KR100244055B1 - Process for producing d-n-carbamoyl-alpha-amino acids - Google Patents

Abstract

5-substituted hydan using a hydantoinase prepared from a microorganism transformed with a recombinant DNA having a DNA and a fragment containing a DNA related to a hydantoinase originating from a specific microorganism belonging to the genus Pseudomonas A process for preparing DN-carbamoyl-α-amino acids from toyne.

Description

Production method of di-ene-carbamoyl-alpha-amino acid {PROCESS FOR PRODUCING D-N-CARBAMOYL-ALPHA-AMINO ACIDS}

The present invention relates to a process for the preparation of D-N-carbamoyl-α-amino acids. More specifically, it is an enzyme that converts 5-substituted hydantoin into the corresponding DN-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis (hereinafter referred to as hydantoinase). It relates to a method for preparing DN-carbamoyl-α-amino acid using a novel transformant having a gene for.

Optically active D-N-carbamoyl-α-amino acids can be converted to the corresponding D-α-amino acids, and optically active D-α-amino acids are important compounds as pharmaceutical intermediates. In particular, examples of industrially useful compounds include D-phenylglycine and D- (p-hydroxyphenyl) glycine as intermediate compounds for the preparation of semisynthetic phenylsilins and semisynthetic sephalosporins.

To prepare DN-carbamoyl-α-amino acids, microbial enzymatic reactions are known, in which asymmetric cleavage hydrolysis converts the 5-substituted hydantoin into the corresponding DN-carbamoyl-α-amino acids. The method is described in JP-A 53-91189, JP-A 53-44690, JP-A 53-69884, JP-A 53-133688 and the like.

In addition, genes associated with hydantoinase, an enzyme for converting 5-substituted hydantoin into the corresponding DN-carbamoyl-α-amino acids, are obtained from thermophilic microorganisms and inserted into mesophilic microorganisms Transformed microorganisms with Dantoinase activity are prepared. Furthermore, in WO94 / 00577, hydantoinase gene segments are isolated from microorganisms belonging to the genus Agrobacterium and introduced into microorganisms of the genus Agrobacterium expressing Escherichia coli or activity.

The above-described method using a microbial enzyme reaction has the disadvantage that the yield of enzymes by microorganisms of known origin is insufficient and the culture medium is expensive.

In addition, in the transformed microorganism described in JP-A 62-87089, the improvement of enzymatic activity is only several times compared to the spore-forming thermophilic microorganism having hydantoinase activity, and the yield of enzyme is insufficient.

Moreover, there is no example in the prior art using a transgenic microorganism described above to asymmetric cleavage hydrolysis of the 5-substituted hydantoin to produce and accumulate the corresponding D-N-carbamoyl-α-amino acids.

The present invention solves this problem, produces microorganisms with very high enzyme productivity, and effectively produces D-N-carbamoyl-α-amino acids using the enzyme source thus obtained.

JP-A 62-87089 and WO94 / 00577 disclose methods for obtaining hydantoinase producing microorganisms using recombinant DNA techniques. However, the microorganism source for providing the hydantoinase gene is completely different from the microorganism used for obtaining the hydantoinase gene in the present invention. In addition, the nucleotide sequence and amino acid sequence of the hydantoinase gene are significantly different from those of the present invention.

The present invention Pseudomonas sp. DNA fragments containing genes for hydantoinase derived from Pseudomonas sp. KNK003A (FERM BP-3181) and recombinant DNA of vector DNA as Escherichia, Pseudomonas, and Flavobacterium ), Transformants obtained by transforming host microorganisms such as Bacillus, Serratia, Agrobacterium, Corynebacterium or Brevibacterium Provided is a method for preparing DN-carbamoyl-α-amino acid, which comprises reacting a 5-substituted hydantoin in an hydantoinase and an aqueous medium.

Transformants with very high hydantoinase productivity as obtained in the present invention have not been known so far in the prior art, and the production of DN-carbamoyl-α-amino acids is highly efficient and economical. It was made possible by the use of an enzymatic reaction of the sieve.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

1 is a restriction map of plasmid pPHD301.

1 is a restriction map of plasmid pPHD301.

DNA fragments comprising the gene used in the present invention is Pseudomonas sp. It can be obtained from KNK003A. Pseudomonas Esp. KNK003A has been deposited by Applicant with the Applicant Number of FERM BP-1381 to the Institute of Biotechnology and Industrial Technology (NIBH), and is a known strain described in detail in WO92 / 10579.

In order to obtain a hydantoinase gene from this strain, the following procedure is usually performed.

Initially, genomic DNA is extracted from microbial cells and then the DNA is partially hydrolyzed with appropriate restriction enzymes such as Sau3AI or the like. The resulting fragments are bound to vector DNA to obtain recombinant DNA molecules with various genomic DNA fragments as gene libraries (cf. JP-A 58-126789 and U.S. Patent 4,237,224).

Then, the recombinant containing the gene of interest is selected from the gene library. For example, selection may be performed by detection of an expression protein using enzymatic activity of the expression protein as a marker, or selection may be performed by analysis of the N-terminal sequence of the protein, synthesis of a corresponding DNA probe, and colony hybridization. This can be done. In addition, the gene of interest can be obtained by polymerase chain reaction (PCR) method or colony hybridization using DNA primers synthesized to be suitable for a portion of a known hydantoinase nucleotide sequence. Once the desired gene is obtained, its nucleotide sequence is analyzed. In addition, a hydantoinase producing microorganism and a recombinant comprising the enzyme gene are cultured. The resulting enzyme is purified and the molecular weight of the resulting protein is measured. At the same time, the amino acid sequence around its N-terminal region is determined by a gaseous protein sequencer or the like.

Next, this DNA nucleotide sequence is compared with the N-terminal amino acid sequence to determine the translation initiation site of the nucleotide sequence portion encoding the hydantoinase protein into the protein, and considering the relationship with the molecular weight of the protein, the enzyme The protein is confirmed to be encoded in the gene portion from this site to the stop codon, demonstrating the gene of interest (Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Chapters 4 & 13). According to SEQ ID No. DNA fragment of 1 is Pseudomonas sp. Obtained from KNK003A. As is known, genes encoding enzymes and / or DNA fragments containing the enzymes thus obtained have amino acid sequences corresponding to the genes and / or DNA fragments, since one amino acid usually corresponds to a plurality of base codons. Identical to a DNA fragment with another nucleotide sequence encoding.

As vectors used in the present invention, plasmids, phages or derivatives thereof can be used, which are derived from microorganisms and are derived from Escherichia, Pseudomonas, Flavobacterium, Bacillus, Serratia, Agrobacterium, Corynebacterium or It can autonomize in cells of bacteria belonging to the genus Brevibacterium.

See, for example, 'Guidelines on Recombinant DNA Experiments, -Commentation, Q & A-' (the Life Science Section of the Research Development Office in the Science and Technology Agency, Ed., Revised September 16, 1987), pages 25 to 27 and 36 to 38 can be used host-vector system.

In addition, recombinant DNA with translational initiation sites linked to appropriate portions of the vector modified to have strong structural promoters can be used to increase the amount of enzyme to produce. Both ends of this fragment can be cleaved with a restriction enzyme to produce a recombinant with a suitable vector. Hydantoinase-producing recombinant microorganisms can be prepared by directly transforming microorganisms belonging to the genus Escherichia, Pseudomonas, Flavobacterium, Bacillus, Serratia, Agrobacterium, Corynebacterium or Brevibacterium.

Hydantoinase producing recombinant microorganisms can also be obtained without direct transformation of the microorganisms of the above genus; That is, the target gene is cloned into the host vector system using other microorganisms such as E. coli, and then recombinant DNA having a suitable vector is produced.

The description in the following literature is applicable broadly to the preparation of recombinants: S. a. yen. U.S. Patent Patent No. The specification of 4,237,244; Idenshi-sousa Jikken-hou (Experimental Method of Gene Manipulation) [Ed., Yasuyuki TAKAGI, Kohdan-sha Scientific (1980)]; Method in Enzymology, 68, Recombinant DNA [Ed., Ray Mv, Academic Press (1970)], JP-A 58-126789 and the like.

The gene of interest is cloned and its unnecessary DNA is removed. Recombinant DNA having a translation initiation site linked to a suitable position of a vector modified to have a strong structural promoter can be used to transform the host cell to increase the amount of enzyme to produce.

The transformants are cultured in conventional nutrient culture medium expressing the introduced recombinant DNA. When properties derived from genetic DNA or vector DNA are transferred to recombinant DNA, appropriate components may be added to the culture medium depending on the specific properties.

In order to take the transformant thus obtained as an enzyme source, it can be cultured using a conventional culture medium. If necessary, it can also be treated for enzyme induction, such as the addition of hydantoin compounds, uracil, isopropyl-1-thio-β-D-galactosid (IPTG), or increased temperatures. Typically, the culture medium used for culturing the transformant may be a conventional culture medium containing a carbon source, a nitrogen source, and an inorganic ion. In many cases, the desired result can be further obtained by adding organic micronutrients such as vitamins, amino acids and the like. As the carbon source, carbohydrates such as glucose and sucrose, organic acid alcohols such as acetic acid, and the like can be commonly used. As a nitrogen source, ammonia gas, ammonia water, ammonium salt, etc. can be used. As inorganic ions, phosphate ions, magnesium ions, potassium ions, iron ions, manganese ions, cobalt ions, nickel ions, copper ions, aluminum ions, molybdenum ions and the like can be used.

While adjusting the pH and temperature to appropriate ranges of 4 to 8 and 25 to 60 ° C., respectively, if the culture is carried out for 1 to 10 days under aerobic conditions, the desired result can be obtained.

The enzymatic activity of the enzyme prepared by the transformant can be exerted, for example, in the form of a culture medium of the transformant, microbial cells thereof, enzymes extracted from microbial cells, immobilized microbial cells, and the like.

As the microbial cells, any culture solution itself, cultured microorganism cells separated from the culture solution, washed microbial cells, etc. can be used. As the treated microbial cells, lyophilized microbial cells, acetone-dried microbial cells, microbial cells in contact with toluene or detergent, lysozyme treated microbial cells, ultrasonically disrupted microbial cells, mechanically crushed microbial cells and the like can be used. . In addition, enzyme extracts having hydantoinase activity obtained from such treated microbial cells, such immobilized microbial cells, insoluble microbial cells, enzyme proteins immobilized on immobilization supports (e.g., ion exchange resins), etc. Can be used. Examples of the immobilization method include specifications such as JP-A 63-185382.

Examples of the immobilization support include phenol-formaldehyde anion exchange resins such as Duolite A568 or DS17186 (Rohm & Hass Co.®) and various anion exchange resins containing various amines, ammonium salts or diethanolamine functional groups, For example, polystyrene resins such as Amberlite IRA935, IRA945, IRA901 (Rohm & Hass Co.®), Lewatit OC1037 (Bayer AG) and Diaion EX-05 (Mitsubishi Chemical Industries, Ltd.) are suitable. Do. Other supports such as DEAE-cellulose can also be used.

In addition, for stronger and more stable adsorption of enzymes, a crosslinking agent is usually used, a preferred example of which is glutaraldehyde. The enzymes used are not only purified enzymes, but also partially purified enzymes, disrupted microbial cells. Those with different degrees of purification, such as suspensions and cell-free extracts, can be used. The preparation of the immobilized enzyme can be carried out by a conventional method, for example, by adsorbing an enzyme on a support, followed by crosslinking treatment.

The 5-substituted hydantoin used as a substrate for the enzymatic reaction of the present invention is not particularly limited and may be any compound commonly used in this kind of reaction.

Examples thereof include those represented by the following formula (1):

The substituent R in the compound of formula (1) can be selected from a wide range. In order to provide industrially useful products such as intermediate compounds of medicaments, preferably R is phenyl, phenyl substituted by hydroxy, alkyl, substituted alkyl, aralkyl or thienyl. In the case of phenyl substituted with hydroxy, the number of hydroxy may be one or more, and may be located in any o-, m- and p- position, a typical example of which is p-hydroxyphenyl. The alkyl group is a group having 1 to 4 carbon atoms, and the corresponding DN-carbamoyl-α-amino acid is DN-carbamoyl-alanine, DN-carbamoyl-valine, DN-carbamoyl-leucine, DN-carbox Bamoyl-Isoleucine and the like. The substituted alkyl group is an alkyl group having 1 to 4 carbon atoms substituted with hydroxy, alkylthio, carboxyl, amino, phenyl, phenyl substituted by hydroxy, amido and the like, and the corresponding DN-carbamoyl-amino acid is DN-carboxyl. Barmoyl-serine, DN-carbamoyl-threonine, DN-carbamoyl-methionine, DN-carbamoyl-cysteine, DN-carbamoyl-asparagine, DN-carbamoyl-glutamine, DN-carbamoyl -Tyrosine, DN-carbamoyl-tryptophan, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN-carbamoyl-histidine, DN-carbamoyl-lysine, DN-carbamoyl- Arginine, DN-carbamoyl-citrulline and the like. Aralkyl groups are groups having 7 or 8 carbon atoms such as benzyl or phenethyl, and the corresponding D-N-carbamoyl-α-amino acid is D-N-carbamoyl-phenylalanine and the like.

As the aqueous medium, a medium containing an organic solvent such as water, buffer or ethanol can be used. In addition, if necessary, nutrients, antioxidants, detergents, coenzymes, hydroxyamines, metals and the like necessary for the growth of microorganisms may also be added to the aqueous medium.

When microbial cells of the aforementioned microorganisms are cultured in a water-soluble medium and the microbial cells are contacted with specific 5-substituted hydantoin, not only 5-substituted hydantoin but also growth of microorganisms such as carbon source, nitrogen source and inorganic source. An aqueous medium containing the necessary nutrients can be used. If you add organic micronutrients, such as vitamins and amino acids, you can get satisfactory results in many cases.

As a carbon source, Carbohydrates, such as glucose and sucrose; Organic acids such as acetic acid; Alcohol is usually used. As a nitrogen source, ammonia gas, ammonia water, ammonium salt, etc. can be used. As inorganic ions, phosphate ions, magnesium ions, potassium ions, iron ions, manganese ions, cobalt ions, nickel ions, copper ions, aluminum ions, molybdenum ions and the like can be used.

Cultivation is carried out under aerobic drying, adjusting the pH and temperature to appropriate ranges of 4 to 8 and 25 to 60 ° C, respectively. If incubation is carried out for 1 to 10 days, 5-substituted hydantoin is converted to D-N-carbamoyl-α-amino acid with high efficiency.

On the other hand, in an aqueous medium containing dissolved or suspended 5-substituted hydantoin, the culture medium of the above-mentioned microorganisms is cultured as it is, microbial cells, treated microbial cells, enzyme extracts, immobilized microbial cells, insoluble microbial cells or immobilized In the case of reacting with an enzyme protein, the reaction mixture can be left to stand or stirred for some time while maintaining an appropriate temperature of 10 to 80 ° C. and a pH of 4 to 9.5. Therefore, if this course is carried out for 5 to 100 hours, the D-specific hydrolysis reaction of the substrate by hydantoinase and the chemical racemization of the substrate proceed and any D-, DL- or L-5- Substituted hydantoin is converted to the corresponding DN-carbamoyl-α-amino acid to accumulate large amounts of amino acids in the aqueous medium. In addition, 5-substituted hydantoin may be added in portions as the reaction proceeds.

The resulting D-N-carbamoyl-α-amino acid can be isolated and purified by conventional methods.

D-α-amino acids are prepared by the reaction mixture obtained by the action of the separated and purified DN-carbamoyl-α-amino acids or the hydantoinase reaction itself obtained by the above-described method, and using DN-carbamoyl It can be obtained by converting the -a-amino acid chemically or enzymatically into the corresponding D-a-amino acid with an enzyme having decarbamylase activity.

The following examples further illustrate the invention in detail.

Example 1

Pseudomonas Esp. Obtaining a Plasmid Containing a Gene for Hydantoinase Derived from KNK003A (FERM BP-3181)

According to the following method, the hydantoinase gene was found to be located in plasmid pPHD301 described in WO92 / 10579.

E. coli JM109 was transformed with pPHD301 according to a conventional method and 50 ml of L-juice (10 g / 1 peptone, 5 g / 1 yeast extract, 5 g / 1 sodium chloride; pH 7.0) containing 100 mg / 1 ampicillin After inoculation, the cells are incubated at 37 ° C. for 16 hours. 50 ml of culture broth was then collected, then the supernatant was removed and 50 ml of substrate solution (0.5% 5- (p-hydroxyphenyl) hydantoin, 0.05% Triton X-100, 0.05 M phosphate butter; The microbial cells are suspended in pH 8.7) and reacted for 3 hours at 37 DEG C under a nitrogen stream. The reaction mixture was deposited on a TLC plate, developed and color developed with a solution of p-dimethylaminolobenzaldehyde in hydrochloride to give a suitable sample of DN-carbamoyl- (p-hydroxyphenyl) glycine. Detect spots In view of the above, it was confirmed that the transformant of pPHD301 expresses a gene encoding hydantoinase.

E. coli HB101 is transformed with pPHD301 to obtain E. coli HP101 pPHD301 (FERM BP-4866). The restriction map of pPHD301 is shown in FIG. 1.

Nucleotide sequencing of the hydantoinase gene is performed using a DNA sequence kit (manufactured by Applied Biosystems). The result is SEQ ID No. 1 is shown.

The deposit of microorganisms

Pseudomonas Esp. KNK003A was deposited on December 1, 1990 under Accession No. of FERM BP-3181; Escherichia coli HB101 pPHD301 was deposited on the following depository under the Budapest Treaty on November 2, 1994, under the accession number of FERM BP-4866.

Name: Biotechnology Research Institute of Industry and Technology

Address: 1-3, Higashi 1-chome, Tsukuba City, 305, Ibaraki, Japan.

SEQ ID NO: 1

Sequence length: 1569

Sequence type: nucleic acid

Sequence:

5-substituted hydan using a hydantoinase prepared from a microorganism transformed with a recombinant DNA having a DNA and a fragment containing a DNA related to a hydantoinase originating from a specific microorganism belonging to the genus Pseudomonas DN-carbamoyl-α-amino acids can be prepared from toyne.

Claims (11)

Pseudomonas Esp. (Pseudomonas sp.) An enzyme that converts 5-substituted hydantoin to the corresponding DN-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis, derived from KNK003A (FERM BP-3181) (hereinafter Hydantoinana) Transformed microorganism obtained by transforming a host microorganism with a recombinant DNA of a DNA fragment and a vector DNA containing a gene encoding a zero). The host microorganism of claim 1, wherein the host microorganism is Escherichia, Pseudomonas, Flavoacterium, Bacillus, Serratia, Agrobacterium, Corynebacterium. Transformed microorganism selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium. The transforming microorganism of claim 2, wherein the transforming microorganism is Escherichia coli HB101 pPHD301 (FERM BP-4866). A method of converting a 5-substituted hydantoin into a corresponding DN-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis, characterized by using the transgenic microorganism according to any one of claims 1 to 3. Manufacturing method. A method for producing D-N-carbamoyl-α-amino acid, characterized by using the enzyme obtained by the method of claim 4. The method of claim 5, wherein the 5-substituted hydantoin is a compound represented by Formula 1 below: [Formula 1] _ Wherein R is phenyl, phenyl substituted by hydroxy, alkyl, substituted alkyl, aralkyl or thienyl. Pseudomonas Esp. Recombinant plasmids obtained by recombination of DNA fragments encoding hydantoinase derived from KNK003A (FERM BP-3181) and having the restriction map of FIG. 1. The recombinant plasmid of claim 7, wherein the recombinant plasmid is pPHD301. For genes for proteins with enzymatic activity that converts 5-substituted hydantoin to a corresponding D-N-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis, the gene is SEQ ID No. A gene encoding the amino acid sequence of the first to 485th amino acids shown in 1. In a DNA fragment comprising a gene for a protein having an enzymatic activity that converts 5-substituted hydantoin to a corresponding D-N-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis, the gene is SEQ ID No. A DNA fragment having the nucleotide sequence of the first to 1569th nucleotides shown in 1. An enzyme comprising the amino acid sequence of the first to 485th amino acids shown in SEQ ID No. 1 and having enzymatic activity for converting 5-substituted hydantoin into the corresponding DN-carbamoyl-α-amino acid by asymmetric cleavage hydrolysis protein.