WO2019062222A1 - 酶法制备r-3-氨基丁酸的方法 - Google Patents

酶法制备r-3-氨基丁酸的方法 Download PDF

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WO2019062222A1
WO2019062222A1 PCT/CN2018/092010 CN2018092010W WO2019062222A1 WO 2019062222 A1 WO2019062222 A1 WO 2019062222A1 CN 2018092010 W CN2018092010 W CN 2018092010W WO 2019062222 A1 WO2019062222 A1 WO 2019062222A1
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aspartase
reaction
aminobutyric acid
mutant
amino acid
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PCT/CN2018/092010
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English (en)
French (fr)
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孙传民
程占冰
焦江华
丁少南
田振华
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上海弈柯莱生物医药科技有限公司
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Priority to US16/652,312 priority Critical patent/US11220701B2/en
Priority to JP2020517977A priority patent/JP7117792B2/ja
Priority to EP18860876.4A priority patent/EP3680340A4/en
Publication of WO2019062222A1 publication Critical patent/WO2019062222A1/zh
Priority to US17/539,414 priority patent/US20220090152A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biotechnology, and in particular to a method for enzymatically preparing R-3-aminobutyric acid.
  • Dolutegravir is a new anti-HIV drug from GlaxoSmithKline, which was approved by the FDA in 2013 and recognizes the breakthrough of this drug.
  • R-3-aminobutyric acid is an important intermediate for the production of Rutwe.
  • the existing preparation methods mainly include chemical synthesis and enzymatic catalysis.
  • CN104370755 a method of condensing ethyl acetoacetate with acetamide, asymmetrically hydrogenating, and then hydrolyzing to obtain R-3-aminobutyric acid is disclosed, but this method requires an expensive asymmetric hydrogenation catalyst. High production costs, heavy metal pollution, and no use of industrialized production.
  • Enzyme catalyzed method such as tert-butyl 3-aminobutyrate as described in ChemCatChem 2016, 8, 1226-1232, based on the stereoselectivity of lipase A (CLA-A) derived from Candida antarctica
  • CLA-A lipase A
  • the R-type 3-butyrylbutyric acid tert-butyl ester is catalyzed and then subjected to CAL-A catalytic hydrolysis to obtain R-3-aminobutyric acid.
  • this method has a low conversion rate and wastes unreacted raw materials.
  • ChemCatChem, 2014, 6, 965-968 reported a method for preparing R-3-aminobutyric acid from butyric acid derived from the aspartase mutant BSASP-C6 of Bacillus sp. YM55-1, but in this method, The conversion rate of the reaction for 100 hours is only 60%, the reaction time is long, and the conversion rate is low; and as the reaction time increases, the ee value of the product decreases.
  • the object of the present invention is to provide a highly efficient and highly stereoselective method for preparing R-3-aminobutyric acid, which can significantly increase the ee value and conversion rate of R-3-aminobutyric acid and shorten the reaction time.
  • the aspartase is derived from Escherichia coli.
  • the aspartase is wild type or mutant.
  • the ee value of the R-3-aminobutyric acid is ⁇ 99.5%, preferably ⁇ 99.7%, more preferably ⁇ 99.8%, optimally ⁇ 99.9%.
  • the conversion of the reaction is ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, still more preferably ⁇ 99%, most preferably 100%.
  • the mutant has an amino acid mutation selected from the group consisting of the amino acid sequence corresponding to the wild-type aspartase: threonine (T) at position 204, and methionine at position 338 (M) ), lysine (K) at position 341, asparagine (N) at position 343, or a combination thereof.
  • the mutation in the mutant is selected from the group consisting of T204C, M338I, K341M, N343C, or a combination thereof.
  • amino acid sequence of the wild type aspartase is as shown in SEQ ID NO.: 5.
  • the aspartase is selected from the group consisting of:
  • the amino acid sequence of the aspartase has at least 70%, preferably at least 75%, 80%, 85% of the sequence set forth in SEQ ID NO.: 5 or SEQ ID NO.: 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the aspartase is in a form selected from the group consisting of a resting cell, a bacterial cell, a crude enzyme solution, a pure enzyme, a crude enzyme powder, an immobilized enzyme, a free enzyme, a fermentation broth, Or a combination thereof.
  • the concentration of the aspartase in the reaction system is from 0.5 to 5 U/ml.
  • the concentration of the butenoic acid in the reaction system is from 100 mM to 1000 mM.
  • an ammonium source is also present in the reaction system.
  • the ammonium source is selected from the group consisting of ammonia, NH 4 + salts (eg, NH 4 Cl), or combinations thereof
  • the molar ratio of the ammonium source to butenoic acid is from 1:1 to 1:3.
  • the pH of the reaction system is 7.0-9.5, preferably 7.5-9.0, more preferably 8.0-8.5;
  • the reaction temperature is 20-60 ° C, preferably 30-50 ° C, more preferably 35-45 ° C.
  • the reaction time is from 0.5 h to 72 h, preferably from 2 h to 48 h, more preferably from 4 h to 24 h.
  • the aspartase is derived from Escherichia coli.
  • the aspartase is wild type or mutant.
  • the aspartase is as defined in the first aspect of the invention.
  • a R-3-aminobutyric acid producing strain which expresses a polypeptide which is exogenous aspartase from Escherichia coli and is used for catalyzing the following three-dimensional Catalytic reaction:
  • the aspartase is wild type or mutant.
  • the mutant has an amino acid mutation selected from the group consisting of the amino acid sequence corresponding to the wild-type aspartase: threonine (T) at position 204, and methionine at position 338 (M) ), lysine (K) at position 341, asparagine (N) at position 343, or a combination thereof.
  • the mutation is selected from the group consisting of T204C, M338I, K341M, N343C, or a combination thereof.
  • the production strain is a bacterium.
  • the production strain is Escherichia coli. More preferably, the production strain is E. coli BL21 (DE3).
  • R-3-aminobutyric acid is isolated from the culture system of 1).
  • an aspartase having a stereoisomerically catalytic activity having a stereoisomerically catalytic activity, the amino acid sequence of the aspartase being as shown in SEQ ID NO.: 3.
  • the aspartase is in a form selected from the group consisting of a bacterial cell, a crude enzyme solution, a pure enzyme, a crude enzyme powder, an immobilized enzyme, a free enzyme, a fermentation broth, or a combination thereof.
  • a polynucleotide comprising the aspartase of the fifth aspect of the invention is provided.
  • polynucleotide is selected from the group consisting of:
  • the nucleotide sequence has a homology of SEQ ID NO.: 4 with a sequence of ⁇ 95% (preferably ⁇ 98%, more preferably ⁇ 99%) and encodes the sequence shown in SEQ ID NO. a polynucleotide of a polypeptide;
  • the inventors have unexpectedly discovered a highly efficient and highly stereoselective preparation method of R-3-aminobutyric acid through extensive and intensive research.
  • This method utilizes an aspartase from Escherichia coli, having stereoisomerically catalytic activity, to efficiently and highly stereoselectively convert butenoic acid to R-3-aminobutyric acid.
  • the mutant aspartase of the present invention has very excellent high stereoselectivity and high conversion, thereby greatly improving conversion efficiency, shortening reaction time, and reducing production cost.
  • the conversion rate is as high as ⁇ 98% and the ee value is ⁇ 99.9% after only 24h.
  • the method of the invention has many advantages such as high yield, high conversion rate, low cost, short production cycle, simple process, easy enlargement, and suitable for large production.
  • the present invention has been completed on this basis.
  • ee value or "enantiomeric excess” is used to characterize the excess value of one enantiomer relative to the other enantiomer in a chiral molecule, usually expressed as a percentage.
  • enzyme as used herein have the same meaning and are used interchangeably herein. , both refer to proteins from Escherichia coli which have stereoisomerized crotonic acid to produce R-3-aminobutyric acid.
  • polypeptide of the invention refers to an enzyme as defined in the first aspect of the invention.
  • Aspartase is a deaminase that is a lytic enzyme that reversibly catalyzes the deamination of L-aspartate to form a fumarate reaction.
  • EC 4.3.1.1 is widely found in bacteria and yeast, and higher plants (such as beans). The buds or leaves of the genus also contain low concentrations of aspartase, which is absent in higher animals.
  • an aspartase derived from Escherichia coli is defined as AspA.
  • the aspartase in Bacillus is defined as AspB, and the mutant is BSASP-C6.
  • the aspartase of the invention is a wild type or a mutant.
  • the mutant has an amino acid mutation selected from the group consisting of the amino acid sequence corresponding to the wild-type aspartase: threonine (T) at position 204, and methionine at position 338 (M) ), lysine (K) at position 341, asparagine (N) at position 343, or a combination thereof.
  • the mutation is selected from the group consisting of T204C, M338I, K341M, N343C, or a combination thereof.
  • amino acid sequence of the wild type aspartase is as shown in SEQ ID NO.: 5.
  • the aspartase is selected from the group consisting of:
  • the amino acid sequence of the aspartase has at least 70%, preferably at least 75%, 80%, 85% of the sequence set forth in SEQ ID NO.: 5 or SEQ ID NO.: 3. 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the aspartase is in a form selected from the group consisting of a bacterial cell, a crude enzyme solution, a pure enzyme, a crude enzyme powder, an immobilized enzyme, a free enzyme, a fermentation broth, or a combination thereof.
  • amino acid sequence of the aspartase is set forth in SEQ ID NO.: 3, and the nucleic acid sequence encoding the polypeptide is set forth in SEQ ID NO.: 4.
  • amino acid sequence of the aspartase is set forth in SEQ ID NO.: 5
  • nucleic acid sequence encoding the polypeptide is set forth in SEQ ID NO.: 6.
  • the aspartase comprises up to 20, preferably up to 10, more preferably than the polypeptide represented by the amino acid sequence of SEQ ID NO.: 5 or SEQ ID NO.: Up to 8, preferably up to 3, more preferably up to 2, optimally up to 1 amino acid replaced by a similar or similar amino acid. Mutants of these conservative variations can be produced according to, for example, amino acid substitutions as shown in the table below.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the homology or sequence identity may be 80% or more, preferably 90% or more, more preferably 95% to 98%, and most preferably 99% or more.
  • Methods for determining sequence homology or identity include, but are not limited to, Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M. Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J.
  • the preferred method of determining identity is to obtain the largest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al, 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the invention provides a high-efficiency, high stereoselective method for preparing R-3-aminobutyric acid.
  • the method of the present invention as described in the first aspect of the present invention, utilizes an aspartase from Escherichia coli having stereoisomerically catalytic activity to efficiently and highly stereoselectively convert butenoic acid to R-3-aminobutyl Acid greatly improves conversion efficiency, shortens reaction time, and reduces production costs.
  • the method of producing R-3-aminobutyric acid comprises the steps of:
  • the aspartase is derived from Escherichia coli.
  • the ee value of the R-3-aminobutyric acid is ⁇ 99.5%, preferably ⁇ 99.7%, more preferably ⁇ 99.8%, optimally ⁇ 99.9%.
  • the conversion of the reaction is ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, still more preferably ⁇ 99%, most preferably 100%.
  • the method for producing R-3-aminobutyric acid comprises:
  • R-3-aminobutyric acid is isolated from the culture system of 1).
  • the inventors have unexpectedly discovered that the aspartase of the present invention can be used to prepare a formulation for catalyzing the following stereoisomeric catalytic reaction:
  • the present inventors also provide an engineered strain expressing the aspartase of the present invention, the engineered strain (or the expressed aspartase of the present invention, or an immobilized enzyme thereof) capable of efficiently and highly high in crotonic acid Stereoisomeric conversion to R-3-aminobutyric acid with a conversion of ⁇ 98% and a chiral ee value of R-3-aminobutyric acid ⁇ 99.9%.
  • the production strain is a bacterium.
  • the production strain is Escherichia coli. More preferably, the production strain is E. coli BL21 (DE3).
  • the invention can convert the butenoic acid into R-3-aminobutyric acid with high efficiency and high stereoisolation, and the conversion rate is up to ⁇ 98% after 24h, and the ee value is ⁇ 99.9%, which greatly improves the conversion efficiency and shortens the reaction time. ,reduce manufacturing cost.
  • the method of the invention has high conversion rate, low cost, high yield, short production cycle, simple process, easy enlargement and suitable for large-scale production, and the obtained ee value of R-3-aminobutyric acid is extremely high. It has great application prospects in the production of R-3-aminobutyric acid and downstream products with R-3-aminobutyric acid as a precursor.
  • a deoxyribonucleic acid sequence (SEQ ID NO.: 6) encoding the AspA wild type enzyme was synthesized, and the enzyme was ligated with pET28a, and the restriction enzyme site NdeI & HindIII was used to link the enzyme.
  • the vector transforms host E. coli BL21 competent cells.
  • the TB medium was inoculated at 37 ° C, shaken at 200 rpm until the OD600 value reached 4.0, and the concentration of IPTG was 0.1 mM.
  • the temperature was adjusted to 28 ° C and the culture was continued for 12 hours.
  • the cells were collected by centrifugation at 4 ° C and buffered with phosphate.
  • the cells were resuspended in liquid (50 mM, pH 7.0) and homogenously disrupted to obtain an AspA wild type enzyme solution.
  • AspA wild type nucleic acid coding sequence AspA wild type nucleic acid coding sequence:
  • the conversion rate is also called the material conversion rate, which is numerically equal to the ratio of the amount of crotonic acid consumed in the fermentation process to the total amount of crotonic acid at the start of fermentation. It is expressed as a percentage, which may be a molar ratio (mol%). It can also be a weight ratio (wt%).
  • the amino acids of the four mutation sites of AspA mutant 1 were all mutated (see Table 1 and Table 2).
  • a deoxyribonucleic acid sequence encoding the AspA mutant 1 enzyme (SEQ ID NO.: 4), an enzyme linked to pET28a, a restriction site NdeI & HindIII, an enzyme A good vector was used to transform host E. coli BL21 competent cells.
  • the TB medium was inoculated at 37 ° C, shaken at 200 rpm until the OD600 value reached 4.0, and the concentration of IPTG was 0.1 mM.
  • the temperature was adjusted to 28 ° C and the culture was continued for 12 hours.
  • the cells were collected by centrifugation at 4 ° C and buffered with phosphate.
  • the cells were resuspended in liquid (50 mM, pH 7.0) and homogenized and disrupted to obtain an AspA mutant 1 enzyme solution.
  • AspA mutant 1 nucleic acid coding sequence AspA mutant 1 nucleic acid coding sequence:
  • the enzyme activity was determined to be 5.1 U/ml.
  • the enzyme activity U of the AspA mutant 1 enzyme is defined as the amount of enzyme that catalyzes the formation of 1 micromole of product R-3-aminobutyric acid per minute from butenoic acid to one enzyme unit, i.e., 1 U.
  • Determination method Take a 100ml flask and add 16mL reaction solution (pH 8.0).
  • the reaction solution contains 300mmol/L butenoic acid, 4mmol/L MgCl 2 , 450mmol/L ammonium chloride, 100mmol/L HEPES buffer, and sealed.
  • the reaction solution and the enzyme solution were respectively placed on a 42-degree shaker for 5-10 min.
  • 4 ml of the AspA mutant enzyme solution was added to the reaction solution, and the reaction was immediately placed in a shaker at a rotation speed of 200 rpm at 42 °C. After 30 min, 1 ml was sampled, the reaction was stopped by adding 1 ml of acetonitrile, and the protein was removed by centrifugation.
  • the supernatant was derivatized with 2,4-dinitrofluorobenzene and analyzed by HPLC (according to the peak area to calculate the enzyme activity).
  • AspA mutants 2-12 Specific mutations of AspA mutants 2-12 are shown in Tables 1 and 2, in which AspA mutants 2-5 are single amino acid mutations, and AspA mutants 6-8 are amino acid mutations at two mutation sites, AspA mutation The 9-12 is a mutation in the amino acid of the three mutation sites.
  • the conversion rate is 30%-70%, and "****" stands for conversion rate >70%.
  • the experimental procedure was the same as in Example 2.2, in which the AspA mutant 1 enzyme solution was replaced with the AspA mutant 2-12 enzyme solution.
  • the AspB mutant enzyme solution was prepared by the method of 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.
  • AspB mutant nucleic acid coding sequence AspB mutant nucleic acid coding sequence:
  • the enzyme activity was measured to be 3.8 U/ml.
  • the enzyme activity U of the AspB mutant enzyme is defined as the amount of enzyme that catalyzes the formation of 1 micromole of product R-3-aminobutyric acid per minute from butenoic acid to one enzyme unit, i.e., 1 U.
  • Determination method Take a 100ml flask and add 16mL reaction solution (pH 8.5).
  • the reaction solution contains 300mmol/L butenoic acid, 4mmol/L MgCl 2 , 450mmol/L ammonium chloride, 100mmol/L HEPES buffer, and sealed.
  • the reaction solution and the enzyme solution were respectively placed on a 42-degree shaker for 5-10 min. 4 ml of the enzyme solution was added to the reaction solution, and the reaction was immediately placed in a shaker at a rotation speed of 200 rpm at 42 °C. After 30 min, 1 ml was sampled, the reaction was stopped by adding 1 ml of acetonitrile, and the protein was removed by centrifugation. The supernatant was derivatized with 2,4-dinitrofluorobenzene and analyzed by HPLC (according to the peak area to calculate the enzyme activity).
  • the progress of the reaction was determined by HPLC, and the reaction was carried out until 24 h, the conversion rate was 42%, the ee value was 99.9%, the reaction was 100 h, the conversion rate was 60%, and the ee value was 99.7%.
  • -Aminobutyric acid greatly improves conversion efficiency, shortens reaction time, and reduces production costs.
  • the method of the invention has many advantages such as high yield, high conversion rate, low cost, short production cycle, simple process, easy enlargement, and suitable for large production. The present invention has been completed on this basis.

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Abstract

一种高效、高立体选择性的R-3-氨基丁酸制备方法，该方法利用来自大肠杆菌、具有立体异构催化活性的天冬氨酸酶，将丁烯酸高效地转化为R-3-氨基丁酸，反应仅24h后转化率高达≥98％，ee值≥99.9％，极大地提高转化效率，缩短反应时间，降低生产成本。所述方法具有收率高、转化率高、成本低、生产周期短、工艺简单、易于放大、适合进行大生产等诸多优点。

Description

酶法制备R-3-氨基丁酸的方法 技术领域
本发明属于生物技术领域，具体地，涉及酶法制备R-3-氨基丁酸的方法。
背景技术
度鲁特韦(Dolutegravir)是葛兰素史克旗下抗HIV新药，2013年FDA批准其上市并承认了这种药物的突破性。R-3-氨基丁酸是生产度鲁特韦的重要中间体，目前现有的制备方法主要有化学合成法和酶催化法。
化学合成法，如Tetrahedron:Asymmetry 18(2007)1554-1566报道了由甲醛为原料，经Horner–Wadsworth–Emmons反应得到2-丁烯酸叔丁酯，再经加成，催化氢化，可得到R-3-氨基丁酸叔丁酯，最后水解得到R-3-氨基丁酸。但是此反应需要-78℃低温，反应条件苛刻，操作困难。
Figure PCTCN2018092010-appb-000001
又如CN104370755公开了由乙酰乙酸乙酯为原料，与乙酰胺缩合，经不对称氢化，再经水解得到R-3-氨基丁酸的方法，但是此方法需要用到昂贵的不对称氢化催化剂，生产成本较高，重金属污染，不利用工业化大生产。
Figure PCTCN2018092010-appb-000002
酶催化法，如ChemCatChem 2016,8,1226-1232报道的以消旋的3-氨基丁酸叔丁基酯为原料，经来源于南极假丝酵母的脂肪酶A(CLA-A)立体选择性催化得到R型3-丁酰胺基丁酸叔丁酯，再经CAL-A催化水解得到R-3-氨基丁酸。但是此方法转化率低，未反应原料浪费。
Figure PCTCN2018092010-appb-000003
另有，ChemCatChem,2014,6,965-968中报道了来源于芽孢杆菌YM55-1的天冬氨酸酶突变体BSASP-C6催化丁烯酸制备R-3-氨基丁酸的方法，但是此方法中，反应100个小时的转化率仅为60％，反应时间长，转化率低；且随着反应时间的增加，产物的ee值降低。
因此，本领域迫切需要开发环境友好、高效、高立体选择性的R-3-氨基丁酸制 备方法。
发明内容
本发明的目的在于提供一种高效、高立体选择性的R-3-氨基丁酸制备方法，所述方法能够显著提高R-3-氨基丁酸的ee值、转化率，缩短反应时间。
本发明的第一方面，提供了一种生产R-3-氨基丁酸的方法，所述方法包括步骤：
(1)在反应体系中，以丁烯酸为底物，在天冬氨酸酶催化下，进行反应式I所示的立体异构催化反应，从而形成R-3-氨基丁酸；
Figure PCTCN2018092010-appb-000004
(2)任选地从所述步骤(1)的反应后的反应体系中分离出R-3-氨基丁酸；
其中，所述天冬氨酸酶来自大肠杆菌。
在另一优选例中，所述天冬氨酸酶为野生型或突变体。
在另一优选例中，所述R-3-氨基丁酸的ee值≥99.5％，较佳地≥99.7％，更佳地≥99.8％，最佳地≥99.9％。
在另一优选例中，所述反应的转化率≥90％，较佳地≥95％，更佳地≥98％，又更佳地≥99％，最佳地100％。
在另一优选例中，所述突变体在对应于野生型天冬氨酸酶的氨基酸序列中存在选自下组的氨基酸突变：第204位苏氨酸(T)、第338位蛋氨酸(M)、第341位赖氨酸(K)、第343位天冬酰胺(N)、或其组合。
在另一优选例中，所述突变体中的突变选自下组：T204C、M338I、K341M、N343C、或其组合。
在另一优选例中，所述野生型天冬氨酸酶的氨基酸序列如SEQ ID NO.:5所示。
在另一优选例中，所述天冬氨酸酶选自下组：
(a)氨基酸序列如SEQ ID NO.:5所示的多肽；
(b)氨基酸序列如SEQ ID NO.:3所示的多肽；或
(c)将SEQ ID NO.:5或SEQ ID NO.:3所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a)或(b)所述多肽功能的由SEQ ID NO.:5或SEQ ID NO.:3所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述天冬氨酸酶的氨基酸序列与SEQ ID NO.:5或SEQ ID NO.:3所示的序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性。
在另一优选例中，所述天冬氨酸酶为选自下组的形式：静息细胞、菌体、粗酶液、纯酶、粗酶粉、固定化酶、游离酶、发酵液、或其组合。
在另一优选例中，所述反应体系中，所述天冬氨酸酶的浓度为0.5-5U/ml。
在另一优选例中，所述反应体系中，所述丁烯酸的浓度为100mM-1000mM。
在另一优选例中，所述反应体系中，还存在铵源。
在另一优选例中，所述铵源选自下组：氨水、NH 4 +盐(如NH 4Cl)、或其组合
在另一优选例中，所述铵源与丁烯酸的摩尔比为1:1-1:3。
在另一优选例中，步骤(a)中，反应体系的pH为7.0-9.5，较佳地，7.5-9.0，更佳地，8.0-8.5；
在另一优选例中，步骤(a)中，反应温度20-60℃，较佳地，30-50℃，更佳地，35-45℃
在另一优选例中，步骤(a)中，反应时间0.5h-72h，较佳地，2h-48h，更佳地，4h-24h。
本发明的第二方面，提供了一种天冬氨酸酶的用途，用于制备一制剂，所述制剂用于催化以下立体异构催化反应：
Figure PCTCN2018092010-appb-000005
其中，所述天冬氨酸酶来自大肠杆菌。
在另一优选例中，所述天冬氨酸酶为野生型或突变体。
在另一优选例中，所述天冬氨酸酶如本发明第一方面所定义的。
本发明的第三方面，提供了一种R-3-氨基丁酸生产菌株，所述菌株表达多肽，所述多肽为外源的来自大肠杆菌的天冬氨酸酶，并用于催化以下立体异构催化反应：
Figure PCTCN2018092010-appb-000006
在另一优选例中，所述天冬氨酸酶为野生型或突变体。
在另一优选例中，所述突变体在对应于野生型天冬氨酸酶的氨基酸序列中存在选自下组的氨基酸突变：第204位苏氨酸(T)、第338位蛋氨酸(M)、第341位赖氨酸(K)、第343位天冬酰胺(N)、或其组合。
在另一优选例中，所述的突变选自下组：T204C、M338I、K341M、N343C、或其组合。
在另一优选例中，所述生产菌株是细菌。优选地，所述生产菌株是大肠杆菌。更优选地，所述生产菌株是E.coli BL21(DE3)。
本发明的第四方面，提供了一种生产R-3-氨基丁酸的方法，所述方法包括步骤：
1)采用生产条件培养本发明第三方面所述的生产菌株，从而得到R-3-氨基丁酸；
2)任选地，从1)的培养体系中分离获得R-3-氨基丁酸。
本发明的第五方面，提供了一种具有立体异构催化活性的天冬氨酸酶，所述天冬氨酸酶的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述天冬氨酸酶为选自下组的形式：菌体、粗酶液、纯酶、粗酶粉、固定化酶、游离酶、发酵液、或其组合。
本发明的第六方面，提供了一种多核苷酸，所述多核苷酸编码本发明第五方面所述的天冬氨酸酶。
在另一优选例中，所述多核苷酸选自下组：
(a)编码如SEQ ID NO.:3所示多肽的多核苷酸。
(b)序列如SEQ ID NO.:4所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:4所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)，且编码SEQ ID NO.:3所示多肽的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地发现一种高效、高立体选择性的R-3-氨基丁酸制备方法。该方法利用来自大肠杆菌、具有立体异构催化活性的天冬氨酸酶，高效、高立体选择性地将丁烯酸转化为R-3-氨基丁酸。尤其是本发明的突变型天冬氨酸酶具有非常优异的高立体选择性和高转化率，从而极大地提高转化效率，缩短反应时间，降低生产成本。实验表明，反应仅24h后转化率高达≥98％，ee值≥99.9％。本发明方法具有收率高、转化率高、成本低、生产周期短、工艺简单、易于放大、适合进行大生产等诸多优点。在此基础上完成了本发明。
术语
ee值
如本文所用，“ee值”或“对映体过量”用来表征手性分子中一个对映异构体相对于另一个对映异构体的过量值，通常用百分数表示。
天冬氨酸酶
本文所用的术语“酶”、“多肽”、“天冬氨酸酶”、“本发明多肽”、“本发明天冬氨酸酶”或“AspA”具有相同的意义，在本文可以互换使用，均是指来自大肠杆菌，具有立体异构催化丁烯酸产生R-3-氨基丁酸活性的蛋白。优选地，所述的本发明多肽指本发明第一方面中所限定的酶。
天冬氨酸酶是脱氨基酶的一种，为可逆地催化L-天冬氨酸脱氨基生成延胡索酸反应的裂解酶，EC 4.3.1.1，广泛存在于细菌、酵母中，高等植物(例如豆类的芽或叶等)中也含有低浓度的天冬氨酸酶，在高等动物中则无此酶。
本发明中，来自大肠杆菌的天冬氨酸酶，定义为AspA。
本发明中，芽孢杆菌中天冬氨酸酶定义为AspB，突变体为BSASP-C6。
基于现有技术的知识，本领域普通技术人员不难知晓，在多肽的某些区域，例如非重要区域改变少数氨基酸残基基本上不会改变生物活性，例如，适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等，Molecular Biology of The Gene，第四版，1987，The Benjamin/Cummings Pub.Co.P224)。因此，本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
在具体的实施方式中，本发明的天冬氨酸酶是野生型或突变体。
在优选的实施方式中，所述突变体在对应于野生型天冬氨酸酶的氨基酸序列中存在选自下组的氨基酸突变：第204位苏氨酸(T)、第338位蛋氨酸(M)、第341位赖氨酸(K)、第343位天冬酰胺(N)、或其组合。
在另一优选例中，所述的突变选自下组：T204C、M338I、K341M、N343C、或其组合。
在另一优选例中，所述野生型天冬氨酸酶的氨基酸序列如SEQ ID NO.:5所示。
在另一优选例中，所述天冬氨酸酶选自下组：
(a)氨基酸序列如SEQ ID NO.:5所示的多肽；
(b)氨基酸序列如SEQ ID NO.:3所示的多肽；或
(c)将SEQ ID NO.:5或SEQ ID NO.:3所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a)或(b)所述多肽功能的由SEQ ID NO.:5或SEQ ID NO.:3所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述天冬氨酸酶的氨基酸序列与SEQ ID NO.:5或SEQ ID NO.: 3所示的序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性。
在另一优选例中，所述天冬氨酸酶为选自下组的形式：菌体、粗酶液、纯酶、粗酶粉、固定化酶、游离酶、发酵液、或其组合。
在具体的实施方式中，所述天冬氨酸酶的氨基酸序列如SEQ ID NO.:3所示，编码该多肽的核酸序列如SEQ ID NO.:4所示。
在具体的实施方式中，所述天冬氨酸酶的氨基酸序列如SEQ ID NO.:5所示，编码该多肽的核酸序列如SEQ ID NO.:6所示。
在本发明中，所述天冬氨酸酶包括与氨基酸序列SEQ ID NO.:5或SEQ ID NO.:3所示的多肽相比，有至多20个、较佳地至多10个，又佳地至多8个，再佳地至多3个，更佳地至多2个，最佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成的突变体。这些保守性变异的突变体可根据，例如下表所示进行氨基酸替换而产生。
表A
初始残基 代表性的取代残基 优选的取代残基
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码本发明多肽的多核苷酸。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
因此，本文所用的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、 “基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
在具体的实施方式中，所述同源性或序列相同性可以是80％以上，优选90％以上，更优选95％-98％，最优选99％以上。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
生产R-3-氨基丁酸的方法
本发明提供一种高效、高立体选择性的R-3-氨基丁酸制备方法。本发明的方法如本发明第一方面所述，利用来自大肠杆菌、具有立体异构催化活性的天冬氨酸酶，高效、高立体选择性地将丁烯酸转化为R-3-氨基丁酸，极大地提高转化效率，缩短反应时间，降低生产成本。
在优选的实施方式中，所述生产R-3-氨基丁酸的方法包括步骤：
(1)在反应体系中，以丁烯酸为底物，在天冬氨酸酶催化下，进行反应式I所示的立体异构催化反应，从而形成R-3-氨基丁酸；
Figure PCTCN2018092010-appb-000007
(2)任选地从所述步骤(1)的反应后的反应体系中分离出R-3-氨基丁酸；
其中，所述天冬氨酸酶来自大肠杆菌。
在另一优选例中，所述R-3-氨基丁酸的ee值≥99.5％，较佳地≥99.7％，更佳地≥99.8％，最佳地≥99.9％。
在另一优选例中，所述反应的转化率≥90％，较佳地≥95％，更佳地≥98％，又更佳地≥99％，最佳地100％。
在另一优选的实施方式中，所述生产R-3-氨基丁酸方法包括：
1)采用生产条件培养本发明的R-3-氨基丁酸生产菌株，从而得到R-3-氨基丁酸；
2)任选地，从1)的培养体系中分离获得R-3-氨基丁酸。
天冬氨酸酶的用途
本发明人意外地发现本发明的天冬氨酸酶可用于制备一制剂，所述制剂用于催化以下立体异构催化反应：
Figure PCTCN2018092010-appb-000008
R-3-氨基丁酸生产菌株
本发明人还提供了表达本发明的天冬氨酸酶的工程菌株，所述工程菌株(或其表达的本发明天冬氨酸酶、或其固定化酶)能够将丁烯酸高效、高立体异构地转化为R-3-氨基丁酸，其转化率≥98％，R-3-氨基丁酸的手性ee值≥99.9％。
在另一优选例中，所述生产菌株是细菌。优选地，所述生产菌株是大肠杆菌。更优选地，所述生产菌株是E.coli BL21(DE3)。
本发明的主要优点：
本发明能够将丁烯酸高效、高立体异构地转化为R-3-氨基丁酸，反应仅24h后转化率高达≥98％，ee值≥99.9％，极大地提高转化效率，缩短反应时间，降低生产成本。
本发明的方法转化率高、成本低、收率高、生产周期短、工艺简单、易于放大、适合进行大生产，获得的R-3-氨基丁酸ee值极高。在R-3-氨基丁酸以及以R-3-氨基丁酸为前体的下游产物的生产中具有极大的应用前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明所用试剂和原料均市售可得。除非另有定义，本文所用的所有技术和科 学术语与本发明所属领域普通技术人员通常理解的意义相同。虽然可利用与本文所述相似或等价的任何方法和材料来实施或检验本发明，但优选本文所述的方法和材料。
实施例
实施例1.AspA野生型催化合成R-3-氨基丁酸及检测
1.1 AspA野生型酶液的制备
根据AspA野生型的氨基酸序列(SEQ ID NO.:5)，合成编码AspA野生型酶的脱氧核糖核酸序列(SEQ ID NO.:6)，酶连pET28a，酶切位点NdeI&HindIII，将酶连好的载体，转化宿主大肠杆菌BL21感受态细胞。菌种接种TB培养基于37℃，摇床200rpm培养至OD600值达到4.0时，加入IPTG浓度0.1mM诱导，温度调至28℃下继续培养12小时，4℃下离心收集菌体，用磷酸盐缓冲液(50mM，pH7.0)重悬细胞，均质破碎，得到AspA野生型酶液。
AspA野生型的氨基酸序列：
Figure PCTCN2018092010-appb-000009
AspA野生型核酸编码序列：
Figure PCTCN2018092010-appb-000010
Figure PCTCN2018092010-appb-000011
1.2 AspA野生型催化合成R-3-氨基丁酸
100ml反应体系中，37℃下，加入100mM HEPES缓冲液,pH 8.0,终浓度为2mM的MgCl 2,300mM丁烯酸,300mM NH 4Cl，AspA野生型酶液20ml。
HPLC检测反应进程，反应至24h结束，转化率＜5％。
转化率的计算：转化率也叫物料转化率，数值上等于发酵过程中所消耗的丁烯酸与发酵开始时丁烯酸的总量之比值，常用百分比表示，可以是摩尔比(mol％)，也可以是重量比(wt％)。
实施例2.AspA突变体1催化合成R-3-氨基丁酸及检测
2.1 AspA突变体1酶液的制备
AspA突变体1的4个突变位点的氨基酸全部突变(参见表1和表2)。根据AspA突变体1的氨基酸序列(SEQ ID NO.:3)，合成编码AspA突变体1酶的脱氧核糖核酸序列(SEQ ID NO.:4)，酶连pET28a，酶切位点NdeI&HindIII，将酶连好的载体，转化宿主大肠杆菌BL21感受态细胞。菌种接种TB培养基于37℃，摇床200rpm培养至OD600值达到4.0时，加入IPTG浓度0.1mM诱导，温度调至28℃下继续培养12小时，4℃下离心收集菌体，用磷酸盐缓冲液(50mM，pH7.0)重悬细胞，均质破碎，得到AspA突变体1酶液。
AspA突变体1的氨基酸序列：
Figure PCTCN2018092010-appb-000012
Figure PCTCN2018092010-appb-000013
AspA突变体1核酸编码序列：
Figure PCTCN2018092010-appb-000014
测定酶活为5.1U/ml。AspA突变体1酶的酶活U定义为：每分钟从丁烯酸催化形成1微摩尔产物R-3-氨基丁酸的酶量为一个酶活单位，即1U。
测定方法：取100ml三角瓶，加入16mL反应液(pH8.0)，反应液中含有300mmol/L丁烯酸，4mmol/L MgCl 2，450mmol/L氯化铵，100mmol/L HEPES缓冲液，密闭，将反应液和酶液分别放于42度摇床温育5-10min。向反应液中加入AspA突变体酶液4ml，立即放于42℃200rpm转速的摇床中开始反应。30min后取样1ml，加入1ml乙腈终止反应，离心除去蛋白，上清液用2，4-二硝基氟苯衍生，HPLC分析(根据峰面积计算酶活)。
2.2 AspA突变体1催化合成R-3-氨基丁酸
100ml反应体系中，37℃下，加入100mM HEPES缓冲液,pH 8.0,终浓度为2mM的MgCl 2,300mM丁烯酸,300mM NH 4Cl，AspA突变体1酶液20ml。
HPLC检测反应进程，反应至24h结束，转化率≥98％，ee值为99.9％。
实施例3.AspA突变体2-12催化合成R-3-氨基丁酸及检测
3.1 AspA突变体2-12酶液的制备
AspA突变体2-12的具体突变情况如表1和表2中所示，其中AspA突变体2-5为单氨基酸突变，AspA突变体6-8为2个突变位点的氨基酸突变，AspA突变体9-12为3个突变位点的氨基酸发生突变。
根据AspA突变体2-12的氨基酸序列，分别合成编码各AspA突变体酶的脱氧核糖核酸序列，酶液制备方法同实施例2.1。
表1突变氨基酸的位置及改变
位置 野生型 突变体
突变位点1(第204位氨基酸) 苏氨酸(T) 半胱氨酸(C)
突变位点2(第338位氨基酸) 蛋氨酸(M) 异亮氨酸(I)
突变位点3(第341位氨基酸) 赖氨酸(K) 蛋氨酸(M)
突变位点4(第343位氨基酸) 天冬酰胺(N) 半胱氨酸(C)
表2各突变体酶催化合成R-3-氨基丁酸的转化率
  突变位点1 突变位点2 突变位点3 突变位点4 转化率
野生型 - - - - *
突变体1 + + + + ****
突变体2 + - - - *
突变体3 - + - - *
突变体4 - - + - *
突变体5 - - - + *
突变体6 + - + - **
突变体7 - + + - **
突变体8 - - + + **
突变体9 + + - + **
突变体10 + - + + ***
突变体11 - + + + ***
突变体12 + + + - ***
注：“+”代表突变，“-”代表未突变；
“*”代表转化率＜10％，“**”代表转化率10％-30％，“***”代表转
化率30％-70％，“****”代表转化率＞70％。
3.2 AspA突变体2-12催化合成R-3-氨基丁酸
实验方法同实施例2.2，用AspA突变体2-12酶液分别替换AspA突变体1酶液。
结果如表2所示。实验结果表明，反应24h后，AspA野生型(实施例1)和突变体1(实施例2)和突变体2-12(实施例3)均具有一定的立体选择性(可选择性地催化形成R-3-氨基丁酸)，并且反应时间均明显缩短。此外，就转化率和反应速度而言，突变体1(四位点的突变体)显著优于三位点的突变体(如突变体9-12)，也优于二位点的突变体(如突变体6-8)、单位点突变体(如突变体2-5)和野生型。
对比例1.来源于芽孢杆菌的AspB突变体催化合成R-3-氨基丁酸及检测
1.1芽孢杆菌AspB突变体酶液的制备
参考ChemCatChem,2014,6,965-968中的方法制备AspB突变体酶液，AspB突变体的氨基酸序列如SEQ ID NO.:1所示，核酸编码序列如SEQ ID NO.:2所示。
AspB突变体的氨基酸序列：
Figure PCTCN2018092010-appb-000015
AspB突变体核酸编码序列：
Figure PCTCN2018092010-appb-000016
Figure PCTCN2018092010-appb-000017
测定酶活为3.8U/ml。AspB突变体酶的酶活U定义为：每分钟从丁烯酸催化形成1微摩尔产物R-3-氨基丁酸的酶量为一个酶活单位，即1U。
测定方法：取100ml三角瓶，加入16mL反应液(pH8.5)，反应液中含有300mmol/L丁烯酸，4mmol/L MgCl 2，450mmol/L氯化铵，100mmol/L HEPES缓冲液，密闭，将反应液和酶液分别放于42度摇床温育5-10min。向反应液中加入酶液4ml，立即放于42℃200rpm转速的摇床中开始反应。30min后取样1ml，加入1ml乙腈终止反应，离心除去蛋白，上清液用2，4-二硝基氟苯衍生，HPLC分析(根据峰面积计算酶活)。
1.2 AspB突变体催化合成R-3-氨基丁酸
100ml反应体系中，37℃下，加入100mM HEPES缓冲液,pH 8.0,终浓度为2mM的MgCl 2,300mM丁烯酸,300mM NH 4Cl，AspB突变体酶液20ml
HPLC检测反应进程，反应至24h，转化率42％，ee值99.9％，反应至100h，转化率60％，ee值99.7％。
结果表明，与对比例中的方法相比，本发明的方法利用来自大肠杆菌、具有立体异构催化活性的天冬氨酸酶，高效、高立体选择性地将丁烯酸转化为R-3-氨基丁酸，极大地提高转化效率，缩短反应时间，降低生产成本。本发明方法具有收率高、转化率高、成本低、生产周期短、工艺简单、易于放大、适合进行大生产等诸多优点。在此基础上完成了本发明。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种生产R-3-氨基丁酸的方法，其特征在于，所述方法包括步骤：
    (1)在反应体系中，以丁烯酸为底物，在天冬氨酸酶催化下，进行反应式I所示的立体异构催化反应，从而形成R-3-氨基丁酸；
    Figure PCTCN2018092010-appb-100001
    (2)任选地从所述步骤(1)的反应后的反应体系中分离出R-3-氨基丁酸；
    其中，所述天冬氨酸酶来自大肠杆菌。
  2. 如权利要求1所述的方法，其特征在于，所述天冬氨酸酶为野生型或突变体。
  3. 如权利要求1所述的方法，其特征在于，所述R-3-氨基丁酸的ee值≥99.5％，和/或所述反应的转化率≥90％。
  4. 如权利要求2所述的方法，其特征在于，所述突变体在对应于野生型天冬氨酸酶的氨基酸序列中存在选自下组的氨基酸突变：第204位苏氨酸(T)、第338位蛋氨酸(M)、第341位赖氨酸(K)、第343位天冬酰胺(N)、或其组合。
  5. 如权利要求2或4所述的方法，其特征在于，所述突变体中的突变选自下组：T204C、M338I、K341M、N343C、或其组合。
  6. 一种天冬氨酸酶的用途，其特征在于，用于制备一制剂，所述制剂用于催化以下立体异构催化反应：
    Figure PCTCN2018092010-appb-100002
    其中，所述天冬氨酸酶来自大肠杆菌。
  7. 一种R-3-氨基丁酸生产菌株，其特征在于，所述菌株表达多肽，所述多肽为外源的来自大肠杆菌的天冬氨酸酶，并用于催化以下立体异构催化反应：
    Figure PCTCN2018092010-appb-100003
  8. 一种生产R-3-氨基丁酸的方法，其特征在于，所述方法包括步骤：
    1)采用生产条件培养权利要求7所述的生产菌株，从而得到R-3-氨基丁酸；
    2)任选地，从1)的培养体系中分离获得R-3-氨基丁酸。
  9. 一种具有立体异构催化活性的天冬氨酸酶，其特征在于，所述天冬氨酸酶的氨基酸序列如SEQ ID NO.:3所示。
  10. 一种多核苷酸，其特征在于，所述多核苷酸编码权利要求9所述的天冬氨酸酶。
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