WO2019148494A1 - 转氨酶突变体及其应用 - Google Patents

转氨酶突变体及其应用 Download PDF

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WO2019148494A1
WO2019148494A1 PCT/CN2018/075272 CN2018075272W WO2019148494A1 WO 2019148494 A1 WO2019148494 A1 WO 2019148494A1 CN 2018075272 W CN2018075272 W CN 2018075272W WO 2019148494 A1 WO2019148494 A1 WO 2019148494A1
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transaminase
enzyme
group
immobilized
mutant
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French (fr)
Inventor
洪浩
詹姆斯·盖吉
卢江平
徐幸福
崔瑜霞
张娜
董学武
于文燕
黄鑫
郝明敏
马玉磊
程逸冰
赵佳东
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Asymchem Life Science Tianjin Co Ltd
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Asymchem Life Science Tianjin Co Ltd
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Priority to PCT/CN2018/075272 priority Critical patent/WO2019148494A1/zh
Priority to US16/966,528 priority patent/US11359219B2/en
Priority to JP2020531690A priority patent/JP7105307B2/ja
Priority to ES18903278T priority patent/ES2931540T3/es
Priority to EP18903278.2A priority patent/EP3733689B1/en
Publication of WO2019148494A1 publication Critical patent/WO2019148494A1/zh
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    • 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)
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals

Definitions

  • the invention relates to the field of enzyme engineering, in particular to a transaminase mutant and an application thereof.
  • ⁇ -transaminase belongs to the class of transferases and, like other transaminase classes, catalyzes the exchange of an amino group with a keto group.
  • ⁇ -transaminase refers to a class of enzymes, which can be called ⁇ -transaminase as long as the substrate or product of the reaction does not contain ⁇ -amino acids in an enzyme-catalyzed transamination reaction.
  • Omega-transaminase uses a ketone compound as a raw material to efficiently produce a chiral amine by stereoselective transamination. Enantiomeric chiral amines are key intermediates in many pharmaceutical compounds with broad biological activity (ChemBioChem.
  • the enzyme When the transaminase used is immobilized, the enzyme can be recovered theoretically, but the recovery rate of the enzyme after the immobilization of the conventional transaminase for catalyzing the substrate 1 or the substrate 2 is still low. Moreover, since most of the existing substrate ketones (amino acceptors) are poor in water solubility, it is impossible to carry out a continuous reaction in a pure aqueous phase. In order to achieve a continuous reaction, it is necessary to add sufficient organic cosolvent to dissolve the substrate, but the existing transaminase is poor in temperature, pH and organic solvent, and the organic solvent is easy to inactivate. Therefore, it is also difficult to achieve its immobilization processing.
  • the main object of the present invention is to provide a transaminase mutant and use thereof to solve the problem that the transaminase activity in the prior art is poorly tolerated in an extreme environment and the application is limited.
  • a transaminase mutant having a sequence in which an amino acid mutation occurs in the sequence of SEQ ID NO: 1 and a site in which an amino acid mutation occurs includes a T7C+S47C position. point.
  • the site where the amino acid mutation occurs further includes any one or more of the following: M356L, F364L, C404L, M430L, R405E/A, K90G, K219T, K304D, K51R, A95P, E368P, Q346E, H333K, D371G, E246A, C328A, N412G, T402P, T107F/A, G110P, K69N, G201C, Q380L, K193I, I297L, R305H, F111Y, K190E, and A286T, where "/" means "or".
  • the site in which the amino acid mutation occurs further includes any one of the following combination mutation sites: K51R+W187Y, R405E+A95P, R405E+A95P+K304D, R405E+A95P+K304D+Q380L, R405E+K90G+A95P+K304D+Q380L+E368P, R405E+K90G+A95P+K304D+Q380L+Q346E, R405E+K90G+A95P+K304D+Q380L+H333K, R405E+K90G+A95P+K304D+Q380L+D371G, R405E+K90G+A95P+K304D+Q380L+E246A, R405E+K90G+A95P+K304D+Q380L+C328A, R405E+K90G+A95G+A95P+K304D+Q380L+C328A, R405E
  • a DNA molecule encoding any one of the above transaminase mutants is provided.
  • a recombinant plasmid linked to any one of the above DNA molecules is provided.
  • an immobilized transaminase comprising any one of the above transaminase mutants.
  • the immobilized transaminase is a transaminase cross-linking enzyme aggregate of a transaminase mutant; preferably, the transaminase mutant is precipitated to obtain a transaminase aggregate, and the free amino group, phenol group, imidazolyl group or sulfhydryl group in the transaminase aggregate is further cross-linked a transaminase cross-linked enzyme aggregate obtained by crosslinking the agent, wherein the crosslinking agent is selected from any one of glutaraldehyde, N, N-methylenebisacrylamide, bismaleimide and dextran;
  • the transaminase cross-linking enzyme aggregate is a cross-linked enzyme aggregate of a transaminase mutant having the following amino acid mutation site based on the amino acid sequence shown in SEQ ID NO: 1: T7C+S47C, T7C+S47C+A95P, T7C +S47C+R405E+
  • the immobilized transaminase is a transaminase-cross-linking immobilized enzyme; preferably, the transaminase-embedded-crosslinking enzyme is a transaminase mutant having the following amino acid mutation site based on the amino acid sequence shown in SEQ ID NO: 1.
  • Embedding-crosslinking immobilized enzyme T7C+S47C, T7C+S47C+R405E+K90G+A95P+K304D+Q380L, T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A, T7C+ S47C+R405E+K90G+A95P+K304D+Q380L+Q346E, and T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L; preferably, the free amino group in the transaminase mutant forms a Schiff base crosslink with glutaraldehyde A transaminase cross-linking enzyme is obtained, and a transaminase cross-linking enzyme is embedded in a polyacrylamide gel grid to obtain a transaminase-embedded immobilized enzyme.
  • the immobilized transaminase is a covalently immobilized enzyme in which a transaminase mutant is covalently linked to a carrier; preferably, the covalent immobilized enzyme is an amino acid mutation site having the following amino acid sequence based on the amino acid sequence shown in SEQ ID NO: 1.
  • Covalently immobilized enzymes of transaminase mutants T7C+S47C, T7C+S47C+A95P, T7C+S47C+Q380L, T7C+S47C+R405E, T7C+S47C+K51R+W187Y, T7C+S47C+R405E+A95P+K304D, T7C+S47C+R405E+K90G+A95P+K304D+Q380L+E368P, T7C+S47C+R405E+K90G+A95P+K304D+Q380L, T7C+S47C+R405E+K90G+A95P+K304D+Q380L, T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L, T7C+S47C+ R405E+K90G+A95P+K304D+Q380L+
  • any one of the resins, the functional group attached to the matrix is selected from a C2 short chain amino group, a C4 medium chain amino group, a C6 long chain amino group or an epoxy group; further preferably, the resin carrier is selected from the group consisting of ECR8309, ECR8315, EC-HFA, LX -1000HA, LX-1000EA, ECR8409, ECR8415, EC-EP, EC EP403, EXE119, LX-1000EP, Immobead-150A, Immobead-150P, Immobead 350A, ECR8206, ECR8209, ECR8215 or ECR8285.
  • the resin carrier is selected from the group consisting of ECR8309, ECR8315, EC-HFA, LX -1000HA, LX-1000EA, ECR8409, ECR8415, EC-EP, EC EP403, EXE119, LX-1000EP, Immobead-
  • the immobilized transaminase is a chelate-immobilized enzyme obtained by chelation of a transaminase mutant with a carrier by a metal ion; preferably, the chelate-immobilized enzyme has the following amino acid based on the amino acid sequence shown in SEQ ID NO: 1.
  • the carrier is a porous glass carrier; further preferably, the porous glass carrier is EziG-101, EziG-102 or EziG-103.
  • the immobilized transaminase is an adsorption-immobilized enzyme in which the transaminase mutant and the carrier are physically adsorbed; preferably, the adsorption-immobilized enzyme is a transaminase having the following amino acid mutation site based on the amino acid sequence shown in SEQ ID NO: 1.
  • the functional group attached to the matrix is octadecyl; further preferably, the resin carrier is selected from the group consisting of ECR8806, ECR1030, ECR1090 , ECR1061, ECR1091, ECR8804, Immobead-EC1, Immobead-S60S, Immobead-S861, X17S0409, EXE120 or Diaion.
  • a process for producing a chiral amine comprising the step of catalytically transaminating a ketone compound and an amino donor using a transaminase, wherein the transaminase is any of the above transaminase mutants or the above Any of the immobilized transaminase.
  • the transaminase is any one of the above transaminase mutants, and the above method is a batch reaction; preferably, the reaction system of the batch reaction is an aqueous phase reaction system.
  • the transaminase is any of the above immobilized transaminase, and the above method is a continuous reaction; preferably, the reaction system of the continuous reaction is an organic phase reaction system.
  • the ketone compound is wherein R 1 and R 2 are each independently a C1-C8 alkyl group, a C5-C10 cycloalkyl group, a C6-C10 aryl group or a C5-C10 heteroaryl group, or R 1 and R 2 are formed together with a carbon on the carbonyl group; a C5-C10 heterocyclic group, a C5-C10 carbocyclic group or a C5-C10 heteroaryl group, and the heteroatoms in the C5-C10 heterocyclic group and the C5-C10 heteroaryl group are each independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • At least one of an aryl group in a C6-C10 aryl group, a heteroaryl group in a C5-C10 heteroaryl group, a carbocyclic group in a C5-C10 carbocyclic group or a heterocyclic group in a C5-C10 heterocyclic group Independently unsubstituted or substituted by at least one of a halogen, an alkoxy group or an alkyl group, preferably a ketone compound is The transamination reaction product is Preferably, the amino donor is isopropylamine.
  • transaminase mutants whose enzyme activity and/or stability are greatly improved by direct directed evolution of the aminotransferase shown by SEQ ID NO: 1 are obtained, and the amino acid sequences of these mutants are obtained.
  • a transaminase mutant comprising the above mutation site can be used in a relatively extreme environment.
  • Site-directed mutagenesis refers to the introduction of a desired change (usually a change in a favorable direction) to a DNA fragment of interest (which may be a genomic or a plasmid) by means of polymerase chain reaction (PCR), etc., including the addition of bases. , delete, point mutation, etc.
  • PCR polymerase chain reaction
  • Site-directed mutagenesis can rapidly and efficiently improve the traits and characterization of the target protein expressed by DNA, which is a very useful means in gene research.
  • the introduction of site-directed mutagenesis using whole-plasma PCR is simple and effective, and currently uses more means.
  • the principle is: a pair of primers containing the mutation site (positive and reverse), and the template plasmid is annealed and then "circularly extended" with the polymerase.
  • the so-called cyclic extension means that the polymerase extends the primer according to the template, and returns after one turn.
  • the end of the primer is terminated at the 5' end, and after repeated heating and annealing, the reaction is different from the rolling circle amplification, and no multiple tandem copies are formed.
  • the extension products of the forward and reverse primers are annealed and paired into a nicked open-loop plasmid. Dpn I digests the extension product.
  • the original stencil plasmid is derived from conventional E. coli, it is methylated by dam, sensitive to Dpn I and chopped, and the plasmid with mutation sequence synthesized in vitro has no methyl group.
  • the cloned plasmid was cloned into a host cell and induced to express the target protein by being successfully transformed in the subsequent transformation.
  • Error-prone PCR which means error-prone PCR, a PCR technique that easily causes errors in the replicated DNA sequence, also known as mismatch PCR or propensity error PCR. Specifically, by using low-fidelity Taq DNA polymerase and changing PCR reaction conditions, the fidelity of DNA replication is reduced, and base mismatches are increased during the synthesis of new DNA strands, thereby causing more point mutations in the amplification products. A method of inducing DNA sequence variation in vitro.
  • Error-prone PCR is the simplest and most effective random mutagenesis technique in vitro.
  • the principle is that the heterogeneity of bases provides a possibility of mismatching.
  • the four bases that make up DNA have tautomers.
  • the three oxygen-containing bases of guanine (G), cytosine (C) and thymine (T) have both ketone and enol tautomers.
  • Adenine (A) and thymine are two nitrogen-containing bases, and there are two tautomers, amine and imine.
  • G, C and T mainly exist in a keto structure, and the ratio of the enol structure is extremely low.
  • the nitrogen atoms on the nitrogenous bases of A and T mainly exist in the amino (NH 2 ) state, and the imine group (NH)
  • the rate at which the state exists is extremely low.
  • the difference in position of hydrogen atoms between different isomers and the deviation of the direction of the electron cloud at the same position can change the pairing form of the base, so that mismatches may occur on the copied sub-chain.
  • thymine is present in a keto structure, it is paired with adenine, and when it exists in an enol structure, it is paired with guanine, so that A can be matched with C, and T can be coupled with unstable base of G. Right, thus causing a mismatch.
  • Taq DNA polymerase has the highest mismatch rate.
  • Taq DNA polymerase is the most active of the thermostable DNA polymerases found, with 5'-3' exonuclease activity and no 3'-5' exonuclease activity, so some single cores are synthesized in the synthesis. Glycosidic mismatches have no corrective function, so there is a higher probability of mismatching than DNA polymerases with 3'-5' proofreading activity.
  • the fidelity of DNA polymerase can be reduced by a variety of methods, including the use of four different concentrations of dNTPs, the addition of Mn 2+ , and the increase in Mg 2+ concentration.
  • MnC1 2 is a mutagenic factor of DNA polymerase. Adding Mn 2+ can reduce the specificity of polymerase to template and increase the mismatch rate. The imbalance of 4 kinds of dNTPs can increase the probability of base misincorporation and achieve mismatch.
  • Mg 2+ has the function of activating Taq enzyme, increasing the concentration of Mg 2+ to exceed the normal dosage, and stabilizing non-complementary base pairs; increasing the amount of Taq DNA polymerase and increasing the extension time per cycle can increase the mismatch The probability of terminal extension; lowering the initial template concentration will increase the proportion of variant templates in the subsequent PCR cycle.
  • Immobilized enzyme refers to an enzyme whose catalytic action can be used repeatedly and continuously in a certain spatial range.
  • the enzyme-catalyzed reaction is carried out in an aqueous solution, and the immobilized enzyme is a state in which the water-soluble enzyme is treated physically or chemically to be insoluble in water but still having an enzyme activity.
  • the general stability is increased, it is easy to separate from the reaction system, easy to control, can be used multiple times, is convenient for transportation and storage, and is advantageous for automated production, but the activity is lowered and the use range is reduced.
  • Immobilized enzyme carrier matrix refers to a material that forms a skeleton of an immobilized enzyme carrier.
  • reference to 1 wt refers to the conversion of 1 g of substrate to 1 g of transaminase mutant recombinant wet cells.
  • the 1 V involved is equal to the volume of the reaction system / the mass of the substrate.
  • a typical embodiment of the present application mutates the R416T site derived from Chromobacterium violaceum.
  • the transaminase was subjected to directed evolution to obtain a transaminase mutant having the sequence-generating amino acid mutation sequence shown in SEQ ID NO: 1, wherein the site in which the amino acid mutation occurred included the T7C+S47C site.
  • the aminotransferase activity of the transaminase mutation that mutated at the R416T+T7C+S47C site was significantly improved compared to the R416T mutant.
  • an aminotransferase derived from Chromobacterium violaceum is engineered to obtain an R416T mutant having improved enzymatic activity, and the amino acid sequence thereof is shown in SEQ ID NO: 1.
  • the activity of the enzyme is good, but the stability is not ideal.
  • five sets of double-point mutations, Q78C+A330C, V137C+G313C, A217C+Y252C, T7C+ were designed using R416T mutant as template.
  • S47C, L295C+328C, primer sequences were designed using the QuikChange Primer Design web page.
  • a mutation site was introduced on the mutant R416T by whole plasmid PCR, and a new mutant site was obtained by using pET-22b(+) as an expression vector.
  • the mutant plasmid was transformed into E. coli cells, and induction was carried out overnight at 25 ° C under the optimum conditions of 0.1 mM IPTG transaminase-induced expression, and then the crude enzyme was obtained by ultrasonically disrupting the cells. After the enzyme solution expressed by the mutant strain was treated under the extreme environment of 45-50 ° C, pH 9.5, 20% DMSO for 1 h, substrate 1 or substrate 2 was added, and the reaction was continued under the conditions for 16 h after using 1 wt of the enzyme amount. Detect conversion rate. In this way, mutants with improved stability were screened, and the T7C+S47C site mutant (R416T+T7C+S47C) activity was significantly improved compared with the R416T mutant. Under this condition, the R416T-catalyzed conversion rate was 15%. The conversion rate catalyzed by the mutant R416T+T7C+S47C was 72%.
  • mutants The enzyme solution expressed by the mutant strain was treated under the extreme environment of 30-45 ° C, pH 9.5-10, 50% DMSO for 1 h, substrate 1 or substrate 2 was added, and the reaction was continued after 16 h of reaction under the conditions. In this way, mutants with enhanced temperature, pH, and tolerance to organic solvents were screened.
  • the screening results were as follows: mutation sites in M356L, F364L, C404L, M430L, R405E, R405A, K90G, K219T, K304D, K51R, A95P, E368P, Q346E, H333K, D371G, E246A, C328A, N412G, T402P, T107F, T107A,
  • the mutant of G110P was more 8%-40% more resistant to the 30°C, pH9.5, 50% DMSO environment than the R416T+T7C+S47C mutant.
  • Some mutants were 1.7- to 2.1-fold more tolerant to the 45°C, pH9.5, 50% DMSO environment than the R416T+T7C+S47C mutant, and some mutants were in the 40°C, pH10, 50% DMSO environment.
  • the tolerance was 3.7-3.9 times higher than that of the R416T+T7C+S47C mutant.
  • Table 1 Single point mutants with increased tolerance to 30 ° C, pH 9.5, 50% DMSO environment obtained by site-directed mutagenesis.
  • Table 2 Single point mutants with increased tolerance to 40 ° C, pH 10, 50% DMSO environment obtained by site-directed mutagenesis.
  • the R416T+T7C+S47C mutant was also randomly mutated using error-prone PCR techniques.
  • the target gene fragment is ligated to the pET-22b vector by an error-prone PCR method to obtain a mutant plasmid carrying the gene of interest.
  • the mutant plasmid was transformed into E. coli cells and induced overnight at 25 ° C under the optimum conditions of 0.1 mM IPTG for transaminase-induced expression. Finally, the crude enzyme was obtained by ultrasonic disruption of cells.
  • mutants with enhanced tolerance to temperature, pH and organic solvents were screened.
  • the screening results showed that mutants with mutation sites at K69N, G201C, Q380L, K193I, I297L, R305H, F111Y, K190E, and A286T were more tolerant to 30°C, pH9.5, and 50% DMSO than R416T+T7C+S47C.
  • the mutants were increased by 16%-45%, and the tolerance of some mutants to 40°C, pH10, 50% DMSO environment was increased by 117% to 537% compared with R416T+T7C+S47C mutant, and some mutants were 30°C, The tolerance of pH 8 and 35% MeOH was 233% to 649% higher than that of the female parent.
  • the R416T+T7C+S47C mutant was used as the female parent, and the specific mutants which were screened by error-prone PCR and improved in tolerance under different extreme environments are shown in Tables 3 to 5 below.
  • Table 3 Single point mutants with improved tolerance to 30 °C, pH 9.5, 50% DMSO environment obtained by error-prone PCR.
  • Mutation site Tolerance improvement Mutation site Tolerance improvement R416T+T7C+S47C no K193I 38% F111Y 16% K190E 31% Q380L 41% R305H 43% I297L 42% A286T 45% K69N twenty four% G201C 28%
  • Table 4 Single point mutants with improved tolerance to 40 ° C, pH 10, 50% DMSO environment obtained by error-prone PCR.
  • Mutation site Tolerance improvement Mutation site Tolerance improvement R416T+T7C+S47C no Q380L 509% K190E 191% K193I 312% F111Y 238% I297L 537% R305H 174% K190E 191% K69N 124% G201C 117%
  • Table 5 Single point mutants with improved tolerance to 30 °C, pH 8, 35% MeOH environment obtained by error-prone PCR.
  • Mutation site Tolerance improvement Mutation site Tolerance improvement R416T+T7C+S47C no R305H 497% F111Y 233% I297L 576% K190E 403% K193I 579% A286T 623% Q380L 649% K69N 288% G201C 294%
  • the present application performs a multi-point combination mutation on the sites of increased stability and tolerance of these transaminase, and then obtains stability and tolerance by directed screening. Increased multipoint mutants.
  • the mutation sites for the combined mutations were derived from K51R, W187Y, R405E, K90G, A95P, K304D, Q380L, E368P, Q346E, H333K, D371G, E246A, C328A, N412G, T402P, T107F, T107A, G110P, I297L, K69N, G201C. , A286T.
  • This combined mutation is any combination of these sites. Specifically, including but not limited to the following mutation combinations: K51R+W187Y, R405E+A95P, R405E+A95P+K304D, R405E+A95P+K304D+Q380L, R405E+K90G+A95P+K304D+Q380L, R405E+K90G+A95P+ K304D+Q380L+E368P, R405E+K90G+A95P+K304D+Q380L+Q346E, R405E+K90G+A95P+K304D+Q380L+H333K, R405E+K90G+A95P+K304D+Q380L+D371G, R405E+K90G+A95P+K304D+ Q380L+E246A, R405E+K90G+A95P+K304D+Q380L+C328A, R405E+K90G+
  • the mutant plasmid was transformed into E. coli cells and induced overnight under optimal conditions of transaminase-induced expression of 0.1 mM IPTG at 25 °C.
  • the crude enzyme is then obtained by ultrasonic disruption of the cells.
  • Table 6 Combined mutants with increased tolerance to 40 ° C, pH 10, 50% DMSO environment.
  • Table 7 Combinatorial mutants with increased tolerance to 30 ° C, pH 8, 35% MeOH environment.
  • Table 8 Combined mutants with increased tolerance to 45 ° C, pH 8, 40% MeOH environment.
  • the transaminase of the parental R416T+T7C+S47C mutant, the single-point mutant selected from the maternal mutant, and the combined mutant transaminase are separately fixed to prepare a transaminase cross-linked enzyme aggregate.
  • cross-linked enzyme aggregates In general, the preparation of cross-linked enzyme aggregates is mainly carried out in two steps: (1) formation of an enzyme protein aggregate precipitate; and (2) cross-linking between precipitates.
  • the enzyme protein can be agglomerated by a salting-out method, an isoelectric precipitation method, a heavy metal salt precipitation method or an organic solvent precipitation method to obtain an enzyme protein precipitate (Aggregates).
  • this enzyme protein precipitates as a reversible precipitate and can be re-dissolved in aqueous solution.
  • a crosslinking agent is added to further connect the protein precipitates by covalent bonds to form a water-insoluble precipitate-crosslinking enzyme aggregate.
  • the cross-linking agent used is a bifunctional or multifunctional reagent, and the bifunctional reagents are glutaraldehyde, N,N-methylenebisacrylamide (MBA), bismaleimide, etc., multifunctional reagent dextran ( The molecular weight is from 6 KDa to 200 KDa).
  • MBA N,N-methylenebisacrylamide
  • the free amino group, phenol group, imidazolyl group and sulfhydryl group of the enzyme protein can participate in the crosslinking reaction.
  • the buffer for preparing the immobilized enzyme solution contains 0.4-1 mg/mL of PLP, and the pH of the enzyme solution is 7.0-8.0.
  • the precipitant used to prepare the enzyme protein precipitate is ethanol, isopropanol and/or ammonium sulfate, and the final concentration of the precipitant is 90%.
  • the crosslinking agent used to prepare the crosslinked aggregate of the enzyme protein is a 25% glutaraldehyde solution, and the final concentration of glutaraldehyde is 200 mM to 500 mM.
  • the prepared cross-linked enzyme aggregate can be directly subjected to catalytic reaction in an aqueous phase by using an aqueous cross-linked enzyme obtained by filtration, or lyophilized with an aqueous cross-linked enzyme to obtain a dry powder.
  • the lyophilized powder can also be applied to the reaction in an organic solvent phase.
  • the cross-linked enzyme aggregate After the cross-linked enzyme aggregate is used once, it can be recovered by centrifugation or filtration, and the number of repeated uses is statistically compared with the first use, and the cross-linked enzyme aggregate is in water.
  • the activity recovery is >80% compared to the free enzyme, and the female R416T+T7C+S47C can be reused 3 times, while the single point mutant and/or combination mutant on the maternal basis is reused. The number of times is significantly higher than that of the female parent, and the best mutants can be reused up to 13 times.
  • the cross-linking enzyme of certain mutants can be reused at least 6 times in a system containing 35% methanol.
  • the stability of the mutant free enzyme activity is improved, and the number of enzyme activities recovered and reused after immobilization is also increased.
  • the cross-linked enzyme is used to catalyze the reaction, and the post-reaction treatment extracts the product from the aqueous phase with an organic solvent, and the emulsification phenomenon is remarkably reduced.
  • the preparation of the cross-linking enzyme does not require a carrier, has low cost, uses a cross-linking enzyme to catalyze the reaction, has a large number of repeated uses, a comprehensive use frequency, a reduced enzyme dosage, and a lower use cost than a free enzyme.
  • the cross-linked enzyme lyophilized powder was reacted in a 100% organic phase solvent, and the activity was >10% compared to the first time when the female was repeatedly used for the second time. Some mutants were used up to 5 times and the activity was ⁇ 5% compared to the first time.
  • CLEAs have no carrier support, the immobilized enzyme particles are small ( ⁇ 10 ⁇ m), and the mechanical strength is poor. In the process of filtering and recovering the enzyme, the enzyme is easy to be kneaded, and the next time it is used, it is not well dispersed in the reaction system.
  • glutaraldehyde can be used as a crosslinking agent in combination with cross-linking and embedding techniques, and glutaraldehyde is added dropwise in a mixture containing an enzyme solution, acrylamide and methylidene bisacrylamide.
  • the free enzyme forms a Schiff base to prepare a cross-linked enzyme aggregate, and the initiator ammonium persulfate is added to form a polyacrylamide gel, and the cross-linked enzyme aggregate is embedded in the polyacrylamide gel matrix, thereby obtaining Stable immobilized enzyme.
  • the parental R416T+T7C+S47C mutant, the single-point mutant selected from the maternal mutant and the transaminase of the combined mutant are immobilized by embedding-crosslinking method to prepare the embedding-crossing method. Binding enzyme.
  • the prepared embedding-cross-linking enzyme, the enzyme activity recovery is >80%, and the aqueous phase reaction is used once, and is easily recovered after being filtered, and the activity loss is less than 5% compared with the first use.
  • the female parent R416T+T7C+S47C can be reused 8 times, and the number of repeated use of single-point mutants and/or combination mutants on the maternal basis is significantly higher than that of the female parent, among which the best The mutants can be reused up to 18 times.
  • the embedding-crosslinking enzyme of certain mutants can be reused at least 12 times in a system containing 35% methanol. After the treatment, the product is extracted from the aqueous phase with an organic solvent, and the emulsification phenomenon is remarkably reduced.
  • the enzyme-immobilized enzyme can be prepared by adsorption and binding of the enzyme molecule and the water-insoluble carrier by electrostatic interaction, hydrogen bonding, hydrophobic interaction or the like.
  • the method has mild conditions and is not easy to cause denaturation of the enzyme.
  • the immobilized enzyme prepared by the adsorption method is mainly applied to the reaction in an organic solvent.
  • the carrier which can be used for adsorbing the immobilized enzyme is divided into two types: an inorganic carrier and a polymer carrier.
  • the inorganic carrier includes activated carbon, porous glass, acid white clay, bleaching earth, kaolinite, alumina, silica gel, bentonite, hydroxyapatite, calcium phosphate, metal oxide, etc.; the polymer carrier has starch, gluten, macroporous Synthetic resin, ceramics, etc.
  • the carrier used in the present application is a macroporous synthetic resin carrier comprising a matrix and optionally a functional group modifying the matrix, wherein the substrate includes, but is not limited to, polystyrene resin, polymethacrylate resin or styrene and methacrylate. Copolymer.
  • various types of carriers can be modified with octadecyl functional groups. Table 9 below lists the carriers suitable for the immobilization of transaminase in the present application.
  • the transaminase and the macroporous resin carrier are directly combined by a physical means such as a hydrophobic bond or a hydrogen bond.
  • the parental R416T+T7C+S47C mutant, the single-point mutant screened on the basis of the maternal mutant, and the transaminase of the combined mutant were fixed by physical adsorption binding method.
  • the buffer used for preparing the enzyme solution contained 0.4-1 mg/mL of PLP, the buffer pH was 7.0-8.0, and the buffer salt was Na 2 HPO 4 -NaH 2 PO 4 , Tris-Cl or boric acid-sodium hydroxide.
  • the prepared adsorption-immobilized enzyme can be dried by nitrogen blowing, vacuum drying, freeze drying or the like.
  • the transaminase is bound to the above carrier in an adsorption manner, and the activity recovery is >80%; the reaction is carried out in an organic solvent, and the immobilized enzyme is recovered by filtration or aspirating the liquid by a syringe, and can be repeatedly used.
  • the number of repeated use of the activity loss ⁇ 5% range, for some mutants of the immobilized enzyme can be repeated 6 times, activity loss ⁇ 5%.
  • the covalent immobilization of the enzyme is that the non-essential group of the enzyme protein forms an irreversible linkage with the water-insoluble carrier through a covalent bond, and the protein groups that can be coupled under mild conditions include: amino group, carboxyl group, cysteine.
  • a group covalently bound to a carrier is generally not a group necessary for the enzyme to exhibit viability.
  • the immobilized enzyme carrier may be an inorganic material such as silica, glass, minerals, diatomaceous earth, or the like, or may be a natural organic material such as carboxymethylcellulose, dextran, agarose, pectin, and shell. Sugars and the like, and non-natural organic synthetic polymers such as polystyrene resins, polymethacrylate resins, or copolymers of styrene and methacrylate. These carriers may further be functionalized to facilitate binding to the same protein molecule, such as a functional group such as an amino group, a hydroxyl group, an epoxy group, or an octadecyl group.
  • a functional group such as an amino group, a hydroxyl group, an epoxy group, or an octadecyl group.
  • the amino group and the hydroxyl group-functionalized carrier may be ionically bonded to the enzyme protein molecule, the amino type carrier may also be covalently bonded to the enzyme protein, the carrier of the epoxy functional group is mainly linked to the enzyme protein by a covalent bond, and the octadecyl group.
  • the carrier of the functional group is bound to the enzyme molecule by hydrophobic action.
  • the carrier can take any shape or form such as a film, tube, sheet, bead, granule, chip, fiber, or the like.
  • the carriers used in the present application are chitosan, resin, and porous glass.
  • Chitosan can be used as a carrier for enzyme immobilization because of its good biocompatibility, high shape plasticity (can be made into gel, film, fiber, etc.), non-toxic, and easy to be chemically modified (ProcessBiochem, 2005; 40: 2833–40). Chitosan itself is soluble in water, and it is required to prepare it into water-insoluble carrier particles by a solvent evaporation method, an emulsification method, and a coacervation method (Macromol Biosci, 2003; 3: 511-220). The carrier particles obtained by the emulsification method are small and uniform, and are usually preferred.
  • the chitosan molecule has active hydroxyl groups and amino groups, etc., and the enzyme can be adsorbed and combined by ionic bond, hydrogen bond and van der Waals force, but the adsorption is weak, the enzyme is easy to fall off, and the commonly used crosslinking agent such as formaldehyde and glutaraldehyde is activated. It is then covalently bound to the enzyme.
  • the resin carrier used in the present application includes a matrix and a functional group modifying the matrix, wherein the matrix includes, but is not limited to, a polystyrene resin, a polymethacrylate resin, and a styrene and methacrylate copolymer.
  • Suitable functional groups carried by such matrices include, but are not limited to, short chain amino groups, long chain amino groups, and epoxy groups. Table 10 below lists the carriers suitable for the transaminase immobilization in this application.
  • a transaminase and a resin having an epoxy functional group are directly bonded by a covalent bond to a resin having an amino functional group activated by glutaraldehyde by a covalent bond.
  • the parental R416T+T7C+S47C mutant, the single-point mutant screened on the basis of the maternal mutant, and the transaminase of the combined mutant were separately immobilized by covalent binding.
  • the buffer used for preparing the enzyme solution contained 0.4-1 mg/mL of PLP, the buffer pH was 7.0-8.0, and the buffer salt was Na 2 HPO 4 -NaH 2 PO 4 , Tris-Cl or boric acid-sodium hydroxide.
  • the molecular weight of chitosan used in the present application includes, but is not limited to, 300-500 KDa.
  • the carrier is prepared by an emulsification method. After the carrier is activated by glutaraldehyde, the enzyme solution is added, and the mixture is incubated at 20 ° C for 6 hours. The precipitate is collected by filtration or centrifugation, and the precipitate is moistened with a buffer solution. wash.
  • the vector was first activated with glutaraldehyde, and then the enzyme solution was added thereto, and incubated at 20 ° C overnight, and the precipitate was collected by filtration, and the precipitate was rinsed with a buffer.
  • the enzyme solution was directly mixed with the carrier, incubated at 20 ° C overnight, then allowed to stand for 20 h, and the precipitate was collected by filtration, and the precipitate was rinsed with a buffer.
  • the immobilized enzyme prepared can be dried by nitrogen drying, vacuum drying, freeze drying or the like.
  • the transaminase is immobilized to the above carrier by covalent bonding, immobilized to a chitosan carrier, and the viability recovery is 50%-60%; immobilized to a short-chain amino type carrier, and the viability recovery is 50%-70%.
  • the immobilized enzyme activity of the mutant was significantly higher than that of the parental immobilized enzyme.
  • the immobilized enzyme is recovered by filtration or by sucking out a liquid from a syringe, and can be reused.
  • Porous glass is very suitable for enzyme immobilization because of its inert material and good water permeability.
  • the silanol group on the surface of the glass and its pores acts as a binding site to bind to the enzyme to achieve immobilization, but the surface of the conventional glass has a limited density and uneven distribution of silanol groups, large steric hindrance to the enzyme, and low protein loading (Science, 2010, 329, 305-309, JChromatogr, 1976, 125, 115-127), and it is easy to inactivate the enzyme.
  • the inner and outer surfaces of the porous glass are covered with an organic polymer film to form an environment more favorable for enzyme immobilization (Langmuir, 2004, 20, 10639-10647).
  • the polymer film can be further modified as needed, and various functional groups suitable for immobilization are added to the surface.
  • the histidine-tagged protein can be purified by solid phase metal affinity chromatography, and the histidine residue in the protein can be chelated with metal ions (Ni 2+ , Co 2+ , on the water-insoluble substrate, Fe 3+ ) is linked by chelation and the target protein can then be eluted with an imidazole-containing buffer (Nature, 1975, 258, 598-599).
  • the histidine-tagged enzyme can be specifically chelated with a terminal-chelated metal ion-carrying carrier to achieve immobilization, and at the same time, the immobilization specificity is high and the heteroprotein is hardly be fixed.
  • the glass carrier used in the present application is coated on the inner and outer surfaces of the porous glass via a polymer film which may be hydrophilic such as acrylic polymer, semi-hydrophilic styrene and acrylonitrile polymer and hydrophobic such as chlorine. Styrene polymer.
  • the surface of the film is modified with an amino group and then acylated with 2,4-dihydroxyacetophenone, and the hydroxyl group of 2,4-dihydroxyacetophenone is chelated with the metal ion to pass the 2,4-dihydroxyacetophenone.
  • the arm acts, one end is bound to the carrier, and the other end is chelated with metal ions, so that the end of the glass carrier carries a metal ion, which can be affinity-activated with a histidine-tagged protein to achieve specific immobilization (Chemical Communications, 2014, 50 (65): 9134-7).
  • Chelated metal ions include, but are not limited to, Ni 2+ , Co 2+ , Fe 3+ . Table 11 below lists the carriers suitable for the immobilization of transaminase in the present application.
  • the parental R416T+T7C+S47C mutant, the single-point mutant screened on the basis of the maternal mutant, and the transaminase of the combined mutant were separately immobilized by covalent binding.
  • the buffer used for preparing the enzyme solution contained 0.4-1 mg/mL of PLP, the buffer pH was 7.0-8.0, and the buffer salt was Na 2 HPO 4 -NaH 2 PO 4 , Tris-Cl or boric acid-sodium hydroxide.
  • the enzyme solution was directly mixed with the carrier, and incubated at 20 ° C for 40-60 min, and the precipitate was collected by filtration, and the precipitate was rinsed with a buffer.
  • the immobilized enzyme prepared can be dried by nitrogen drying, vacuum drying, freeze drying or the like.
  • the transaminase is bound to the above carrier in a chelate manner, and the viability recovery is about 70%-80%; the immobilized enzyme is recovered by filtration or aspirating the liquid by a syringe, and can be repeatedly used.
  • Reacts in an aqueous solvent the number of statistical re-uses in the range of ⁇ 5% loss of viability compared to the first use, for 12 times of immobilized enzymes of certain mutants, activity loss ⁇ 5%;
  • the organic solvent the number of statistical re-uses in the range of ⁇ 5% of viability loss compared to the first use, and 8 times for the immobilized enzyme of some mutants.
  • the immobilized transaminase of the present application converts the amino acceptor represented by substrate 1 and substrate 2 to the corresponding primary amine, and the amino donor used is isopropylamine.
  • the immobilized transaminase of the present application can be used in a solvent of 100% aqueous solution, a solvent containing 20% to 50% DMSO, a solvent containing 35% methanol or a 100% water-saturated organic solvent (for example, it can be 100% water saturated). Methyl tert-butyl ether or 100% water saturated isopropyl acetate).
  • the immobilized enzymes of the present application can be applied to batch reactions in a stirred form as well as to continuous flow reactions in a pipeline reactor.
  • the batch stirring reaction operation mode is as follows: the raw material, that is, the amino acceptor, the amino donor, the immobilized enzyme, the coenzyme PLP and the solvent are added to the reaction container at one time, and the reaction is carried out for 16 hours or more by mechanical stirring. After the reaction is completed, the immobilized enzyme is recovered by filtration and applied to the next round of the reaction.
  • the continuous reaction is operated by filling the immobilized enzyme into a tubular reactor, and completely dissolving the raw material, that is, the amino acceptor, the amino donor, and the coenzyme PLP, in a suitable solvent to prepare a reaction solution, and using a plunger pump to transfer the reaction solution. It is injected into a tubular reactor filled with an immobilized enzyme at a suitable flow rate, and the product solution is received at the outlet with a solvent.
  • the solvent may be a 35% methanol solution or 100% water saturated methyl tertiary ether.
  • Example 1 Mutant 30 ° C, pH 9.5, 50% DMSO tolerance test
  • the crude enzyme was treated at 30 ° C, pH 9.5, DMSO concentration of 50% for 1 h, then 0.1 g of substrate 1 was added to a 10 mL reaction flask, and 4 eq of isopropylamine hydrochloride and 0.6-1 mg of PLP were added ( 5'-pyridoxal phosphate), 5 mg of the enzyme after the above treatment was added, and the mixture was stirred at a constant temperature of 30 ° C, pH 9.5, and a DMSO concentration of 50% for 16 hours.
  • the conversion was measured by HPLC, and the mutant reaction data is shown in Table 12 below.
  • Example 2 Mutant 45 ° C, pH 10, 50% DMSO tolerance test
  • the crude enzyme was treated at 45 ° C, pH 10, DMSO concentration of 50% for 1 h, then in a 10 mL reaction flask, 0.1 g of substrate 1 was added, and 4 eq of isopropylamine hydrochloride and 0.6-1 mg of PLP (5'- Pyridoxal phosphate), 5 mg of the enzyme after the above treatment was added, and the mixture was stirred at 45 ° C, pH 10, and a DMSO concentration of 50% for 16 hours.
  • the conversion rate of the system was determined by HPLC, and the mutant reaction data is shown in Table 13 below.
  • Example 3 Mutant 30 ° C, pH 8, 35% methanol tolerance test
  • the crude enzyme was treated at 30 ° C, pH 8, 35% MeOH for 1 h, then in a 10 mL reaction flask, 0.1 g of substrate 1 was added, and 4 eq of isopropylamine hydrochloride and 0.6-1 mg of PLP (5'- Pyridoxal phosphate), 5 mg of the enzyme after the above treatment was added, and stirring was continued at a constant temperature of 30 ° C, pH 8, 35% MeOH for 16 h.
  • the conversion was measured by HPLC, and the mutant reaction data is shown in Table 14 below.
  • Example 4 Mutant 45 ° C, pH 8, 40% methanol tolerance test
  • the crude enzyme was treated at 45 ° C, pH 8, 40% MeOH for 1 h, then in a 10 mL reaction flask, 0.1 g of substrate 1 was added, and 4 eq of isopropylamine hydrochloride and 0.6-1 mg of PLP (5'- Pyridoxal phosphate), 5 mg of the enzyme after the above treatment was added, and stirring was continued at a constant temperature of 45 ° C, pH 8, 40% MeOH for 16 h.
  • the conversion rate of the system was determined by HPLC, and the mutant reaction data is shown in Table 15 below.
  • 0.1 g of enzyme powder was dissolved in 2 mL of phosphate buffer (0.1 MPB, pH 7.0-8.0, containing 0.4-1 mg/mL PLP (5'-pyridoxal phosphate)), and slowly added 18 mL of ethanol or 18 mL of isothermal stirring in an ice water bath.
  • phosphate buffer 0.1 MPB, pH 7.0-8.0, containing 0.4-1 mg/mL PLP (5'-pyridoxal phosphate)
  • Propyl alcohol, or ammonium sulfate (final saturation 90%) as a precipitant after adding, stirring for 10min, then adding 1.1 ⁇ 2.7mL 25% glutaraldehyde solution (final concentration 200-500mM), stirring in ice water bath for 30-40min After centrifugation or filtration, the precipitate is washed 3 times with phosphate buffer and stored at 4 ° C, and can be directly applied to the aqueous phase reaction. Or the cross-linked enzyme aggregates are lyophilized, and the lyophilized powder of the cross-linked enzyme aggregate obtained after lyophilization can be applied in the reaction of the aqueous phase and the organic phase.
  • Example 8 Transaminase-intercalation immobilized enzyme aqueous phase reaction test
  • Example 9 Reaction of a transaminase-embedded immobilized enzyme in a 35% aqueous methanol solution
  • Example 10 Transaminase adsorption immobilization method
  • Example 11 Transaminase adsorption immobilized enzyme organic phase reaction inactivation
  • Example 12 Chitosan as a carrier immobilized transaminase
  • chitosan carrier 5 g of chitosan (molecular weight 300KDa-500KDa) was added to 250 mL of 1% acetic acid solution, and dissolved in a microwave oven to prepare an aqueous phase. 300 mL of toluene was mixed with 2.2 g of Span 80, 1.2 mL of n-hexanol, and stirred at room temperature for 2 hours to obtain an oil phase. The aqueous phase was slowly added dropwise to the oil phase under stirring to prepare an emulsion.
  • the emulsion was poured into 1.5 L of a 12% NaOH solution, stirred for 3 hours, 1 L of ethanol was added, and the filter cake was thoroughly washed with purified water to obtain an approximate amount. 40 g of wet carrier, the wet carrier was immersed in 140 mL of pure water and stored at 4 °C.
  • Activated carrier 1.1 mL of 25% glutaraldehyde (2.5% final concentration of glutaraldehyde) per ml of wet carrier.
  • Immobilization 0.2 g of enzyme was added to the activated carrier, stirred at 20-25 ° C for 6 h, the carrier was washed, centrifuged, and the supernatant was removed. The precipitate was immobilized and stored at 4 ° C.
  • Activated carrier 1 g of carrier ECR8409 was washed 1-2 times with 20 mM low ionic strength buffer, the supernatant was removed, and 4 mL of 2% glutaraldehyde (diluted with 20 mM low ionic strength buffer reagent 25% glutaraldehyde) was added. It was activated at 20 ° C, 80 rpm for 1 h, washed with 20 mM buffer for 1-2 times, and the supernatant was removed.
  • Immobilization 4 mL of PB buffer (20 mM, pH 7.0) containing 0.4 mg/m LPLP was added to the activated carrier, and 0.1 to 0.2 g of the enzyme was added thereto, and stirred at a low speed of 80 rpm at 20 ° C overnight. The supernatant was removed, and the precipitate was washed 3-4 times with buffer, the supernatant was removed, the precipitate was dried with nitrogen, or dried by freeze drying, and stored at 4 °C.
  • Example 15 Covalent immobilization of transaminase aqueous phase reaction test
  • Example 16 Reactive activation of covalent immobilized transaminase in 35% aqueous methanol solution
  • Example 18 Covalently immobilized enzyme transaminase packed bed continuous reaction (solvent with methanol as substrate)
  • Example 19 Glass carrier chelate immobilized transaminase
  • EziG-101, EziG-101 or EziG-103 porous glass carrier was washed 1-2 times with buffer (20 mM Tris-Cl 8.5), the supernatant was removed, 20 mL of Buffer was added to the carrier, and enzyme powder or enzyme solution was added. Stir at 20 ° C, 80 rpm for 1 h at low speed, remove the supernatant, wash the precipitate with Buffer for 3-4 times, filter, vacuum dry, and store at 4 ° C.
  • Example 20 EziG-101 porous glass carrier chelate immobilized transaminase aqueous phase reaction test
  • Example 21 Porous glass carrier chelation immobilized transaminase organic phase reaction inactivation
  • the activity, stability, and tolerance to temperature, pH and organic solvent tolerance obtained by directed evolution screening not only reduce the amount of enzyme used in production applications, but also greatly improve the number of enzymes used in production applications.
  • the possibility of preparing various immobilized enzymes not only reduce the amount of enzyme used in production applications, but also greatly improve the number of enzymes used in production applications.
  • the present application immobilizes (self-crosslinking or covalently binding with a carrier) by the above-described directed evolution of a transaminase to realize the application of the immobilized transaminase in the aqueous phase reaction and the organic phase reaction, so that the enzyme and the reaction system are easily separated, and Reduces the emulsification caused by residual enzyme protein during the post-reaction treatment, and the immobilized transaminase mutant can tolerate various extreme environments, with low activity loss and high reutilization times, thus realizing substrate 1 and substrate 2 Continuous transamination reaction.

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Abstract

提供了一种转氨酶突变体及其应用，该转氨酶突变体的氨基酸序列是SEQ ID NO：1所示氨基酸序列发生突变的氨基酸序列，突变的氨基酸位点包括T7C+S47C位点。具有上述突变位点的转氨酶突变体可以进一步通过固定化技术制备成固定化酶，且其固定化酶的活力和稳定性较高，能够被多次回收再利用，适合应用于填充床连续流反应。

Description

转氨酶突变体及其应用 技术领域
本发明涉及酶工程领域，具体而言，涉及一种转氨酶突变体及其应用。
背景技术
ω-转氨酶(ω-TA)属于转移酶类，同其他的转氨酶类一样，催化一个氨基与酮基互换的过程。大部分情况下ω-转氨酶是指一类酶，只要某个酶催化的转氨反应中，反应的底物或产物中不含有α-氨基酸，就可以称该酶为ω-转氨酶。ω-转氨酶以酮类化合物为原料，通过立体选择性地转氨基作用，可以高效生产手性胺。对映体手性胺是具有广泛生物活性的许多药物化合物的关键中间体(ChemBioChem.9，2008，363-365，Chem.Commun.46，2010，5569-5571，Biotechnol.Bioeng.108，2011，1479-1493)。因其底物相对廉价、产物纯度高的特点，受到研究人员越来越多的关注(Green Chemistry,2017,19，2:333-360.)。并且转氨酶已经显现出应用于生产手性胺的希望(Organic Process Research&Development,2010,14,234-237)。
尽管使用转氨酶生产手性胺的进展已被高度关注，但酶促方法在放大生产应用中存在很多问题。如酶活性低及酶用量大导致发酵成本增加；易受反应体系中有机溶剂的影响而变性失活等。
另外，在分离产品胺的过程中，只能通过使酶变性失活形成沉淀后除去并丢弃，无法再利用；或将产品用有机溶剂从水溶液中萃取出，酶继续存在于水溶液中，但此时由于pH及溶剂等众多苛刻条件的影响使酶失活，无法再利用。
现有技术中有报道通过固定化技术将酶进行固定化以提高酶的回收再利用的问题。然而，目前固定化技术在脂肪酶、青霉素酰化酶、淀粉酶等领域的研究较多，因为这些酶相比其他酶有更好的稳定性，固定化后酶活性的损失较低。而对于大部分转氨酶而言，其稳定性较差，尤其是当体系中存在有机相时，固定化操作过程中容易引起酶活损失，因而，对转氨酶的固定化的研究较少，适合连续化反应的固定化的转氨酶的研究更少。
对于能够催化下列底物1和底物2进行氨基转换反应的转氨酶而言，如果用游离的转氨酶催化该反应，游离酶不能回收，只能使用一次。且由于反应体系中酶蛋白的存在使得后处理乳化现象极其严重，产物分离困难。
Figure PCTCN2018075272-appb-000001
Figure PCTCN2018075272-appb-000002
若将所用转氨酶固定化后，理论上可以实现酶的回收利用，但现有的用于催化底物1或底物2的转氨酶固定化后的酶活回收率依旧很低。而且，由于现有的底物酮(氨基受体)大多都水溶性不好，无法在纯水相中进行连续化反应。若要实现连续化反应，则需要加入足够的有机助溶剂使底物溶解，但现有的转氨酶对温度、pH及有机溶剂的耐受性差，有机溶剂易使其失活。因而也难以实现其固定化处理。
因此，需要对现有的能够催化上述底物的转氨酶进行改进，以改善其在有机溶剂等极端环境中稳定性差而应用受限的问题。
发明内容
本发明的主要目的在于提供一种转氨酶突变体及其应用，以解决现有技术中的转氨酶活性在极端环境中耐受性差而使得应用受限的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种转氨酶突变体，该转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列，发生氨基酸突变的位点包括T7C+S47C位点。
进一步地，发生氨基酸突变的位点还包括如下任意一个或多个：M356L、F364L、C404L、M430L、R405E/A、K90G、K219T、K304D、K51R、A95P、E368P、Q346E、H333K、D371G、E246A、C328A、N412G、T402P、T107F/A、G110P、K69N、G201C、Q380L、K193I、I297L、R305H、F111Y、K190E以及A286T，其中“/”表示“或”。
进一步地，发生氨基酸突变的位点还包括如下任一种组合突变位点：K51R+W187Y、R405E+A95P、R405E+A95P+K304D、R405E+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L+E368P、R405E+K90G+A95P+K304D+Q380L+Q346E、R405E+K90G+A95P+K304D+Q380L+H333K、R405E+K90G+A95P+K304D+Q380L+D371G、R405E+K90G+A95P+K304D+Q380L+E246A、R405E+K90G+A95P+K304D+Q380L+C328A、R405E+K90G+A95P+K304D+Q380L+N412G、R405E+K90G+A95P+K304D+Q380L+T402P、R405E+K90G+A95P+K304D+Q380L+T107F、R405E+K90G+A95P+K304D+Q380L+T107A、R405E+K90G+A95P+K304D+Q380L+G110P、R405E+K90G+A95P+K304D+Q380L+I297L、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、R405E+K90G+A95P+K304D+Q380L+I297L+A286T R405E+K90G+A95P+K304D+Q380L+I297L+E368P R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+K69N、 R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C及R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T。
为了实现上述目的，根据本发明的第二个方面，提供了一种DNA分子，该DNA分子编码上述任一种转氨酶突变体。
根据本发明的第三个方面，提供了一种重组质粒，该重组质粒连接有上述任一种DNA分子。
根据本发明的第四个方面，提供了一种固定化转氨酶，该固定化转氨酶包括上述任一种转氨酶突变体。
进一步地，固定化转氨酶为转氨酶突变体的转氨酶交联酶聚集体；优选地，转氨酶突变体经沉淀得到转氨酶聚集体，转氨酶聚集体中游离的氨基、酚基、咪唑基或巯基进一步与交联剂交联得到的转氨酶交联酶聚集体，其中，交联剂选自戊二醛、N,N-亚甲基双丙烯酰胺、双马来酰亚胺及右旋糖苷中的任意一种；优选地，转氨酶交联酶聚集体为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的交联酶聚集体：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+K69N、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+E368P、T7C+S47C+K51R+W187Y、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+C328A及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L；优选地，右旋糖苷的分子量为6KDa～200KDa；优选地，转氨酶聚集体为转氨酶突变体经乙醇沉淀得到；优选地，转氨酶聚集体中游离的氨基与戊二醛交联得到转氨酶交联酶聚集体。
进一步地，固定化转氨酶为转氨酶包埋-交联固定化酶；优选地，转氨酶包埋-交联酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的包埋-交联固定化酶：T7C+S47C、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L；优选地，转氨酶突变体中游离氨基与戊二醛形成席夫碱交联得到转氨酶交联酶，转氨酶交联酶包埋至聚丙烯酰胺凝胶网格中得到转氨酶包埋-交联固定化酶。
进一步地，固定化转氨酶为转氨酶突变体与载体共价连接的共价固定化酶；优选地，共价固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的共价固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+Q380L、T7C+S47C+R405E、 T7C+S47C+K51R+W187Y、T7C+S47C+R405E+A95P+K304D、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+E368P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T；优选地，载体为壳聚糖载体、树脂载体；更优选地，壳聚糖载体通过羟基和/或氨基与转氨酶突变体共价结合形成共价固定化酶；更优选地，树脂载体包括基质及与基质连接的官能团，基质选自苯乙烯和甲基丙烯酸酯共聚物、聚苯乙烯树脂以及聚甲基丙烯酸酯树脂中的任意一种，与基质连接的官能团选自C2短链氨基、C4中链氨基、C6长链氨基或环氧基；进一步优选地，树脂载体选自ECR8309、ECR8315、EC-HFA、LX-1000HA、LX-1000EA、ECR8409、ECR8415、EC-EP、EC EP403、EXE119、LX-1000EP、Immobead-150A、Immobead-150P、Immobead350A、ECR8206、ECR8209、ECR8215或ECR8285。
进一步地，固定化转氨酶为转氨酶突变体与载体经金属离子螯合而成的螯合固定化酶；优选地，螯合固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的螯合固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T；优选地，载体为多孔玻璃载体；进一步优选地，多孔玻璃载体为EziG-101、EziG-102或EziG-103。
进一步地，固定化转氨酶为转氨酶突变体与载体经物理吸附结合的吸附固定化酶；优选地，吸附固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的共价固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+N412G、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C；优选地，载体为树脂载体；更优选地，树脂载体包括基质及与基质连接的官能团，基质选自苯乙烯和甲基丙烯酸酯共聚物、聚苯乙烯树脂以及聚甲基丙烯酸酯树脂中的任意一种，与基质连接的官能团为十八烷基；进一步优选地，树脂载体选自ECR8806、ECR1030、ECR1090、ECR1061、ECR1091、ECR8804、Immobead-EC1、Immobead-S60S、Immobead-S861、X17S0409、EXE120或Diaion。
根据本发明的第五个方面，提供了一种生产手性胺的方法，包括采用转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，转氨酶为上述任一种转氨酶突变体或上述任一种固定化转氨酶。
进一步地，转氨酶为上述任一种转氨酶突变体，上述方法为批次反应；优选批次反应的反应体系为水相反应体系。
进一步地，转氨酶为上述任一种固定化转氨酶，上述方法为连续化反应；优选连续化反应的反应体系为有机相反应体系。
进一步地，酮类化合物为
Figure PCTCN2018075272-appb-000003
其中，R 1和R 2各自独立地为C1～C8烷基、C5～C10环烷基、C6～C10芳基或C5～C10杂芳基，或者R 1和R 2与羰基上的碳共同形成C5～C10杂环基、C5～C10碳环基或C5～C10杂芳基，C5～C10杂环基和C5～C10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，C6～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，酮类化合物为
Figure PCTCN2018075272-appb-000004
Figure PCTCN2018075272-appb-000005
转氨基反应产物为
Figure PCTCN2018075272-appb-000006
优选地，氨基供体为异丙胺。
应用本发明的技术方案，通过对SEQ ID NO：1所示氨基酸序列的转氨酶进行定向进化，筛选得到了一系列其酶活性和/或稳定性大大提高的转氨酶突变体，这些突变体的氨基酸序列是在SEQ ID NO：1所示氨基酸序列的基础上发生突变的氨基酸序列，其中突变的氨基酸位点包括T7C+S47C位点。包含上述突变位点的转氨酶突变体，能够在相对极端的环境中应用。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
名称解释：
定点突变：是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点 突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。
利用全质粒PCR引入定点突变是简单有效，且目前使用较多的手段。其原理是：一对包含突变位点的引物(正、反向)，和模版质粒退火后用聚合酶“循环延伸”，(所谓的循环延伸是指聚合酶按照模版延伸引物，一圈后回到引物5’端终止，再经过反复加热退火延伸的循环，这个反应区别于滚环扩增，不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。Dpn I酶切延伸产物，由于原来的模版质粒来源于常规大肠杆菌，是经dam甲基化修饰的，对Dpn I敏感而被切碎，而体外合成的带突变序列的质粒由于没有甲基化而不被切开，因此在随后的转化中得以成功转化，即可得到突变质粒的克隆。突变质粒转化至宿主细胞，诱导表达出目标蛋白。
易错PCR：意为易错条件下的PCR,即容易使复制出的DNA序列出现错误的PCR技术，又称错配PCR或倾向错误PCR。具体是指通过利用低保真度TaqDNA聚合酶和改变PCR反应条件，降低DNA复制的保真度，在新DNA链合成过程中增加碱基错配，从而使扩增产物出现较多点突变的一种体外诱导DNA序列变异的方法。
易错PCR是目前最简单、有效的基因体外随机诱变技术，其原理是：碱基的异构为错配提供了可能，组成DNA的4种碱基都有互变异构体存在，其中鸟嘌呤(G)、胞嘧啶(C)和胸腺嘧啶(T)3种含氧碱基有酮式和烯醇式两种互变异构体。腺嘌呤(A)和胸腺嘧啶两种含氮碱基，有胺式、亚胺式两种互变异构体。G、C和T主要以酮式结构存在，烯醇式结构的比率极低，A和T两种含氮碱基上的氮原子主要以氨基(NH 2)状态存在，以亚胺基(NH)状态存在的比率极低。不同同分异构体之间氢原子位置的不同及同一位置电子云偏离方向的不同，可使得碱基的配对形式发生改变，这样在复制后的子链上就可能出现错配。例如当胸腺嘧啶以酮式结构存在时，与腺嘌呤配对，而以烯醇式结构存在时，与鸟嘌呤配对，这样就出现了A能配上C，T能配上G的不稳定碱基对，从而造成错配。
在已知的几种耐热DNA聚合酶中，TaqDNA聚合酶的错配率最高。Taq DNA聚合酶是发现的耐热DNA聚合酶中活性最高的一种，具有5'-3'外切酶活性，不具3'-5'外切酶活性，因此在合成中对某些单核苷酸错配没有校正功能，所以比有3'-5'校对活性的DNA聚合酶发生错配的概率较高。DNA聚合酶的保真性可以通过多种方法来降低，包括使用4种浓度不同dNTP、添加Mn 2+、提高Mg 2+浓度等。几种诱变方法导致扩增DNA链碱基变异的机理各不相同。MnC1 2是DNA聚合酶的诱变因子，加入Mn 2+可以降低聚合酶对模板的特异性，提高错配率；4种dNTPs浓度的不平衡可以提高碱基错误掺入的概率，实现错配；Mg 2+具有激活Taq酶的作用，增加Mg 2+浓度，使之超过正常用量，能稳定非互补的碱基对；提高Taq DNA聚合酶用量、增加每个循环延伸时间，可以增加错配终端延伸的概率；降低起始模板浓度，会使后面PCR循环的变异模板比例增加。
固定化酶：是指在一定的空间范围内，其催化作用能反复和连续使用的酶。通常酶催化反应都是在水溶液中进行的，而固定化酶是将水溶性酶用物理或化学方法处理，使之成为不 溶于水的，但仍具有酶活性的状态。酶固定化之后，一般稳定性增加，易从反应体系中分离，易于控制，能多次使用，便于运输和储存，有利于自动化生产，但活性降低，使用范围减小。
固定化酶载体基质：指形成固定化酶载体骨架的材料。
本申请中，所涉及到的1wt均指转化1g底物需要1g转氨酶突变体重组湿细胞。
本申请中，所涉及的1V等于反应体系的体积/底物的质量。
本申请为了解决现有技术中的转氨酶活性在极端环境中耐受性差而使得应用受限的问题，本申请一种典型的实施方式对来源于紫色杆菌(Chromobacterium violaceum)的R416T位点发生突变的转氨酶进行了定向进化，得到了一种转氨酶突变体，该转氨酶突变体具有SEQ ID NO:1所示的序列发生氨基酸突变的序列，其中，发生氨基酸突变的位点包括T7C+S47C位点。在相对极端的环境下，R416T+T7C+S47C位点发生突变的转氨酶突变的转氨酶活性较R416T突变体有明显提高。
以下将结合试验对上述技术方案和技术效果进行说明。
一、对极端环境耐受性提高的突变体的筛选
本申请对来源于紫色杆菌(Chromobacterium violaceum)的氨基转移酶进行改造，得到酶活性提高的R416T突变体，其氨基酸序列如SEQ ID NO：1所示。该酶活性好，但是稳定性还不理想，为了提高该酶的稳定性，又以R416T突变体为模板，设计了五组双点突变，Q78C+A330C，V137C+G313C，A217C+Y252C，T7C+S47C，L295C+328C，引物序列是利用QuikChange Primer Design网页设计得到。通过全质粒PCR的方式在突变体R416T上引入突变位点，以pET-22b(+)为表达载体，获得新的突变位点的突变质粒。
将突变质粒转化至大肠杆菌细胞内，在25℃，0.1mM IPTG的转氨酶诱导表达最佳条件下诱导过夜，然后通过超声破碎细胞的方法获得粗酶。将突变菌株表达的酶液在45-50℃，pH9.5，20％DMSO的极端环境下处理1h后，加入底物1或底物2，使用1wt的酶量继续在此条件下反应16h后检测转化率。利用此方式筛选出稳定性提高的突变体，其中T7C+S47C位点的突变体(R416T+T7C+S47C)活性较R416T突变体有明显提高，在此条件下，R416T催化的转化率为15％，而突变体R416T+T7C+S47C催化的转化率为72％。
进一步地，以R416T+T7C+S47C突变体为母本，设计了33对定点突变(具体引物采用QuikChange Primer Design网页设计得到)(M356L、W360L、F364L、C404L、M430L、M438L、C445A、F449V、R405E、R405A、K90G、K190R、K219T、K304D、K51R、W187Y、K193E、K143R、N151M、S8P、A33P、A95P、E368P、Q346E、H333K、D371G、E246A、C328A、N412G、T402P、T107F、T107A、G110P)，经QuikChange Primer Design网页设计引物序列，利用定点突变手段，以pET-22b(+)为表达载体获得带有目的基因的突变质粒。将突变质粒转化至大肠杆菌细胞内，并在25℃、0.1mM IPTG的转氨酶诱导表达最佳条件下诱导过夜。然后通过超声破碎细胞的方法获得粗酶。
将突变菌株表达的酶液在30-45℃、pH 9.5-10、50％DMSO的极端环境下处理1h后、加入底物1或底物2、继续在此条件下反应16h后检测转化率。以此方式筛选出温度、pH及有机溶剂耐受性增强的突变体。筛选结果为：突变位点在M356L、F364L、C404L、M430L、R405E、R405A、K90G、K219T、K304D、K51R、A95P、E368P、Q346E、H333K、D371G、E246A、C328A、N412G、T402P、T107F、T107A、G110P的突变体、对30℃、pH9.5、50％DMSO环境的耐受性较R416T+T7C+S47C突变体提高8％-40％。某些突变体对45℃、pH9.5、50％DMSO环境的耐受性较R416T+T7C+S47C突变体提高1.7倍～2.1倍、某些突变体对40℃、pH10、50％DMSO环境的耐受性较R416T+T7C+S47C突变体提高3.7倍～3.9倍。
以R416T+T7C+S47C突变体为母本，定点突变所筛选到的分别在不同极端环境下耐受性得到提高的具体突变体见下表1和表2。
表1：定点突变所获的对30℃、pH 9.5、50％DMSO环境的耐受性提高的单点突变体。
Figure PCTCN2018075272-appb-000007
表2：定点突变所获的对40℃、pH10、50％DMSO环境的耐受性提高的单点突变体。
Figure PCTCN2018075272-appb-000008
为简单有效地筛选出更理想的突变体、本申请还采用易错PCR技术对R416T+T7C+S47C突变体进行随机突变。
本申请经易错PCR方法，将获得目的基因片段连接至pET-22b载体上，获得带有目的基因的突变质粒。将突变质粒转化至大肠杆菌细胞内，并在25℃、0.1mMIPTG的转氨酶诱导表达最佳条件下诱导过夜。最后通过超声破碎细胞的方法获得粗酶。
将突变菌株表达的酶液在30-45℃、pH9-10、有机溶剂浓度为50％DMSO或35％MeOH的极端环境下处理1h后，加入底物1或2，继续在此条件下反应16h后检测转化率。以此方式筛选出对温度、pH及有机溶剂耐受性增强的突变体。筛选结果显示：突变位点在K69N、 G201C、Q380L、K193I、I297L、R305H、F111Y、K190E、A286T的突变体对30℃、pH9.5、50％DMSO环境的耐受性较R416T+T7C+S47C突变体提高16％-45％、某些突变体对40℃、pH10、50％DMSO环境的耐受性较R416T+T7C+S47C突变体提高117％～537％、某些突变体对30℃、pH8、35％MeOH环境的耐受性较母本提高233％～649％。
以R416T+T7C+S47C突变体为母本，易错PCR所筛选到的分别在不同极端环境下耐受性得到提高的具体突变体见下表3至表5。
表3：易错PCR所获对30℃、pH9.5、50％DMSO环境的耐受性提高的单点突变体。
突变位点 耐受性提高程度 突变位点 耐受性提高程度
R416T+T7C+S47C 无 K193I 38％
F111Y 16％ K190E 31％
Q380L 41％ R305H 43％
I297L 42％ A286T 45％
K69N 24％ G201C 28％
表4：易错PCR所获对40℃、pH10、50％DMSO环境的耐受性提高的单点突变体。
突变位点 耐受性提高程度 突变位点 耐受性提高程度
R416T+T7C+S47C 无 Q380L 509％
K190E 191％ K193I 312％
F111Y 238％ I297L 537％
R305H 174％ K190E 191％
K69N 124％ G201C 117％
表5：易错PCR所获对30℃、pH 8、35％MeOH环境的耐受性提高的单点突变体。
突变位点 耐受性提高程度 突变位点 耐受性提高程度
R416T+T7C+S47C 无 R305H 497％
F111Y 233％ I297L 576％
K190E 403％ K193I 579％
A286T 623％ Q380L 649％
K69N 288％ G201C 294％
为进一步进化出稳定性和耐受性更好的转氨酶，本申请将这些转氨酶稳定性和耐受性提高的位点进行多点组合突变，然后通过定向筛选的方法获得稳定性和耐受性进一步提高的多点突变体。
进行组合突变的突变位点来自于K51R、W187Y、R405E、K90G、A95P、K304D、Q380L、E368P、Q346E、H333K、D371G、E246A、C328A、N412G、T402P、T107F、T107A、G110P、I297L、K69N、G201C、A286T。
该组合突变是对这些位点进行任意组合。具体而言，包括但不限于如下突变组合：K51R+W187Y、R405E+A95P、R405E+A95P+K304D、R405E+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L+E368P、R405E+K90G+A95P+K304D+Q380L+Q346E、R405E+K90G+A95P+K304D+Q380L+H333K、R405E+K90G+A95P+K304D+Q380L+D371G、R405E+K90G+A95P+K304D+Q380L+E246A、R405E+K90G+A95P+K304D+Q380L+C328A、R405E+K90G+A95P+K304D+Q380L+N412G、R405E+K90G+A95P+K304D+Q380L+T402P、R405E+K90G+A95P+K304D+Q380L+T107F、R405E+K90G+A95P+K304D+Q380L+T107A、R405E+K90G+A95P+K304D+Q380L+G110P、R405E+K90G+A95P+K304D+Q380L+I297L、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、R405E+K90G+A95P+K304D+Q380L+I297L+A286T、R405E+K90G+A95P+K304D+Q380L+I297L+E368P、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+K69N、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C及R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T。
将突变质粒转化至大肠杆菌细胞内，在25℃、0.1mMIPTG的转氨酶诱导表达最佳条件下诱导过夜。然后通过超声破碎细胞的方法获得粗酶。
在更极端的条件下、如45℃、pH9.5-10、含50％DMSO或35％MeOH的环境下将酶液处理1h后、加入底物1、继续在此条件下反应16h、检测转化率。某些组合突变体对40℃、pH10、50％DMSO环境的耐受性较母本提高231％～610％、某些组合突变体对30℃、pH8、35％MeOH环境的耐受性较母本提高213％～990％、某些组合突变体对45℃、pH8、40％MeOH环境的耐受性较母本提高3000％之多。具体的耐受性提高的组合突变体见下表6至表8。
表6：对40℃、pH10、50％DMSO环境的耐受性提高的组合突变体。
Figure PCTCN2018075272-appb-000009
表7：对30℃、pH8、35％MeOH环境的耐受性提高的组合突变体。
Figure PCTCN2018075272-appb-000010
表8：对45℃、pH8、40％MeOH环境的耐受性提高的组合突变体。
Figure PCTCN2018075272-appb-000011
二、对本申请的转氨酶突变体进行固定化
2.1转氨酶交联酶聚集体(CLEAs)的制备
本申请中，分别对母本R416T+T7C+S47C突变体、在母本突变体基础上筛选得到的单点突变体及组合突变体的转氨酶进行交联法固定，制备转氨酶交联酶聚集体。
一般地，交联酶聚集体的制备主要分两步进行：(1)酶蛋白聚集沉淀体的形成；(2)沉淀体之间的交联。
酶蛋白可以通过盐析法、等电点沉淀法、重金属盐沉淀法或有机溶剂沉淀法等方法凝聚沉淀，得到酶蛋白沉淀体(Aggregates)。一般情况下，这种酶蛋白沉淀为可逆沉淀，能重新在水溶液中溶解。
在蛋白沉淀后、加入交联剂使蛋白沉淀体间进一步通过共价键相连、形成水不溶性沉淀-交联酶聚集体。使用的交联剂为双功能或多功能试剂，双功能试剂有戊二醛、N,N-亚甲基双丙烯酰胺(MBA)、双马来酰亚胺等、多功能试剂右旋糖苷(分子量为6KDa～200KDa)。酶蛋白的游离氨基、酚基、咪唑基及巯基均可参与交联反应。
制备固定化酶液的缓冲液中含有0.4-1mg/mL的PLP、酶液pH为7.0-8.0。制备酶蛋白沉淀所用的沉淀剂为乙醇、异丙醇和/或硫酸铵，沉淀剂终浓度为90％。制备酶蛋白的交联聚集体所用的交联剂为25％戊二醛溶液，戊二醛终浓度为200mM-500mM。
制备的交联酶聚集体、可以直接使用过滤得到的含水交联酶在水相进行催化反应、或将含水的交联酶冻干得到干粉后再应用。冻干粉还可以应用于有机溶剂相中的反应。交联酶聚集体使用一次后、可以通过离心或过滤等方式回收后再使用，在与第一次使用相比，活力损失＜5％的范围内统计重复使用次数，交联酶聚集体在水相反应中应用，与游离酶相比，活力回收＞80％，母本R416T+T7C+S47C可重复使用3次，而在母本基础上的单点突变体和/或组合突变体的重复使用次数较母本明显提高，其中最好的突变体的重复使用次数可达13次。
某些突变体的交联酶在含35％甲醇的体系中反应可重复使用至少6次。突变体游离酶活性稳定性提高，固定化后酶活回收及重复使用次数也提高。
使用交联酶催化反应，反应后处理从水相中用有机溶剂萃取产品，乳化现象明显减轻。制备交联酶无需载体、成本低、使用交联酶催化反应、重复使用次数多、综合使用次数、酶用量减少、使用成本较游离酶低。交联酶冻干粉在100％的有机相溶剂中反应，母本重复使用第二次时，其活性与第一次相比损失＞10％。而某些突变体的重复使用可达5次，活性与第一次相比损失＜5％。
2.2转氨酶的包埋-交联固定化方法
CLEAs无载体支撑，固定化酶颗粒小(＜10μm)，机械强度较差，过滤回收酶的过程中，酶容易板结，下次使用时不能很好地分散在反应体系中。为解决这一问题，可以结合交联和包埋两种技术先采用戊二醛作为交联剂，在含有酶液、丙烯酰胺和甲叉双丙烯酰胺的混合液中，滴加戊二醛使其和自由酶形成希夫碱制得交联酶聚集体，加入引发剂过硫酸铵形成聚丙烯酰胺凝胶，而交联酶聚集体便被包埋至聚丙烯酰胺凝胶矩阵中，从而得到稳定的固定化酶。
本申请中，分别对母本R416T+T7C+S47C突变体、在母本突变体基础上筛选得到的单点突变体及组合突变体的转氨酶进行包埋-交联法固定，制备包埋-交联酶。
制备的包埋-交联酶，酶活回收＞80％，水相反应使用一次后、经过滤等方式极易回收后再使用，在与第一次使用相比，活力损失＜5％的范围内统计重复使用次数，母本R416T+T7C+S47C可重复使用8次，而在母本基础上的单点突变体和/或组合突变体的重复使用次数较母本明显提高，其中最好的突变体的重复使用次数可达18次。
某些突变体的包埋-交联酶在含35％甲醇的体系中反应可重复使用至少12次。后处理从水相中用有机溶剂萃取产品，乳化现象明显减轻。
2.3转氨酶的吸附固定化方法
酶分子与水不溶性载体通过静电作用、氢键、疏水作用等方式吸附结合可制备得到吸附固定化酶。该方法条件温和，不易引起酶的变性，但在水溶液中酶很容易与载体脱离，无法回收再利用，因此吸附法制备的固定化酶主要应用于有机溶剂中的反应。
可用于吸附固定化酶的载体分为无机载体和高分子载体两大类。无机载体有活性炭、多孔玻璃、酸性白土、漂白土、高岭石、氧化铝、硅胶、膨润土、羟基磷灰石、磷酸钙、金属氧化物等；高分子载体有淀粉、谷蛋白、大孔型合成树脂、陶瓷等。
本申请所用载体为大孔合成树脂载体，包括基质以及可选的修饰该基质的官能团，其中，基质包括但不限于聚苯乙烯树脂、聚甲基丙烯酸酯树脂或苯乙烯与甲基丙烯酸酯的共聚物。除此之外，各类载体可经十八烷基官能团修饰。下表9所列为适用于本申请中转氨酶吸附固定化的载体。
表9.吸附型载体
载体名称 基质 官能团
Diaion 聚甲基丙烯酸酯 无
X17S0401 聚甲基丙烯酸酯 无
ECR8806 聚甲基丙烯酸酯 无
EXE120 聚甲基丙烯酸酯 十八烷基
ECR-1030 聚甲基丙烯酸酯 无
ECR-1090 聚苯乙烯 无
本申请将转氨酶与大孔树脂载体直接通过疏水键、氢键等物理方式结合。
分别对母本R416T+T7C+S47C突变体、在母本突变体基础上筛选得到的单点突变体及组合突变体的转氨酶进行物理吸附结合法固定。
制备酶液所用的缓冲液中含有0.4-1mg/mL的PLP、缓冲液pH为7.0-8.0、缓冲盐为Na 2HPO 4-NaH 2PO 4、Tris-Cl或硼酸-氢氧化钠。
制备得到的吸附固定化酶可以通过氮气吹干、真空干燥、冷冻干燥等方式进行干燥处理。
本申请将转氨酶以吸附的方式结合至上述载体，活力回收＞80％；在有机溶剂中反应，固定化酶经过滤或注射器吸出液体等方式回收，可重复使用。与第一次使用相比，活力损失＜5％的范围内统计重复使用次数，对于某些突变体的固定化酶，可重复使用6次，活性损失＜5％。
2.4转氨酶的共价固定化方法
酶的共价固定化，是酶蛋白的非必需基团与水不溶性载体通过共价键形成不可逆的连接，在温和的条件下能偶联的蛋白质基团包括：氨基、羧基、半胱氨酸的巯基、组氨酸的咪唑基、 酪氨酸的酚基、丝氨酸和苏氨酸的羟基。与载体共价结合的基团，通常不能是酶表现活力所必需的基团。
2.4.1共价固定化的酶载体
固定化酶载体可以是无机材料的如二氧化硅、玻璃、矿物质及硅藻土等、也可以是天然有机材料、如羧甲基纤维素、右旋糖苷、琼脂糖、果胶、壳聚糖等、还有非天然有机合成聚合物如聚苯乙烯树脂、聚甲基丙烯酸酯树脂、或苯乙烯与甲基丙烯酸酯的共聚物。这些载体还可以进一步官能团化、以利于同蛋白分子的结合、如在载体上加入氨基、羟基、环氧基、十八烷基等官能团。其中氨基和羟基官能团化的载体可以和酶蛋白分子通过离子键结合、氨基型载体还可以与酶蛋白共价结合、环氧基官能团的载体主要和酶蛋白通过共价键相连、十八烷基官能团的载体与酶分子通过疏水作用结合。载体可以采用任何形状或形式、如薄膜、管、片、珠、颗粒、芯片、光纤等。
本申请所用载体为壳聚糖、树脂以及多孔玻璃。
壳聚糖因其生物相容性好、形状可塑性高(可以做成凝胶、薄膜、纤维等形状)、无毒、、以及易于被化学修饰等特点、可以作为酶固定化的载体(ProcessBiochem,2005；40:2833–40)。壳聚糖本身可溶于水、需要先将其制备成水不溶性的载体颗粒、制备方法有溶剂蒸发法、乳化法、以及凝聚法等(MacromolBiosci,2003；3:511–20)。乳化法制得的载体颗粒较小且均一、通常为首选。壳聚糖分子中有活泼的羟基和氨基等、可将酶通过离子键、氢键和范德华力等作用吸附结合、但吸附作用弱、酶易脱落、常用交联剂甲醛、戊二醛等活化后再与酶共价结合。
本申请所用树脂载体，包括基质以及修饰该基质的官能团，其中，基质包括但不限于聚苯乙烯树脂、聚甲基丙烯酸酯树脂及苯乙烯和甲基丙烯酸酯共聚物。此类基质带有的合适的官能团包括但不限于短链氨基、长链氨基和环氧基。下表10所列为适用于本申请中转氨酶固定化的载体。
表10：
载体名称 基质 官能团
ECR8309 聚甲基丙烯酸酯 短链氨基(C2)
ECR8409 聚甲基丙烯酸酯 长链氨基(C6)
ECR8285 聚甲基丙烯酸酯 环氧基
本申请将转氨酶与具有环氧基官能团的树脂直接通过共价键结合、与经戊二醛活化后的具有氨基官能团的树脂通过共价键结合。
2.4.2共价固定化的方法
本申请分别对母本R416T+T7C+S47C突变体、在母本突变体基础上筛选得到的单点突变体及组合突变体的转氨酶进行共价结合法固定。
制备酶液所用的缓冲液中含有0.4-1mg/mL的PLP、缓冲液pH为7.0-8.0、缓冲盐为Na 2HPO 4-NaH 2PO 4、Tris-Cl或硼酸-氢氧化钠。本申请所用壳聚糖分子量包括但不限于300-500KDa，通过乳化法制备载体，载体经戊二醛活化后，加入酶液，20℃孵育6h，过滤或离心后收集沉淀，沉淀用缓冲液润洗。
共价固定化至氨基型载体，首先将载体用戊二醛活化，再加入酶液，20℃孵育过夜，过滤收集沉淀，沉淀用缓冲液润洗。共价固定化至环氧基型载体，直接将酶液与载体混合，20℃孵育过夜，随后静置20h，过滤收集沉淀，沉淀用缓冲液润洗。
制备得到的固定化酶可以通过氮气吹干，真空干燥，冷冻干燥等方式进行干燥处理。
本申请将转氨酶以共价结合的方式固定化至上述载体，固定化至壳聚糖载体，活力回收为50％-60％；固定化至短链氨基型载体，活力回收为50％-70％；固定化至长链氨基型载体，活力回收为70％-80％；固定化至环氧基型载体，活力回收为40％-60％。突变体的固定化酶活性与母本固定化酶比，有明显提高。固定化酶经过滤或注射器吸出液体等方式回收，可重复使用。在与第一次使用相比，活力损失＜5％的范围内统计重复使用次数，对于某些固定化至壳聚糖载体的酶，可重复使用3次，活性损失＜5％；固定化至短链氨基型载体，某些酶可以重复使用5次；固定化至长链氨基型载体，对于某些突变体的酶，重复使用次数可达11次；固定化至环氧基型载体，某些酶可以重复使用6次。突变体固定化酶的可重复使用次数比母本固定化酶明显提高。共价固定化后的某些转氨酶，在100％有机溶剂中可以发挥催化作用，一些突变体固定化后在有机溶剂中反应，重复使用3次，活性损失＜5％。在35％甲醇溶液中发挥催化作用，一些突变体在固定化后，重复使用5次，活性损失＜5％。
2.5金属离子螯合固定化方法
多孔玻璃因其材质惰性、透水性好等特点、非常适用于做酶固定化的基质。玻璃及其孔道表面的硅烷醇基作为结合位点跟酶结合、实现固定化、但是传统玻璃其表面硅烷醇基密度有限而且分布不均、与酶结合位阻大、蛋白负载量低(Science,2010,329,305-309、JChromatogr,1976,125,115-127)、而且容易使酶失活。在多孔玻璃内外表面覆盖一层有机聚合物薄膜、可形成更有利于酶固定化的环境(Langmuir,2004,20,10639-10647)。聚合物薄膜可根据需要经进一步修饰、在表面加上适合于固定化的各种官能团。
带有组氨酸标签的蛋白可以经固相金属亲和层析法进行纯化、蛋白中的组氨酸残基可以与螯合在水不溶性基质上的金属离子(Ni 2+、Co 2+、Fe 3+)经螯合作用相连、然后可用含咪唑的缓冲液将目标蛋白洗脱下来(Nature,1975,258,598-599)。基于这项技术、可以将带有组氨酸标签的酶与末端螯合有金属离子的载体特异性螯合、达到固定化的目的、同时这种方法固定化特异性高、杂蛋白几乎不会被固定。
本申请所用的玻璃载体是经聚合物薄膜在多孔玻璃内外表面进行涂层，聚合物薄膜可以是亲水的如丙烯酸聚合物、半亲水的苯乙烯与丙烯腈聚合物和疏水的如氯甲基苯乙烯聚合物。薄膜表面经氨基修饰，再经2,4-二羟基苯乙酮酰基化，而2,4-二羟基苯乙酮的羟基再与金属离子螯合，从而通过2,4-二羟基苯乙酮的手臂作用，一端与载体结合，另一端与金属离子螯合， 使得玻璃载体末端带有金属离子，可以与带有组氨酸标签的蛋白亲和结合，实现特异性固定化(ChemicalCommunications,2014,50(65):9134-7)。螯合的金属离子包括但不限于为Ni 2+、Co 2+、Fe 3+。下表11所列为适用于本申请中转氨酶固定化的载体。
表11：
载体名称 性质 金属离子
EziG-101 亲水 Fe 3+
EziG-102 半亲水 Fe 3+
EziG-103 疏水 Fe 3+
本申请分别对母本R416T+T7C+S47C突变体、在母本突变体基础上筛选得到的单点突变体及组合突变体的转氨酶进行共价结合法固定。
制备酶液所用的缓冲液中含有0.4-1mg/mL的PLP、缓冲液pH为7.0-8.0、缓冲盐为Na 2HPO 4-NaH 2PO 4、Tris-Cl或硼酸-氢氧化钠。
共价固定化至多孔玻璃载体，直接将酶液与载体混合，20℃孵育40-60min，过滤收集沉淀，沉淀用缓冲液润洗。
制备得到的固定化酶可以通过氮气吹干，真空干燥，冷冻干燥等方式进行干燥处理。
本申请将转氨酶以螯合的方式结合至上述载体，活力回收约70％-80％；固定化酶经过滤或注射器吸出液体等方式回收，可重复使用。在水相溶剂中反应，与第一次使用相比，活力损失＜5％的范围内统计重复使用次数，对于某些突变体的固定化酶，可重复使用12次，活性损失＜5％；有机溶剂中反应，第一次使用相比，活力损失＜5％的范围内统计重复使用次数，对于某些突变体的固定化酶，可重复使用8次。
三、固定化转氨酶的使用方法
本申请的固定化转氨酶可将底物1和底物2所示的氨基受体转化为对应的伯胺，所用的氨基供体为异丙胺。
本申请的固定化转氨酶可以在如下溶剂中应用：100％水溶液、含20％-50％DMSO的溶剂、含35％甲醇的溶剂或100％的水饱和有机溶剂(比如，可以是100％水饱和甲基叔丁基醚或100％水饱和乙酸异丙酯)。
本申请的固定化酶可以应用于搅拌形式的批次反应中，也适用于填充于管道反应器中的连续流反应。
批次搅拌反应操作方式为：将原料即氨基受体、氨基供体、固定化酶、辅酶PLP及溶剂一次性加入到反应容器中，通过机械搅拌的方式，反应16h以上。反应结束后，通过过滤的方式将固定化酶回收，应用到下一轮的反应。
连续反应的操作方式为：将固定化酶填充到管式反应器中，将原料即氨基受体、氨基供体及辅酶PLP用合适的溶剂完全溶解配制成反应液，用柱塞泵将反应液以合适的流速注入到填充了固定化酶的管式反应器中，出口处用溶剂接收产品溶液。
连续反应操作时，溶剂可以为35％的甲醇溶液，或100％的水饱和甲叔醚。
下面将结合具体的实施例来进一步说明本申请的有益效果。
实施例一：突变体30℃，pH9.5，50％DMSO耐受性检测
将粗酶在30℃，pH9.5，DMSO浓度为50％的环境下处理1h，然后在10mL的反应瓶中，加入0.1g底物1，并加入4eq异丙胺盐酸盐和0.6-1mgPLP(5’-磷酸吡哆醛)，再加入上述处理后的酶5mg，在30℃，pH9.5，DMSO浓度为50％的环境恒温搅拌16h。体系经HPLC检测转化率，突变体反应数据如下表12。
表12：
Figure PCTCN2018075272-appb-000012
实施例二：突变体45℃，pH10，50％DMSO耐受性检测
粗酶在45℃，pH10，DMSO浓度为50％的环境下处理1h，然后在10mL的反应瓶中，加入0.1g底物1，并加入4eq异丙胺盐酸盐和0.6-1mgPLP(5’-磷酸吡哆醛)，再加入经上述处理后的酶5mg，在45℃，pH10，DMSO浓度为50％的环境恒温搅拌16h。体系经HPLC检测转化率，突变体反应数据如下表13。
表13：
Figure PCTCN2018075272-appb-000013
Figure PCTCN2018075272-appb-000014
实施例三：突变体30℃，pH8，35％甲醇耐受性检测
将粗酶在30℃，pH8，35％MeOH浓度的环境下处理1h，然后在10mL的反应瓶中，加入0.1g底物1，并加入4eq异丙胺盐酸盐和0.6-1mgPLP(5’-磷酸吡哆醛)，再加入经上述处理后的酶5mg，继续在30℃，pH8，35％MeOH浓度的环境恒温搅拌16h。体系经HPLC检测转化率，突变体反应数据如下表14。
表14：
Figure PCTCN2018075272-appb-000015
Figure PCTCN2018075272-appb-000016
实施例四：突变体45℃，pH8，40％甲醇耐受性检测
将粗酶在45℃，pH8，40％MeOH浓度的环境下处理1h，然后在10mL的反应瓶中，加入0.1g底物1，并加入4eq异丙胺盐酸盐和0.6-1mgPLP(5’-磷酸吡哆醛)，再加入经上述处理后的酶5mg，继续在45℃，pH8，40％MeOH浓度的环境恒温搅拌16h。体系经HPLC检测转化率，突变体反应数据如下表15。
表15：
Figure PCTCN2018075272-appb-000017
实施例五：交联固定化
0.1g酶粉用2mL磷酸缓冲液(0.1MPB，pH7.0-8.0，含0.4-1mg/mL PLP(5’-磷酸吡哆醛))溶解，冰水浴搅拌下缓慢加入18mL乙醇，或18mL异丙醇，或硫酸铵(终饱和度90％)作为沉淀剂，加毕，搅拌10min后，再加入1.1～2.7mL25％戊二醛溶液(终浓度200-500mM)，冰水浴搅拌30-40min后离心或过滤，沉淀用磷酸缓冲液洗3次后，4℃保存，可直接应用于水相反应。或将交联酶聚集体冻干，冻干后得到的交联酶聚集体冻干粉可在水相和有机相反应中应用。
实施例六：交联酶聚集体水相反应验活
在10mL的反应瓶中，加入0.3mLDMSO，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和1.0mgPLP(5’-磷酸吡哆醛)，补加0.1MPB7.0至反应液终体积为1mL，再加入5mg酶或由5mg酶制备的交联酶聚集体湿酶或交联酶聚集体冻干粉，在45℃搅拌16h。体系经HPLC检测转化率，反应数据如下表16。
表16：
Figure PCTCN2018075272-appb-000018
Figure PCTCN2018075272-appb-000019
实施例七：交联酶有机相反应验活
10mL的反应瓶中，加入1mL水饱和甲基叔丁基醚，再加入10mg底物1及4eq异丙胺，然后加入由10mg酶粉制备的交联酶聚集体冻干粉，30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表17。
表17：
Figure PCTCN2018075272-appb-000020
Figure PCTCN2018075272-appb-000021
实施例八：转氨酶包埋-交联固定化酶水相反应验活
在10mL的反应瓶中，加入0.4mL DMSO(DMSO终浓度为40％)，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和1.0mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入5mg酶或由5mg酶制备的包埋-交联固定化酶，在45℃搅拌16h。体系经HPLC检测转化率，反应数据如下表18。
表18：
Figure PCTCN2018075272-appb-000022
实施例九：转氨酶包埋-交联固定化酶35％甲醇水溶液中反应验活
在10mL的反应瓶中，加入0.35mL甲醇(甲醇终浓度为40％)，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和1.0mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入10mg酶或由10mg酶制备的包埋-交联固定化酶，在30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表19。
表19：
Figure PCTCN2018075272-appb-000023
Figure PCTCN2018075272-appb-000024
实施例十：转氨酶吸附固定化方法
向载体中入4mL含0.4mg/mLPLP的PB缓冲液(100mM，pH7.0)，同时加入0.1g酶，20℃，80rpm低速搅拌过夜。去上清，沉淀用缓冲液清洗3-4遍，去上清，沉淀用氮气吹干，或冷冻干燥法干燥，4℃保存。
实施例十一：转氨酶吸附固定化酶有机相反应验活
在10mL的反应瓶中，加入1mL水饱和甲基叔丁基醚，再加入10mg底物1及4eq异丙胺，然后加入由20mg酶制备的固定化转氨酶，30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表20。
表20：
Figure PCTCN2018075272-appb-000025
Figure PCTCN2018075272-appb-000026
实施例十二：壳聚糖为载体固定化转氨酶
壳聚糖载体制备：5g壳聚糖(分子量300KDa-500KDa)加入到250mL1％乙酸溶液中，微波炉加热溶解，制得水相。300mL甲苯与2.2g司盘80，1.2mL正己醇混匀，室温搅拌2h，制得油相。将水相在搅拌下缓慢滴加至油相中，制得乳剂，再将乳剂倒入至1.5L12％NaOH溶液中，搅拌3h，加入1L乙醇，过滤，用纯化水彻底清洗滤饼，得到约40g湿载体，将湿载体浸泡在140mL纯水中，4℃保存。
活化载体：每毫升湿载体，加入1.1mL25％戊二醛(戊二醛终浓度2.5％)。
固定化：向活化好的载体中加入0.2g酶，20-25℃搅拌6h，清洗载体，离心，去上清，沉淀即为固定化酶，4℃保存。
实施例十三：C6型氨基型载体固定化转氨酶
活化载体：1g载体ECR8409用20mM低离子强度的缓冲液清洗1-2遍，除去上清液，加入4mL2％戊二醛(由20mM低离子强度的缓冲液稀释试剂级25％戊二醛配制)，20℃，80rpm活化1h，用20mM的缓冲液清洗1-2遍，去上清。
固定化：向活化好的载体中入4mL含0.4mg/mLPLP的PB缓冲液(20mM，pH7.0)，同时加入0.1～0.2g酶，20℃，80rpm低速搅拌过夜。去上清，沉淀用缓冲液清洗3-4遍，去上清，沉淀用氮气吹干，或冷冻干燥法干燥，4℃保存。
实施例十四：环氧基型载体固定化转氨酶
1g载体ECR8285用100mM PB缓冲液清洗1-2遍，除去清液，加入4mL Buffer(100mM PB，pH7.0，含1M NaCl)，同时加入0.1g～0.2g酶，20℃，80rpm低速搅拌过夜(18-20h)，再于4℃静置20h。去上清，沉淀用Buffer清洗3-4遍，用氮气吹干，4℃保存
实施例十五：共价固定化转氨酶水相反应验活
在10mL的反应瓶中，加入0.4mL DMSO(DMSO终浓度为40％)，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和1.0mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入5mg酶或由5mg酶制备的固定化转氨酶，在45℃搅拌16h。体系经HPLC检测转化率，反应数据如下表21。
表21：
Figure PCTCN2018075272-appb-000027
Figure PCTCN2018075272-appb-000028
实施例十六：共价固定化转氨酶35％甲醇水溶液中反应验活
在10mL的反应瓶中，加入0.35mL甲醇(甲醇终浓度为40％)，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和1.0mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入10mg酶或由10mg酶制备的固定化转氨酶，在30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表22。
表22：
Figure PCTCN2018075272-appb-000029
实施例十七：共价固定化转氨酶有机相反应验活
在10mL的反应瓶中，加入1mL水饱和乙酸异丙酯，再加入10mg底物1及4eq异丙胺，然后加入20mg酶或由20mg酶制备的固定化转氨酶，30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表23。
表23：
Figure PCTCN2018075272-appb-000030
实施例十八：共价固定化酶转氨酶填充床连续反应(以甲醇作为底物的助溶剂)
75g突变体R405E+K90G+A95P+K304D+Q380L+I297L固定化至ECR8409载体的固定化酶填充至反应器中，柱体积(CV)150mL，用柱塞泵将2CV缓冲液(0.1M的PB7.0，含5mg/mL PLP，2M异丙胺盐酸盐)注入填充床中。将配制的反应液(0.5M底物1，2M异丙胺盐酸 盐，5mg/mL PLP，35％MeOH)用柱塞泵注入填充床，40℃水浴,流速0.25mL/min，保留时间约600min，转化率＞98％，连续操作240h，转化率无降低。
实施例十九：玻璃载体螯合固定化转氨酶
1g EziG-101、EziG-101或EziG-103多孔玻璃载体用缓冲液(20mM Tris-Cl 8.5)清洗1-2遍，除去上清，向载体中加入20mL Buffer，同时加入酶粉或酶液，20℃，80rpm低速搅拌1h，去上清，沉淀用Buffer清洗3-4遍，过滤，真空干燥，4℃保存。
实施例二十：EziG-101多孔玻璃载体螯合固定化转氨酶水相反应验活
在10mL的反应瓶中，加入0.2mL DMSO，溶解0.1g底物1，并加入4eq异丙胺盐酸盐和0.5mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入0.1g酶粉或由0.1g酶粉经EziG-101载体螯合法制备的固定化酶，在45℃搅拌16h。体系经HPLC检测转化率，反应数据如下表24。
表24：
Figure PCTCN2018075272-appb-000031
实施例二十一：多孔玻璃载体螯合固定化转氨酶有机相反应验活
在10mL的反应瓶中，加入1mL水饱和甲基叔丁基醚，再加入10mg底物1及4eq异丙胺，然后加入20mg酶或由20mg酶制备的固定化转氨酶，30℃搅拌16h。体系经HPLC检测转化率，反应数据如下表25。
表25：
Figure PCTCN2018075272-appb-000032
Figure PCTCN2018075272-appb-000033
从以上实施例可以看出，本申请通过定向进化筛选得到的活性、稳定性、对温度、pH及有机溶剂耐受性提高的突变体，不仅降低生产应用中的用酶量，而且大大提高了制备成各种固定化酶的可能性。而且，本申请通过上述定向进化后的转氨酶进行固定化(自身交联或与载体共价结合)，实现固定化转氨酶在水相反应和有机相反应的应用，使酶与反应体系易于分离，并减少反应后处理过程中因残留酶蛋白而造成的乳化现象，同时固定化转氨酶突变体能够耐受各种极端环境，活性损失小，重复利用次数高，从而实现了底物1和底物2的连续化转氨反应。
以上所述仅为本申请的优选实施例而已、并不用于限制本申请、对于本领域的技术人员来说、本申请可以有各种更改和变化。凡在本申请的精神和原则之内、所作的任何修改、等同替换、改进等、均应包含在本申请的保护范围之内。

Claims (15)

  1. 一种转氨酶突变体，其特征在于，所述转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点包括T7C+S47C位点。
  2. 根据权利要求1所述的转氨酶突变体，其特征在于，所述发生氨基酸突变的位点还包括如下任意一个或多个：M356L、F364L、C404L、M430L、R405E/A、K90G、K219T、K304D、K51R、A95P、E368P、Q346E、H333K、D371G、E246A、C328A、N412G、T402P、T107F/A、G110P、K69N、G201C、Q380L、K193I、I297L、R305H、F111Y、K190E以及A286T，其中“/”表示“或”。
  3. 根据权利要求1所述的转氨酶突变体，其特征在于，所述发生氨基酸突变的位点还包括如下任一种组合突变位点：K51R+W187Y、R405E+A95P、R405E+A95P+K304D、R405E+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L、R405E+K90G+A95P+K304D+Q380L+E368P、R405E+K90G+A95P+K304D+Q380L+Q346E、R405E+K90G+A95P+K304D+Q380L+H333K、R405E+K90G+A95P+K304D+Q380L+D371G、R405E+K90G+A95P+K304D+Q380L+E246A、R405E+K90G+A95P+K304D+Q380L+C328A、R405E+K90G+A95P+K304D+Q380L+N412G、R405E+K90G+A95P+K304D+Q380L+T402P、R405E+K90G+A95P+K304D+Q380L+T107F、R405E+K90G+A95P+K304D+Q380L+T107A、R405E+K90G+A95P+K304D+Q380L+G110P、R405E+K90G+A95P+K304D+Q380L+I297L、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、R405E+K90G+A95P+K304D+Q380L+I297L+A286T、R405E+K90G+A95P+K304D+Q380L+I297L+E368P、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+K69N、R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C及R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T。
  4. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1至3中任一项所述的转氨酶突变体。
  5. 一种重组质粒，其特征在于，所述重组质粒连接有权利要求4所述的DNA分子。
  6. 一种固定化转氨酶，其特征在于，所述固定化转氨酶包括权利要求1至3中任一项所述的转氨酶突变体。
  7. 根据权利要求6所述的固定化转氨酶，其特征在于，所述固定化转氨酶为所述转氨酶突变体的转氨酶交联酶聚集体；
    优选地，所述转氨酶突变体经沉淀得到转氨酶聚集体，所述转氨酶聚集体中游离的氨基、酚基、咪唑基或巯基通过与交联剂交联得到所述转氨酶交联酶聚集体，其中，所述交联剂选自戊二醛、N,N-亚甲基双丙烯酰胺、双马来酰亚胺及右旋糖苷中的任意一种；
    优选地，所述转氨酶交联酶聚集体为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的交联酶聚集体：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+K69N、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+E368P、T7C+S47C+K51R+W187Y、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+C328A及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L；
    优选地，所述右旋糖苷的分子量为6KDa～200KDa；
    优选地，所述转氨酶突变体经乙醇沉淀得到所述转氨酶聚集体；
    优选地，所述转氨酶聚集体中游离的氨基与戊二醛交联得到所述转氨酶交联酶聚集体。
  8. 根据权利要求6所述的固定化转氨酶，其特征在于，所述固定化转氨酶为转氨酶包埋-交联固定化酶；
    优选地，所述转氨酶包埋-交联固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的包埋-交联固定化酶：T7C+S47C、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L；
    优选地，所述转氨酶突变体中游离氨基与戊二醛形成席夫碱交联得到转氨酶交联酶，所述转氨酶交联酶包埋至聚丙烯酰胺凝胶网格中得到所述转氨酶包埋-交联固定化酶。
  9. 根据权利要求6所述的固定化转氨酶，其特征在于，所述固定化转氨酶为所述转氨酶突变体与载体共价连接的共价固定化酶；
    优选地，所述共价固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的共价固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+Q380L、T7C+S47C+R405E、T7C+S47C+K51R+W187Y、T7C+S47C+ R405E+A95P+K304D、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+E368P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T；
    优选地，所述载体为壳聚糖载体或树脂载体；
    更优选地，所述壳聚糖载体通过羟基和/或氨基与所述转氨酶突变体共价结合形成所述共价固定化酶；
    更优选地，所述树脂载体包括基质及与所述基质连接的官能团，所述基质选自苯乙烯和甲基丙烯酸酯共聚物、聚苯乙烯树脂以及聚甲基丙烯酸酯树脂中的任意一种，与所述基质连接的官能团选自C2短链氨基、C4中链氨基、C6长链氨基或环氧基；进一步优选地，所述树脂载体选自ECR8309、ECR8315、EC-HFA、LX-1000HA、LX-1000EA、ECR8409、ECR8415、EC-EP、EC EP403、EXE119、LX-1000EP、Immobead-150A、Immobead-150P、Immobead350A、ECR8206、ECR8209、ECR8215或ECR8285。
  10. 根据权利要求6所述的固定化转氨酶，其特征在于，所述固定化转氨酶为所述转氨酶突变体与载体经金属离子螯合而成的螯合固定化酶；
    优选地，所述螯合固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的螯合固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T；
    优选地，所述载体为多孔玻璃载体；
    进一步优选地，所述多孔玻璃载体为EziG-101、EziG-102或EziG-103。
  11. 根据权利要求6所述的固定化转氨酶，其特征在于，所述固定化转氨酶为所述转氨酶突变体与载体经物理吸附结合的吸附固定化酶；
    优选地，所述吸附固定化酶为在SEQ ID NO:1所示氨基酸序列基础上含有如下氨基酸突变位点的转氨酶突变体的吸附固定化酶：T7C+S47C、T7C+S47C+A95P、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+H333K、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+Q346E、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+N412G、 T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A、T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+A286T及T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L+E368P+T107A+G201C；
    优选地，所述载体为树脂载体；
    更优选地，所述树脂载体包括基质及与所述基质连接的官能团，所述基质选自苯乙烯和甲基丙烯酸酯共聚物、聚苯乙烯树脂以及聚甲基丙烯酸酯树脂中的任意一种，与所述基质连接的官能团为十八烷基；进一步优选地，所述树脂载体选自ECR8806、ECR1030、ECR1090、ECR1061、ECR1091、ECR8804、Immobead-EC1、Immobead-S60S、Immobead-S861、X17S0409、EXE120或Diaion HP2MG。
  12. 一种生产手性胺的方法，包括采用转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，其特征在于，所述转氨酶为权利要求1至3中任一项所述的转氨酶突变体或权利要求6至11中任一项所述的固定化转氨酶。
  13. 根据权利要求12所述的方法，其特征在于，所述转氨酶为权利要求1至3中任一项所述的转氨酶突变体，所述方法为批次反应；优选所述批次反应的反应体系为水相反应体系。
  14. 根据权利要求12所述的方法，其特征在于，所述转氨酶为权利要求6至11中任一项所述的固定化转氨酶，所述方法为连续化反应；优选所述连续化反应的反应体系为有机相反应体系。
  15. 根据权利要求12至14中任一项所述的方法，其特征在于，所述酮类化合物为
    Figure PCTCN2018075272-appb-100001
    其中，R 1和R 2各自独立地为C1～C8烷基、C5～C10环烷基、C6～C10芳基或C5～C10杂芳基，或者R 1和R 2与羰基上的碳共同形成C5～C10杂环基、C5～C10碳环基或C5～C10杂芳基，所述C5～C10杂环基和C5～C10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，所述C6～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，所述酮类化合物为
    Figure PCTCN2018075272-appb-100002
    Figure PCTCN2018075272-appb-100003
    其中转氨基反应的产物为
    Figure PCTCN2018075272-appb-100004
    Figure PCTCN2018075272-appb-100005
    优选地，氨基供体为异丙胺。
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JP2023514862A (ja) * 2020-02-26 2023-04-11 アシムケム ラボラトリーズ （ターンジン） カンパニー リミテッド キラルジアミン化合物の合成方法
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EP4116289A4 (en) * 2020-02-26 2023-09-27 Asymchem Laboratories (Tianjin) Co., Ltd. METHOD FOR SYNTHESIS OF A FLUORINE-CONTAINING CHIRAL AMINE COMPOUND
KR20220076193A (ko) * 2020-11-30 2022-06-08 (주)네오크레마 고정화된 이눌리나제를 이용한 돼지감자로부터 이눌린의 제조 방법
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US20240124853A1 (en) * 2021-02-04 2024-04-18 Asymchem Laboratories (Tianjin) Co., Ltd Transaminase mutants and uses thereof

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