METHOD FOR ENZYMATIC PREPARATION OF R-3 AMINOBUTYRIC ACID

Abstract

An R-3-aminobutyric acid preparation method with high efficiency and high stereoselectivity. The method comprises using aspartase with stereoisomerization catalytic activity derived from Escherichia coli to efficiently convert butenoic acid into R-3-aminobutyric acid. After only 24 h of reaction, the conversion rate is as high as ≥98%, and the ee value is ≥99.9%. The conversion efficiency is greatly improved, the reaction time is shortened, and the production costs are reduced. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like.

Claims

1. An R-3-aminobutyric acid production strain expressing a polypeptide, wherein the polypeptide is an exogenous aspartase derived from E. coli and is used to catalyze the stereoisomeric catalytic reaction as below: ##STR00009## wherein the exogenous aspartase is a mutant having 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, which has two to four amino acid mutations in the amino acid sequence corresponding to the wild type aspartase shown in SEQ ID NO: 5; or wherein the exogenous aspartase is a mutant having an amino acid mutation selected from the group consisting of threonine (T) at position 204, methionine (M) at position 338, Lysine (K) at position 341, asparagine (N) at position 343, or a combination thereof.

2. The R-3-aminobutyric acid production strain of claim 1, wherein the mutant has one or more mutations selected from the group consisting of T204C, M338I, K341M, N343C, or a combination thereof.

3. The R-3-aminobutyric acid production strain of claim 1, wherein the exogenous aspartase has the amino acid sequence shown in SEQ ID NO: 3.

4. An aspartase having stereoisomeric catalytic activity, wherein the amino acid sequence of the aspartase is shown in SEQ ID NO: 3.

5. A polynucleotide encoding a polypeptide, wherein the polypeptide is an exogenous aspartase derived from E. coli and is used to catalyze the stereoisomeric catalytic reaction as below: ##STR00010## wherein the exogenous aspartase is a mutant having 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, which has two to four amino acid mutations in the amino acid sequence corresponding to the wild type aspartase shown in SEQ ID NO: 5; or wherein the exogenous aspartase is a mutant having an amino acid mutation selected from the group consisting of threonine (T) at position 204, methionine (M) at position 338, Lysine (K) at position 341, asparagine (N) at position 343, or a combination thereof.

6. The polynucleotide of claim 5, wherein the exogenous aspartase has the amino acid sequence shown in SEQ ID NO: 3.

7. The polynucleotide of claim 5, wherein the polynucleotide has a sequence as shown in SEQ ID NO: 4.

8. The polynucleotide of claim 5, wherein the polynucleotide has over 95% homologous to the sequence of SEQ ID NO: 4, and encodes a polypeptide as shown in SEQ ID NO: 3.

Description

EXAMPLES

Example 1. Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Wild-Type and the Detection Thereof

[0101] 1.1 Preparation of AspA Wild Type Enzyme Solution

[0102] Based on the amino acid sequence of AspA wild-type (SEQ ID NO: 5), a DNA sequence (SEQ ID NO: 6) encoding the AspA wild-type enzyme was synthesized and linked to pET28a by enzymes, wherein the restriction enzyme cutting sites were NdeI and HindIII. The linked vector was transformed into the host E. coli BL21 competent cells. The strain was inoculated in the TB medium, and cultured in a shaker at 37° C., 200 rpm until the OD600 value reached 4.0. Then IPTG having a concentration of 0.1 mM was added for induction, the temperature was adjusted to 28° C. and the culture was continued for 12 hours. The bacterial cells were collected by centrifugation at 4° C., and resuspended with phosphate buffer (50 mM, pH 7.0). The cells were homogenized and disrupted to obtain AspA wild-type enzyme solution.

[0103] Amino acid sequence of AspA wild type:

TABLE-US-00002 (SEQ ID NO: 5) CLKQIIGSLKKKVHMSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYI SNNKISDIPEFVRGMVMVKKAAAMANKELQTIPKSVANAIIAACDEVLNN GKCMDQFPVDVYQGGAGTSVNMNTNEVLANIGLELMGHQKGEYQYLNPND HVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILK MGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGATAIG TGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKR LAVKMSKICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNVVPEVVN QVCFKVIGNDTTVTMAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNLL EKCINGITANKEVCEGYVYNSIGIVTYLNPFIGHHNGDIVGKICAETGKS VREVVLERGLLTEAELDDIFSVQNLMHPAYKAKRYTDESEQ

[0104] Nucleic acid coding sequence of AspA wild type:

TABLE-US-00003 (SEQ ID NO: 6) TGCCTGAAACAGATCATCGGTTCTCTGAAAAAAAAAGTTCACATGTCTAA CAACATCCGTATCGAAGAAGACCTGCTGGGTACCCGTGAAGTTCCGGCTG ACGCTTACTACGGTGTTCACACCCTGCGTGCTATCGAAAACTTCTACATC TCTAACAACAAAATCTCTGACATCCCGGAATTCGTTCGTGGTATGGTTAT GGTTAAAAAAGCTGCTGCTATGGCTAACAAAGAACTGCAGACCATCCCGA AATCTGTTGCTAACGCTATCATCGCTGCTTGCGACGAAGTTCTGAACAAC GGTAAATGCATGGACCAGTTCCCGGTTGACGTTTACCAGGGTGGTGCTGG TACCTCTGTTAACATGAACACCAACGAAGTTCTGGCTAACATCGGTCTGG AACTGATGGGTCACCAGAAAGGTGAATACCAGTACCTGAACCCGAACGAC CACGTTAACAAATGCCAGTCTACCAACGACGCTTACCCGACCGGTTTCCG TATCGCTGTTTACTCTTCTCTGATCAAACTGGTTGACGCTATCAACCAGC TGCGTGAAGGTTTCGAACGTAAAGCTGTTGAATTCCAGGACATCCTGAAA ATGGGTCGTACCCAGCTGCAGGACGCTGTTCCGATGACCCTGGGTCAGGA ATTCCGTGCTTTCTCTATCCTGCTGAAAGAAGAAGTTAAAAACATCCAGC GTACCGCTGAACTGCTGCTGGAAGTTAACCTGGGTGCTACCGCTATCGGT ACCGGTCTGAACACCCCGAAAGAATACTCTCCGCTGGCTGTTAAAAAACT GGCTGAAGTTACCGGTTTCCCGTGCGTTCCGGCTGAAGACCTGATCGAAG CTACCTCTGACTGCGGTGCTTACGTTATGGTTCACGGTGCTCTGAAACGT CTGGCTGTTAAAATGTCTAAAATCTGCAACGACCTGCGTCTGCTGTCTTC TGGTCCGCGTGCTGGTCTGAACGAAATCAACCTGCCGGAACTGCAGGCTG GTTCTTCTATCATGCCGGCTAAAGTTAACCCGGTTGTTCCGGAAGTTGTT AACCAGGTTTGCTTCAAAGTTATCGGTAACGACACCACCGTTACCATGGC TGCTGAAGCTGGTCAGCTGCAGCTGAACGTTATGGAACCGGTTATCGGTC AGGCTATGTTCGAATCTGTTCACATCCTGACCAACGCTTGCTACAACCTG CTGGAAAAATGCATCAACGGTATCACCGCTAACAAAGAAGTTTGCGAAGG TTACGTTTACAACTCTATCGGTATCGTTACCTACCTGAACCCGTTCATCG GTCACCACAACGGTGACATCGTTGGTAAAATCTGCGCTGAAACCGGTAAA TCTGTTCGTGAAGTTGTTCTGGAACGTGGTCTGCTGACCGAAGCTGAACT GGACGACATCTTCTCTGTTCAGAACCTGATGCACCCGGCTTACAAAGCTA AACGTTACACCGACGAATCTGAACAG

[0105] 1.2 Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Wild-Type

[0106] The synthesis reacts in a 100 ml reaction system at 37° C. 100 mM HEPES buffer of pH 8.0 was added. 2 mM MgCl.sub.2, 300 mM butenoic acid, 300 mM NH.sub.4Cl and 20 ml AspA wild-type enzyme solution were added, wherein the above concentrations were final concentrations.

[0107] The progress of the reaction was detected by HPLC. The reaction was completed at 24 h, and the conversion rate was <5%.

[0108] Calculation of conversion rate: the conversion rate is also referred to material conversion rate, which is numerically equal to the ratio of the butenoic acid consumed in the fermentation process to the total amount of butenoic acid at the beginning of the fermentation. It is usually expressed as a percentage and can be a molar ratio (mol %), can also be a weight ratio (wt %).

Example 2. Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Mutant 1 and the Detection Thereof

[0109] 2.1 Preparation of AspA Mutant 1 Enzyme Solution

[0110] All of the amino acids at the 4 mutation sites of AspA mutant 1 were mutated (see Tables 1 and 2). Based on the amino acid sequence of AspA mutant 1 (SEQ ID NO: 3), a DNA sequence (SEQ ID NO: 4) encoding the AspA mutant 1 enzyme was synthesized and linked to pET28a by enzymes, wherein the restriction enzyme cutting sites were NdeI and HindIII. The linked vector was transformed into the host E. coli BL21 competent cells. The strain was inoculated in the TB medium, and cultured in a shaker at 37° C., 200 rpm until the OD600 value reached 4.0. Then IPTG having a concentration of 0.1 mM was added for induction, the temperature was adjusted to 28° C. and the culture was continued for 12 hours. The bacterial cells were collected by centrifugation at 4° C., and resuspended with phosphate buffer (50 mM, pH 7.0). The cells were homogenized and disrupted to obtain AspA mutant 1 enzyme solution.

[0111] Amino Acid Sequence of AspA Mutant 1:

TABLE-US-00004 (SEQ ID NO: 3) CLKQIIGSLKKKVHMSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYI SNNKISDIPEFVRGMVMVKKAAAMANKELQTIPKSVANAIIAACDEVLNN GKCMDQFPVDVYQGGAGTSVNMNTNEVLANIGLELMGHQKGEYQYLNPND HVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILK MGRCQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGATAIG TGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKR LAVKMSKICNDLRLLSSGPRAGLNEINLPELQAGSSIIPAMVCPVVPEVV NQVCFKVIGNDTTVTMAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNL LEKCINGITANKEVCEGYVYNSIGIVTYLNPFIGHHNGDIVGKICAETGK SVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKRYTDESEQ

[0112] Nucleic Acid Coding Sequence of AspA Mutant 1:

TABLE-US-00005 (SEQ ID NO: 4) TGCCTGAAACAAATCATTGGTAGCCTGAAGAAAAAAGTGCACATGAGCAA TAACATTCGCATCGAAGAGGATCTGCTGGGTACACGTGAAGTGCCGGCAG ATGCCTACTACGGTGTGCATACACTGCGCGCCATCGAAAATTTTTACATC AGCAATAATAAAATCAGCGATATCCCGGAATTCGTGCGCGGCATGGTTAT GGTGAAAAAAGCCGCCGCAATGGCCAACAAGGAACTGCAGACCATTCCGA AGAGTGTGGCAAACGCCATTATCGCCGCCTGTGATGAAGTGCTGAACAAT GGTAAATGCATGGATCAGTTTCCGGTGGACGTGTATCAAGGCGGCGCCGG TACCAGCGTGAACATGAACACCAATGAGGTGCTGGCCAACATTGGTCTGG AGCTGATGGGTCACCAGAAAGGCGAATACCAGTACCTGAACCCGAACGAT CACGTGAACAAGTGTCAGAGCACAAATGACGCATACCCGACAGGCTTTCG TATTGCCGTGTACAGTAGCCTGATCAAGCTGGTGGATGCCATCAATCAGC TGCGTGAAGGCTTCGAGCGTAAGGCCGTTGAATTTCAGGACATCCTGAAA ATGGGTCGTTGTCAGCTGCAGGATGCAGTGCCGATGACCCTGGGTCAGGA ATTTCGCGCATTCAGCATCCTGTTAAAAGAGGAAGTGAAAAACATCCAGC GTACCGCCGAACTGCTGCTGGAAGTTAACCTGGGTGCCACCGCCATCGGC ACAGGCCTGAATACCCCGAAAGAGTATAGCCCGCTGGCCGTTAAAAAACT GGCAGAGGTGACCGGTTTCCCGTGTGTGCCGGCAGAGGATCTGATCGAAG CAACCAGCGATTGCGGTGCTTATGTTATGGTGCATGGTGCCCTGAAACGC CTGGCCGTTAAGATGAGTAAAATCTGTAATGACCTGCGTCTGCTGAGCAG CGGTCCTCGTGCAGGCCTGAACGAGATCAACCTGCCGGAACTGCAGGCCG GCAGTAGCATCATCCCGGCCATGGTTTGCCCTGTGGTGCCGGAGGTGGTG AATCAGGTGTGCTTCAAGGTGATCGGCAATGACACCACCGTGACAATGGC CGCAGAGGCAGGCCAGCTGCAACTGAACGTGATGGAGCCGGTGATTGGCC AGGCCATGTTTGAAAGCGTGCACATCTTAACCAACGCCTGCTACAACCTG CTGGAGAAATGCATCAATGGTATTACCGCCAACAAAGAAGTTTGCGAGGG TTACGTGTACAACAGCATTGGCATCGTGACCTATCTGAATCCGTTTATTG GCCATCACAACGGCGACATTGTGGGCAAGATTTGCGCAGAGACCGGCAAA AGTGTTCGCGAAGTGGTTCTGGAGCGCGGTTTACTGACCGAGGCCGAACT GGATGACATTTTCAGCGTTCAAAATCTGATGCACCCGGCCTACAAAGCCA AACGCTACACAGACGAAAGCGAGCAA

[0113] The measured enzyme activity was 5.1 U/ml. The enzyme activity U of the AspA mutant 1 enzyme is defined as: the amount of enzyme catalyzing the formation of 1 micromole of product R-3-aminobutyric acid from butenoic acid per minute is one enzyme unit, that is, 1 U.

[0114] Determination method is: 16 mL reaction solution (pH8.0) was added to a 100 ml Erlenmeyer flask, wherein the reaction solution contains 300 mmol/L butenoic acid, 4 mmol/L MgCl.sub.2, 450 mmol/L ammonium chloride, 100 mmol/L HEPES buffer. The flask was sealed and the reaction solution and enzyme solution were placed in a 42° C. shaker respectively and incubated for 5-10 minutes. 4 ml of AspA mutant enzyme solution was added to the reaction solution, and immediately placed in a shaker at 42° C., 200 rpm to start the reaction. After 30 min, 1 ml of reaction solution was sampled, and 1 ml of acetonitrile was added to stop the reaction. The protein was removed by centrifugation. The supernatant was derivatized with 2, 4-dinitrofluorobenzene, and analyzed by HPLC (the enzyme activity was calculated based on the peak area).

[0115] 2.2 Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Mutant1

[0116] The synthesis reacts in a 100 ml reaction system at 37° C. 100 mM HEPES buffer of pH 8.0 was added. 2 mM MgCl.sub.2, 300 mM butenoic acid, 300 mM NH.sub.4Cl and 20 ml AspA mutant 1 enzyme solution were added, wherein the above concentrations were final concentrations.

[0117] The progress of the reaction was detected by HPLC. The reaction was completed in 24 h, and the conversion rate was >98%, and the ee value was 99.9%.

Example 3. Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Mutants 2-12 and the Detection Thereof

[0118] 3.1 Preparation of AspA Mutants 2-12 Enzyme Solutions

[0119] The specific mutations of AspA mutants 2-12 are shown in Tables 1 and 2. AspA mutants 2-5 are single amino acid mutation, AspA mutants 6-8 have amino acid mutations at two mutation sites, and AspA mutants 9-12 have amino acid mutations at three mutation sites.

[0120] Based on the amino acid sequences of AspA mutants 2-12, DNA sequences encoding the enzymes of each AspA mutant were synthesized respectively. The preparation method of the enzyme solution was the same as in Example 2.1.

TABLE-US-00006 TABLE 1 Positions and changes of mutant amino acids position Wild type Mutant Mutation site 1 Threonine (T) Cysteine (C) (amino acid at position 204) Mutation site 2 Methionine (M) Isoleucine (I) (amino acid at position 338) Mutation site 3 Lysine (K) Methionine (M) (amino acid at position 341) Mutation site 4 Asparagine (N) Cysteine (C) (amino acid at position 343)

TABLE-US-00007 TABLE 2 Conversion rate of R-3-aminobutyric acid synthesis catalyzed by each mutant enzyme Mutation Mutation Mutation Mutation Conversion site 1 site 2 site 3 site 4 rate Wild type − − − − * Mutant 1 + + + + **** Mutant 2 + − − − * Mutant 3 − + − − * Mutant 4 − − + − * Mutant 5 − − − + * Mutant 6 + − + − ** Mutant 7 − + + − ** Mutant 8 − − + + ** Mutant 9 + + − + ** Mutant 10 + − + + *** Mutant 11 − + + + *** Mutant 12 + + + − *** Note: “+” represents mutation and “−” represents no mutation; “*” represents a conversion rate of <10%, “**” represents a conversion rate of 10%-30%, “***” represents a conversion rate of 30%-70%, and “****” represents a conversion rate of >70%.

[0121] 3.2 Catalytic Synthesis of R-3-Aminobutyric Acid with AspA Mutants 2-12

[0122] The experimental method was the same as in Example 2.2, and the AspA mutants 2-12 enzyme solutions were used to replace the AspA mutant 1 enzyme solution, respectively.

[0123] The results are shown in Table 2. The experimental results show that after 24 hours of reaction, AspA wild type (Example 1), mutant 1 (Example 2) and mutants 2-12 (Example 3) all have a certain stereoselectivity (selectively catalyzed to form R-3-aminobutyric acid), and the reaction time is significantly shortened. In addition, in terms of conversion rate and reaction speed, mutant 1 (four-site mutant) is significantly better than three-site mutants (such as mutants 9-12) and also better than two-site mutants (such as mutants 6-8), single site mutants (such as mutants 2-5) and wild type.

Comparative Example 1. Catalytic Synthesis of R-3-Aminobutyric Acid with AspB Mutant Derived from Bacillus and the Detection Thereof

[0124] 1.1 Preparation of Bacillus AspB Mutant Enzyme Solution

[0125] The AspB mutant enzyme solution was prepared by reference to the method in ChemCatChem, 2014, 6, 965-968. The amino acid sequence of the AspB mutant is shown in SEQ ID NO: 1, and the nucleic acid coding sequence is shown in SEQ ID NO: 2.

[0126] Amino Acid Sequence of AspB Mutant:

TABLE-US-00008 (SEQ ID NO: 1) NTDVRIEKDFLGEKEIPKDAYYGVQTIRATENFPITGYRIHPELIKSLGI VKKSAALANMEVGLLDKEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGT SINMNANEVIANRALELMGEEKGNYSKISPNSHVNMSQSTNDAFPTATHI AVLSLLNQLIETTKYMQQEFMKKADEFAGVIKMGRCHLQDAVPILLGQEF EAYARVIARDIERIANTRNNLYDINMGATAVGTGLNADPEYISIVTEHLA KFSGIIPLRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMAS GPRAGLSEIVLPARQPGSSIIPGMVCPVMPEVMNQVAFQVFGNDLTITSA SEAGQFELNVMEPVLFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKE YVEKSIGIITAINPHVGYETAAKLAREAYLTGESIRELCIKYGVLTEEQL NEILNPYEMIHPGIAGRK

[0127] Nucleic Acid Coding Sentience of AspB Mutant:

TABLE-US-00009 (SEQ ID NO: 2) AACACCGATGTGCGCATTGAGAAGGACTTCCTGGGTGAAAAGGAAATCCC GAAGGATGCCTATTACGGCGTGCAGACCATCCGTGCCACAGAGAACTTTC CTATCACCGGCTACCGCATCCATCCGGAACTGATTAAGAGCCTGGGCATT GTGAAGAAAAGCGCCGCACTGGCAAACATGGAGGTGGGTCTGCTGGATAA GGAAGTGGGTCAGTACATCGTGAAGGCCGCCGACGAAGTTATTGAAGGTA AGTGGAACGATCAGTTTATCGTGGACCCGATTCAGGGCGGCGCAGGTACA AGCATTAATATGAACGCCAACGAAGTGATCGCAAACCGCGCCCTGGAACT GATGGGTGAGGAAAAGGGCAACTATAGCAAGATCAGCCCGAACAGCCACG TTAACATGAGCCAGAGCACCAATGATGCATTTCCGACCGCAACCCATATT GCCGTGCTGAGTCTGCTGAATCAGCTGATCGAGACCACCAAGTACATGCA GCAGGAGTTTATGAAGAAGGCCGACGAATTCGCCGGCGTTATTAAAATGG GCCGCTGCCATCTGCAAGACGCCGTTCCGATTCTGCTGGGTCAGGAGTTT GAGGCTTATGCTCGTGTGATCGCACGTGACATTGAGCGCATCGCCAATAC CCGTAACAACCTGTATGATATCAACATGGGCGCAACCGCCGTTGGCACAG GCCTGAATGCAGACCCGGAGTACATTAGCATCGTTACCGAGCACCTGGCC AAATTTAGCGGTCATCCGCTGCGTAGTGCCCAGCATCTGGTTGATGCCAC CCAGAATACAGATTGCTACACCGAGGTGAGCAGTGCCCTGAAAGTGTGCA TGATCAATATGAGTAAGATTGCCAACGACCTGCGCTTAATGGCAAGTGGC CCGCGCGCAGGCCTGAGCGAAATTGTTCTGCCTGCACGCCAACCGGGCAG CAGCATCATCCCTGGTATGGTGTGTCCGGTGATGCCGGAAGTGATGAACC AGGTTGCCTTCCAGGTGTTCGGTAACGACCTGACCATCACAAGCGCAAGC GAAGCAGGCCAGTTCGAGTTAAACGTGATGGAACCTGTGCTGTTTTTTAA CTTAATTCAGAGCATCAGTATTATGACAAATGTTTTTAAGTCTTTTACCG AAAACTGTCTGAAAGGTATCAAGGCCAACGAGGAACGCATGAAAGAGTAT GTGGAAAAAAGCATTGGCATCATCACCGCCATCAACCCGCATGTGGGCTA TGAGACAGCCGCCAAACTGGCCCGCGAAGCCTATTTAACCGGCGAGAGTA TTCGCGAGCTGTGTATCAAGTACGGCGTGCTGACCGAAGAGCAGCTGAAC GAGATCCTGAATCCGTACGAGATGATCCATCCTGGCATTGCAGGTCGCAA A

[0128] The measured enzyme activity was 3.8 U/ml. The enzyme activity U of the AspB mutant enzyme is defined as: the amount of enzyme catalyzing the formation of 1 micromole of product R-3-aminobutyric acid from butenoic acid per minute is one enzyme unit, that is, 1 U.

[0129] Determination method is: 16 mL reaction solution (pH8.5) was added to a 100 ml Erlenmeyer flask, wherein the reaction solution contains 300 mmol/L butenoic acid, 4 mmol/L MgCl.sub.2, 450 mmol/L ammonium chloride, 100 mmol/L HEPES buffer. The flask was sealed and the reaction solution and enzyme solution were placed in a 42° C. shaker respectively and incubated for 5-10 minutes. 4 ml of enzyme solution was added to the reaction solution, and immediately placed in a shaker at 42° C., 200 rpm to start the reaction. After 30 min, 1 ml of reaction solution was sampled, and 1 ml of acetonitrile was added to stop the reaction. The protein was removed by centrifugation. The supernatant was derivatized with 2, 4-dinitrofluorobenzene, and analyzed by HPLC (the enzyme activity was calculated based on the peak area).

[0130] 1.2 Catalytic Synthesis of R-3-Aminobutyric Acid with AspB Mutant

[0131] The synthesis reacts in a 100 ml reaction system at 37° C. 100 mM HEPES buffer of pH 8.0 was added. 2 mM MgCl.sub.2, 300 mM butenoic acid, 300 mM NH.sub.4Cl and 20 ml AspB mutant enzyme solution were added, wherein the above concentrations were final concentrations.

[0132] The progress of the reaction was detected by HPLC. The reaction was performed for 24 hours, the conversion rate was 42% and the ee value was 99.9%. The reaction was performed for 100 hours, the conversion rate was 60%, and the ee value was 99.7%.

[0133] The results show that, compared with the method in the comparative example, the method of the present invention utilizes an aspartase derived from E. coli that has stereoisomeric catalytic activity to efficiently and highly stereoselectively convert butenoic acid to R-3-aminobutyric acid. The method greatly improves conversion efficiency, shortens reaction time, and reduces production costs. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like. The present invention has been completed on the basis of this.

[0134] All literatures mentioned in the present application are incorporated herein by reference, as though each one is individually incorporated by reference. In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications, equivalents of which falls in the scope of claims as defined in the appended claims.