WO2022088528A1 - 转氨酶突变体及其在制备西他列汀中间体中的应用 - Google Patents

转氨酶突变体及其在制备西他列汀中间体中的应用 Download PDF

Info

Publication number
WO2022088528A1
WO2022088528A1 PCT/CN2021/072993 CN2021072993W WO2022088528A1 WO 2022088528 A1 WO2022088528 A1 WO 2022088528A1 CN 2021072993 W CN2021072993 W CN 2021072993W WO 2022088528 A1 WO2022088528 A1 WO 2022088528A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
trifluorophenyl
final concentration
sitagliptin
substrate
Prior art date
Application number
PCT/CN2021/072993
Other languages
English (en)
French (fr)
Inventor
柳志强
程峰
张晓健
贾东旭
郑裕国
何人宝
金逸中
邵鸿鸣
林娇华
张峰
Original Assignee
浙江永太科技股份有限公司
浙江工业大学
浙江永太药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江永太科技股份有限公司, 浙江工业大学, 浙江永太药业有限公司 filed Critical 浙江永太科技股份有限公司
Priority to US17/778,627 priority Critical patent/US20220396816A1/en
Priority to EP21884273.0A priority patent/EP4050102A4/en
Publication of WO2022088528A1 publication Critical patent/WO2022088528A1/zh

Links

Images

Classifications

    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • C12P13/001—Amines; Imines
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01018—Beta-alanine-pyruvate transaminase (2.6.1.18)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention relates to the technical field of biochemical industry, in particular to a method for preparing an optically pure sitagliptin intermediate of transaminase and its mutant enzyme, including transaminase, mutant, encoding gene, recombinant vector containing the gene, the Recombinant genetic engineering bacteria and recombinase obtained by transformation of recombinant vector, and application.
  • Sitagliptin is the first dipeptidyl peptidase-IV (DPP-IV) inhibitor approved by the FDA for the treatment of type 2 diabetes, developed and developed by Merck and Codexis in the United States. Sitagliptin can increase insulin secretion in a blood sugar-dependent manner, with moderate hypoglycemic effect, without causing hypoglycemia, and without side effects such as weight gain, nausea, and vomiting.
  • the trade name of sitagliptin is Januvia, which has been approved for use in more than 70 countries around the world, and is the top 20 drug sales in the world.
  • Sitagliptin and its intermediates can be prepared by a combination of chemical-enzymatic methods, and have gradually become the first choice for the synthesis of chiral pharmaceutical chemicals and their intermediates.
  • the key to the chemo-enzymatic method is to obtain a transaminase that can catalyze the asymmetric transamination reaction to obtain an optically pure sitagliptin intermediate.
  • US Patent US8293507 discloses a biocatalyst obtained by the modification of Arthrobacter-derived transaminase (ATA117) by Codexis Company, and the ee value of the transamination product reaches 99%.
  • the present invention provides a transaminase mutant, an encoding gene, a recombinant vector, a recombinant transaminase, and a
  • the genetic engineering bacteria and the application of obtaining sitagliptin or sitagliptin ester intermediates through asymmetric transamination have high raw material conversion rate, low production cost and high yield.
  • the present invention provides a transaminase mutant, wherein the tyrosine at position 74 of the amino acid sequence shown in SEQ ID NO: 2 is substituted with proline, and the glutamic acid at position 228 is substituted with aspartic acid , the leucine at the 254th position is replaced by alanine, the methionine at the 290th position is replaced by threonine and obtained, the amino acid sequence of the transaminase mutant is SEQ ID NO: 4, and the nucleotide sequence of the encoding gene is SEQ ID NO: 4 ID NO: 3.
  • the transaminase mutant of the present invention is obtained by mutating a transaminase derived from Mycobacterium (Mycolici bacterrium), the amino acid sequence of the transaminase is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO:1 Show.
  • the amino acid sequence identity of the transaminase shown in SEQ ID No: 2 of the present invention and the Arthrobacter-derived transaminase ATA117-Rd11 (US Pat. No. 8,293,507) is only 42%, with significant differences.
  • the present invention also relates to a recombinant vector constructed by the encoding gene of the transaminase mutant and a recombinant genetically engineered bacterium prepared by transforming the recombinant vector.
  • the recombinant genetically engineered bacterium is preferably prepared as follows: transaminase gene (or mutant gene) Connected with the expression vector pET28b to construct a heterologous expression recombinant plasmid pET28b-MbTA (or pET28b-MbTAmut1) containing the transaminase gene; the expression recombinant plasmid pET28b-MbTA (or pET28b-MbTAmut1) was transformed into Escherichia coli BL21 (DE3), A recombinant E. coli/pET28b-MbTA (or pET28b-MbTAmut1) containing the recombinant plasmid pET28b-
  • the present invention also provides an application of the transaminase mutant in the biocatalysis of a sitagliptin intermediate precursor ketone to synthesize a sitagliptin intermediate.
  • Wet cells obtained by fermentation and culture of engineering bacteria (preferably recombinant Escherichia coli) or pure enzymes extracted from wet cells after ultrasonication are used as biocatalysts, and the intermediate precursor ketone of sitagliptin ([1-piperidine-4- (2,4,5-trifluorophenyl)-1,3-dibutanone]) as substrate, dimethyl sulfoxide (DMSO) as cosolvent, pyridoxal phosphate as coenzyme, isopropylamine
  • a reaction system is formed with pH 8-9 triethanolamine buffer as a reaction medium, and the reaction is carried out under the conditions of a temperature of 30-45 °C (preferably 35 °C) and a stirring speed of 100-250 r/min (preferably 150 r/min
  • the reaction solution is separated and purified to obtain sitagliptin intermediate ((R)-3-amino-1-piperidine-4-(2,4,5-trifluorophenyl)- 1-butanone);
  • the amount of wet cells is 10-50g/L (preferably 50g/L)
  • the amount of pure enzyme is 0.01-1.0g/L (preferably 0.07g/L)
  • the substrate is added
  • the final concentration is 2-50g/L (preferably 20g/L)
  • the final concentration of dimethyl sulfoxide is 10-40% (v/v) (preferably 20%) by volume
  • the final concentration of pyridoxal phosphate is 0.5g/ L
  • isopropylamine was added to the final concentration of 10g/L.
  • the present invention also provides an application of the transaminase mutant in biocatalysis of latent chiral carbonyl compounds to synthesize sitagliptin ester intermediates.
  • (preferably recombinant Escherichia coli) wet cells obtained by fermentation and culture are biocatalysts, with latent chiral carbonyl compounds as substrates, dimethyl sulfoxide as cosolvent, pyridoxal phosphate as coenzyme, and isopropylamine as Co-substrate, using pH 8-9 triethanolamine buffer as the reaction medium to form a reaction system, under the conditions of temperature 25-35 °C (preferably 35 °C), stirring speed 100-250r/min (preferably 150r/min) Catalytic reaction, after the reaction is completed, the reaction solution is separated and purified to obtain sitagliptin ester intermediates; in the reaction system, the amount of wet bacteria is 10-100g/L (preferably 50g/L), and the substrate is added at the end.
  • the concentration is 2 ⁇ 60g/L (preferably 20g/L), the final concentration of dimethyl sulfoxide is 10-40% (preferably 20%), the final concentration of pyridoxal phosphate is 0.5g/L, and the final concentration of isopropylamine is added.
  • the substrate is one of the following: 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester, 3-carbonyl-4-(2,4,5 -Trifluorophenyl)-butyric acid propyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid isopropyl ester, 3-carbonyl-4-(2,4,5- Trifluorophenyl)-butyric acid ethyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid isobutyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl) Fluorophenyl)-benzyl butyrate.
  • the method for separating and purifying (R)-3-amino-1-piperidine-4-(2,4,5-trifluorophenyl)-1-butanone of the reaction solution of the present invention is as follows: after the reaction is completed, use concentrated hydrochloric acid (mass fraction is 36%-38%) The pH of the reaction solution is adjusted to 1.0-2.0, diatomaceous earth is added to adsorb cells, stirred for 10-30min, filtered to obtain filtrate a and filter residue a, and 1M hydrochloric acid (amount of The filtrate a and the filtrate b are combined and extracted once with dichloromethane to obtain organic Phase a and aqueous phase a, organic phase a was extracted with 1M hydrochloric acid to obtain organic phase b and aqueous phase b, combined aqueous phase a and aqueous phase b and adjusted pH to 12 with sodium hydroxide, then extracted with dichloromethane to obtain organic phase Phase c and water phase c, add dichlorome
  • the wet cells of the present invention are prepared as follows: the recombinant Escherichia coli containing the transaminase encoding gene or the transaminase mutant encoding gene is inoculated into LB liquid medium containing 50 ⁇ g/ml kanamycin, and cultured at 37° C.
  • the method for extracting pure enzyme from wet cells of the present invention after ultrasonication is as follows: the wet cells are resuspended in a binding buffer (50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazole), and then ultrasonically crushed.
  • a binding buffer 50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazole
  • Proteins whose amino acid sequences of the present invention are substituted, deleted or added with one or several amino acid residues and have amino acid sequences derived from aminotransferase activity with at least 95% identity belong to the protection scope of the present invention.
  • the protein composed of the amino acid sequence shown in SEQ ID No: 2 can be isolated and obtained from Mycobacterium (Mycolicibacterium), can also be isolated and obtained from the expression transformant that recombinantly expresses the protein, or can be obtained by artificial synthesis.
  • Mycobacterium Mycolicibacterium
  • the identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the art, preferably calculated by NCBI Blastp and Blastn software according to default parameters.
  • the nucleic acid sequences encoding the amino acid sequences of SEQ ID No: 2 and SEQ ID No 4 are not limited to SEQ ID No: 1 and SEQ ID No: 3.
  • the transaminase gene of the present invention may also be a homolog of a polynucleotide provided by appropriate introduction of substitutions, deletions, or insertions in SEQ ID No: 1, SEQ ID No: 3.
  • the invention also relates to the application of the transaminase gene in the preparation of recombinant transaminase, specifically: constructing a recombinant vector containing the transaminase gene (or transaminase mutant gene), transforming the recombinant vector into Escherichia coli, and obtaining a recombinant genetic engineering
  • the bacteria are induced and cultured, and the culture medium is separated to obtain bacterial cells containing recombinant transaminase, the crushed crude transaminase enzyme liquid and the purified transaminase enzyme (or transaminase mutant).
  • the catalysts of the invention include transaminase and its mutant pure enzymes, corresponding wet cells of recombinant genetically engineered bacteria, crude enzyme liquid, crude enzyme powder, pure enzyme liquid, pure enzyme powder and other forms.
  • transaminase of the present invention in the biocatalytic synthesis of sitagliptin intermediates, uses wet cells obtained by fermentation and culture of recombinant Escherichia coli containing transaminase-encoding genes as biocatalysts, and sitagliptin intermediates are used as biocatalysts.
  • the precursor ketone [1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone] was used as the substrate, and dimethyl sulfoxide (DMSO) was used as the auxiliary Solvent, pyridoxal phosphate as coenzyme, isopropylamine as cosubstrate, pH 8-9 triethanolamine buffer as reaction medium to form reaction system, under the conditions of temperature 30-45 °C, stirring speed 100-250r/min
  • the biocatalytic reaction is carried out under the following conditions, and after the reaction is completed, the reaction solution is separated and purified to obtain a sitagliptin intermediate (to obtain (R)-3-amino-1-piperidine-4-(2,4,5-trifluorobenzene) base)-1-butanone); in the reaction system, the amount of wet cells is 10-50 g/L (preferably 50 g/L), the final concentration of the substrate is 2-50 g/L, and the final volume of dimethyl sulfoxide
  • the beneficial effects of the present invention are mainly reflected in: the total yield of the reported asymmetric synthesis of sitagliptin and its intermediates is not high (generally lower than 50%), and the stereoselectivity is low. (The product e.e. value is generally lower than 90%), the metal catalyst is expensive, and the biocatalyst cannot directly use the sitagliptin precursor ketone as the substrate.
  • the present invention provides a transaminase mutant derived from Mycolicibacterium.
  • sitagliptin intermediate precursor ketone eg: 1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone
  • sitagliptin Ester Intermediate Carbonyl Substrate 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl) -Propyl butyrate, 3-carbonyl-4-(2,4,5-trifluorophenyl)-isopropyl butyrate, 3-carbonyl-4-(2,4,5-trifluorophenyl)- Ethyl butyrate, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid isobutyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butane acid benzyl ester) as substrate, isopropylamine as amino donor, pyr
  • Figure 1 is a schematic diagram of the reaction scheme for the biocatalytic synthesis of sitagliptin intermediates by transaminase mutants.
  • the total genomic DNA of the transaminase of Mycobacterium Mycolicibacterium was extracted with a nucleic acid rapid extractor, and the genomic DNA was used as a template. 2(GTAGCAGATATCTTCGA) was used for PCR amplification.
  • PCR reaction system total volume 50 ⁇ L: 5 ⁇ L of 10 ⁇ Pfu DNA Polymerase Buffer, 1 ⁇ L of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), 1 ⁇ L each of cloning primer 1 and primer 2 at a concentration of 50 ⁇ M, genomic DNA 1 ⁇ L, Pfu DNA Polymerase 1 ⁇ L, nucleic acid-free water 40 ⁇ L.
  • PCR reaction conditions pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30s, annealing at 65°C for 45s, extension at 72°C for 1 min, a total of 30 cycles, and a final extension at 72°C for 10 min.
  • the PCR reaction solution was detected by 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel, and base A was introduced into the 5' end of the fragment using Taq DNA polymerase.
  • the fragment was ligated with the pMD18-T vector under the action of T4 DNA ligase to obtain the cloned recombinant plasmid pMD18-T-MbTA.
  • the recombinant plasmid was transformed into Escherichia coli JM109, screened by the basket white spot screening system, randomly selected white clones for sequencing, and the sequencing results were analyzed by software.
  • the length of the nucleotide sequence amplified by primer 1 and primer 2 It is 1011bp (MbTA gene, its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2), and this sequence encodes a complete open reading frame.
  • Example 2 Construction of recombinant Escherichia coli BL21/pET28b-MbTA
  • Primer 3 (CCG GAATTC GGTATCGACACCGGTACCTC) and primer 4 (TTGGG ATCC GT ACTGGATAGCTTCGATCAGC) were designed according to the MbTA gene sequence in Example 1, and EcoR I and BamH I restriction sites were introduced into primer 3 and primer 4 respectively ( underscore). Under the initiation of primer 3 and primer 4, high-fidelity Pfu DNA polymerase was used for amplification, and the recombinant plasmid pMD18-T-MbTA was used as a template (obtained in Example 1) to obtain the MbTA gene sequence.
  • coli BL21(DE3) (Invitrogen) (42°C, 90s), spread on LB plates containing 50 ⁇ g/ml kanamycin resistance, and cultured at 37°C for 8- 12h, randomly picked clones and extracted plasmids for sequencing and identification, and screened to obtain recombinant Escherichia coli BL21(DE3)/pET28b-MbTA containing the expression recombinant plasmid pET28b-MbTA.
  • the recombinant Escherichia coli BL21(DE3)/pET28b-MbTA obtained in Example 2 was inoculated into LB liquid medium containing 50 ⁇ g/ml kanamycin resistance, cultured at 37° C.
  • the inoculation amount was inoculated into fresh LB liquid medium containing 50 ⁇ g/ml kanamycin resistance, cultivated at 37°C and 150 rpm until the OD 600 of the cells reached 0.6-0.8, and IPTG with a final concentration of 0.1 mM was added
  • centrifuge at 4°C and 5000rpm for 25min discard the supernatant, and collect the precipitate to obtain recombinant Escherichia coli BL21/pET28b-MbTA wet cells containing the expression recombinant plasmid.
  • the bacterial cells can be directly used as biocatalysts or used for protein purification.
  • the wet cells obtained in Example 3 were resuspended with binding buffer (50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazole), and then crushed by ultrasonic (under ice bath conditions, 240W for 10min, working for 2s) Pause for 2 s), centrifuge at 12,000 rpm for 40 min, incubate the supernatant with Ni affinity chromatography resin equilibrated with the above binding buffer, and then rinse with buffer (50 mM, pH 8.0 sodium phosphate buffer, containing 300 mM NaCl, 20 mM imidazole) Rinse until there is basically no impurity protein, then elute with elution buffer (50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 250mM imidazole) and collect the eluate to obtain the target protein.
  • binding buffer 50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 10m
  • Dialysis buffer 50mM, pH 8.0 sodium phosphate buffer
  • the cut-off solution was taken as transaminase enzyme solution.
  • the protein content was determined to be 1.8 mg/mL by the Coomassie brilliant blue method, and the enzyme solution (enzyme activity was about 150 U/mg) was diluted with 50 mM, pH 8.0 sodium phosphate buffer to a final concentration of 0.5 mg/mL. 80°C.
  • the amount of enzyme required for transaminase MbTA to catalyze 1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone to produce 1 ⁇ mol of product per hour is one unit of enzyme activity. It is represented by U.
  • PCR reaction system total volume 50 ⁇ L: 5 ⁇ L of 10 ⁇ Pfu DNA Polymerase Buffer, 1 ⁇ L of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), 0.5 ⁇ M each of cloning primer 1 and primer 2 at a concentration of 50 ⁇ M, plasmid Template 0.8ng/ ⁇ L, Taq DNA Polymerase 2.5U, MnCl 2 0.2mM, deionized water to make up 50 ⁇ L.
  • a BioRad PCR instrument was used.
  • PCR reaction conditions pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 45 s, extension at 72°C for 1 min, a total of 30 cycles, and a final extension at 72°C for 10 min.
  • mega-primer PCR was performed using the product as a primer and the plasmid pET28b-MbTA constructed in Example 2 as a template to obtain a mega-primer PCR product (ie, mutant library 1).
  • PCR system megaprimer 10ng/ ⁇ L, plasmid template 1ng/ ⁇ L, Pfu DNA Polymerase 2.5U.
  • PCR reaction conditions A tail was removed at 72°C for 5 min, pre-denaturation at 96°C for 2 min, denaturation at 96°C for 30s, annealing at 60°C for 45s, extension at 72°C for 4 min, a total of 25 cycles, and a final extension at 72°C for 10 min.
  • the gene library 1 of Example 5 was transformed into competent cells of Escherichia coli BL21 (DE3), the transformation conditions were 42°C, heat shock for 90 seconds, and single clones were picked on LB resistant plates containing 50 ⁇ g/ml kanamycin 9501 cells were respectively inoculated into LB medium containing 50 ⁇ g/ml kanamycin for induction expression.
  • the induction conditions were as in Example 3, and 9501 recombinant Escherichia coli wet cells containing mutant genes were obtained, namely, mutant 1 wet cells. Bacteria.
  • the biotransformation screening of the intermediate precursor ketone of sitagliptin at a low concentration of 20 g/L was carried out.
  • the final concentration of the catalytic system (15 ml) and the catalytic conditions were as follows: Mutant 1 wet cell 0.75 g , pH8-8.5 triethanolamine buffer, 20g/L substrate sitagliptin intermediate precursor ketone 1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone , the final concentration of DMSO was 10% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L.
  • Reaction conditions temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as blank control, and the wet cells of Escherichia coli BL21/pET28b containing empty vector were used instead of the wet cells of mutant 1 as negative control.
  • sampling was carried out for HPLC detection (conditions were the same as in Example 15) (50:50 acetonitrile: water, 10mM ammonium acetate, 0.8ml/min flow rate, 205nm detection wavelength), and the conversion rate of the substrate was selected from 6503 proteins
  • the highest mutant, pET28b-MbTAmut1 had a conversion rate of 96% and an e.e. value of 99%.
  • mutant pET28b-MbTAmut1 The nucleotide sequence and amino acid sequence of the mutant pET28b-MbTAmut1 are shown in SEQ ID No: 3 and SEQ ID No: 4 in the sequence listing.
  • Mutant 1 replaces tyrosine at position 74 shown in SEQ ID NO: 2 with proline, glutamic acid at position 228 is substituted with aspartic acid, and leucine at position 254 is substituted with alanine Acid, methionine at position 290 was substituted with threonine.
  • the wet cell of mutant 1 was obtained by the method of Example 3, and the pure enzyme of mutant 1 was obtained by the method of Example 4 (enzyme activity was about 150 U/mg).
  • Example 7 Application of recombinant aminotransferase MbTA in the preparation of sitagliptin intermediate (R)-3-amino-1-piperidine-4-(2,4,5-trifluorophenyl)-1-butanone
  • the recombinant Escherichia coli BL21/pET28b-MbTA wet cell containing the expression plasmid obtained by the method of Example 3 or the pure MbTA enzyme obtained by the method of Example 4 was used as a biological catalyst, and the intermediate precursor ketone of sitagliptin [1- Using piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone] as substrate, biocatalytic reaction to synthesize sitagliptin intermediate (R)-3-amino-1 - Piperidine-4-(2,4,5-trifluorophenyl)-1-butanone.
  • the final concentration composition and catalytic conditions of the low substrate concentration catalytic system (15ml) are as follows: wet cell 0.75g or MbTA pure enzyme 1mg, pH 8-8.5 triethanolamine buffer, substrate sitagliptin precursor ketone 2g/L, The final concentration of DMSO was 10% (v/v), pyridoxal phosphate 0.5 g/L, and isopropylamine 10 g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTA.
  • samples were taken for HPLC detection the conditions were the same as those in Example 15
  • the substrate conversion rate was 2.3%
  • the e.e. value was 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • the final concentration composition and catalytic conditions of the high substrate concentration catalytic system (15ml) are as follows: wet cell 0.75g or MbTA pure enzyme 1mg, pH 8-8.5 triethanolamine buffer, substrate sitagliptin precursor ketone 50g/L, The final concentration of DMSO was 40% (v/v), pyridoxal phosphate 0.5 g/L, and isopropylamine 10 g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTA.
  • sampling was carried out for HPLC detection (conditions were the same as in Example 15), and the substrate conversion rate was less than 1%.
  • Escherichia coli cells without transaminase MbTA as a catalyst, the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression recombinant plasmid obtained by the method of Example 3 in Example 6 or the pure enzyme of MbTA mutant 1 obtained by the method of Example 4 was used as a biocatalyst, and sitagliptin
  • the intermediate precursor ketone [1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone] was used as the substrate, and the biocatalytic reaction was carried out to synthesize sitagliptin intermediate ( R)-3-Amino-1-piperidine-4-(2,4,5-trifluorophenyl)-1-butanone.
  • the final concentration composition and catalytic conditions of the low substrate concentration catalytic system (15ml) are as follows: wet cell 0.75g or MbTA mutant 1 pure enzyme 1mg, pH 8-8.5 triethanolamine buffer, 2g/L substrate before sitagliptin Body ketones, the final concentration of DMSO is 10% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (conditions were the same as those in Example 15).
  • the substrate concentration of the reaction system was 2 g/L
  • the conversion rate of the substrate was 95.5%
  • the e.e. value was 99%.
  • Escherichia coli cells without transaminase MbTA mut1 as a catalyst, the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • the final concentration composition and catalytic conditions of the high substrate concentration catalytic system (15ml) are as follows: wet cell 0.75g or MbTA mutant 1 pure enzyme 1mg, pH 8-8.5 triethanolamine buffer, 50g/L substrate sitagliptin intermediate Precursor ketone, the final concentration of DMSO is 40% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (conditions were the same as those in Example 15).
  • the conversion of the substrate was 58% with an e.e. value of 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Examples 9-14 introduce the application of recombinant transaminase MbTA mutant 1 in the preparation of sitagliptin ester intermediates
  • Example 9 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression plasmid obtained by the method of Example 3 in Example 6 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl )-butyric acid methyl ester was used as the substrate, and (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester was prepared by biocatalytic reaction.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-methyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (conditions were the same as in Example 15), and 0.12mol of the substrate (3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester) was obtained about 0.12mol of product (R)-3-Amino-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester (28.3 g), substrate conversion 90%, e.e. value 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Example 10 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid ethyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression plasmid obtained by the method of Example 3 in Example 6 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid ethyl ester was prepared by biocatalytic reaction using )-butyric acid ethyl ester as substrate.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-ethyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (the conditions were the same as in Example 15), the conversion rate of the substrate was 90%, and the e.e. value was 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Example 11 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid propyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression plasmid obtained by the method of Example 3 in Example 6 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl )-butyric acid propyl ester was used as the substrate, and (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid propyl ester was prepared by biocatalytic reaction.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-propyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (the conditions were the same as those in Example 15), the conversion rate of the substrate was 88%, and the e.e. value was 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Example 12 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid isopropyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbAmut1 wet cell containing the expression recombinant plasmid obtained in Example 6 by the method of Example 3 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl )-isopropyl butyrate was used as substrate to prepare (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid isopropyl ester by biocatalytic reaction.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-isopropyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L.
  • Reaction conditions temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (the conditions were the same as those in Example 15), the conversion rate of the substrate was 84%, and the e.e. value was 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Example 13 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid isobutyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression plasmid obtained by the method of Example 3 in Example 6 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid isobutyl ester was prepared by biocatalytic reaction using )-isobutyl butyrate as substrate.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-isobutyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L.
  • Reaction conditions temperature 35°C, stirring speed 150r/min, reaction time 36h. The enzyme was verified to be able to transaminate with high selectivity (e.e.
  • Example 14 Application of recombinant aminotransferase MbTA mutant 1 in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid benzyl ester
  • the recombinant Escherichia coli BL21/pET28b-MbTAmut1 wet cell containing the expression plasmid obtained by the method of Example 3 in Example 6 was used as a biocatalyst, and 3-carbonyl-4-(2,4,5-trifluorophenyl (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid benzyl ester is prepared by biocatalytic reaction as substrate.
  • the final concentration composition of the catalytic system (15ml) and the catalytic conditions are as follows: wet cell 0.75g, pH 8-8.5 triethanolamine buffer, 20g/L substrate 3-carbonyl-4-(2,4,5-trifluorophenyl) )-benzyl butyrate, the final concentration of DMSO is 20% (v/v), pyridoxal phosphate 0.5g/L, and isopropylamine 10g/L. Reaction conditions: temperature 35°C, stirring speed 150r/min, reaction time 36h.
  • the reaction solution added with sterile body was used as a blank control, and Escherichia coli BL21/pET28b wet cells were used as a negative control instead of the above recombinant Escherichia coli BL21/pET28b-MbTAmut1.
  • sampling was carried out for HPLC detection (the conditions were the same as those in Example 15), the conversion rate of the substrate was 84%, and the e.e. value was 99%.
  • the catalytic reaction was carried out under the same conditions, and the substrate conversion rate was less than 0.01%.
  • Example 15 Liquid phase detection method of sitagliptin intermediate precursor ketone, sitagliptin (R) type intermediate and (S) type enantiomer of sitagliptin.
  • HPLC instruments Shimadzu LC-16 system-SPD-16 UV detector and Hitachi 8DD-0801 system-1410 UV detector.
  • the retention time of sitagliptin intermediate precursor ketone was 4.0 min.
  • the retention times of sitagliptin intermediates were 2.8 min, respectively.
  • the retention time of sitagliptin intermediate precursor ketone and sitagliptin intermediate (R) enantiomer is about 10 and 5 min.
  • the retention time of the (S) enantiomer of the sitagliptin intermediate was 9.5 min.
  • the liquid phase is Shimadzu LC-20AD system-SPD20A detector
  • CR is the peak area of sitagliptin and Cs is the peak area of the S-enantiomer.
  • Example 16 Separation and purification from the reaction system to obtain high-purity sitagliptin intermediate (R)-3-amino-1-piperidine-4-(2,4,5-trifluorophenyl)-1-butane ketone

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种转氨酶突变体及其制备西他列汀中间体的应用，所述突变体是SEQ ID NO：2所示氨基酸序列第74位的酪氨酸取代为脯氨酸、第228位的谷氨酸取代为天冬氨酸、第254位的亮氨酸取代为丙氨酸、第290位的蛋氨酸取代为苏氨酸获得的。以含有该转氨酶突变体编码基因的工程菌经发酵培养获得的湿菌体或纯酶作为生物催化剂，以西他列汀中间体前体酮或潜手性羰基化合物为底物，制备西他列汀中间体或西他列汀酯类中间体，总收率约82％，产物e.e.值达到99％。

Description

转氨酶突变体及其在制备西他列汀中间体中的应用 (一)技术领域
本发明涉及生物化工技术领域，具体地说，是转氨酶及其突变体酶在制备光学纯西他列汀中间体中的方法，包括转氨酶、突变体、编码基因、含有该基因的重组载体、该重组载体转化得到的重组基因工程菌和重组酶、以及应用。
(二)背景技术
西他列汀(sitagliptin)是由美国Merck公司和Codexis公司研制和开发，是首个获得FDA批准用于治疗Ⅱ型糖尿病的二肽基肽酶-IV(DPP-IV)抑制剂。西他列汀能够血糖依赖性地增加胰岛素的分泌，降糖作用较为适中，不会引发低血糖的发生，并且无增加体重、恶心、呕吐等副作用。西他列汀的商品名为捷诺维(Januvia)，目前已在全世界70多个国家批准使用，是国际上药物销售额前20强的药物。
西他列汀及其中间体可由化学-酶法相结合制得，逐步成为合成手性医药化学品及其中间体的首选方案。对于化学-酶法而言关键就是要获得一种转氨酶，其能够催化不对称转氨反应得到光学纯的西他列汀中间体。美国专利US8293507公开了Codexis公司对节杆菌来源的转氨酶(ATA117)进行改造得到的生物催化剂，转氨得到的产物ee值达到99％。
但是目前，对于R-型选择性转氨的天然转氨酶报道很少，且这些转氨酶催化的底物谱较窄，往往是为特定反应筛选的最适生物催化剂，因此大大限制了其应用范围，导致能够用于合成西他列汀中间的酶寥寥无几。随着定向进化技术的发展，蛋白质工程越来越多地被用于改造酶的底物特异性，筛选具有较宽底物谱的新型转氨酶，研究其可以高效高选择性催化的手性药物及其中间体，不仅可以拓宽其应用范围，提升其应用潜力，也为实现工业化生产奠定基础。
(三)发明内容
本发明针对现有生产西他列汀中间体工艺中存在的缺陷(立体选择性不佳、催化剂价格昂贵、溶剂难以回收等问题)，提供了一种转氨酶突变体、编码基因、重组载体、重组基因工程菌，以及在不对称转氨得到西他列汀或西他列汀酯类中间体的应用，该方法原料转化率高、生产成本低、收率高。
本发明采用的技术方案是：
本发明提供一种转氨酶突变体，所述突变体是将SEQ ID NO：2所示氨基酸序列第74位的酪氨酸取代为脯氨酸、第228位的谷氨酸取代为天冬氨酸、第254位的亮氨酸取代为丙氨酸、第290位的蛋氨酸取代为苏氨酸获得的，所述转氨酶突变体的氨基酸序列为SEQ IDNO：4，编码基因的核苷酸序列为SEQ ID NO：3所示。
本发明所述转氨酶突变体是将源于分枝杆菌(Mycolici bacterrium)的转氨酶进行突变获得的，所述转氨酶氨基酸序列为SEQ ID NO.2所示，核苷酸序列为SEQ ID NO：1所示。本发明SEQ ID No:2所示的转氨酶和节杆菌来源的转氨酶ATA117-Rd11(美国专利US8293507)的氨基酸序列一致性仅为42％，具有显著差异性。
本发明还涉及所述转氨酶突变体的编码基因构建的重组载体及所述重组载体转化制备的重组基因工程菌，所述重组基因工程菌优选按如下方法制备：将转氨酶基因(或者突变体基因)同表达载体pET28b连接，构建了含有转氨酶基因的异源表达重组质粒pET28b-MbTA(或pET28b-MbTAmut1)；将表达重组质粒pET28b-MbTA(或pET28b-MbTAmut1)转化至大肠杆菌BL21(DE3)中，获得含有重组质粒pET28b-MbTA的重组大肠杆菌/pET28b-MbTA(或pET28b-MbTAmut1)。
本发明还提供一种所述转氨酶突变体在生物催化西他列汀中间体前体酮合成西他列汀中间体中的应用，所述的应用为：以含有转氨酶突变体编码基因的重组基因工程菌(优选重组大肠杆菌)经发酵培养获得的湿菌体或湿菌体经超声破碎后提取的纯酶作为生物催化剂，以西他列汀中间体前体酮([1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮])为底物，以二甲基亚砜(DMSO)为助溶剂、以磷酸吡哆醛为辅酶、以异丙胺为辅底物，以pH 8-9三乙醇胺缓冲液为反应介质构成反应体系，在温度30-45℃(优选35℃)、搅拌速度100-250r/min(优选150r/min)的条件下进行生物催化反应，反应结束后，将反应液分离纯化，获得西他列汀中间体((R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮)；所述反应体系中，湿菌体用量为10～50g/L(优选50g/L)，纯酶用量为0.01-1.0g/L(优选0.07g/L)，底物加入终浓度为2～50g/L(优选20g/L)，二甲基亚砜体积加入终浓度为10-40％(v/v)(优选20％)，磷酸吡哆醛加入终浓度0.5g/L，异丙胺加入终浓度10g/L。
本发明还提供一种所述转氨酶突变体在生物催化潜手性羰基化合物合成西他列汀酯类中间体中的应用，所述的应用为：以含有转氨酶突变体编码基因的重组基因工程菌(优选重组大肠杆菌)经发酵培养获得的湿菌体为生物催化剂，以潜手性羰基化合物为底物，以二甲基亚砜为助溶剂、以磷酸吡哆醛为辅酶、以异丙胺为辅底物，以pH 8-9三乙醇胺缓冲液为反应介质构成反应体系，在温度25-35℃(优选35℃)、搅 拌速度100-250r/min(优选150r/min)的条件下进行生物催化反应，反应结束后，将反应液分离纯化，获得西他列汀酯类中间体；所述反应体系中，湿菌体用量为10～100g/L(优选50g/L)，底物加入终浓度为2～60g/L(优选20g/L)，二甲基亚砜体积加入终浓度为10-40％(优选20％)，磷酸吡哆醛加入终浓度0.5g/L，异丙胺加入终浓度10g/L；所述底物为下列之一：3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸乙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丁酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸卞酯。
本发明所述反应液分离纯化(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮方法为：反应结束后，用浓盐酸(质量分数为36％-38％)将反应液pH调至1.0-2.0，加入硅藻土吸附细胞，搅拌10-30min，过滤，获得滤液a和滤渣a，向滤渣a中加入1M盐酸(用量以能浸没滤渣a即可，优选与反应液体积比1.5:1)，搅拌10-30min，抽滤，获得滤液b和滤渣b；合并滤液a和滤液b，用二氯甲烷萃取一次，获得有机相a和水相a，有机相a用1M盐酸萃取，获得有机相b和水相b，合并水相a和水相b并用氢氧化钠调节pH至12，再用二氯甲烷萃取，获得有机相c和水相c，水相c中再加入二氯甲烷萃取，获得有机相d和水相d，合并有机相c和有机相d并用饱和氯化钠水洗二次，加入无水硫酸钠干燥，抽滤去除硫酸钠，45℃下旋转蒸发，得到西他列汀中间体；所述硅藻土用量以反应液体积计为0.18g/mL。以400mL反应液为例：I.用浓盐酸(质量分数为36％-38％)将反应液pH调至1.5，加入72g硅藻土吸附细胞，搅拌20min，抽滤，获得滤液a和滤渣a，向滤渣a中加入600ml的1M盐酸，搅拌20min，抽滤，获得滤液b和滤渣b；II.合并滤液a和滤液b总计共约1.0L，以500mL二氯甲烷萃取一次，获得水相a和有机相a，有机相a用100ml的1M盐酸萃取，获得水相b和有机相b，合并水相a和水相b并用氢氧化钠调节pH至12再加入1.2L二氯甲烷萃取，获得有机相c和水相c，水相c中再加入800mL二氯甲烷萃取，获得有机相d和水相d，合并有机相c和有机相d；III.有机相c和有机相d用饱和氯化钠(36g/L)水洗二次，加入无水硫酸钠干燥，抽滤去除硫酸钠，45℃下旋转蒸发，最后得到高纯度西他列汀中间体白色粉末23.1g，(白色粉末状)纯化收率95％，西他列汀中间体纯度大于99％。本发明所述滤液a-滤液c，滤渣a-滤渣c，有机相a-有机相d，水相a-水相d中的字母均无含义，为了便于表述而命名。
本发明所述湿菌体按如下方法制备：将含有转氨酶编码基因或转氨酶突变体编码 基因的重组大肠杆菌接种至含有50μg/ml卡那霉素的LB液体培养基，37℃，200rpm下培养12h，再以体积浓度1％接种量接种至新鲜的含有50μg/ml卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD 600达0.6-0.8，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、5000rpm离心20min，弃去上清液，收集沉淀，即获得所述的湿菌体。
本发明所述湿菌体经超声破碎后提取纯酶的方法为：将湿菌体以结合缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，10mM咪唑)重悬后，经超声破碎(冰浴条件下，240W破碎10min，工作2s暂停2s)，12000rpm离心40min，上清与经上述结合缓冲液平衡过的Ni亲和层析树脂孵育后，再用冲洗缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，20mM咪唑)冲洗至基本无杂蛋白，随后以洗脱缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，250mM咪唑)洗脱并收集洗脱液获得目的蛋白，电泳鉴定纯度后合并目的蛋白并以透析缓冲液(50mM，pH 8.0磷酸钠缓冲液)透析48h(透析袋分子截留量14KD)，取截留液，即为转氨酶纯酶。
本发明所述氨基酸序列经过取代、缺失或添加一个或几个氨基酸残基且具有转氨酶活性的衍生的氨基酸序列具有至少95％同一性的蛋白质，均属于本发明的保护范围。SEQID No:2所示的氨基酸序列组成的蛋白质可以从分枝杆菌(Mycolicibacterium)中分离获得，也可以从重组表达该蛋白质的表达转化体中分离获得，也可以人工合成获得。两条氨基酸序列或两条核苷酸序列之间的同一性均可通过本领域常用的算法得到，优选采用NCBI Blastp和Blastn软件根据默认参数计算得到。
本领域技术人员所知，由于密码子的兼并性，编码SEQ ID No:2、SEQ ID No4的氨基酸序列的核酸序列不仅仅局限于SEQ ID No:1、SEQ ID No:3。本发明的转氨酶基因还可以是通过在SEQ ID No:1、SEQ ID No:3适当引入替换、缺失、或插入来提供一个多聚核苷酸的同系物。
本发明还涉及转氨酶基因在制备重组转氨酶中的应用，具体为：构建含有所述转氨酶基因(或转氨酶突变体基因)的重组载体，将所述重组载体转化至大肠杆菌中，获得的重组基因工程菌进行诱导培养，培养液分离得到含有重组转氨酶的菌体细胞，破碎后的转氨酶粗酶液以及纯化后转氨酶纯酶(或转氨酶突变体)。
本发明所述催化剂包括转氨酶及其突变体纯酶、相应的重组基因工程菌湿菌体、粗酶液、粗酶粉、纯酶液、纯酶粉等其他形态。
本发明所述转氨酶在生物催化合成西他列汀中间体中的应用，所述的应用以含有 转氨酶编码基因的重组大肠杆菌经发酵培养获得的湿菌体作为生物催化剂，以西他列汀中间体的前体酮([1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮])为底物，以二甲基亚砜(DMSO)为助溶剂、以磷酸吡哆醛为辅酶、以异丙胺为辅底物，以pH 8-9三乙醇胺缓冲液为反应介质构成反应体系，在温度30-45℃、搅拌速度100-250r/min的条件下进行生物催化反应，反应结束后，将反应液分离纯化，获得西他列汀中间体(得(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮)；所述反应体系中，湿菌体用量为10～50g/L(优选50g/L)，底物终浓度为2-50g/L，二甲基亚砜体积终浓度为10-20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。
与现有技术相比，本发明的有益效果主要体现在：针对己报道的不对称合成西他列汀及其中间体的总收率不高(一般低于50％)，立体选择性偏低(产物e.e.值普遍低于90％)、金属催化剂昂贵、生物催化剂不能直接以西他列汀前体酮为底物的问题，本发明提供了来源于一种分枝杆菌(Mycolicibacterium)的转氨酶突变体(生物催化剂)，以西他列汀中间体前体酮(如：1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮)或西他列汀酯类中间体羰基底物(3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸乙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丁酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸卞酯)为底物，同时异丙胺为氨基供体，磷酸吡多醛为辅酶，进行生物催化反应、分离纯化制备高光学纯度的西他列汀中间体或西他列汀酯类中间体，该方法的总收率达到82％(包括转化得率及分离纯化收率)，产物e.e.值达到99％(立体选择性高)。
(四)附图说明
图1为转氨酶突变体生物催化合成西他列汀中间体的反应式示意图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：转氨酶基因MbTA的扩增
根据Genbank收录的来自分枝杆菌(Mycolicibacterium)的转氨酶基因测序信息为依据，用核酸快速提取仪提取分枝杆菌Mycolicibacterium的转氨酶总基因组DNA，以该基因组DNA为模板，在引物1(ATGGGCATCGATACC)、引物2(GTAGCAGATATCTTCGA)的作用下进行PCR扩增。PCR反应体系(总体积50μL)：10×Pfu DNA Polymerase Buffer 5μL，10mM dNTP mixture(dATP、dCTP、dGTP和dTTP 各2.5mM)1μL，浓度均为50μM的克隆引物1、引物2各1μL，基因组DNA 1μL，Pfu DNA Polymerase 1μL，无核酸水40μL。
采用BioRad的PCR仪，PCR反应条件：预变性95℃5min，95℃变性30s，65℃退火45s，72℃延伸1min，共30个循环，最后72℃延伸10min。
PCR反应液用0.9％琼脂糖凝胶电泳检测并切胶回收纯化该片段，利用Taq DNA聚合酶向片段5’端引入碱基A。在T4DNA连接酶作用下将该片段同pMD18-T载体进行连接，得到克隆重组质粒pMD18-T-MbTA。将该重组质粒转化至大肠杆菌JM109中，利用篮白斑筛选系统进行筛选，随机挑取白色克隆测序，利用软件分析测序结果，结果表明：经引物1和引物2扩增到的核苷酸序列长度为1011bp(MbTA基因，其核苷酸序列如SEQ ID NO：1所示，编码蛋白的氨基酸序列为SEQ ID NO：2所示)，该序列编码一个完整的开放阅读框。
实施例2：重组大肠杆菌BL21/pET28b-MbTA的构建
根据实施例1中MbTA基因序列设计引物3(CCG GAATTC GGTATCGACACCGGTACCTC)、引物4(TTGGG ATCC  GTACTGGATAGCTTCGATCAGC))，并分别在引物3和引物4中引入了EcoR I和BamH I限制性酶切位点(下划线标记)。在引物3和引物4的引发下，利用高保真Pfu DNA聚合酶进行扩增，以重组质粒pMD18-T-MbTA为模板(实施例1中获得)，获MbTA基因序列，测序后利用EcoR I和BamH I限制性内切酶(TaKaRa)对扩增片段进行处理，并利用T4DNA连接酶(TaKaRa)将该片段同用相同的限制性内切酶处理的商业化载体pET28b(Invitrogen)进行连接，构建表达载体pET28b-MbTA。将构建的表达载体pET28b-MbTA转化至大肠杆菌BL21(DE3)(Invitrogen)中(42℃，90s)，涂布于含有50μg/ml卡那霉素抗性的LB平板，37℃下培养8-12h，随机挑取克隆抽提质粒进行测序鉴定，筛选获得含有表达重组质粒pET28b-MbTA的重组大肠杆菌BL21(DE3)/pET28b-MbTA。
实施例3：转氨酶(MbTA)的诱导表达
将实施例2获得的重组大肠杆菌BL21(DE3)/pET28b-MbTA接种至含有50μg/ml卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/ml卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD 600达0.6-0.8，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、5000rpm离心25min，弃去上清液，收集沉淀，即获得含有表达重组质粒的重组 大肠杆菌BL21/pET28b-MbTA湿菌体。该菌体可直接作为生物催化剂或者用于蛋白纯化。
实施例4：转氨酶(MbTA)的分离纯化
将实施例3中获得的湿菌体以结合缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，10mM咪唑)重悬后，经超声破碎(冰浴条件下，240W破碎10min，工作2s暂停2s)，12000rpm离心40min，上清与经上述结合缓冲液平衡过的Ni亲和层析树脂孵育后，再用冲洗缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，20mM咪唑)冲洗至基本无杂蛋白，随后以洗脱缓冲液(50mM，pH 8.0磷酸钠缓冲液，含300mM NaCl，250mM咪唑)洗脱并收集洗脱液获得目的蛋白，电泳鉴定纯度后合并目的蛋白并以透析缓冲液(50mM，pH 8.0磷酸钠缓冲液)透析48h(透析袋分子截留量14KD)，取截留液，即为转氨酶酶液。采用考马斯亮蓝法测定蛋白含量为1.8mg/mL，将酶液(酶活约150U/mg)用50mM，pH 8.0磷酸钠缓冲液稀释至终浓度为0.5mg/mL分装，冻存于-80℃。
转氨酶MbTA每小时催化底物1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮生成1μmol产物所需要的酶量即为1个酶活力单位，用U表示。
实施例5：MbTA基因突变文库的建立
以实施例2中构建质粒pET28b-MbTA为模板，进行易错PCR。在引物1(ATGGGCATCGATACC)、引物2(GTAGCAGATATCTTCGA)的作用下进行易错PCR。PCR反应体系(总体积50μL)：10×Pfu DNA Polymerase Buffer 5μL，10mM dNTP mixture(dATP、dCTP、dGTP和dTTP各2.5mM)1μL，浓度均为50μM的克隆引物1、引物2各0.5μM，质粒模板0.8ng/μL，Taq DNA Polymerase 2.5U，MnCl 2 0.2mM，去离子水补足50μL。采用BioRad的PCR仪，PCR反应条件：预变性95℃5min，95℃变性30s，65℃退火45s，72℃延伸1min，共30个循环，最后72℃延伸10min。纯化易错PCR产物后，以该产物作为引物、实施例2中构建质粒pET28b-MbTA为模板进行大引物PCR，获得大引物PCR产物(即突变体文库1)。PCR体系：大引物10ng/μL，质粒模板1ng/μL，Pfu DNA Polymerase 2.5U。PCR反应条件：去A尾72℃5min，预变性96℃2min，96℃变性30s，60℃退火45s，72℃延伸4min，共25个循环，最后72℃延伸10min。
实施例6：MbTA基因突变文库1的筛选获得突变体1
将实施例5基因文库1转入大肠杆菌BL21(DE3)的感受态细胞中，转化条件42℃， 热击90秒，在含有50μg/ml卡那霉素的LB抗性平板上挑取单克隆9501个，分别接种于含有50μg/ml卡那霉素的LB培养基中进行诱导表达，诱导条件如实施例3，获得9501个含有突变体基因的重组大肠杆菌湿菌体，即突变体1湿菌体。
得到含有突变蛋白的大肠杆菌后，对20g/L低浓度西他列汀中间体前体酮进行生物转化筛选，催化体系(15ml)终浓度组成及催化条件如下：突变体1湿菌体0.75g，pH8-8.5三乙醇胺缓冲液，20g/L底物西他列汀中间体前体酮1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮，DMSO终浓度为10％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以含空载体的大肠杆菌BL21/pET28b湿菌体代替上述突变体1湿菌体作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)(50:50乙腈：水，10mM乙酸铵，0.8ml/min流速，205nm检测波长)，从6503个蛋白中挑选出底物的转化率最高的一个突变体pET28b-MbTAmut1，转化率为96％，e.e.值99％。突变体pET28b-MbTAmut1的核苷酸序列与氨基酸序列如序列表中的SEQ ID No:3和SEQ ID No:4所示。突变体1是将SEQ ID NO：2所示第74位的酪氨酸取代为脯氨酸、第228位的谷氨酸取代为天冬氨酸、第254位的亮氨酸取代为丙氨酸、第290位的蛋氨酸取代为苏氨酸。
采用实施例3方法获得突变体1湿菌体，采用实施例4方法获得突变体1纯酶(酶活约150U/mg)。
实施例7：重组转氨酶MbTA在制备西他列汀中间体(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮的应用
以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTA湿菌体或通过实施例4的方法得到MbTA纯酶作为生物催化剂，以西他列汀中间体前体酮[1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮]为底物，进行生物催化反应合成西他列汀中间体(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮。
低底物浓度催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g或MbTA纯酶1mg，pH 8-8.5三乙醇胺缓冲液，底物西他列汀前体酮2g/L，DMSO终浓度为10％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTA作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物转化率为2.3％，e.e.值99％。 以不含转氨酶MbTA的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
高底物浓度催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g或MbTA纯酶1mg，pH 8-8.5三乙醇胺缓冲液，底物西他列汀前体酮50g/L，DMSO终浓度为40％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTA作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物转化率小于1％。以不含转氨酶MbTA的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例8：重组转氨酶MbTA突变体1在制备西他列汀中间体中(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTAmut1湿菌体或通过实施例4的方法得到MbTA突变体1纯酶作为生物催化剂，以西他列汀中间体前体酮[1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮]为底物，进行生物催化反应合成西他列汀中间体(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮。
低底物浓度催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g或MbTA突变体1纯酶1mg，pH 8-8.5三乙醇胺缓冲液，2g/L底物西他列汀前体酮，DMSO终浓度为10％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)。反应体系的底物浓度为2g/L时，底物的转化率为95.5％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
高底物浓度催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g或MbTA突变体1纯酶1mg，pH 8-8.5三乙醇胺缓冲液，50g/L底物西他列汀中间体前体酮，DMSO终浓度为40％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)。底物的转化率 为58％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
进一步，实施例9-14介绍了重组转氨酶MbTA突变体1在制备西他列汀酯类中间体中的应用
实施例9：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸甲酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸甲酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，0.12mo1底物(3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯)约得到0.12mol产物(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸甲酯(28.3g)，底物的转化率为90％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例10：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸乙酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸乙酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸乙酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸乙酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物的转化率为90％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化 反应，底物转化率小于0.01％。
实施例11：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸丙酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸丙酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸丙酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸丙酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物的转化率为88％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例12：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸异丙酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸异丙酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸异丙酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸异丙酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTA mut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物的转化率为84％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例13：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸异丁酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌 BL21/pET28b-MbTAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸异丁酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸异丁酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸异丁酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。经验证该酶能够高选择性(e.e.值；99％)转氨生成(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸异丁酯。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物的转化率为86％，e.e.99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例14：重组转氨酶MbTA突变体1在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸卞酯中的应用
将实施例6中以实施例3方法获得的含有表达重组质粒的重组大肠杆菌BL21/pET28b-MbTAmut1湿菌体作为生物催化剂，以3-羰基-4-(2,4,5-三氟苯基)-丁酸卞酯为底物，进行生物催化反应制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸卞酯。
催化体系(15ml)终浓度组成及催化条件如下：湿菌体0.75g，pH 8-8.5三乙醇胺缓冲液，20g/L底物3-羰基-4-(2,4,5-三氟苯基)-丁酸卞酯，DMSO终浓度为20％(v/v)，磷酸吡哆醛0.5g/L，异丙胺10g/L。反应条件：温度35℃、搅拌速度150r/min，反应时间36h。同样条件下，以无菌体加入的反应液作为空白对照，以大肠杆菌BL21/pET28b湿菌体代替上述重组大肠杆菌BL21/pET28b-MbTAmut1作为阴性对照。反应结束后，取样进行HPLC检测(条件同实施例15)，底物的转化率为84％，e.e.值99％。以不含转氨酶MbTA mut1的大肠杆菌细胞为催化剂，在相同条件下进行催化反应，底物转化率小于0.01％。
实施例15：西他列汀中间体前体酮、西他列汀(R)型中间体以及西他列汀的(S)型对映体的液相检测方法。
高效液相色谱仪器：Shimadzu LC-16系统-SPD-16紫外检测器和Hitachi 8DD-0801系统-1410紫外检测器。
检测转化率是色谱柱为ZORBAX Eclipse XDB-C18(4.6mm×250mm，5μm)，流动相：水：乙腈＝50:50(v/v)，水相加乙酸铵10mM，流速0.8mL/min，柱温40℃，检 测波长：205nm。西他列汀中间体前体酮的保留时间为4.0min。西他列汀中间体的保留时间分别为2.8min。
检测e.e.手性色谱柱为Chiralpak AD-H(150×4.6mm,5μm)，流动相为乙醇/正庚烷/二乙胺＝60:40:0.1(v/v/v)，流速0.8mL/min，柱温35℃，检测波长：205nm。西他列汀中间体前体酮和西他列汀中间体(R)型对映体的保留时间为10和5min左右。西他列汀中间体的(S)型对映体的保留时间为9.5min。(液相是Shimadzu LC-20AD系统-SPD20A检测器)
产物e.e. p计算公式：
e.e. p＝(C R-C S)/(C R+C S)×100％
C R为西他列汀的峰面积，Cs为S-对映体的峰面积。
实施例16：从反应体系中分离纯化得到高纯度西他列汀中间体(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮
取实施例8中低底物浓度催化体系的400mL反应液，用浓盐酸(质量分数为36％-38％)将pH调至1.5，加入72g硅藻土(中位粒径19.6μm)吸附细胞，搅拌20min。抽滤，获得滤液a和滤渣a，向滤渣a中加入600ml的1M盐酸，搅拌20min，抽滤，获得滤液b和滤渣b；合并滤液a和滤液b总计共约1.0L，以500mL二氯甲烷(纯度99.5％)萃取一次，获得水相a和有机相a，有机相a用100ml的1M盐酸萃取，获得水相b和有机相b，合并水相a和水相b并用氢氧化钠调节pH至12再加入1.2L二氯甲烷萃取，获得有机相c和水相c，水相c再加入800mL二氯甲烷萃取，获得水相d和有机相d，合并有机相c和有机相d用饱和氯化钠(36g/L)水洗二次，加入无水硫酸钠干燥，抽滤去除硫酸钠，45℃下旋转蒸发，最后得到白色粉末20.5g，经实施例15中的液相检测，收率为93％，西他列汀中间体纯度为99.5％。西他列汀中间体的总收率为82％。
应理解，在阅读了本发明的上述内容之后，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. 一种转氨酶突变体，其特征在于所述突变体是SEQ ID NO：2所示氨基酸序列第74位的酪氨酸取代为脯氨酸、第228位的谷氨酸取代为天冬氨酸、第254位的亮氨酸取代为丙氨酸、第290位的蛋氨酸取代为苏氨酸获得的。
  2. 一种权利要求1所述转氨酶突变体的编码基因，其特征在于所述编码基因的核苷酸序列为SEQ ID NO：3所示。
  3. 一种权利要求2所述转氨酶突变体的编码基因转化制备的重组基因工程菌。
  4. 一种权利要求1所述转氨酶突变体在生物催化西他列汀中间体前体酮合成西他列汀中间体中的应用。
  5. 如权利要求4所述的应用，其特征在于所述的应用为：以含有转氨酶突变体编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体经超声破碎后提取的纯酶为生物催化剂，以1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮为底物，以二甲基亚砜为助溶剂、以磷酸吡哆醛为辅酶、以异丙胺为辅底物，以pH 8-9三乙醇胺缓冲液为反应介质构成反应体系，在温度30-45℃、搅拌速度100-250r/min的条件下进行生物催化反应，反应结束后，将反应液分离纯化，获得(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮。
  6. 如权利要求5所述的应用，其特征在于所述反应体系中，湿菌体用量为10-100g/L，纯酶用量为0.01-1.0g/L，底物加入终浓度为2～50g/L，二甲基亚砜体积加入终浓度为10-40％，磷酸吡哆醛加入终浓度0.5g/L，异丙胺加入终浓度10g/L。
  7. 一种权利要求1所述转氨酶突变体在生物催化潜手性羰基化合物合成西他列汀酯类中间体中的应用，其特征在于所述潜手性羰基化合物为下列之一：3-羰基-4-(2,4,5-三氟苯基)-丁酸甲酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸乙酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸异丁酯、3-羰基-4-(2,4,5-三氟苯基)-丁酸卞酯。
  8. 如权利要求7所述的应用，其特征在于所述的应用为：以含有转氨酶突变体编码基因的重组基因工程菌经发酵培养获得的湿菌体为生物催化剂，以潜手性羰基化合物为底物，以二甲基亚砜为助溶剂、以磷酸吡哆醛为辅酶、以异丙胺为辅底物，以pH 8-9三乙醇胺缓冲液为反应介质构成反应体系，在温度25-35℃、搅拌速度100-250r/min的条件下进行生物催化反应，反应结束后，将反应液分离纯化，获得西他列汀酯类中间体。
  9. 如权利要求8所述的应用，其特征在于所述反应体系中，湿菌体用量为10～100g/L，底物终浓度为2～60g/L，二甲基亚砜体积加入终浓度为10-40％，磷酸吡哆醛加入终浓度0.5g/L，异丙胺加入终浓度10g/L。
  10. 如权利要求5或8所述的应用，其特征在于所述湿菌体按如下方法制备：将含有转氨酶突变体编码基因的重组大肠杆菌接种至含有50μg/ml卡那霉素的LB液体培养基，37℃，200rpm下培养12h，再以体积浓度1％接种量接种至新鲜的含有50μg/ml卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD 600达0.6-0.8，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、5000rpm离心20min，弃去上清液，收集沉淀，即获得所述的湿菌体。
PCT/CN2021/072993 2020-10-26 2021-01-21 转氨酶突变体及其在制备西他列汀中间体中的应用 WO2022088528A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/778,627 US20220396816A1 (en) 2020-10-26 2021-01-21 A transaminase mutant and application thereof in preparation of sitagliptin intermediates
EP21884273.0A EP4050102A4 (en) 2020-10-26 2021-01-21 TRANSAMINASE MUTANT AND ITS USE IN THE PREPARATION OF SITAGLIPTIN INTERMEDIATE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011158149.4A CN112522229B (zh) 2020-10-26 2020-10-26 转氨酶突变体及其在制备西他列汀中间体中的应用
CN202011158149.4 2020-10-26

Publications (1)

Publication Number Publication Date
WO2022088528A1 true WO2022088528A1 (zh) 2022-05-05

Family

ID=74980476

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/072993 WO2022088528A1 (zh) 2020-10-26 2021-01-21 转氨酶突变体及其在制备西他列汀中间体中的应用

Country Status (4)

Country Link
US (1) US20220396816A1 (zh)
EP (1) EP4050102A4 (zh)
CN (1) CN112522229B (zh)
WO (1) WO2022088528A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113373126B (zh) * 2021-06-17 2022-08-23 天津法莫西生物医药科技有限公司 一种转氨酶突变体及其编码基因和应用
CN113481254B (zh) * 2021-06-29 2023-05-26 台州酶易生物技术有限公司 西他列汀中间体的制备方法
CN117467733B (zh) * 2023-12-27 2024-03-12 北京元延医药科技股份有限公司 高手性纯度西格列汀及使用固定化转氨酶制备的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011026556A1 (en) * 2009-09-02 2011-03-10 Lonza Ag A process for the identification and preparation of a (r)-specific omega-transaminase
US8293507B2 (en) 2009-02-26 2012-10-23 Codexis, Inc. Transaminase biocatalysts
CN108866021A (zh) * 2018-05-30 2018-11-23 浙江工业大学 一种转氨酶突变体及在制备西他列汀中间体中的应用
CN111411094A (zh) * 2020-04-09 2020-07-14 浙江工业大学 一种(R)-ω-转氨酶突变体及其应用
CN111534494A (zh) * 2020-04-09 2020-08-14 浙江工业大学 (R)-ω-转氨酶突变体及其在制备西他列汀中间体中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110540975B (zh) * 2019-08-28 2021-04-30 浙江工业大学 ω-转氨酶突变体及其在制备西他列汀中间体中的应用
CN111235127B (zh) * 2020-04-29 2020-08-14 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8293507B2 (en) 2009-02-26 2012-10-23 Codexis, Inc. Transaminase biocatalysts
WO2011026556A1 (en) * 2009-09-02 2011-03-10 Lonza Ag A process for the identification and preparation of a (r)-specific omega-transaminase
CN108866021A (zh) * 2018-05-30 2018-11-23 浙江工业大学 一种转氨酶突变体及在制备西他列汀中间体中的应用
CN111411094A (zh) * 2020-04-09 2020-07-14 浙江工业大学 一种(R)-ω-转氨酶突变体及其应用
CN111534494A (zh) * 2020-04-09 2020-08-14 浙江工业大学 (R)-ω-转氨酶突变体及其在制备西他列汀中间体中的应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHENG FENG; CHEN XIU-LING; XIANG CHAO; LIU ZHI-QIANG; WANG YA-JUN; ZHENG YU-GUO: "Fluorescence-based high-throughput screening system for-ω-transaminase engineering and its substrate scope extension", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 104, no. 7, 17 February 2020 (2020-02-17), Berlin/Heidelberg, pages 2999 - 3009, XP037057307, ISSN: 0175-7598, DOI: 10.1007/s00253-020-10444-y *
DATABASE Protein 23 July 2021 (2021-07-23), ANONYMOUS: "MULTISPECIES: aminotransferase class IV [Mycolicibacterium]", XP055926362, retrieved from Genbank Database accession no. WP_011781668 *
FENG CHENG, CHAO XIANG, YAJUN WANG: "ω-Transaminase for asymmetric synthesis of chiral amines and unnatural amino acids", CHINESE JOURNAL OF BIOPROCESS ENGINEERING, vol. 16, no. 3, 15 May 2018 (2018-05-15), CN , pages 1 - 11, XP055926357, ISSN: 1672-3678, DOI: 10.3969/j.issn.1672-3678.2018.03.001 *
LU WANYI , WU JIANPING , XU GANG , YANG LIRONG: "Mining, functional expression and application of novel aminotransferase genes", ABSTRACTS OF 2015 NATIONAL SYMPOSIUM ON ENZYME ENGINEERING AND GLYCOBIOTECHNOLOGY, 23 August 2015 (2015-08-23), pages 132, XP009536543 *
See also references of EP4050102A1

Also Published As

Publication number Publication date
CN112522229B (zh) 2021-07-27
CN112522229A (zh) 2021-03-19
EP4050102A1 (en) 2022-08-31
US20220396816A1 (en) 2022-12-15
EP4050102A4 (en) 2023-03-08

Similar Documents

Publication Publication Date Title
WO2022088528A1 (zh) 转氨酶突变体及其在制备西他列汀中间体中的应用
CN107384887B (zh) 一种氨基转移酶、突变体及其制备西他列汀的应用
CN108866021B (zh) 一种转氨酶突变体及在制备西他列汀中间体中的应用
CN102405281B (zh) 转氨酶生物催化剂
CN108823179B (zh) 一种源自放线菌的转氨酶、突变体、重组菌及应用
CN109837317B (zh) 一种手性双芳基醇化合物的合成方法
CN113774036B (zh) 一种亚胺还原酶突变体及其应用
CN107858340B (zh) 高催化活性的d-果糖-6-磷酸醛缩酶a突变体、重组表达载体、基因工程菌及其应用
CN107858384B (zh) 一种利用活性包涵体制备光学纯l-叔亮氨酸的方法
CN113481254B (zh) 西他列汀中间体的制备方法
CN110387359B (zh) 羰基还原酶突变体及其应用
CN106834246B (zh) 石蒜科植物忽地笑细胞色素p450还原酶2及其编码基因与应用
CN113930404B (zh) 一种酶法合成手性枸橼酸托法替布中间体的方法
CN106754774B (zh) 一种反式肉桂酸-4-羟化酶及其编码基因与应用
WO2021057014A1 (zh) 马克斯克鲁维酵母醛酮还原酶KmAKR突变体及其应用
WO2023245933A1 (zh) 高效生产l-高苯丙氨酸的方法及一种产l-高苯丙氨酸的菌株
CN111100851B (zh) 醇脱氢酶突变体及其在手性双芳基醇化合物合成中的应用
CN114908075B (zh) 一种酶法合成布瓦西坦手性中间体的方法
CN115838697A (zh) 亚胺还原酶突变体及其在拉罗替尼手性中间体合成中的应用
WO2011071058A1 (ja) 光学活性２－ヒドロキシシクロアルカンカルボン酸エステルの製造方法
CN116121216A (zh) 羰基还原酶和葡萄糖脱氢酶的融合酶、编码基因、工程菌及应用
JP2007529232A (ja) 新規のアルコールデヒドロゲナーゼ
CN106701696B (zh) 石蒜科植物忽地笑细胞色素p450还原酶1及其编码基因与应用
CN114277023A (zh) 重组腈水合酶及其在耦合离子交换树脂制备烟酰胺中的应用
WO2024125533A1 (zh) 一种ω-转氨酶突变体及其应用

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 21 884 273.0

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21884273

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021884273

Country of ref document: EP

Effective date: 20220523

NENP Non-entry into the national phase

Ref country code: DE