include a phenol group and a 4-hydroxyphenol group. Examples of the substituent include an amino group, a hydroxyl group, an alkyl group, an aralkyl group, an aryl group, an alkanol group, an alkyl group, an alkyl group, an alkoxyl group, or a halogen atom.

[0037] In addition, examples of the hydantoin having a small substituent at the 5-position include a 5-substituted hydantoin represented by the above formula (1), and the substituent represented by R has a substituent. There can also be mentioned those having an alkyl group of 1 to 3 carbon atoms. Although it has a substituent! /, The alkyl group having 1 to 3 carbon atoms is not particularly limited, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and a rubamoylmethyl group. , 2-force rubermoylethyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, (1 mercapto-1-methyl) ethyl, chloromethyl, methyl Examples thereof include a toximethyl group, a 3-aminopropyl group, a 2-cyanoethyl group, a 3-cyanopropyl group, and a 3-guano-propyl group.

[0038] In this reaction as an embodiment, the above-mentioned hydantoinase is allowed to act on the substrate, and the reaction is carried out in an aqueous solvent. The substrate charge concentration in this reaction is preferably 0.1% (WZV) or more and 90% (wZv) or less, more preferably 1% (WZV) or more and 60% (wZv) or less. The substrate is reacted in a dissolved or suspended state, and the reaction temperature is preferably adjusted to an appropriate temperature of 10 ° C or higher and 80 ° C or lower, more preferably 20 ° C or higher and 60 ° C or lower. _The pH is preferably kept at a pH of 4 or more and 12 or less, more preferably at a pH of 6 or more and 10 or less, and may be allowed to stand for a while or stirred. Further, the substrate may be added all at once, dividedly or continuously. In addition, this reaction can be performed by a batch method or a continuous method.

[0039] This reaction can also be performed using an immobilized enzyme, a membrane reactor, or the like. Examples of the aqueous medium include water, a buffer solution, an aqueous medium containing a water-soluble organic solvent such as ethanol, or an organic solvent that is difficult to dissolve in water, for example, ethyl acetate, butyl acetate, toluene, chloro, and the like. A suitable solvent such as a two-phase system with an aqueous medium containing an organic solvent such as oral form or n-hexane can be used. Furthermore, antioxidants, surfactants, coenzymes, metals, etc. can be added as necessary. The metal is not particularly limited, but manga , Nickel, zinc, iron, magnesium, calcium, copper, etc., or a salt thereof, preferably, a metal of manganese or conoret or a salt thereof. These metals may be used alone, or two or more metals may be used in combination.

8. Product isolation

The produced N-strength D-amino acid can be isolated and purified by a conventional separation method, for example, a separation method such as extraction, concentration, crystallization or column chromatography, or a combination thereof. In addition, the N-strength rubamoyl D-amino acid obtained by the method described above can be easily converted to the corresponding D-amino acid by the action of an enzyme having chemical or weak rubamoyl activity.

Example

[0040] Specific examples of the present invention are shown below. However, the present invention is not limited to these examples.

[0041] (Example 1) Isolation of hydantoinase gene

Sterilized Aneurinibacillus sp. KNK491C¾ (FERM BP—10735) in a 500 mL Sakaguchi flask. LOOmL medium A (meat extract 1.0%, peptone 1.0%, yeast extract 0.5 Inoculated to pH 7.5) before sterilization, and cultured with shaking at 37 ° C for 30 hours. After completion of the culture, the cells were collected by centrifugation, and chromosomal DNA was prepared using the Marnmr method.

[0042] To 100 μg of the chromosomal DNA thus obtained, 10 U of restriction enzyme Sau3AI was added and allowed to act at 37 ° C for 30 minutes for partial degradation. Subsequently, the partially decomposed chromosomal DNA was electrophoresed on a garose gel, and a DNA fragment having a size estimated to be about 4 to 9 Kbp was recovered from the garose gel. On the other hand, as it was completely digested with restriction enzymes BamHI separately plasmid _P UC18, previously linked by T4DNA ligase The recovered DNA fragment from Agarosugeru to give a mixture of plasmids with a variety of chromosomal DNA fragments. This plasmid mixture was transformed with a combination cell of Escherichia coli JM109 to perform transformation, and agar medium B (trypton 10 g, yeast extract 5 g, sodium chloride sodium 10 g, manganese chloride 20 mg, isopropyl 1 1-Cho 1 j8-D-Galatatoside (IPTG) 24m g, agar 15 g, ampicillin 100 mg, made up to 1 L with deionized water, pH 7.0 before sterilization, but ampicillin and IPTG were added after sterilization. And transformants containing various chromosomal DNA fragments were obtained as colonies.

[0043] A colony of the obtained transformant was replicated on a filter paper, and the reaction solution (0.1 M potassium phosphate buffer (PH 7.0), 5-methylhydantoin 1.0%, phenol 0.1%, D —Amino acid oxidase (manufactured by SIGMA) 1. 4 UZmL, decarbamoylase crude enzyme solution (see Reference Example 1 below) 50% (vZv), Peroxidase (manufactured by TOYOBO) 0. 07 U / mL, 0 (5 mM 4-aminoaminopyrine) and left at 37 ° C. for 5 hours. Select red mouthpiece and sterilize medium C in a test tube (trypton 10g, yeast extract 5g, sodium chloride sodium 10g, ampicillin 100mg, deionized water up to 1L, before sterilization pH 7.0, but ampicillin should be added after sterilization) and cultured under aerobic shaking at 37 ° C for 24 hours. After completion of the culture, the cells were collected by centrifugation to obtain a plasmid, and then the base sequence of an about 4.4 Kbp chromosomal DNA fragment inserted into the BamHI site of pUC18 was determined. The nucleotide sequence of the ORF of the hydantoinase gene contained in this chromosomal DNA fragment is shown in SEQ ID NO: 2 in the sequence listing. The cells of the above transformant are suspended in 0.1M Tris-HCl buffer (PH8.5), and the cells are crushed by ultrasonic waves. Then, insoluble matters derived from the cells are removed by centrifugation, and the cells are transformed. A crude enzyme solution of the converted product was obtained. When the hydantoinase activity was measured using the obtained crude enzyme solution, the activity was confirmed and it was confirmed that this ORF is the target hydantoinase gene. The activity of hydantoinase was measured according to the following method. Mix 0.1 mL of enzyme solution diluted appropriately with 4 mL of substrate solution consisting of 500 mM DL-5-methylhydantoin, 0. ImM manganese sulfate, 0.1 M sodium carbonate Z sodium bicarbonate buffer (PH8.7) After 10 minutes of reaction at ° C, 2 mL of 2N hydrochloric acid was added to stop the reaction. After adding 10 mL (wZv) p-dimethylaminobenzaldehyde Z35% hydrochloric acid solution (1 mL) to this reaction solution and mixing, the absorbance at 440 nm was measured, and based on a calibration curve prepared in advance, The amount of N-power ruberamolylanin produced was quantified.

[Example 2] Preparation of recombinant plasmid expressing hydantoinase gene

Using the plasmid having the hydantoinase gene obtained in Example 1 as a saddle, A primer that binds the cleavage site of the restriction enzyme Ndel to the N-terminus of the toinase gene (Primer-1: SEQ ID NO: 3 in the sequence listing) and the Ndel cleavage site at base 1228 of the hydantoinase gene are amino acid substituted. DNA primers were amplified by PCR using a primer (Primer-2: SEQ ID NO: 4 in the sequence listing) changed as described above to obtain an N-terminal DNA fragment of about 1.2 Kbp. Similarly, a primer having a sequence in which the cleavage site of the restriction enzyme Hindi II is bound to the C-terminal part of hydantoinase (Primer-3: SEQ ID NO: 5 in the sequence listing) and the cleavage site of Ndel present at the 1228th base of the hydantoinase gene The DNA was amplified by PCR using a primer (Primer-4: SEQ ID NO: 6 in the Sequence Listing) that was changed so that amino acid substitution did not occur, and a C-terminal DNA fragment of about 0.2 Kbp was obtained. DNA amplification by PCR using Primer-1 and Primer-3 is performed using a mixture of the N-terminal DNA fragment and the C-terminal DNA fragment as a saddle type as shown above! /, SEQ ID NO: 7 in the Sequence Listing. The DNA fragment shown in 1 was obtained. The obtained DNA fragment was cleaved with restriction enzymes Ndel and Hindlll, and vector plasmid pUCNT (which can be produced by those skilled in the art based on the description in the International Publication No. WO94Z03613) and T4 DNA ligase was used. Thus, the plasmid pALHIOl, which is represented by the restriction enzyme map of Fig. 1 and designed to express a large amount of the hydantoinase gene, was obtained.

[0045] Example 3) Including hydantoinase transmission ¾ ^ Production of deformed body using ^ body DNA

Transformation was performed by mixing the plasmid pALHIOl obtained in Example 2 with a competent cell of Escherichia coli HBl 01, and agar medium C (trypton 10 g, yeast extract 5 g, salted sodium 10 g, Agar 15 g, ampicillin 100 mg, made up to 1 L with deionized water, pH 7.0 before sterilization, but ampicillin was added after sterilization.) Plated to contain recombinant DNA containing hydantoinase gene Transformant Escherichia coli HB101 (pALHIOl) (FERM P-20712) was obtained as a colony.

[0046] The colony of the obtained transformant was sterilized in a test tube in 6 mL of medium D (tryptone 16 g, yeast extract 10 g, salted sodium 5 g, salted sodium 400 mg, ampicillin 100 mg, deionized water. Up to 1L, pH 7.0 before sterilization, but ampicillin after sterilization After inoculation, the cells were cultured under aerobic shaking at 37 ° C for 22 hours. The bacterial cells are collected from the obtained culture broth by centrifugation, suspended in 0.1 M Tris-HCl buffer (pH 8.5), disrupted by ultrasonic waves, and centrifuged to obtain the cells derived from the bacterial cells. Insoluble matter was removed, and a crude enzyme solution of transformant was obtained. Using the obtained crude enzyme solution, hydantoinase activity was measured in the same manner as in Example 1, and the activity was confirmed.

(Example 4) Synthesis of N-strength rubamoyl D-alanine using Aneurinibacillus bacteria

Aneurinibacillus sp. KNK491C strain sterilized in 500 mL Sakaguchi Flasco E (meat extract 2.0%, yeast extract 1.0%, sodium chloride 0.3%, uracil 0 5%, manganese chloride tetrahydrate 0.002%, pH 7.5 before sterilization was inoculated and cultured at 37 ° C for 20 hours under aerobic shaking. To the obtained culture broth, 10.0 g of 5-methylhydantoin was added, and the mixture was stirred at 55 ° C. for 20 hours while maintaining the pH at 8.7 with a 6M aqueous sodium hydroxide solution. The conversion rate of the produced N-force rubermoyl-D-alanine was analyzed by HPLC, and it was 96.3%. In addition, the N-type ruberamoyl D-alanine was weakened by reacting sodium nitrite with sulfuric acid in the presence of sulfuric acid, and the optical purity of the resulting D-alalanine was analyzed using HPLC. It was% ee. HP LC analysis was performed under the following conditions.

(Conversion rate analysis) Column: COSMOSIL 5C18ARII (4.6 mm x 250 mm, manufactured by Nacalai Tesque), mobile phase: 10 mM potassium phosphate buffer (pH 2.0), flow rate: 1. OmL / min., Column temperature: 20 ° C, detection: 210nm

(Optical purity analysis) Column: SUMICHIRAL OA—5000 (4.6 mm X 250 mm, manufactured by Sumika Chemical Analysis Co., Ltd.), mobile phase: 0.5 mM copper sulfate aqueous solution, flow rate: 0.5 mL / min., Column temperature: 10 ° C, detection: 254nm

(Example 5) Synthesis of N-force rubermoyl D-alalanine using hydantoinase genotype

Transformant Escherichia coli HB101 (pALH 101) having hydantoinase activity obtained in Example 3 was inoculated into 1 OOmL of medium D sterilized in a Sakaguchi flask and incubated at 37 ° C. The culture was shaken aerobically for a time. Centrifuge the 10 ml culture broth The cells are collected, suspended in 10 mL of 0. ImM manganese sulfate, 0.1 M Tris-hydrochloric acid buffer (pH 8.5), disrupted by sonication, centrifuged, and the supernatant is crude enzyme. It was collected as a liquid. To 40 L of the resulting crude enzyme solution, add 0.1 M Tris-hydrochloric acid buffer (pH 8.7) 1. 96 mL, 5-methylhydantoin 400 mg, and adjust the pH to 8 with 10 M aqueous sodium hydroxide. While maintaining at 7, the mixture was stirred at 50 ° C for 29 hours. When the conversion rate of the produced N-force rubymoyl D-alan was analyzed in the same manner as in Example 4, it was 97.0%. In addition, the obtained N-strength D-alanine was weakened by reacting sodium nitrite with sodium nitrite in the presence of sulfuric acid, and the resulting D-alanine was optically purified in the same manner as in Example 4. Compared with the reaction using the hydantoinase gene transformant derived from Bacillus sp. KNK245 strain (FERM BP-4863) described in Comparative Example 1 described later. An optically pure N-strength rubermoyl D-alanine was obtained.

[0048] (Example 6) A special case of hydantoinase cattle in early transmission

Using the crude enzyme solution of Escherichia coli HB101 (pALHIOl) obtained in Example 5, the activity against various hydantoins was measured. The activity of hydantoinase was measured as follows. Substrate solution 4 mL (30 mM various hydantoins (but 15 benzylhydantoin only 15 mM), 0.ImM manganese chloride, 50 mM sodium carbonate Z sodium bicarbonate buffer (PH8.7)), and crude enzyme solution diluted appropriately. ImL was mixed and reacted at 40 ° C for 15 minutes. Then, 2mL of 5N HC1 was added to stop the reaction. Subsequently, 10% (wZv) p dimethylaminobenzaldehyde Z35% hydrochloric acid solution ImL was mixed, and the absorbance at 440 nm of the supernatant obtained by centrifugation was measured. Based on a calibration curve prepared in advance, N-force rubamoyl amino acid produced in the reaction solution was quantified. Table 1 shows the relative activity values when the activity when 5-methylhydantoin is used as a substrate is 100%.

[0049] [Table 1] Relative activity value

Substrate Relative activity (%)

5 Methylhydantoin 100.0

5— (2 —Methylthioethyl) hydantoin 31.1

5-Benzylhydantoin 8.6

5- (4-Hydroxyphenyl) hydantoin 31.0

(Comparative Example 1) Synthesis of N-strength rubermoyl D-alanine using hydantoinase gene transformant derived from Bacillus sp. KNK245

Transformant Escherichia coli HB101 (p TH104) (FERM BP-4864) with a hydantoinase gene derived from Bacillus sp. KNK245 (FERM BP-4863) in a Sakaguchi flask Sterile lOOmL medium D was inoculated and cultured with shaking at 37 ° C for 24 hours. The culture fluid force was also collected by centrifugation, suspended in 10 mL of 0. ImM manganese sulfate, 0.1 M Tris-HCl buffer (pH 8.5), disrupted by ultrasonic waves, centrifuged, and centrifuged. Kiyoshi was recovered as a crude enzyme solution. To 160 L of the obtained crude enzyme solution, add 0.1 M Tris-hydrochloric acid buffer (pH 8.7) 1. 84 mL, 5-methylhydantoin 400 mg, and adjust the pH with 10 M aqueous sodium hydroxide solution. 8. While maintaining at 7, the mixture was stirred at 50 ° C for 29 hours. The conversion rate of the produced N-force rubermoyl-D-alanine was analyzed in the same manner as in Example 4. As a result, it was 97.5%. Further, the obtained N-stralbumoyl-D-alanine was decarbamoylated by the action of sodium nitrite in the presence of sulfuric acid, and the optical purity of the resulting D-alanine was analyzed in the same manner as in Example 4. It was 7% ee.

(Reference Example 1) Preparation of decarbamoylase ffi enzyme solution using transformed microorganism Escherichia coli ΗΒ101 (ΌΝΤ4553)

The crude decarbamoylase solution used in the selection of colonies described in Example 1 was prepared by the following method. E. coli HB101 (pNT4553) (FERM BP-4368) containing the weak ruvalamylase gene derived from Agrobacterium sp. Sterilized with 10 mL of medium F (tryptone 16 g, yeast extract 10 g, sodium chloride 5g, ampicillin 100mg, make up to 1L with deionized water, inoculate pH 7.0 before sterilization, but add ampicillin after sterilization), at 37 ° C for 12 hours, The culture was shaken vigorously. 3.5 mL of this culture solution was inoculated into 350 mL of medium C sterilized in a Sakaguchi flask, and cultured with shaking at 37 ° C for 30 hours. The obtained culture solution was subjected to ultrasonic treatment to disrupt the cells, kept at 50 ° C for 30 minutes, and then centrifuged, and the supernatant was recovered as a crude enzyme solution.

Brief Description of Drawings

FIG. 1 is a diagram showing the structure of a recombinant plasmid pALHIOl containing a hydantoinase gene as an embodiment of the present invention.

Copy on paper (Caution Electronic data is the original)

[This form does not form part of the international application and is not included in the number of international application forms]

The following indications appear in the detailed description of the invention

In relation to microbial or biological material

-1 paragraph number 0011

-3 Display of deposit

-3-1 Name of Depositary Institution IP0D National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (IP0D)

-3-2 Name of depositary institution Japan 1 1-chome East, Tsukuba, Ibaraki 305-8566, Japan 1

Central 6th

-3-3 Date of deposit November 20, 2006 (20.11.2006)

-3-4 Accession number IPOD FERM BP-10735

-5 Designated countries for making this indication All designated countries

-4 This form was accepted along with the international application

(Yes, No)

-4-1 Authorized staff Entry field of the International Bureau -5 Date when this form was received by the International Bureau -5-1 Authorized staff

Replacement paper (Rules)

Claims

The scope of the claims

[1] The following polypeptide (a), (b) or (c):

(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

(b) It consists of an amino acid sequence in which one or several amino acids are substituted, inserted, deleted and Z or added to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and has hydantoinase activity. A polypeptide;

[2] The polypeptide according to claim 1, which is derived from a microorganism belonging to the genus Aneurinibacillus.

[3] Aneurinibacillus sp. KNK491C (FERM BP— 10

735).

[4] DNA encoding the polypeptide of claim 1.

[5] DNA of (d), (e), (f), or (g) below:

(d) DNA consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing,

(g) A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing and encodes a polypeptide having hydantoinase activity.

[6] A recombinant plasmid obtained by inserting the DNA according to claim 4 or 5 into a vector.

[7] A transformant obtained by transforming a host microorganism with the recombinant plasmid according to claim 6.

[8] has the ability to produce a polypeptide according to claim 1, and § Neu linear Bacillus (A replacement sheet (Rule ² 6) A microorganism belonging to the genus (neurinibacillus).

[9] A hydantoinase produced by culturing a microorganism capable of producing a polypeptide as described in claim 1, accumulating the polypeptide in the culture, and collecting the polypeptide. Method.

[10] The method for producing hydantoinase according to claim 9, wherein the microorganism is the transformant according to claim 7 or the microorganism according to claim 8.

[11] An N_strengthening D-amino acid is produced by reacting the polypeptide according to claim 1, the transformant according to claim 7, or the microorganism according to claim 8 with a 5-substituted hydantoin. Method.

Replacement paper (Rule 26)