KR20110029697A - Enantioselective amidase specific to l-amino acid amide and dna encoding the same - Google Patents

Abstract

PURPOSE: A novel enantio-selective amidase which is specific to L-type amino acid amide and a gene encoding the same are provided to ensure remarkably high activity and thermal resistance. CONSTITUTION: An enantio-selective amidase which enatio-selectively hydrolyses amide bond of L-type amino acid amide contains an amino acid sequence of sequence number 6, or an amino acid sequence in which one or plural amino acid(s) is/are deleted, substituted, or added in the amino acid sequence of sequence number 6. An enantio-selective amidase DNA contains a base sequence encoding a protein with an enzyme activity of enantio-selective amidase. A vector in which the DNA is introduced is pFRPT of deposit number KCCM-10320. A recombinant microorganism transformed by the recombinant vector is deposited by number KCCM11021P.

Description

Enantioselective amidase specific to L-amino acid amide and DNA encoding the same}

The present invention relates to enantio-selective amidases specific for L-type amino acid amides, and DNA encoding them.

Some of the amidases have the activity of hydrolyzing amino acids to convert them to amino acids and ammonia. Most amino acids present in nature are optically active around alpha carbon, and enantio-selective amidase can only hydrolyze one type of optical isomer in the mixture of amino acid amide isomers.

Chemical synthesis and enzyme conversion are known as methods for preparing optically active amino acids from racemic amino acids. Chemical synthesis involves the difficulty of complex optical purification because the product is prepared in the form of racemic mixtures. Thus, it is known that industrially advantageous methods for producing specific optically active amino acids in a one step process using microorganisms comprising enantio-selective amidase or cell processing materials of the microorganisms.

Optically active amino acids or derivatives thereof are widely used in various industries such as pharmaceutical industry and agriculture. As an example of the optically active amino acid, tert-leucine is used as an intermediate of pesticides and pharmaceuticals, and specific examples include the following.

Optical isomer amino acids or derivatives thereof are widely used in various industries, such as pharmaceutical industry and agriculture. As an example of the optical isomer amino acid, tert-leucine is used as an intermediate of pesticides and pharmaceuticals, and specific examples include the following.

L-tert-leucineol, a derivative of L-tert-leucine, is used to prepare chiral 1,3,2-oxazaphospholidine 2-sulfide, which is used as an antiparasitic agent. Used. (Wu et al., J. Pesticide Sci., 12, 221-227, 1987)

L-tert-leucine is also used as an intermediate for HIV protease inhibitors. Substances in which amide bonds of phenylnorstatine or its derivatives are substituted with hydroxyethylene or dihydroxyethylene have good activity as HIV-protease inhibitors (Kato et al., Chem. Pharm. Bull., 42, 176-178, 1994), a method of optimizing antiviral activity by protecting the dihydroxyethylene portion of the HIV protease inhibitor with tert-leucine has been used (Ettmayer et al., Bioorg. Med). Lett., 4, 2851-2856, 1994).

L-tert-leucine-N-methylamide (trade name: RO 31-9790, manufactured by Roche) is used as a collagenase inhibitor to treat arthritis. Brown et al., EP Appl. 0497 192, 1992

In addition, L-tert-leucine or L-tert-leucineol can be used as an intermediate of modified peptides developed to inhibit Herpes Simplex type virus (HSV) ribonucleotide reductase (Deziel et al, EP). Appl. 0560274, 1993).

In addition, L-tert-leucine or its derivatives can be used as an intermediate of gelatinase inhibitors or thymidylate synthase inhibitors associated with anticancer mechanisms (Spector et al., Biochem. Phrmacol., 42, 91-96, 1991).

The production method of L-type tert-leucine is largely chemical synthesis method and enzyme conversion method, and the representative enzyme conversion method known to date is α-N-phenylacetyl using penicillin G acylase (EC 3.5.1.4) Method for separating optical isomers of -D, L-tert-leucine (α-N-phenacetyl-D, L-tert-leucine) (Garbley et al., EP 0 141 223, 1987), enantio-selective D- D, L-5-tert-butyl-hydantoin was converted to N-carbamoyl-D-tert-leucine (N) using hydantoinase. -carbamoyl-D-tert-leucine (Bommarius et al., patent application DE 195 29 211.1, 1995), leucine dehydrogenase and formate dehydrogenase And trimethyl pyruvate using L-tert-leucine (Kragl et al., Chem. Ing. Tech., 64, 499-509, 1992).

In addition, Ocrobacterium anthrophyll NCIB 40321 ( Ochrobacterium .... anthropi NCIB40321) ( Sonke et al, Pat US 7,241,602 B2, 2007) and xanthosine bakteo Plastic bus NR303 (Xanthobacter flavus NR303) (Asano et al, Pat WO / 2006/008872, 2006) derived from the enantiomers - It is known to produce L-tert-leucine from L-tert-leucine amide using selective L-amidase.

It is an object of the present invention to provide a novel high activity novel L-type enantio-selective amidase, useful for the preparation of optical isomeric amino acids, in particular L-type tert-leucine, and DNAs encoding the same, recombinants containing recombinant DNA containing said DNA. It provides a microorganism and a method for producing an optically active amino acid using the same.

The inventors have screened novel microorganisms showing enantio-selective amidase enzyme activity from soil to achieve the above object, and enantio-selective amidase enzymes from Bacillus cereus E306, thus screened microorganisms. The gene encoding the protein having the activity was identified, its DNA was isolated, recombinant DNA containing the DNA, and a recombinant microorganism incorporating the recombinant DNA were prepared to complete the present invention.

That is, the present invention is as shown below.

(1) an amide bond of an L-type amino acid amide comprising an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 6; A protein having enantio-selective amidase enzymatic activity that enantio-selectively hydrolyzes.

(2) The protein according to (1), wherein the protein having an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 6 has at least 95% or more homology with SEQ ID NO: 6 protein.

(3) The protein according to (1) or (2), wherein the L-type amino acid amide is L-tert-leucine amide.

(4) DNA comprising a nucleotide sequence encoding the protein of (1).

(5) The DNA according to the above (4), comprising a nucleotide sequence represented by SEQ ID NO: 5.

(6) The recombinant vector which introduce | transduced the DNA of said (4).

(7) The recombinant vector of (6), wherein the expression vector is pFRPT (Accession Number: KCCM-10320).

(8) A recombinant microorganism transformed with the recombinant vector of (6) or (7).

(9) The recombinant microorganism according to the above (8), wherein the recombinant microorganism is Escherichia coli.

(10) The recombinant microorganism according to the above (9), which is accession number KCCM 11021P.

(11) A method for producing an L-type amino acid, in which an L-type amino acid amide is converted to an L-type amino acid by reacting the protein of (1) with a racemic amino acid amide mixture.

(12) The method for producing an L-type amino acid according to the above (11), wherein the L-type amino acid amide is represented by the following Formula (I):

Formula (I)

(Wherein R ₁ is hydrogen; alkyl group having 1 to 4 carbon atoms; hydroxy group, methoxy group, mercapto group, methyl mercapto group, amino group, guanyl group, carboxyl group, carboxamide group, halogen group, phenyl group, hydroxyphenyl group, Or an alkyl group having 1 to 4 carbon atoms substituted with one or more groups selected from imidazolyl groups; one or more selected from hydroxy, methoxy, mercapto, methylmercapto, amino, guanyl, carboxyl, carboxamide or halogen groups A phenyl group substituted with a group; a heterocyclic group of an indole group or an imidazolyl group; or substituted with one or more groups selected from hydroxy group, methoxy group, mercapto group, methyl mercapto group, amino group, guanyl group, carboxyl group, carboxamide group or halogen group Indole group or imidazolyl group).

(13) The method for producing an L-type amino acid according to the above (12), wherein in formula (I), R ₁ is a tert-butyl group.

(14) Bacillus cereus E306, which has the expression ability of the protein containing the amino acid sequence represented by SEQ ID NO: 6 of (1), and has accession number KCCM11020P.

Hereinafter, the present invention will be described in more detail.

The present invention provides an amino acid sequence represented by SEQ ID NO: 6 encoding L-Enantiospecific Amino Acid Amidase (hereinafter referred to as "LEAA"), which is an L-type amino acid asymmetric hydrolase, or substantially the same. To provide a protein comprising the same amino acid sequence (hereinafter referred to as "LEAA protein").

An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 may include an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added in SEQ ID NO: 6, which is at least 95% or more than SEQ ID NO: 6 It has homology and substantially equivalent physiological and biological activity.

The LEAA protein can be found in Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 or Current Protocols in Molecular Biology (1994) (Wiley- Interscience). For example, the DNA encoding the LEAA protein may be introduced into a host cell to express the LEAA protein.

That is, based on the full length DNA encoding the LEAA protein, a recombinant vector is prepared by arbitrarily preparing a DNA fragment of an appropriate length and inserting the DNA fragment or the full length DNA downstream of a promoter of a suitable expression vector. The LEAA protein can be prepared by introducing a recombinant vector into an appropriate host cell to prepare a transformant that produces the LEAA protein. Here, the nucleotide sequence encoding the protein can be prepared as base-substituted DNA so as to be most suitable for expression in the host.

Deletion, substitution or addition of amino acids in the amino acid sequence represented by SEQ ID NO: 6 may be performed by site-specific potential mutagenesis, which is well known, for example, Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J ., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

In addition, the present invention includes a DNA fragment having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 6 encoding the LEAA (SEQ ID NO: 5) or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 It provides a DNA fragment having a nucleotide sequence encoding a protein.

As a cloning method of the DNA encoding the LEAA protein, genomic DNA of the microorganism producing the LEAA protein of the present invention is amplified by PCR using a synthetic DNA primer having a partial sequence thereof as a template, or combined into a suitable vector. One DNA can be selected by hybridization with labeled DNA fragments or synthetic DNA encoding some or all regions of the protein of the invention. The hybridization method can be carried out, for example, according to the method described in Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell D. W., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

The present invention also provides a recombinant vector into which DNA encoding the LEAA protein of the present invention is introduced for introduction into an appropriate host cell known in the art.

The recombinant vector can be prepared by inserting the DNA fragment or full length DNA downstream of the promoter of a suitable expression vector. As the expression vector, it is possible to use a known appropriate vector which can autonomously replicate in the host cell, be inserted into the chromosome and include a promoter at a suitable position for transcription of the LEAA protein gene, preferably by the applicant. PFRPT (Accession Number: KCCM-10320) produced and deposited in the Korea Culture Center of Microorganisms (KCCM) can be used (see FIG. 1). The promoter may be any appropriate promoter corresponding to the host used for gene expression, and any promoter known in the art may be used. For example, when the host is Escherichia coli, a trp promoter, a lac promoter, a recA promoter, a λPL promoter, an lpp promoter, a T7 promoter and the like can be used. In addition to the above-described expression vector, those containing a selection marker known in the art, a replication origin, and the like may be used as desired. The transformant may be prepared using the recombinant vector thus prepared.

The present invention also provides a transformant transformed with the recombinant vector.

If the LEAA protein is expressed in a cell, any cell, for example, prokaryotic, yeast, animal, plant or insect cell, can be used as a host cell, preferably prokaryotic, more preferably E. coli. have. Prokaryotic transformation can be performed by methods known in the art, such as calcium chloride methods [Molecular cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 1.117, Sambrook J., Russell DW,, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001], electroporation (electroporation) and the like can be carried out easily.

The present invention also provides a method for preparing L-type amino acid from racemic amino acid amide mixture using the LEAA protein. In particular, there is provided a method for preparing L-tert-leucine from racemic tert-leucineamide mixtures using the LEAA protein of the invention.

An illustration of the reaction of the L-tert-leucine to produce L-tert-leucine using the L-type enantio-selective amidase is as follows.

A method for separating L-tert-leucine from a mixture of L-tert-leucine and D-tert-leucine amide, the product of the enzymatic reaction, wherein the mixture is centrifuged or filtered using conventional microbial cell removal means such as a filtration membrane. After removal of the solution, it is concentrated under reduced pressure, and then an organic solvent is added to precipitate an amino acid, thereby recovering pure L-tert-leucine (Katoh et al., Pat. US 6,949,658 B2, 2005). In addition, purification methods using recrystallization, column chromatography, ion exchange electrodialysis, or adsorption and desorption by ion exchange resins can be used (Asano et al., WO / 2006/008872).

The L-type enantio-selective amidase of the present invention has significantly higher activity than the conventional L-type enantio-selective amidase, exhibits excellent high temperature stability, exhibits even activity even at neutral to weak alkali pH conditions, and has a high concentration of substrate. Very fast reaction rates under conditions. It shows the effect of producing L-type amino acids with high purity from a mixture of D- and L-type amino acid amides. With the enantio-selective amidase of the present invention having excellent stability to external reaction conditions, it is possible to produce optically active amino acids, especially L-tert-leucine, which are widely useful compounds in the agricultural and pharmaceutical fields, inexpensively and effectively.

Hereinafter, although an Example and a comparative example demonstrate this invention further more concretely, this invention is not limited to these examples.

Example  1: L- tert - Leucineamide  Screening for Soil Microbial Strains that Specificly Hydrolyze

In order to screen microorganisms having enzymatic activity that enantio-selectively hydrolyzes amide bonds of L-tert-leucineamide, samples were taken from soils in various forests and coastal areas of Korea. After diluting the collected soil sample with 50mM potassium phosphate buffer (pH7.4) solution, spread it on tryptic soybean agar (TSA; Tryptic Soybean agar) containing 1.5% agar powder in the composition as shown in Table 1. Incubated for 1-7 days at harvest.

The collected microorganisms were cultured for 7 days at 25 ° C. and 200 rpm in a yeast carbon base (BD, NJ USA) supplied with 1% racemic tert-leucineamide as a nitrogen source. I identified it. After culturing the identified microorganisms in the medium of the composition of Table 1 and recovered by centrifugation at 4 ℃, 8000rpm for 20 minutes using a JA-20 rotor in a high-speed centrifuge (Beckman, California USA, www.beckman, com) And frozen at -20 ° C.

The recovered microorganisms were reacted at 40 ° C. and 200 rpm for 7 days after adding 0.1 g of a wet cell to a reaction solution of 900 ul of 50 mM potassium phosphate buffer of pH 7.4 containing 10 mg of racemic tert-leucineamide. After the reaction, cells were removed using a 0.2um syringe filter, and 30 kinds of microorganisms generating tert-leucine were selected through HPLC condition A of Table 2 below. The reaction solution of the selected microorganism was selected from two microorganisms that selectively hydrolyze L-type tert-leucineamide through the following HPLC conditions.

Table 1 Medium Composition

pH 7.3 Casein Pancreatic Digest of Casein 1.7% Enzymatic digest of Soybean Meal 0.3% Dextrose 0.25% Sodium chloride 0.5% Dipotassium Phosphate 0.25%

TABLE 2 HPLC Conditions A

column Inercil ODS-3 (150mm X 4.6mm, GL science) Column temperature 30 ℃ Mobile phase 70 v / v% 10 mM potassium phosphate buffer (pH 7.4) + 30 v / v% methanol Flow rate 0.5 mL / min wavelength 208 nm Detector UV-1575 (JASCO co.)

TABLE 3 HPLC Conditions B

column Sumichiral OA-5000 (Simika Chemical Analysis Service, Ltd.) Column temperature 30 ℃ Mobile phase 85 v / v% 2mM aqueous solution of copper (II) + 15 v / v% methanol Flow rate 1 mL / min wavelength 254nm Detector UV-1575 (JASCO co.)

As a result, the selected microbial strain was estimated to belong to the classification group using about 500bp of partial base sequence of 16S rDNA (16S rRNA gene), and the partial nucleotide sequence was consistent with Bacillus cereus with 99% homology. And biochemical patterns using the API kit 50CHB was confirmed that the most similar to Bacillus cereus. The selected strain was identified as Bacillus cereus and named E306 strain. This was deposited with the Korea Culture Center of Microorganisms (KCCM) (Accession No. KCCM 11020P). The bacteriological properties of the identified Bacillus cereus E306 strain were shown in Table 4 below.

[Table 4] Mycological Properties

Gram Dye + Bacillus Chain arrangement Possession of flagella (with movement) Aerobic Heat-resistant apoptosis Colony form (TSA; tryptic soy agar, 37 degrees, 24 hours) circle Light brown No gloss Rough surface Growth temperature 5-50 degrees Optimum temperature 30-37 degrees Growth pH 4.9 ~ 9.3

Example  2: L type Enantio -Optional Amidase  Coded DNA  Preparation of Fragments

Tert-leucineamide is identified as an enantio-selective singer by searching the GenBank accession number (AE016877.1) of B.cereus at the National Center for Biological Information (www.ncbi.nlm.nih.gov). A gene predicted to encode an enzyme to degrade (BC_2537) was selected. Bacillus cereus E306 strain identified in the preparation example was inoculated in 3 ml of Tryptic Soybean Broth (TSB, Soy Casein Digestive Medium) medium of Table 1 above at 37 ° C, 200 rpm, pH7 Cells were harvested by incubating overnight under conditions and centrifugation. The harvested microorganisms were separated by genomic purification kit (Genome purification kit; Promega, WI USA, www.promega.com) and used as a template for PCR.

Based on the nucleotide sequence of BC_2537 gene of B. cereus, previously selected oligonucleotides of SEQ ID NOs: 1 and 2 were designed as primers for PCR. The primer was prepared by a request from Bionics (Korea, Seoul, www.bionicsro.co.kr).

SEQ ID NO: 5 'CAT ATG TAT CGA ATT CAT AAA GAC C 3'

SEQ ID NO: 5 'CGA GCT CTA TTT AAT GTG AAA TTT AC 3'

The manipulations required for the acquisition of amidase genes, production of recombinant plasmids and transgenic microorganisms are described in Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ] According to the method described.

To a microorganism genomic DNA isolated from the above as a template (0.1μg), primers of dNTP, 20 pmol of 200μM (SEQ ID NO: 1 and SEQ ID NO: 2), 1 × Taq DNA polymerase buffer solution (2 mM Mg ^{+ 2)} and 2 The reaction was repeated 30 times at 94 ° C. for 30 seconds, at 55 ° C. for 30 seconds, and at 72 ° C. for 30 seconds in a 50 μl reaction solution in which Taq DNA polymerase of the unit was present. The resulting amplified 924 bp base pairs (SEQ ID NO: 5) amidase gene fragments were identified by agarose gel electrophoresis, power gel extraction kit (Dynebio, Korea, Seongnam, www.dynebio.co.kr) was used to recover the DNA from the gel.

Example  3: LEAA  Preparation of Recombinant Vectors for Protein Expression

PGEM-T easy vector (Promega, Wisconsin, www.promega.com) was used as an intermediate vector for cloning the amidase DNA fragment prepared in Example 1.

The binding of the prepared DNA fragment of Example 1 to the pGEM-T easy vector is described in Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. It was carried out according to the ligase reaction method described in. The two DNA fragments (20-25ng) contain 3 units of T4 DNA ligase (Promega) and 1X ligase buffer (66 mM Tris-HCl, 6.6 mM MgCl ₂ , 10 mM DTT, 0.1 mM ATP). The reaction solution was added to 10 µl and reacted at 20 ° C for 18 hours. The reaction solution according to the E. coli transformation method using CaCl ₂ specified in [Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001]. After transforming DH5α Escherichia coli cells (TAKARA, Shiga Japan, www.takara-bio.com), LB agar plate medium containing ampicillin (tryptone 1%, yeast extract 0.5%, sodium chloride 1%, agar 0.8%, ampicillin) 25 μg / ml) and then colonies with antibiotic resistance after 14-18 hours incubation at 37 ℃ conditions were selected. From this, the recombinant plasmid was isolated and identified, and the recombinant microorganism thus produced was named pLEAA-T / DH5α.

Subsequently, in order to insert the amidase gene into the pFRPT vector (Korean Patent No. 0449639, Accession No. KCCM-10320), which is an expression vector for Escherichia coli, the pFRPT vector was digested with restriction enzymes NdeI and SalI and the cleaved plasmid Analysis by agarose gel electrophoresis, 6.4kbp DNA fragments were recovered from the gel. On the other hand, plasmid pLEAA-T was cleaved using SalI and then partially cleaved with NdeI (the 100bp lower gene NdeI site was present from the initiation codon) and analyzed by agarose gel electrophoresis, and then 955 bp amidase DNA fragment was removed from the gel. Recovered. The two DNA fragments obtained through the above method were prepared according to the method described in Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. The cross-linking reaction was carried out using an aze.

Example  4: LEAA  Preparation of Transgenic Strains for Protein Expression

E. coli transformation method using CaCl ₂ using the cross-linking reaction solution of the DNA fragment prepared in Example 3 (Molecular cloning: A Laboratory Manual, 3rd Edition, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold JM109 Escherichia coli cells (TAKARA, shiga Japan, www.takara-bio.com) were transformed according to Spring Harbor, New York, 2001, followed by LB agar plate medium containing kanamycin (tryptone 1%, yeast extract 0.5%). , 1% sodium chloride, 0.8% agar, containing 25 μg / ml of kanamycin), and colonies resistant to antibiotics were selected after 14-18 hours of incubation at 37 ° C. Single colony was inoculated in 3 ml of LB medium (containing tryptone 1%, yeast extract 0.5%, sodium chloride 1%, kanamycin 25 μg / ml) and cultured overnight under shaking at 37 ° C. A Laboratory Manual, 3rd Edition, Vol. 1, 1-32, Sambrook J., Russell DW, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001], followed by cleavage of the plasmid with restriction enzymes NdeI and SalI. The fragment of the plasmid pFRPT and the amidase DNA fragment were confirmed by agarose electrophoresis. The recombinant plasmid for enzyme expression obtained here was named pFRPT-LEAA (FIG. 1).

To identify the nucleotide sequence of the cloned pFRPT-LEAA, based on the nucleotide sequence upstream and downstream of the cloning position of pFRPT, oligonucleotide of SEQ ID NO: 3,4 was entrusted to Bionics (Korea, Seoul, www.bionicsro. com). It was confirmed that the gene having the nucleotide sequence of SEQ ID NO: 5 was cloned by requesting nucleotide sequence translation using the oligonucleotide as the template of pFRPT-LEAA as a template.

SEQ ID NO: 5 'GAG CGG ATA ACA ATT CCC ACC 3'

SEQ ID NO: 5 'ACT CAT CCG CCA AAA CAG CCA 3'

The transformed strain thus prepared was named pFRPT-LEAA / JM109. This was deposited with the Korea Culture Center of Microorganisms (KCCM) (Accession No. KCCM 11021P).

On the other hand, analysis of the genetic information of SEQ ID NO: 5 showed 96% (887bp / 917bp) gene homology with BC_2537, and as a result of amino acid sequence translation it was confirmed that it is a novel gene showing 96% (293/304) amino acid sequence homology.

Example  5: L type tert - Leucine  Produce

(One) LEAA  Culture of Protein Expression Transgenic Strains

To prepare a medium of the composition of Table 5, put 200mL of this medium in a 1L Erlenmeyer flask, sterilize and add so that the concentration of kanamycin is 25ug / mL and the transformed strain pFRPT-LEAA / JM109 prepared in Example 4 Was inoculated, followed by shaking culture at 37 ° C for 4 hours, and 200 ug / mL of IPTG (isopropyl-β-D-1-thiogalactopyranoside) was added thereto, followed by shaking culture for 10 hours.

[Table 5] Medium composition

pH 7.0 Trypton 1% Yeast Extract 0.5% Sodium chloride 0.5% Glycerol 0.5%

The cells were recovered from the culture by centrifugation at 4 ℃ and 8000rpm for 20 minutes using JA-14 rotor in a high speed centrifuge (Beckman, USA, California). Was stored frozen at 20 ℃.

(2) tert Leucine and tert - Leucineamide  Quantitative Analysis

Quantitative analysis of tert-leucine and tert-leucineamide was performed by High Performance Liquid Chromatography (HPLC) under the conditions of Table 2 above.

tert-leucine was detected between 4.5 and 4.7 minutes and tert-leucineamide was detected between 8.2 and 8.4 minutes. The area of the detected peak was quantified by concentration calculation using any detectable standard.

(3) tert - Leucine Chiral analysis

Chiral analysis of tert-leucine was performed by high performance liquid chromatography under the conditions of Table 3 above.

L-tert-leucine was detected between 13-14 minutes, and D-tert-leucine was detected between 22-24 minutes (Figure 2A).

(4) Confirmation of Enzyme Activity of Transgenic Strains

Into a 500 ml Erlenmeyer flask, 75 ml of the reaction solution dissolved in a distilled water at a temperature of 50 ° C. so that the concentration of tert-leucineamide racemic mixture was 150 g / L (the initial mixture had a pH of 7.8 and no other pH adjustment) was added to (1) The transgenic strain pFRPT-LEAA / JM109 cultured at was further added at a concentration of 10 g / L to perform a hydrolysis reaction at 50 ° C. for 2 hours.

After the reaction, the reaction solution was centrifuged at 15 ° C., 8000 rpm for 20 minutes using a J-14 rotor in an ultrafast centrifuge (Beckman) to obtain a cell removal solution. The cell removal solution was quantitatively analyzed by HPLC conditions A and quantification method of Table 2 described in Example 4, and it was confirmed that 47% of the tert-leucineamide racemic mixture was converted to tert-leucine. In addition, chiral analysis of the HPLC conditions B and the chiral analysis method of Table 3 described in Example 4 showed that the optical purity of L-tert-leucine was 100% e.e. (FIG. 2B).

(5) L- tert - Leucine  refine

The cell removal solution prepared above was removed by distillation of water using a vacuum concentrator (Eyela, Japan, Tokyo). After adding 5 times the volume of isopropyl alcohol with respect to the weight of the reactor, stirring at elevated temperature to 50 ° C, 0.05% of 25% ammonia water was slowly added dropwise and stirred for 1 hour, followed by cooling at 15 ° C for 2 hours to precipitate the crystals. The Buchner funnel was filtered under reduced pressure using a filter paper and dried to obtain 4.3 g of L-tert-leucine. The recovered L-tert-leucine showed 99% purity and the optical purity was 100% e.e.

Experimental Example  One: Bacillus Cereus (B. cereus ) E306 of tert - Leucineamide Racemic  Of mixture Enantio Selective hydrolysis confirmation

A medium having the composition shown in Table 1 was prepared, 200 mL of this medium was placed in a 1 L Erlenmeyer flask, sterilized, inoculated with B. cereus E306 selected in Example 1, and shaken at 30 ° C. for 48 hours. Incubated. After incubation, the wet cells were recovered by centrifugation at 4 ° C., 8000 rpm for 20 minutes using a JA-14 rotor in a high speed centrifuge (Beckman) from the culture.

10 ml of the tert-leucineamide racemic mixture was stirred and dissolved at 50 ° C. in a distilled water at a concentration of 10 g / L (initial mixed solution had a pH of 7.8 and no other pH control) in a 50 mL flask, followed by Bacillus cereus E306 wet. Cells were further added at a concentration of 50 g / L and stirred at 50 ° C. for 24 hours to carry out the enzyme reaction.

After the reaction, the reaction solution was removed by using a 0.2um syringe filter, the cell removal solution was analyzed by HPLC condition A of Table 2. As a result, it was confirmed that about 25% of the tert-leucineamide racemic mixture was converted to tert-leucine. In addition, analysis by HPLC Condition B of Table 3 showed that the optical purity of L-tert-leucine was 100% e.e.

Experimental Example  2: pH  Confirmation of Transgenic Strain Activity According to Changes

The concentration of the tert-leucineamide racemic mixture and the transforming strain pFRPT-LEAA / JM109 cultured in Example 5 (1) was dissolved in distilled water to 10 g / L and 1 g / L, respectively, and then hydrochloric acid and sodium hydroxide solution were used. After adjusting the pH to 4-9 to make a total volume of 10ml into a 50ml Erlenmeyer flask and stirred at 40 ° C and 200rpm for 30 minutes and 1 hour, the reaction solution was removed from the cells using a 0.2um syringe filter. The solution was analyzed by HPLC condition A of Table 2. As a result, as shown in Figure 4, the transformed strain of the present invention was confirmed to exhibit a stable enzyme activity at pH 7-9. The enzyme activity unit was defined as the produced tert-leucine (umol) / reaction time (min). In addition, the reaction solution was analyzed by HPLC Condition B in Table 3, and the optical activity of L-tert-leucine was 100% e.e. in all pH ranges 4-9.

Experimental Example  3: Confirmation of Activity of Transgenic Strains by Temperature Change

10 g / L and 1 g / L of tert-leucineamide racemic mixture and the transformed strain pFRPT-LEAA / JM109 cultured in Example 5 (1) were dissolved in distilled water, respectively, and then total volume of 10 ml (initial mixed solution) PH of 7.8 was adjusted to no other pH control) was put into a 50ml Erlenmeyer flask and fixed at different temperatures from 15 ℃ to 60 ℃ in 200rpm shaking reactor to start the stirring reaction. After 30 minutes and 1 hour after the start of the reaction, the samples were taken, cells were removed using a 0.2um syringe filter, and analyzed by HPLC condition A of Table 2. As a result, as shown in Figure 5 transgenic strain of the present invention showed a high enzyme activity as the reaction temperature increases up to 60 ℃. In addition, the optical purity of the resulting tert-leucine was 100% e.e. in the reaction at all temperatures.

Therefore, by using the L-type enantio-selective amidase of the present invention, it is possible to increase the solubility of the reactants during the high temperature reaction, thereby enabling a high concentration reaction, and the reaction rate is increased at high temperature, thereby increasing the productivity of L-tert-leucine. It is advantageous in terms of cost.

Experimental Example  4: Substrate concentration  See how your conversion rate changes with changes

The concentration of the tert-leucineamide racemic mixture was changed to 100, 200, 300 and 400 g / L, respectively, and the concentrations of the transformed strain pFRPT-LEAA / JM109 cultured in Example 5 (1) were respectively 5, 10, 15, 20g / L (0.05 times the amount of substrate) was dissolved in distilled water and then adjusted to a total volume of 10ml into a 50ml Erlenmeyer flask, pH 7.8 (the pH of the initial mixed solution was 7.8), 50 ℃, 200rpm stirring reaction. As the enzyme reaction proceeds, the pH rises as ammonia is produced. Thus, pH 7.8 to pH 8 was maintained using acetic acid. As a result of analyzing the reaction solution from which the bacteria were removed by using a 0.2um syringe filter by the reaction time according to the reaction time, as shown in FIG. 5, even in a high concentration substrate reaction of 400 g / L (about 3.08M) within 5 hours The conversion rate was 50%, which is consistent with the value. In addition, the results were analyzed by HPLC condition B of Table 3, the optical range was 100% e.e.

As described above, in the Examples and Experimental Examples, the LEAA protein of the present invention has a significantly higher activity than the conventional L-type enantio-selective amidase, and even at a high concentration of substrate of about 3.07 M (400 g / L) Very fast response rate, 100% ee The high purity optical specificity of was shown, and the conversion rate of 50% of the theoretical racemic tert-leucineamide was shown for the high concentration of racemic tert-leucineamide (400 g / L, about 3 M concentration). In addition, it exhibits high temperature stability in which the reaction proceeds rapidly even at a high temperature of 60 ° C., and has even activity at neutral to weak alkali pH. Since the LEAA protein of the present invention shows excellent stability against external reaction conditions, it can be seen that it can be advantageously used for the preparation of enantio-selective amino acids.

1 is a diagram showing the structure of a recombinant plasmid of the present invention.

Figure 2 is an HPLC comparison graph of L-tert-leucine after L-entio-selective hydrolysis of racemic tert-leucine amide using tert-leucine racemic mixture and the amidase of the present invention.

Figure 3 is a diagram showing the change in activity according to the pH of the recombinant enzyme strain of the present invention.

Figure 4 is a diagram showing the change in activity according to the temperature of the recombinant enzyme strain of the present invention.

5 is a diagram showing the substrate conversion rate according to the substrate concentration of the recombinant cells of the present invention.

<110> Samchully Pharm. Co., Ltd. <120> Enantioselective amidase specific to L-amino acid amide and DNA          encoding the same <130> P09-073-SAM <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 catatgtatc gaattcataa agacc 25 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cgagctctat ttaatgtgaa atttac 26 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gagcggataa caattcccac c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 actcatccgc caaaacagcc a 21 <210> 5 <211> 915 <212> DNA <213> Bacillus cereus <220> <221> gene (222) (1) .. (915) <223> DNA of L-specific enantioselective amidase <400> 5 atgtatcgaa ttcataaaga ccatatcatt tactcaatgt caccagaaaa taagccttgt 60 atggaagtgg aaattgggag cagtcttgta tttgaaacat atgattgctt tgaaaatcaa 120 atcgattctg aagatgttgt gtttcaagaa ttagattgga accgaattaa tccagcgact 180 ggacctgtat atgttaaagg agcagaacct ggcgatatat tagccgtaac gattgaaaag 240 attaaaattg cagagcaggg cgttttaact acaggtgcaa acctcggtgt aatagggaat 300 gagttaaatg aaaatacagt gaaaattgtc ccaatacata atgaacatgt tttgttttca 360 agtgaactac aaatcccgat taatccaatg attggggtaa ttggtattgc accgaaagaa 420 gagagcattt catgtggcac accgcatgat cacggcggga atatggactg taaagaaatt 480 aaagaaggaa caacattact actaccagtt aatgtcccag gagcactatt agcgttaggt 540 gatttacacg cagcaatggg ggatggtgaa attggggtta gtggagtaga ggttgctggt 600 gaggtaactg taacagttca aattataaaa gggaaaaaat ggccactacc aatggctatt 660 caaaaagaaa aagtaatgac gattgcttca gaaaaattgc tagatgatgc agcaaatcgt 720 gctgtacgta atatggtgac atttttacat gaagaattag caatgtctaa agctgatgca 780 acgcttttat tatcggcagc aggtaattta aaagtttgcc aagttgtgga tccgttgaaa 840 acggcgcgta tggaattagg gatggattat gtggagaaac taggatttac ttttagtaaa 900 tttcacatta aatag 915 <210> 6 <211> 304 <212> PRT <213> Bacillus cereus <220> <221> CHAIN (222) (1) .. (304) <223> polypeptide of L-specific enantioselective amidase <400> 6 Met Tyr Arg Ile His Lys Asp His Ile Ile Tyr Ser Met Ser Pro Glu   1 5 10 15 Asn Lys Pro Cys Met Glu Val Glu Ile Gly Ser Ser Leu Val Phe Glu              20 25 30 Thr Tyr Asp Cys Phe Glu Asn Gln Ile Asp Ser Glu Asp Val Val Phe          35 40 45 Gln Glu Leu Asp Trp Asn Arg Ile Asn Pro Ala Thr Gly Pro Val Tyr      50 55 60 Val Lys Gly Ala Glu Pro Gly Asp Ile Leu Ala Val Thr Ile Glu Lys  65 70 75 80 Ile Lys Ile Ala Glu Gln Gly Val Leu Thr Thr Gly Ala Asn Leu Gly                  85 90 95 Val Ile Gly Asn Glu Leu Asn Glu Asn Thr Val Lys Ile Val Pro Ile             100 105 110 His Asn Glu His Val Leu Phe Ser Ser Glu Leu Gln Ile Pro Ile Asn         115 120 125 Pro Met Ile Gly Val Ile Gly Ile Ala Pro Lys Glu Glu Ser Ile Ser     130 135 140 Cys Gly Thr Pro His Asp His Gly Gly Asn Met Asp Cys Lys Glu Ile 145 150 155 160 Lys Glu Gly Thr Thr Leu Leu Leu Pro Val Asn Val Pro Gly Ala Leu                 165 170 175 Leu Ala Leu Gly Asp Leu His Ala Ala Met Gly Asp Gly Glu Ile Gly             180 185 190 Val Ser Gly Val Glu Val Ala Gly Glu Val Thr Val Thr Val Gln Ile         195 200 205 Ile Lys Gly Lys Lys Trp Pro Leu Pro Met Ala Ile Gln Lys Glu Lys     210 215 220 Val Met Thr Ile Ala Ser Glu Lys Leu Leu Asp Asp Ala Ala Asn Arg 225 230 235 240 Ala Val Arg Asn Met Val Thr Phe Leu His Glu Glu Leu Ala Met Ser                 245 250 255 Lys Ala Asp Ala Thr Leu Leu Leu Ser Ala Ala Gly Asn Leu Lys Val             260 265 270 Cys Gln Val Val Asp Pro Leu Lys Thr Ala Arg Met Glu Leu Gly Met         275 280 285 Asp Tyr Val Glu Lys Leu Gly Phe Thr Phe Ser Lys Phe His Ile Lys     290 295 300  

Claims (11)

L-type amino acid amide comprising (1) an amino acid sequence represented by SEQ ID NO: 6 or (2) an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 6 A protein having enantio-selective amidase enzymatic activity that enantio-selectively hydrolyzes an amide bond. The protein of claim 1, wherein the protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 6 has at least 95% homology with SEQ ID NO. 6. DNA comprising a nucleotide sequence encoding the protein of claim 1. Recombinant vector in which the DNA of claim 3 is introduced. The recombinant vector of claim 4, wherein the expression vector is pFRPT of Accession No. KCCM-10320. A recombinant microorganism transformed with the recombinant vector of claim 4. The recombinant microorganism according to claim 6, which is Accession No. KCCM11021P. A method for producing an L-type amino acid by converting an L-type amino acid amide into an L-type amino acid by acting a mixture of the protein of claim 1 and the racemic amino acid amide. The method for producing an L-type amino acid according to claim 8, wherein the L-type amino acid amide is represented by the following Formula (I): Formula (I)

(Wherein R ₁ is hydrogen; alkyl group having 1 to 4 carbon atoms; hydroxy group, methoxy group, mercapto group, methyl mercapto group, amino group, guanyl group, carboxyl group, carboxamide group, halogen group, phenyl group, hydroxyphenyl group, Or an alkyl group having 1 to 4 carbon atoms substituted with one or more groups selected from imidazolyl groups; one or more selected from hydroxy, methoxy, mercapto, methylmercapto, amino, guanyl, carboxyl, carboxamide or halogen groups A phenyl group substituted with a group; a heterocyclic group of an indole group or an imidazolyl group; or substituted with one or more groups selected from hydroxy group, methoxy group, mercapto group, methyl mercapto group, amino group, guanyl group, carboxyl group, carboxamide group or halogen group Indole group or imidazolyl group). 9. The method of claim 8 wherein the racemic amino acid amide is racemic tert-leucine amide and the L-type amino acid is L-tert-leucine. Bacillus cereus E306 which has the expression ability of the protein containing the amino acid sequence shown by sequence number 6 of Claim 1, and has accession number KCCM 11020P.

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