PVA membrane immobilized enzyme and its manufacturing method

Abstract

The present invention provides a PVA membrane-immobilized enzyme and a method for its preparation. The PVA membrane-immobilized enzyme comprises a PVA porous membrane and an enzyme embedded in the PVA porous membrane. The PVA porous membrane is a three-dimensionally structured PVA porous membrane, and the enzyme is selected from aminotransferase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, alkene reductase, nitrile hydrolase, ammonialytic enzyme, amino acid dehydrogenase, imine reductase, alcohol dehydrogenase, ammonium formate dehydrogenase, glucose 1-dehydrogenase, and their variants. The three-dimensionally structured PVA porous membrane is used as a carrier to immobilize the enzyme by embedding. The embedding process is simple, the conditions are mild, and the specific surface area is large. The enzyme embedded in the PVA porous membrane is relatively stable and less likely to leach during use. The porous structure of the PVA porous membrane allows for better delivery of reactants and products, making it suitable for use in continuous-flow biocatalysis and compatible with a wide range of enzymes.

Description

[Technical field]

[0001] The present invention relates to the technical field of enzyme immobilization, and more particularly to a PVA membrane-immobilized enzyme and a method for producing the same. [Background technology]

[0002] Biocatalysis is becoming an integral part of chemical, intermediate, fine chemical and final drug molecule manufacturing programs. However, as process demands continue to expand, efficiency and economy in enzyme usage are imperative. To this end, there is a need to improve enzyme activity, specificity and productivity, as well as shelf life and recyclability, especially to promote the economic viability of commercial-scale use.

[0003] Enzyme immobilization platforms provide an excellent tool for the proper integration of enzymes in the production process. Over the years, several natural and synthetic support pairs have been evaluated for enzyme immobilization efficiency, e.g., each platform has been professionally evaluated based on its uses, economics, and advantages. Immobilized biocatalysts are widely used in the fields of organic synthesis, pollution prevention, and diagnostics (Enzyme Microb Technol, 31, 171-8; J Pharm Sci, 89, 979-90).

[0004] Immobilization is achieved by immobilizing the enzyme on or in a solid support, thus obtaining a heterogeneous immobilized enzyme system. Enzymes can be immobilized in a variety of ways, including physical (weak interactions exist between the support and the enzyme) and chemical (the support and the enzyme form a covalent bond) (Analyst, 133, 697-701; Chem Soc Rev, 40, 2567-92; Berlin Heidelberg: Springer, 95-126) or a combination of both, thus including a variety of functionally active supports.

[0005] Physical methods of immobilization of enzymes include in membranes or membrane reactors, adsorption in water-insoluble matrices (physical methods by ions, e.g., adsorption in mesoporous materials), encapsulation (or gel embedding), microencapsulation with solid membranes, microencapsulation with liquid membranes, formation of enzyme-catalyzed Langmuir-Blodgett films (Anal Chem, 1994; 66, 1120A-7A), etc. In the case of membrane embedding or encapsulation, the obtained enzyme catalyst is determined by the properties of the membrane support, such as hydrophilicity, hydrophobicity, density of reactive functional groups, porosity, pore size distribution, membrane thickness, reactor configuration, etc. Immobilization is based on the localization of the enzyme in the membrane, the purpose of which is to achieve a higher expression of the enzyme as well as a high stability under operating conditions.

[0006] At present, there are many studies on immobilizing enzymes using porous membranes, but the structure and active sites of different enzymes are different, so the immobilization methods are also different. For example, when lipase is immobilized using a PVA membrane (polyvinyl alcohol membrane), glutaraldehyde must be used for crosslinking in order to achieve the purpose of improving the stability and activity of lipase through immobilization. In addition, when enzymes such as xylanase, catalase, cellulase, β-galactosidase, and ascorbic acid oxidase are immobilized using a porous membrane, it is common to covalently bond the porous membrane and the enzyme using groups such as amino groups, carboxy groups, thiol groups, hydroxy groups, imidazole groups, or phenol groups. As can be seen from the above, when enzymes are immobilized using a crosslinker in the conventional technology, it is necessary to use high-purity enzymes, which makes the immobilization method complicated and limits the enzyme loading capacity. Summary of the Invention [Problem to be solved by the invention]

[0007] The main objective of the present invention is to provide a PVA membrane immobilized enzyme and its preparation method, thereby solving the problem in the prior art that the process of immobilizing enzyme by adopting a porous membrane is complicated. [Means for solving the problem]

[0008] In one embodiment of the present invention for achieving the above object, a PVA membrane-immobilized enzyme is provided, the PVA membrane-immobilized enzyme comprising a PVA porous membrane and an enzyme embedded in the PVA porous membrane, the PVA porous membrane being a three-dimensionally structured PVA porous membrane, and the enzyme being selected from aminotransferase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, alkene reductase, nitrile hydrolase (nitrilase), ammonia decomposition enzyme (ammonia lyase), amino acid dehydrogenase, imine reductase, alcohol dehydrogenase, ammonium formate dehydrogenase, glucose 1-dehydrogenase, and mutants thereof.

[0009] Furthermore, the enzyme may be a free enzyme or a cross-linked enzyme aggregate.

[0010] Further, the aminotransferase is an aminotransferase derived from Chromobacterium violaceum DSM30191, an aminotransferase derived from Arthrobacter citreus, or an aminotransferase derived from Bacillus thuringiensis, the ketoreductase is a ketoreductase derived from Acetobacter sp. CCTCC M209061, or a ketoreductase derived from Candida macedoniensis AKU4588, and the cyclohexanone monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans. petroleovorans or Rhodococcus ruber-SD1, the ammonia decomposition enzyme is Aspergillus niger CBS 513.88 or Solenostemon scutellarioides, and the alkene reductase is Saccharomyces cerevisiae or Chryseobacterium sp.The alkene reductase is from Bacillus sp. CA49, the imine reductase is from Streptomyces sp. and from Bacillus cereus, the amino acid dehydrogenase is from Bacillus cereus leucine dehydrogenase and from Bacillus sphaericus phenylalanine dehydrogenase, and the nitrile hydrolase is from Aspergillus niger CBS 513.88 and from Neurospora crassa OR74A.

[0011] Furthermore, the aminotransferase derived from Chromobacterium violaceum DSM30191 has the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the aminotransferase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes at least one mutation site at positions 7, 47, 90, 95, 297, 304, 380, 405, and 416, and threonine at position 7 is mutated to cysteine, serine at position 47 is mutated to cysteine, and lysine at position 90 is mutated to glycine. alanine at position 95 is mutated to proline, isoleucine at position 297 is mutated to leucine, lysine at position 304 is mutated to aspartic acid, glutamine at position 380 is mutated to leucine, arginine at position 405 is mutated to glutamic acid, and arginine at position 416 is mutated to threonine; or the amino acid sequence of the aminotransferase mutant has a mutation site of the amino acid sequence obtained by mutation and has a homology of 80% or more to the amino acid sequence obtained by mutation.

[0012] Furthermore, the aminotransferase derived from Arthrobacter citreus has the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the aminotransferase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 2, and the mutation includes at least one mutation site at positions 3, 5, 60, 164, 171, 178, 180, 186, 187, 252, 370, 384, 389, 404, 411, 423, and 424, and the leucine at position 3 is mutated to serine, the valine at position 5 is mutated to serine, the cysteine ​​at position 60 is mutated to tyrosine, the phenylalanine at position 164 is mutated to leucine, the glutamic acid at position 171 is mutated to aspartic acid, and the alanine at position 178 is mutated to leucine. the amino acid sequence of the aminotransferase mutant is an amino acid sequence having a mutation site of the amino acid sequence obtained by mutation and having an identity of 80% or more with the amino acid sequence obtained by mutation, the amino acid sequence of the aminotransferase mutant is different from the amino acid sequence obtained by mutation, the isoleucine at position 180 is mutated to valine, the serine at position 186 is mutated to glycine, the serine at position 187 is mutated to alanine, the valine at position 252 is mutated to isoleucine, the leucine at position 370 is mutated to alanine, the tyrosine at position 384 is mutated to phenylalanine, the isoleucine at position 389 is mutated to phenylalanine, the leucine at position 404 is mutated to glutamine, the glycine at position 411 is mutated to aspartic acid, the methionine at position 423 is mutated to lysine, and the glutamic acid at position 424 is mutated to glutamine;

[0013] Furthermore, the ketoreductase derived from Acetobacter sp. CCTCC M209061 has the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 3, the mutation including at least one of the mutation sites at positions 94, 144, and 156, and wherein alanine at position 94 is mutated to asparagine, glutamic acid at position 144 is mutated to serine, and asparagine at position 156 is mutated to threonine or valine, or the amino acid sequence of the ketoreductase mutant has a mutation site of the amino acid sequence obtained by mutation and has 80% or more homology to the amino acid sequence obtained by mutation.

[0014] Furthermore, the Rhodococcus Cyclohexanone monooxygenase derived from P. sp. Phi1 has the amino acid sequence shown in SEQ ID NO: 4, and the amino acid sequence of the mutant cyclohexanone monooxygenase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 4, and the mutation includes at least one mutation site at positions 280, 435, 436, 438, 441, 508, and 510, in which phenylalanine at position 280 is mutated to tyrosine, phenylalanine at position 435 is mutated to asparagine, phenylalanine at position 436 is mutated to serine, leucine at position 438 is mutated to alanine, serine at position 441 is mutated to valine, and leucine at position 510 is mutated to valine. Alternatively, the amino acid sequence of the mutant cyclohexanone monooxygenase has a mutation site of the amino acid sequence obtained by mutation and has 80% or more homology to the amino acid sequence obtained by mutation.

[0015] Furthermore, the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 has the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of the mutant cyclohexanone monooxygenase is an amino acid sequence obtained by causing a mutation in the amino acid sequence shown in SEQ ID NO:5, and the mutation includes at least one mutation site at positions 45, 190, 249, 257, 393, 504, and 559, and methionine at position 45 is mutated to threonine and proline at position 190 is mutated to threonine. phosphorus is mutated to leucine, cysteine ​​at position 249 is mutated to valine, cysteine ​​at position 257 is mutated to alanine, cysteine ​​at position 393 is mutated to valine, proline at position 504 is mutated to valine, and tyrosine at position 559 is mutated to methionine, or the amino acid sequence of the cyclohexanone monooxygenase mutant has a mutation site of the amino acid sequence obtained by mutation and has a homology of 80% or more to the amino acid sequence obtained by mutation.

[0016] Furthermore, the PVA membrane-immobilized enzyme further contains a coenzyme and a cofactor for each enzyme, and the coenzyme and the cofactor are embedded in the PVA porous membrane.

[0017] Furthermore, the PVA porous membrane further contains polyethylene glycol and / or polyethyleneimine, the molecular weight of the polyethylene glycol being PEG400 to PEG6000, and the molecular weight of the polyethyleneimine being 3 KDa to 70 KDa.

[0018] Furthermore, the mass ratio of the polyethylene glycol to the PVA porous membrane is 5:4 to 75:4, and the mass ratio of the polyethyleneimine to the PVA porous membrane is 1:12 to 1:240.

[0019] Furthermore, the enzyme is a crude enzyme.

[0020] Furthermore, the enzyme loading capacity is 0.05 to 0.4 g of free enzyme / cm 2 membrane or 0.03-0.06 g of dried cross-linked enzyme aggregates / cm 2 It is a membrane.

[0021] In another aspect of the present invention, there is provided a method for producing any one of the above PVA membrane-immobilized enzymes, the method comprising the steps of: mixing raw materials including an enzyme and a PVA solution for a predetermined period of time to obtain a mixed system, step S1, adding the mixed system to a mold and drying the mixed system to obtain a membrane-embedded enzyme, the mold being a three-dimensionally structured mold for forming a three-dimensionally structured PVA porous membrane, and step S3, immersing the membrane-embedded enzyme in a phosphate buffer solution to wash it, and then obtaining the PVA membrane-immobilized enzyme.

[0022] Furthermore, the pH of the mixture is 6.0 to 6.5.

[0023] Furthermore, the step S1 includes preparing an enzyme suspension or enzyme solution, in which the enzyme in the suspension is a cross-linked enzyme aggregate and the enzyme in the enzyme solution is a free enzyme from which cells have been removed, and mixing the suspension or enzyme solution with a PVA solution for a predetermined time to obtain a mixed system.

[0024] Furthermore, the predetermined time is 10 to 60 minutes, and the molecular weight of the PVA in the PVA solution is 20 KDa to 200 KDa.

[0025] Furthermore, the content of PVA in the PVA solution is 10 to 50 g / 100 mL.

[0026] Additionally, acetic acid, methanol and sulfuric acid are dispersed in the PVA solution.

[0027] Furthermore, the pH of the PVA solution is 5.5 to 6.5.

[0028] Furthermore, the ratio of the enzyme to the PVA solution is 1 to 50 g / 100 mL.

[0029] Additionally, the suspension or enzyme solution further comprises a phosphate buffer, optional cofactors and optional coenzymes.

[0030] Furthermore, the weight ratio of the coenzyme to the enzyme is 10:1 to 1:10.

[0031] Furthermore, the step S1 includes mixing the PVA aqueous solution and the cross-linked enzyme particles to form a mixed system.

[0032] Furthermore, the ratio of the crosslinked enzyme particles to the aqueous PVA solution is 1 to 50 g / 100 mL.

[0033] Furthermore, the predetermined time is 10 to 60 minutes.

[0034] Additionally, the crosslinked enzyme particles include an enzyme, an optional cofactor, and an optional coenzyme.

[0035] Furthermore, step S1 includes mixing the PVA aqueous solution and the modifier solution for a first predetermined time to form a second mixed system, and mixing the second mixed system and the enzyme system for a second predetermined time to form a mixed system.

[0036] Furthermore, the concentration of the aqueous PVA solution is 5 to 30 g / 100 mL, and preferably, the modifier solution contains an aqueous polyethylene glycol solution in which a cofactor is dispersed and / or an aqueous polyethyleneimine solution in which a cofactor is dispersed.

[0037] Furthermore, the molecular weight of the polyethylene glycol is PEG400 to PEG6000, and the concentration of the polyethylene glycol in the mixed system is 3 to 10 g / 100 mL.

[0038] Furthermore, the molecular weight of the polyethyleneimine is from 3 KDa to 70 KDa, and preferably from 3 KDa to 50 KDa.

[0039] Furthermore, the concentration of polyethyleneimine in the mixed system is 0.1 to 1 g / 100 mL, and preferably 0.1 to 0.3 g / 100 mL.

[0040] Additionally, the enzyme system comprises an enzyme, an optional cofactor, an optional coenzyme, and a phosphate buffer.

[0041] Additionally, the enzyme may be free enzyme or cross-linked enzyme aggregates from which cells have been removed.

[0042] Furthermore, the concentration of the cofactor in the enzyme system is 1 to 20 mg / mL.

[0043] Furthermore, the weight ratio of the coenzyme to the enzyme in the enzyme system is 10:1 to 1:10.

[0044] Furthermore, the ratio of the enzyme to the PVA solution is 1 to 50 g / 100 mL.

[0045] Furthermore, step S2 includes placing the mixed system in a mold and leaving it to stand for a third predetermined time, and then adding a dehydration accelerator to the mold to perform a drying process, and the dehydration accelerator is one or more selected from the group consisting of acetonitrile, ethanol, and acetone.

[0046] Furthermore, the volume ratio of the dehydration promoter to the mixture is 1:10 to 5:1.

[0047] Furthermore, the third predetermined time period is 2 to 4 hours.

[0048] Furthermore, the mold is a three-dimensionally structured mold, and the three-dimensionally structured mold has a protrusion or a recessed groove.

[0049] Furthermore, the step S3 includes immersing the membrane-embedded enzyme in a phosphate buffer for 2 to 16 hours, and then washing the membrane-embedded enzyme with a fresh phosphate buffer to obtain a PVA membrane-immobilized enzyme. Effect of the Invention

[0050] The technical solution of the present invention uses a PVA porous membrane as a carrier to immobilize the enzyme by embedding, the embedding immobilization process is simple, the conditions are mild, and it has a good immobilization effect on both purified and crude enzymes, and the enzyme is relatively stable when embedded and immobilized in the PVA porous membrane, and is not easily leached during use, and the porous structure of the PVA porous membrane used can better deliver reactants and products, making it suitable for use in continuous flow biocatalysis. The embedding immobilization is a mechanical fixation, so it has a wide range of compatibility with enzymes. The PVA porous membrane is structured three-dimensionally to provide a three-dimensional structure, and has a larger surface area, which provides more embedding sites, and increases the enzyme loading capacity while ensuring high enzyme activity and stability. [Brief description of the drawings]

[0051] The drawings are provided as part of the present application for a better understanding of the present invention, and the illustrative embodiments of the present invention and the description thereof are not construed as limitations on the present invention but are used to interpret the present invention.

[0052] [Figure 1] 1 shows stability curves of the PVA membrane-immobilized enzyme according to Example 2 of the present invention with and without the addition of PLP. [Diagram 2] 1 shows the stability curves of two types of PVA membrane-immobilized enzymes according to Example 3 of the present invention. [Diagram 3] 1 shows stability curves of the PVA membrane-immobilized enzyme according to Example 5 of the present invention with and without the addition of PLP. [Figure 4] 1 shows stability curves of the PVA membrane-immobilized enzyme according to Example 6 of the present invention with and without the addition of PLP. [Diagram 5] 1 shows the stability curves of the PVA membrane-immobilized enzyme of Example 8 of the present invention when PLP and NAD+ are added. [Figure 6] 1 shows the stability curves of the PVA membrane-immobilized enzyme of Example 8 of the present invention when PLP and NAD+ are added. [Figure 7] 1 shows the stability curves of the PVA membrane-immobilized enzyme according to Example 9 of the present invention with and without the addition of PLP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] In addition, the embodiments and features of the embodiments of the present application can be combined with each other unless a contradiction occurs. MODE FOR CARRYING OUT THE PRESENTLY PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the drawings in conjunction with the embodiments.

[0054] As described in the background of this application, the conventional technique of immobilizing an enzyme using a porous membrane involves a complicated process. In order to solve this problem, this application provides a PVA membrane-immobilized enzyme and a method for producing the same.

[0055] In a typical embodiment of the present application, a PVA membrane-immobilized enzyme is provided, the PVA membrane-immobilized enzyme comprising a PVA porous membrane and an enzyme embedded in the PVA porous membrane, the PVA porous membrane being a three-dimensionally structured PVA porous membrane, and the enzyme being selected from the group consisting of aminotransferase (e.g., ω-aminotransferase), D-lactate dehydrogenase, formate dehydrogenase, carbonyl reductase, cyclohexanone monooxygenase, ketene reductase, nitrile hydrolase, ammonia decomposition enzyme, amino acid dehydrogenase, imine reductase, and variants thereof.

[0056] The present application uses a PVA porous membrane as a carrier to immobilize enzymes by embedding, the process of embedding and immobilizing is simple, the conditions are mild, and it has good immobilization effect on both purified and crude enzymes. When the enzyme is embedded and immobilized in the PVA porous membrane, it is more stable and less likely to leach during use compared with the immobilization in a flat PVA membrane, and the porous structure of the PVA porous membrane used can better deliver reactants and products, making it suitable for use in continuous flow biocatalysis. The embedding and immobilization is a mechanical fixation, so it has a wide compatibility with enzymes. The PVA porous membrane is structured three-dimensionally to provide a three-dimensional structure, and has a larger surface area, which provides more embedding sites, and increases the enzyme loading capacity while ensuring high enzyme activity and stability.

[0057] In the PVA membrane-immobilized enzyme of the present invention, the enzyme to be embedded may be a free enzyme or a cross-linked enzyme aggregate, and whether it is a free enzyme or a cross-linked enzyme aggregate, both can effectively exhibit catalytic effect after embedding.

[0058] Crosslinked enzyme aggregates are insoluble enzyme aggregates obtained by precipitating free enzyme with a precipitating agent such as ammonium sulfate, ethanol, acetonitrile, acetone, propanol, or PEG, as in conventional techniques, and then covalently crosslinking the precipitate by adding a bifunctional reagent such as glutaraldehyde, glyoxal, or dextran aldehyde.

[0059] As described above, the PVA membrane-immobilized enzyme of the present invention has a wide compatibility with enzymes, and is particularly suitable for the following enzymes: an aminotransferase derived from Chromobacterium violaceum DSM30191 or an aminotransferase derived from Arthrobacter citreus or an aminotransferase derived from Bacillus thuringiensis; a ketoreductase derived from Acetobacter sp. CCTCC M209061 or a ketoreductase derived from Candida macedoniensis AKU4588; a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans; ammonia decomposition enzyme, which is derived from Aspergillus niger CBS 513.88 and from Solenostemon scutellarioides; an alkene reductase, which is derived from Saccharomyces cerevisiae and from Chryseobacterium sp. CA49; an imine reductase, which is derived from Streptomyces sp and from Bacillus cereus; cereus, an imine reductase; and an amino acid dehydrogenase, leucine dehydrogenase derived from Bacillus cereus and Bacillus sphaericus.phenylalanine dehydrogenase derived from Aspergillus niger CBS 513.88 and nitrile hydrolase derived from Neurospora crassa OR74A, preferably Chromobacterium violaceum. The amino acid sequence of the amino acid transferase derived from DSM30191 is shown in SEQ ID NO:1, and the amino acid sequence of the amino acid transferase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO:1, and the mutation includes at least one mutation site at positions 7, 47, 90, 95, 297, 304, 380, 405, and 416, and includes a mutation of threonine at position 7 to cysteine, a mutation of serine at position 47 to cysteine, a mutation of lysine at position 90 to glycine, and a mutation of alanine at position 95 to proline. The amino acid sequence of the aminotransferase mutant has a mutation site of the amino acid sequence obtained by the mutation and has a homology of 80% or more with the amino acid sequence obtained by the mutation, and is preferably Arthrobacter citreus (Arthrobactercitreus) has the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the mutant aminotransferase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 2, and the mutation includes at least one mutation site at positions 3, 5, 60, 164, 171, 178, 180, 186, 187, 252, 370, 384, 389, 404, 411, 423, and 424, and includes a mutation of leucine at position 3 to serine, a mutation of valine at position 5 to serine, a mutation of cysteine ​​at position 60 to tyrosine, a mutation of phenylalanine at position 164 to leucine, a mutation of glutamic acid at position 171 to aspartic acid, a mutation of alanine at position 178 to leucine, and a mutation of isoleucine at position 180 to 200. to valine, serine at position 186 to glycine, serine at position 187 to alanine, valine at position 252 to isoleucine, leucine at position 370 to alanine, tyrosine at position 384 to phenylalanine, isoleucine at position 389 to phenylalanine, leucine at position 404 to glutamine, glycine at position 411 to aspartic acid, methionine at position 423 to lysine, and glutamic acid at position 424 to glutamine, or the amino acid sequence of the amino acid transferase mutant has a mutation site of the amino acid sequence obtained by mutation and has 80% or more homology with the amino acid sequence obtained by mutation, and is preferably an amino acid sequence of the genus Acetobacter sp. CCTCC M209061 has the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 3, and the mutation includes at least one mutation site at positions 94, 144, and 156, and alanine at position 94 is mutated to asparagine, glutamic acid at position 144 is mutated to serine, and asparagine at position 156 is mutated to threonine or valine, or the amino acid sequence of the ketoreductase mutant has a mutation site of the amino acid sequence obtained by mutation and has 80% or more homology with the amino acid sequence obtained by mutation, and is preferably a ketoreductase derived from Rhodococcus sp. CCTCC M209061.Cyclohexanone monooxygenase derived from Phi1 has the amino acid sequence shown in SEQ ID NO: 4, and the amino acid sequence of the mutant cyclohexanone monooxygenase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 4, wherein the mutation includes at least one mutation site at positions 280, 435, 436, 438, 441, 508, and 510, and the phenylalanine at position 280 is mutated to tyrosine and the phenylalanine at position 435 is mutated to aspartate. a cyclohexanone monooxygenase mutant in which the amino acid sequence of the cyclohexanone monooxygenase mutant has a mutation site in the amino acid sequence obtained by the mutation and has 80% or more homology with the amino acid sequence obtained by the mutation, and is preferably ... cyclohexanone monooxygenase derived from C. ruber-SD1) has the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of the mutant cyclohexanone monooxygenase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO:5, and the mutation includes at least one mutation site at positions 45, 190, 249, 257, 393, 504, and 559, and the methionine at position 45 is mutated to threonine and the proline at position 190 is mutated to threonine. the amino acid sequence of the cyclohexanone monooxygenase mutant has a mutation site of the amino acid sequence obtained by the mutation and has a homology of 80% or more to the amino acid sequence obtained by the mutation, or ..., or the amino acid sequence of the cyclohexanone monooxygenase mutant has a mutation site of the amino acid sequence obtained by the mutation and has a homology of 80% or more to the amino acid sequence obtained by the mutation.

[0060] In order to improve the catalytic efficiency of the PVA membrane-immobilized enzyme, the PVA membrane-immobilized enzyme preferably further contains coenzymes and cofactors for each enzyme, i.e., if the enzyme has a corresponding coenzyme and cofactor, the PVA membrane-immobilized enzyme further contains the coenzyme and cofactor for the enzyme, and if the enzyme does not have a coenzyme or cofactor, the PVA membrane-immobilized enzyme does not contain other coenzymes or cofactors.

[0061] The PVA membrane-immobilized enzyme of the present invention is suitable not only for purified enzymes but also for crude enzymes, and preferably the enzyme is crude enzyme in order to simplify the process. In addition, since the immobilization method of the present invention is embedding, the amount of enzyme that can be supported is relatively large, and preferably the enzyme loading capacity is 0.05 to 0.4 g of free enzyme / cm. 2 membrane or 0.03-0.06 g of dried cross-linked enzyme aggregates / cm 2 It is a membrane.

[0062] In another embodiment, the PVA porous membrane further comprises polyethylene glycol and / or polyethyleneimine, the molecular weight of the polyethylene glycol being PEG400-PEG6000, and the molecular weight of the polyethyleneimine being 3KDa-70KDa, preferably 3KDa-50KDa. Furthermore, preferably, the mass ratio of the polyethylene glycol to the PVA porous membrane is 5:4-75:4, and the mass ratio of the polyethyleneimine to the PVA porous membrane is 1:12-1:240. The polyethylene glycol and polyethyleneimine increase the pore structure in the PVA porous membrane.

[0063] In another exemplary embodiment of the present application, there is provided a method for producing any one of the above PVA membrane-immobilized enzymes, the method including: step S1 of mixing raw materials containing an enzyme and a PVA solution for a predetermined time to obtain a mixed system; step S2 of adding the mixed system to a mold and drying the mixed system to obtain a membrane-embedded enzyme, the mold being a three-dimensionally structured mold for forming a three-dimensionally structured PVA porous membrane; and step S3 of immersing the membrane-embedded enzyme in a phosphate buffer solution to wash it, and then obtaining the PVA membrane-immobilized enzyme, and the pH of the mixed system is preferably 6.0 to 6.5.

[0064] The present invention can form a PVA membrane-immobilized enzyme by simply mixing, drying and post-treatment, and the process is simple and easy to operate, without the need for cross-linking or covalent fixation using glutaraldehyde, amino groups, carboxyl groups, etc. The PVA porous membrane in the formed PVA membrane-immobilized enzyme is used as a carrier to immobilize the enzyme by embedding, and has good immobilization effect on both purified and crude enzymes, and the enzyme is relatively stable when embedded and immobilized in the PVA porous membrane and is not easily leached during use. The porous structure of the PVA porous membrane used can better deliver reactants and products, making it suitable for use in continuous flow biocatalysis. The embedding and immobilization is a mechanical fixation, so it has a wide compatibility with enzymes. The three-dimensionally structured mold is used to make the PVA porous membrane three-dimensionally structured, which gives it a three-dimensional structure and a larger surface area, providing more embedding sites, and increasing the enzyme loading capacity while ensuring high enzyme activity and stability.

[0065] The method of forming the mixed system may vary depending on the form of the enzyme provided. Hereinafter, some preferred methods of forming the mixed system will be provided, and the following description of step S1 does not limit the scope of step S1.

[0066] In one embodiment of the present application, the step S1 includes preparing an enzyme suspension or enzyme solution, in which the enzyme in the suspension is a cross-linked enzyme aggregate, and the enzyme in the enzyme solution is a cell-free free enzyme, and mixing the suspension or enzyme solution with a PVA solution for a predetermined time to obtain a mixed system. Either the enzyme solution or the enzyme suspension can be mixed with the PVA solution, and the mixing process can be performed by mechanical stirring or magnetic stirring.

[0067] The PVA solution is a mixed solution containing PVA, water, acetic acid, methanol and sulfuric acid. The mixed solution preferably has a pH of 5.5 to 6.5. When acetic acid, methanol and sulfuric acid are combined, the resulting membrane has more micropores.

[0068] In order to improve the uniformity of the dispersion of the enzyme in the PVA solution, the predetermined time is preferably 2 to 4 hours.

[0069] In order to form a thin gel film with sufficient mechanical strength, the molecular weight of the PVA in the PVA solution is 20 KDa to 200 KDa. Furthermore, in order to form a PVA porous film with high porosity and to facilitate the dispersion of the enzyme therein, the content of PVA in the PVA solution is preferably 5 to 30 g / 100 mL, and more preferably 10 to 50 g / 100 mL.

[0070] The mechanical immobilization method of embedding an enzyme in a PVA membrane can increase the enzyme loading capacity, and preferably the enzyme concentration in the suspension or enzyme solution is 0.1 to 0.5 g / mL, and the ratio of the enzyme to the PVA solution is preferably 1 to 50 g / 100 mL, and more preferably 5 to 40 g / 100 mL.

[0071] In order to maintain high activity of the enzyme in the mixing step, the suspension or enzyme solution preferably further comprises a phosphate buffer, an optional cofactor and an optional coenzyme, preferably the concentration of the cofactor is 1 to 20 mg / mL, and the weight ratio of the coenzyme to the enzyme is preferably 10:1 to 1:10.

[0072] In another embodiment of the present application, the step S1 includes mixing the PVA aqueous solution and the cross-linked enzyme particles to form a mixed system. In this embodiment, the cross-linked enzyme is mixed with the PVA aqueous solution in the form of dry particles, so that the cross-linked enzyme particles are easy to disperse. In order to ensure the enzyme loading capacity, the ratio of the cross-linked enzyme particles to the PVA aqueous solution is preferably 1-50 g / 100 mL. Similarly, in order to improve the uniformity of the dispersion of the cross-linked enzyme particles in the PVA aqueous solution, the predetermined time is preferably 10-60 minutes. Furthermore, if necessary, the cross-linked enzyme particles preferably include an enzyme, an optional cofactor, and an optional coenzyme.

[0073] In another embodiment of the present application, the step S1 includes mixing the PVA aqueous solution and the modifier solution for a first predetermined time to form a second mixed system, and mixing the second mixed system and the enzyme system for a second predetermined time to form a mixed system. The modifier is used to promote the film formation of the PVA and increase the pore structure therein.

[0074] Furthermore, in order to form a PVA porous membrane with high porosity and to facilitate the dispersion of the enzyme therein, the concentration of the aqueous PVA solution is preferably 5 to 30 g / 100 mL. The modifier used in the present application can be selected from modifiers commonly used in the formation of PVA films. In order to avoid the effect of the modifier on the enzyme, the modifier solution preferably contains a mixed solution in which acetic acid, methanol, and sulfuric acid are dispersed, and / or a polyethylene glycol aqueous solution in which a cofactor is dispersed, and / or a polyethyleneimine aqueous solution in which a cofactor is dispersed. Preferably, the mixed solution has an acetic acid content of 2 to 4 g / 100 mL, a methanol content of 5 to 9 g / 100 mL, and a sulfuric acid content of 0.5 to 1 g / 100 mL. Preferably, the molecular weight of the polyethylene glycol is PEG400 to PEG6000. Preferably, the concentration of the polyethylene glycol in the mixed system is 3 to 10 g / 100 mL. Preferably, the molecular weight of the polyethyleneimine is 3 KDa to 70 KDa, more preferably 3 KDa to 50 KDa. Preferably, the concentration of the polyethyleneimine in the mixed system is 0.1 to 1 g / 100 mL, more preferably 0.1 to 0.3 g / 100 mL.

[0075] As described above, when the modifier solution is added, there is no obvious effect on the mixing of the enzyme system and the second mixed system, so the enzyme system may be in the general form of conventional enzyme provision, preferably the enzyme system includes an enzyme, an optional cofactor, an optional coenzyme and a phosphate buffer, and preferably the enzyme is a cell-free free enzyme or a cross-linked enzyme aggregate. When it is necessary to use a cofactor and a coenzyme to increase the enzyme loading capacity, the concentration of the cofactor is 1-20mg / mL, preferably the weight ratio of the coenzyme to the enzyme in the enzyme system is 10:1-1:10, and the ratio of the enzyme to the PVA solution is 1-50g / 100mL.

[0076] After the mixed system is formed, the mixed system may be left to dry. In order to accelerate the film formation process, preferably, the step S2 is to place the mixed system in a mold and leave it for a third predetermined time, and then add a dehydration promoter to the mold to perform a drying process, and the dehydration promoter is selected from the group consisting of acetonitrile, ethanol, and acetone, and preferably, the volume ratio of the dehydration promoter to the mixed system is 1:10 to 5:1. In the film formation process, the enzyme is embedded by the formed PVA porous film to form a stable immobilized enzyme structure. In order to prevent the enzyme from being completely covered due to too fast film formation, preferably, the third predetermined time is 2 to 4 hours. In addition, in order to simplify the structure of the three-dimensionally structured mold, preferably, the three-dimensionally structured mold has protrusions or recessed grooves.

[0077] In order to further ensure the immobilization of the enzyme after the membrane formation, the step S3 preferably includes immersing the membrane-embedded enzyme in a phosphate buffer for 2 to 16 hours and then washing the membrane-embedded enzyme with fresh phosphate buffer to obtain a PVA membrane-immobilized enzyme.

[0078] In the preparation method described herein, the enzyme used may be a purified enzyme or a crude enzyme, preferably the enzyme is a crude enzyme in order to save costs.

[0079] The PVA membrane-immobilized enzyme obtained in the present application may be further resuspended in a buffer solution and modified by adding glutaraldehyde, so that the enzyme molecules crosslink with each other by the covalent bond between the amino group and the aldehyde group of glutaraldehyde to form larger aggregates that will not leak from the PVA membrane, and the enzyme attached to the surface of the PVA membrane is more firmly immobilized by covalent bonding with PVA through the arm action of glutaraldehyde, thereby increasing the number of times it can be used. Preferably, the amount of glutaraldehyde is 1 to 2 g / 100 mL of suspension.

[0080] The beneficial effects of the present invention will be further illustrated by the following examples and comparative examples.

[0081] See Table 1 for the enzymes used in the following examples and their origins.

[0082] [Table 1]

[0083] The amino acid sequence of the TA-Cv aminotransferase is SEQ ID NO:1: MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFFKTTHPAVVELSSLLAEVTPAGFDRVF YTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGA GGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMVQAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRACGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA.

[0084] The mutation site and amino acid mutation situation of mutant 1 (TA-Cv-V1) are R416T+T7C+S47C+Q380L, and the mutation site and amino acid mutation situation of mutant 2 (TA-Cv-V2) are R416T+T7C+S47C+R405E+K90G+A95P+K304D+Q380L+I297L.

[0085] The amino acid sequence of TA-Ac aminotransferase is SEQ ID NO:2: MGLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLITPGGTRLLDFFNQLCCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVST GSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSCSSAVLMAPSSNTFQDSNGNYLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQ GVGSTMPPYEYVPQIRKMTKELGVLWISDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVVVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENLVEQAKNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFRHGMNPNQIPTQIIMEKALEKGVLIGGAMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQQS.

[0086] The mutation site and amino acid mutation status of mutant 1 (TA-Ac-V1) are L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+L404Q+E171D, and the mutation site and amino acid mutation status of mutant 2 (TA-Ac-V2) are L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+E424Q+M423K.

[0087] The amino acid sequence of KRED-Ac ketoreductase is SEQ ID NO:3: MARVAGKVAIVSGAANGIGKATAQLLAKEGAKVVIGDLKEEDGQKAVAEIKAAGGEAAFVKLNVTDEAAWKAAIGQTLKLYGRLDIAVNNAGINYSGSVESTSLEDWRRVQSINLDGVFLGTQVAIEA MKKSGGGSIVNLSSISGLIGDPMLAAYVASKGGVRLFTKSAALHCAKSGYKIRVNSVHPGYIWTPMVAGLTKEDAAARQKLVDLHPIGHLGEPNDIAYGILYLASDESKFVTGSELIVIDGGYTAQ.

[0088] The mutation site and amino acid mutation situation of the mutant 1 (KRED-Ac-V1) are E144S+A94N+N156V, and the mutation site and amino acid mutation situation of the mutant 2 (KRED-Ac-V2) are E144S+A94T+N156T.

[0089] The amino acid sequence of CHMO-Rs cyclohexanone monooxygenase is SEQ ID NO:4: It is.

[0090] The mutation site and amino acid mutation situation of mutant 1 (CHMO-Rs-Cv-V1) are F508Y+F435N+L438A+T436S+F280V+S441V, and the mutation site and amino acid mutation situation of mutant 2 (CHMO-Rs-Cv-V2) are F508Y+F435N+L438A+T436S+F280V+S441V+L510V.

[0091] The amino acid sequence of CHMO-Rr cyclohexanone monooxygenase is set forth in SEQ ID NO:5: It is.

[0092] The mutation site and amino acid mutation status of mutant 1 (CHMO-Rr-V1) are P190L+Y559M+C249V+C393V+C257A+M45T, and the mutation site and amino acid mutation status of mutant 2 (CHMO-Rr-V2) are Y559M+P190L+P504V.

[0093] The phosphate buffer (PB) used in the following examples is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0094] Example 1 Immobilization of aminotransferase TA-CV CLEA (cross-linking enzyme) in PVA membrane: Preparation of PVA I solution: 50 mL of 10% (w / v) PVA (200 KDa) and 30 mL of 10% (w / v) acetic acid, 50% (v / v) methanol, 10% (w / v) sulfuric acid were mixed. The encapsulation was performed by PVA membrane. The pH of the PVA I solution was adjusted to 6.0, and 20 mL was taken out from the solution and mixed with a suspension of TA-CV cross-linked enzyme aggregates (CLEA) (the composition of the suspension of TA-CV cross-linked enzyme aggregates CLEA was 0.5 g of TA-CV cross-linked enzyme in 2 mL of 0.1 M phosphate buffer (PB, pH 7.0), and each mL contained 2 mg of PLP (pyridoxal phosphate)). The mixture was stirred for 20 minutes to form a mixed system. It was poured into a 3D porous silica gel template, and the wells of each template were square, the volume was about 0.1-0.2 cubic centimeters, and the surface area of ​​the wells was about 2-5 square centimeters. The mixture was dried at 37 °C to obtain the membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1 M PB (pH 7.0) + 0.5 M NaCl buffer for 3 hours to remove the membrane-embedded enzyme from the buffer, and then washed three times with 0.1 M PB (pH 7.0) to obtain the PVA membrane-immobilized enzyme of Example 1.

[0095] Comparative Example 1 The 3D porous silica gel template in Example 1 was replaced with a high-temperature resistant silica gel disk to form a planar PVA membrane-embedded enzyme, and the bottom area of ​​the silica gel disk was 80 square centimeters. At the same time, the effects of parameters such as different molecular weight PVA, PVA concentration, and enzyme to PVA ratio on the activity and stability of PVA-immobilized enzyme were investigated. Conversion and stability studies: Model reactions employed in the conversion studies: [ka] In the above reaction formula, R1 and R2 are each independently selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, and a substituted or unsubstituted heterocycloalkyl group, and R1 and R2 may be joined to form a ring. [ka] 0.1g of ketone matrix 1 was dissolved in 0.35mL of methanol, 3.0 molar equivalents of isopropylamine hydrochloride was added as an amino donor, 5mg of PLP was added to the reaction system, and then diluted with 0.3mL of 0.1M PB 7.0 to form the target reaction system. 3mg of TA-CV CLEA or the three-dimensionally structured PVA membrane immobilized enzyme of Example 1 containing 3mg of TA-CV CLEA or the planar PVA membrane immobilized enzyme of Comparative Example 1 containing 3mg of TA-CV CLEA was used as a catalyst. After reacting at 30°C for 20 hours, the conversion rate was detected by HPLC method, and the immobilized enzyme was separated after one reaction was completed and used repeatedly in the next reaction, and the number of repeated uses was examined, and the conversion rate was measured after 11 repeated reactions and recorded in Table 2.

[0096] [Table 2]

[0097] Furthermore, Table 3 shows the effects of the molecular weight of PVA, the concentration of PVA, and the ratio of enzyme to PVA solution on the activity and stability of the PVA-immobilized enzyme.

[0098] [Table 3]

[0099] The effect of pH of the mixed system on enzyme activity and stability is shown in Table 4.

[0100] [Table 4]

[0101] Example 2 Immobilization of aminotransferase TA-CV CLEA in PVA membrane: Preparation of PVA II solution: 12%–15% (w / v) PVA in water. Embedding with PVA membrane. 3g of CLEA wet particles and 50mg of PLP were added to 30mL of PVA solution, and stirred uniformly to form a mixed system, then poured into 3D porous silica gel template, each template well was circular, the volume was about 0.15-0.2 cubic centimeters, the surface area of ​​the well was about 3-5 square centimeters, and dried at 37°C to obtain membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 buffer overnight to remove the membrane-embedded enzyme from the buffer, and then washed twice with 0.1M PB 7.0 buffer to obtain the PVA membrane-immobilized enzyme of Example 2. A certain amount of PEG400–PEG6000 was dissolved in the PVA II solution, respectively, before adding CLEA to perform a control experiment. Conversion and stability studies on the type of substrates in Example 1: 0.1g of ketone matrix 1 was dissolved in 0.35mL of methanol, and 3.0 molar equivalents of isopropylamine hydrochloride was added as an amino donor. 5mg of PLP was added to the reaction system, and then diluted with 0.3mL of 0.1M PB 7.0 to form a target reaction system. Each PVA membrane immobilized enzyme of Example 2, which has a specific surface area of ​​about 6 square centimeters and has 6mg of TA-Cv CLEA embedded therein, was cut out as a catalyst, and a control reaction was performed without adding PLP. After reacting at 30°C for 4 hours, the conversion rate was detected by HPLC method, and one reaction was 20 hours long. After one reaction was completed, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times of repeated use was examined. The test results are shown in Figure 1.

[0102] As can be seen from Figure 1, the PVA membrane immobilized enzyme of Example 2 has excellent activity and stability, and the activity did not decrease after 14 cycles of use. When PLP was not added, the activity was slightly lower, but the stability was as excellent as when PLP was added. Also, as can be seen from Figure 1, the reaction rate can be increased by adding PEG400 or PEG6000.

[0103] Furthermore, the effects of the molecular weight of PEG and the concentration of PEG in the mixed system on the enzyme activity and stability were examined, and the results are shown in Table 5.

[0104] [Table 5]

[0105] Example 3 Embedding and fixing of TA-CV wet cell free enzyme or TA-CV CLEA with PVA-organic solvent membrane: 7.0mL of 10% (w / v) PVA solution was mixed with 5mL of TA-CV free enzyme solution (containing 5mg / mL PLP) or 1g of TA-CV CLEA, and stirred for 30 minutes to form a mixed system, which was poured into a glass or high-temperature resistant plate and left at room temperature for 3 hours, and then 15mL of organic solvent acetonitrile was gently poured into the 3D porous silica gel template, each template well was circular, the volume was about 0.15-0.2 cubic centimeters, the surface area of ​​the well was about 3-5 square centimeters, and dried at 37°C to form a membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1M PB 7.0+0.5M NaCl buffer for 2-3 hours to remove the membrane-embedded enzyme from the buffer, and then the membrane-embedded enzyme was washed three times with 0.1M PB (pH 7.0) to obtain two kinds of PVA membrane-immobilized enzymes in Example 3. Conversion and stability studies on the type of substrates in Example 1: 0.1g of ketone matrix 1 was dissolved in 0.35mL of methanol, 3.0 molar equivalents of isopropylamine was added as an amino donor, 5mg of PLP was added to the reaction system, and then diluted with 0.3mL of 0.1M PB 7.0 to form the target reaction system. A PVA membrane immobilized enzyme with a specific surface area of ​​about 6 square centimeters and prepared by embedding TA-CV wet cells or CLEA was cut out and used as a catalyst. After reacting at 30°C for 20 hours, the conversion rate was detected by HPLC method, and the immobilized enzyme was separated after one reaction was completed and used repeatedly in the next reaction, and the number of times of repeated use was examined. The test results are shown in Table 6 and Figure 2.

[0106] [Table 6]

[0107] After 20 hours of reaction, the conversion rate still reached 98%, and after 10 cycles of use, the catalyst was stable and did not lose activity.

[0108] Example 4 Preparation of immobilized TA-CV with large specific surface area by embedding TA-CV in PVA-organic solvent: 5.0 mL of 10% (w / v) PVA solution and 5 mL of TA-CV free enzyme solution (enzyme concentration is 0.1 g / mL, containing 5 mg / mL PLP) were mixed and stirred for 20 minutes to form a mixed system, air bubbles were removed, and 0.1 mL of the mixed system was added dropwise to a 3D porous silica gel template, each template well was circular or rectangular, the volume was about 0.15-0.2 cubic centimeters, and the surface area of ​​the well was about 3-5 square centimeters. It was left to stand at room temperature for 3 hours, and then 0.06 mL of an organic solvent acetone or acetonitrile was gently added dropwise to the well and dried at 37 °C to form a hollow block-shaped membrane-embedded enzyme in each well. These hollow block-shaped membrane-embedded enzymes were immersed in 0.1 M PB 7.0 + 0.5 M NaCl buffer for 2-3 hours. Next, the buffer solution was removed, and the hollow block-shaped membrane-embedded enzyme was washed three times with 0.1 M PB 7.0 to obtain each PVA membrane-immobilized enzyme of Example 4. A part of the membrane-embedded enzyme was further modified with glutaraldehyde. The membrane-embedded enzyme was resuspended in 0.1M PB 7.0, and glutaraldehyde was added dropwise, 1-2 g of glutaraldehyde per 100 mL of suspension, and gently stirred at room temperature for 2 hours. The buffer was removed, and the hollow block-shaped membrane-embedded enzyme after glutaraldehyde modification was washed three times with 0.1M PB 7.0 to obtain the glutaraldehyde-modified PVA membrane-immobilized enzyme. Reactivity and stability tests on the type of substrates in Example 1: 0.1 g of ketone matrix was dissolved in 0.35 mL of methanol, 3.0 molar equivalents of isopropylamine hydrochloride was added as an amino donor, 5 mg of cofactor PLP was added to the reaction system, and then diluted with 0.3 mL of 0.1 M PB 7.0 to form the target reaction system. A PVA membrane immobilized enzyme having a specific surface area of ​​about 6 square centimeters and embedded with TA-CV free enzyme (acetone dried), a PVA membrane immobilized enzyme having a specific surface area of ​​about 6 square centimeters and embedded with TA-CV free enzyme (acetone dried and modified with glutaraldehyde), a PVA membrane immobilized enzyme having a specific surface area of ​​about 6 square centimeters and embedded with TA-CV free enzyme (acetonitrile dried), and a PVA membrane immobilized enzyme having a specific surface area of ​​about 6 square centimeters and embedded with TA-CV free enzyme (acetonitrile dried and modified with glutaraldehyde) were cut out to be used as catalysts. After reacting at 30°C for 20 hours, 10 cycles were repeated, and the conversion rate after 20 hours of reaction was detected by HPLC. After one reaction, the immobilized enzyme was separated and reused in the next reaction. The number of times of reuse was examined, and the test results are shown in Table 7.

[0109] [Table 7]

[0110] Furthermore, the effect of the ratio of the dehydrating agent and the PVA-enzyme mixture on the enzyme activity and stability was investigated, and the results are shown in Table 8.

[0111] [Table 8]

[0112] As can be seen from the data in Table 7, when the PVA membrane-immobilized enzyme of Example 4 was used as a catalyst, the activity did not decrease after 10 cycles. In the case of GA modification, the enzyme activity was slightly lower, but the stability was as good as that without GA modification.

[0113] Example 5 Immobilization of TA-CV free enzyme in PVA-CFP membrane: PVA solution: A 12% (w / v) solution was prepared in water. Cofactor-polymer solution (CFP solution): Dissolve 2% w / v PEI (polyethyleneimine) (3 KDa to 70 KDa) and add cofactor (PLP) at 5 mg / mL. Mix at room temperature for 0.5 to 3 hours.

[0114] 35mL of PVA solution and 5mL of CFP solution were thoroughly mixed for 30 minutes, then 5mL of enzyme solution (enzyme concentration 0.1g / mL) and 3-5mg of cofactor PLP were added and mixed at room temperature for 30 minutes to form a mixed system. The mixed system was then poured into a 3D structured porous silica gel template and dried at 37°C to obtain a membrane-embedded enzyme, where the wells of each template were circular or rectangular, the volume was about 0.15-0.2 cubic centimeters, and the surface area of ​​the wells was about 3-5 square centimeters. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 buffer overnight. After removing the buffer, the membrane-embedded enzyme was washed twice with 0.1M PB 7.0 to obtain the PVA membrane-immobilized enzyme of Example 5. Activity and stability tests on the type of substrates in Example 1: 0.1g of ketone matrix 1 was dissolved in 0.35mL of methanol, 3.0 molar equivalents of isopropylamine was added as an amino donor, 5mg of PLP was added to the reaction system, and then diluted with 0.3mL of 0.1M PB 7.0 to form a target reaction system. The PVA membrane immobilized enzyme of Example 4, which has a specific surface area of ​​about 3-4 square centimeters and has 3mg of TA-CV free enzyme embedded therein, was cut out as a catalyst, and a control reaction was performed without adding PLP. After reacting at 30°C for 20 hours, 14 cycles were repeated, and the conversion rate after 4 hours of reaction was measured by HPLC, and the test results are shown in Figure 3.

[0115] As can be seen from Figure 3, the activity and stability of the PVA membrane-immobilized enzyme formed was very good, and the activity did not decrease after 14 cycles of use. When PLP was not added, the activity was slightly lower, but the stability was as good as when PLP was added. Furthermore, the effects of the molecular weight of PEI and the concentration of PEI in the mixed system on the enzyme activity and stability were examined, and the results are shown in Table 9.

[0116] [Table 9]

[0117] Example 6 Embedding and fixing of TA-CV CLEA in PVA-CFP membrane: PVA solution: A 12% w / v solution was prepared in water. CFP solution: Prepare a 2% w / v PEI (3 KDa-70 KDa) solution in water and add cofactor (PLP) at 5 mg / mL and mix for 0.5-3 h at room temperature. 3g of TA-CV CLEA was suspended in 10mL of CFP solution and thoroughly mixed with 30mL of PVA solution to form a mixed system. The mixed system was then poured into a porous silica gel template and dried at 37°C to form a membrane-embedded enzyme, with the wells of each template being circular or rectangular, the volume being about 0.15-0.2 cubic centimeters, and the surface area of ​​the wells being about 3-5 square centimeters. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 buffer overnight. The membrane-embedded enzyme was removed from the buffer and then washed twice with 0.1M PB 7.0 to obtain the PVA membrane-embedded immobilized enzyme of Example 6. Activity and stability tests on the type of substrates in Example 1: 0.1 g of ketone matrix 1 was dissolved in 0.35 mL of methanol, 3.0 molar equivalents of isopropylamine was added as an amino donor, 5 mg of PLP was added to the reaction system, and then diluted with 0.3 mL of 0.1 M PB 7.0 to form the target reaction system. The specific surface area of ​​6 mg of TA-CV CLEA embedded in the reaction system was approximately 5 cm 2 A control reaction was carried out without adding PLP, using the PVA membrane-embedded immobilized enzyme of Example 6 as a catalyst. After 20 hours of reaction at 30°C, 14 cycles were repeated, and the conversion rate was detected by HPLC. After one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction. The number of times of repeated use was examined, and the test results are shown in Figure 4.

[0118] As can be seen from Figure 4, the activity and stability of the PVA membrane-embedded immobilized enzyme were excellent, and the activity did not decrease after 14 cycles of use. When PLP was not added, the activity was slightly lower, but the stability was as good as when PLP was added.

[0119] Example 7 Immobilization of TA-Ac-releasing enzyme in PVA-CFP membrane: PVA solution: A 12% w / v solution was prepared in water. CFP solution: Prepare a 2% w / v PEI (3 KDa-70 KDa) solution in water and add cofactor (PLP) at 5 mg / mL and mix for 0.5-3 h at room temperature. 35mL of PVA solution and 5mL of CFP solution were thoroughly mixed for 30 minutes, then 5mL of TA-Ac enzyme solution (enzyme concentration 0.1) and 3-5mg of cofactor PLP were added and mixed at room temperature for 30 minutes to obtain a mixed system. The mixed system was then poured into a 3D porous silica gel template, each template well was circular or rectangular, the volume was about 0.15-0.2 cubic centimeters, the surface area of ​​the well was about 3-5 square centimeters, and dried at 37°C to obtain a membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 overnight. The membrane-embedded enzyme was removed from the buffer solution and then washed twice with 0.1M PB 7.0 to obtain the PVA membrane-embedded immobilized enzyme of Example 7. Activity and stability tests on the type of substrates in Example 1: [ka] Specifically, ketone matrix 2 (see immediately preceding formula) replaced ketone matrix 1 used in Example 1. The test was conducted in an aqueous buffer system. 0.1g of ketone matrix 2 was suspended in 1mL of 0.1M PB 7.0 buffer, 3.0 molar equivalents of isopropylamine hydrochloride was added as an amino donor, 5mg of PLP was added to the reaction system, and the PVA membrane-embedded immobilized enzyme of Example 7 containing 10mg of TA-Ac liberating enzyme was used as a catalyst. After reacting at 30°C for 20 hours, the reaction was repeated for 5 cycles, the conversion rate was detected by HPLC, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction. The number of times of repeated use was examined, and the test results are shown in Table 10.

[0120] This was a test in a biphasic system. 0.1g of ketone matrix 2 was dissolved in 1mL of MTBE as a non-aqueous phase, 1mL of 0.1M PB 7.0 was added as an aqueous phase, 3.0 molar equivalents of isopropylamine were added as an amino donor, and 5mg of PLP was added to the reaction system to form a target reaction system, and the PVA membrane immobilized enzyme of Example 7 containing 10mg of TA-Ac liberating enzyme was used as a catalyst. After reacting for 20 hours at 30°C, five cycles were repeated, the conversion rate was detected by HPLC, and the immobilized enzyme was separated after one reaction was completed and used repeatedly in the next reaction, and the number of repeated uses was examined, and the test results are shown in Table 10.

[0121] [Table 10]

[0122] As can be seen from the data in Table 10, the PVA membrane-immobilized enzyme of Example 7 had excellent activity and stability, and the activity did not decrease after five cycles of use.

[0123] The effects of the molecular weight of PVA, the concentration of PVA, and the ratio of enzyme to PVA solution on the activity and stability of the enzyme were examined, and the results are shown in Table 11.

[0124] [Table 11]

[0125] Example 8 Encapsulation of ketoreductase in PVA-CFP membrane: PVA solution: A 12% (w / v) solution was prepared in water. CFP solution: Prepare by dissolving 2% (w / v) PEI (3KDa to 70KDa) in water, and optionally adding cofactor (NAD) at 5mg / mL. + or PLP) was added and mixed at room temperature for 0.5 to 3 hours. 35 mL of PVA solution and 5 mL of CFP solution were mixed thoroughly for 30 min, then 5 mL of KRED-Ac or KRED-Cm enzyme solution (enzyme concentration 0.1 g / mL) and 4 mg of cofactor NAD+ and mixed at room temperature for 30 minutes to form a mixed system. The mixed system was then poured into a 3D siloxane template, where each template well was circular or square, had a volume of about 0.15-0.2 cubic centimeters, and had a surface area of ​​about 3-5 square centimeters. The mixture was dried at 37°C to form a membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 overnight. The membrane-embedded enzyme was removed from the buffer and then washed twice with 0.1M PB 7.0 to obtain the NAD of Example 8. + The enzyme immobilized in PVA membrane was obtained. PLP and the cofactor NAD + The above steps were repeated with the replacement of the above, to obtain the PLP-containing PVA membrane-immobilized enzyme of Example 8. Activity and stability studies: Model reactions employed in the conversion studies: [ka] In the above reaction scheme, R1 and R2 are each independently selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, and a substituted or unsubstituted heterocycloalkyl group; or R1 and R2 may be joined to form a ring. [ka] Dissolve 0.1 g of ketone matrix 3 or 4 in 0.5 mL of isopropanol and add 0.5 mL of the cofactor NAD to the reaction system. +A target reaction system was formed by adding 0.1M PB 7.0 containing 5 mg of the above, and the PVA membrane-embedded immobilized enzyme of Example 8 in which 30 mg of ketoreductase KRED-Ac (activity confirmed with ketone matrix 3) or ketoreductase KRED-Cm (activity confirmed with ketone matrix 4) was embedded was added to the target reaction system as a catalyst. After reacting for 20 hours at 30°C, 11 cycles were repeated, the conversion rate was detected by GC method, and the immobilized enzyme was separated after one reaction was completed and used repeatedly in the next reaction. The number of repeated uses was examined, and the results are shown in Figures 5 and 6. The substrate corresponding to Figure 5 was ketone matrix 3, and the substrate corresponding to Figure 6 was ketone matrix 4.

[0126] As can be seen from FIG. 5, the PVA membrane immobilized enzyme KRED-Ac of Example 8 has excellent activity and stability against the ketone matrix 3, with a conversion rate of up to 99% and no loss of activity after 11 cycles of repeated use. When the CFP solution was prepared with PLP, the activity was NAD + far exceeded that.

[0127] As can be seen from FIG. 6, the PVA membrane-embedded immobilized enzyme KRED-Cm obtained in Example 8 has excellent activity and stability, with a conversion rate of up to 99% and no loss of activity after 10 cycles of repeated use. When the CFP solution was prepared with PLP, the activity was increased by 10% compared to NAD. + far exceeded that.

[0128] The concentration of the cofactor in the enzyme system was examined, and the results are shown in Table 12.

[0129] [Table 12]

[0130] Example 9 Hereinafter, the PVA membrane-embedded immobilized aminotransferase TA-Bt and its coenzymes LDH and FDH co-crosslinking enzyme, and the aminotransferase TA-Bt and its coenzymes LDH and FDH co-crosslinking enzyme are abbreviated as co-crosslinking enzyme. Preparation of PVA I solution: 50 mL of 10% (w / v) PVA (200 KDa) and 30 mL of 10% (w / v) acetic acid, 50% (v / v) methanol, 10% (w / v) sulfuric acid were mixed. The encapsulation was performed by PVA membrane. The pH of the PVA I solution was adjusted to 4-6.5, and 20 mL was taken out of the solution and mixed with the co-crosslinked enzyme suspension (the composition of the co-crosslinked enzyme suspension was 0.5 g of TA-Bt and co-crosslinked enzymes of LDH and FDH in 2 mL of 0.1 M phosphate buffer (PB, pH 7.0), and 1 mL contained 2 mg of PLP (pyridoxal phosphate)). The mixture was poured into a 3D porous silica gel template, and the wells of each template were square, the volume was about 0.1-0.2 cubic centimeters, and the surface area of ​​the wells was about 2-5 square centimeters. The mixture was dried at 37 °C to obtain the membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1 M PB (pH 7.0) + 0.5 M NaCl buffer for 3 hours to remove the membrane-embedded enzyme from the buffer, and then washed three times with 0.1 M PB (pH 7.0) to obtain the PVA membrane-immobilized enzyme of Example 1. Activity and stability studies: Reaction models employed in the conversion study: [ka] In the above reaction scheme, R may be selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted heterocycloalkyl group, and a halogen. [ka] 5 mL of 0.1 M PB (pH 8.0) was placed in a 10 mL reaction flask, followed by the addition of 100 mg of the substrate 5, 80 mg of ammonium formate and 5 mg of PLP to adjust the pH to 7.5-8.0, and then 5 mg of NAD. +and 10 mg of membrane-immobilized enzyme (wet material containing 50-80% water) were added. The reaction was carried out at 30°C for 20 hours to detect the conversion rate, and the immobilized enzyme was separated after one reaction and repeatedly used in the next reaction to examine the number of repeated uses. Specifically, the effect of the ratio of main enzyme to coenzyme in the co-immobilized enzyme of TA-Bt and coenzyme on the enzyme activity and stability was examined, and the results are shown in Table 13.

[0131] [Table 13]

[0132] Example 10 Co-immobilization of TA-Bt with coenzymes D-LDH and FDH by embedding in PVA-CFP membrane: PVA (200 KDa) solution: A 12% (w / v) solution was prepared in water. CFP solution: Prepare an aqueous solution of 2% (w / v) PEI (3 KDa - 70 KDa), add a cofactor (PLP) at 5 mg / mL, and mix at room temperature for 0.5 - 3 h. 35 mL of PVA solution and 5 mL of CFP solution were mixed thoroughly for 30 min, and then 5 mL of TA-Bt enzyme solution (enzyme concentration 0.08 g / mL), 0.08 g of coenzyme D-LDH, 0.1 g of coenzyme FDH, and 4 mg of cofactor NAD were added. + and mixed at room temperature for 30 minutes to form a mixed system. The mixed system was then poured into a 3D siloxane template, where each template well was circular or square, had a volume of about 0.15-0.2 cubic centimeters, and had a surface area of ​​about 3-5 square centimeters. The mixture was dried at 37°C to form a membrane-embedded enzyme. The membrane-embedded enzyme was immersed in 0.1M PB 7.0 overnight. The membrane-embedded enzyme was removed from the buffer and then washed twice with 0.1M PB 7.0 to obtain the PLP and NAD of Example 10. + The enzyme immobilized in the PVA-CFP membrane was obtained. Activity and stability testing using the model of Example 9: 5 mL of 0.1 M PB (pH 8.0) was placed in a 10 mL reaction flask, followed by the addition of 100 mg of the substrate 5, 80 mg of ammonium formate and 5 mg of PLP to adjust the pH to 7.5-8.0, and then NAD + 5mg, PLP and NAD + A PVA-CFP membrane immobilized enzyme (wet material containing 50-80% water) containing 10 mg of PLP was added. The reaction was carried out at 30°C for 20 hours to detect the conversion rate. After one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction. The number of times of repeated use was examined, and a control reaction was carried out without adding PLP. After reacting at 30°C for 4 hours, 9 cycles were repeated, and the conversion rate was detected by HPLC. After one reaction, the immobilized enzyme was separated and reused in the next reaction. The number of times of reuse was examined, and the test results are shown in Figure 7.

[0133] As can be seen from FIG. 7, the obtained PVA membrane-immobilized enzyme had excellent activity and stability. There was no obvious loss of activity after 9 cycles of use, and the activity and stability were as excellent when PLP was not added to the reaction system as when PLP was added.

[0134] Example 11 Immobilization of cyclohexanone monooxygenase on PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to cyclohexanone monooxygenase CHMO-Rs or CHMO-Bp, or it could be a mixed enzyme of cyclohexanone monooxygenase CHMO and its coenzyme alcohol dehydrogenase ADH-Tb or glucose dehydrogenase GDH. The specific enzyme composition is shown in Table 14. Activity and stability studies: Reaction models employed in the conversion study: [ka] In the above reaction scheme, R may be selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted heterocycloalkyl group, or R forms a fused ring system with the heterocycle to which it is attached. The activity of the PVA-CFP membrane-immobilized enzyme of CHMO was detected by a reaction using the following substrate 6. [ka] 3 mL of 0.1 M PB (pH 8.0) was placed in a 10 mL reaction flask, followed by 50 mg of substrate 6, 100 mg of glucose and 5 mg of NADP. + 50 mg of alcohol dehydrogenase ADH-Tb and 5 mg of glucose dehydrogenase GDH were added, followed by the addition of 20 mg of the PVA-CFP membrane-immobilized enzyme containing cyclohexanone monooxygenase. The reaction was carried out at 30°C for 20 hours to detect the conversion rate. After the completion of one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. The activity of the PVA-CFP membrane immobilized enzymes of CHMO and coenzyme GDH was detected under the following reaction conditions. 3 mL of 0.1 M PB (pH 8.0) was placed in a 10 mL reaction flask, followed by 50 mg of substrate 6, 100 mg of glucose and 5 mg of NADP. + Then, 30 mg of the co-immobilized enzyme of CHMO and GDH was added. The reaction was carried out at 30°C for 20 hours to detect the conversion rate. After the completion of one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times of repeated use was examined. The activity of the PVA-CFP membrane immobilized enzymes of CHMO and coenzyme ADH-Tb was detected under the following reaction conditions. Add 3 mL of 0.1 M PB (pH 8.0) to a 10 mL reaction flask, followed by 50 mg of substrate 6, 200 μL of isopropanol, and 5 mg of NADP. +Then, 30 mg of the co-immobilized enzyme of CHMO and ADH was added. The reaction was carried out at 30°C for 20 hours to detect the conversion rate. After the completion of one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. The test results are shown in Table 14.

[0135] [Table 14]

[0136] Example 12 Immobilization of alkene reductase on PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to alkene reductase ERED-Sc or ERED-Chr, and could also be a mixed enzyme of alkene reductase and its coenzyme glucose dehydrogenase GDH or ammonium formate dehydrogenase FDH, with the weight ratio of the two enzymes being ERED:GDH (or FDH) = 5:1. Activity and stability studies: Reaction models employed in the conversion study: [ka] In the above reaction scheme, R1 and R2 are each independently selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, and a substituted or unsubstituted heterocycloalkyl group; or R1 and R2 form a ring. The activity of the PVA-CFP membrane-immobilized ERED enzyme was detected in the next reaction with substrate 7. [ka] Add 3 mL of 0.1 M PB (pH 7.0-8.0) to a 10 mL reaction flask, followed by 100 mg of substrate 7, followed by 20 mg of NAD(P). +To the reaction mixture, 80 mg of ammonium formate, 5 mg of FDH, and 30 mg of an alkene reductase-containing PVA-CFP membrane-immobilized enzyme were added. The reaction was allowed to proceed at 30°C for 16 hours, and the conversion rate was detected. After one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. In the next reaction with substrate 7, the activity of the PVA-CFP membrane-immobilized enzymes ERED and FDH was detected. Add 3 mL of 0.1 M PB (pH 7.0-8.0) to a 10 mL reaction flask, followed by 100 mg of substrate 7, followed by 20 mg of NAD(P). + , 80 mg of ammonium formate, and 40 mg of a PVA-CFP membrane-immobilized enzyme containing a mixture of alkene reductase and FDH were added. The reaction was carried out at 30°C for 16 hours, and the conversion rate was detected. After one reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. In the next reaction, the activity of the PVA-CFP membrane-immobilized enzymes ERED and GDH was detected. Add 3 mL of 0.1 M PB (pH 7.0-8.0) to a 10 mL reaction flask, followed by 100 mg of substrate 7, followed by 20 mg of NAD(P). + 120 mg of glucose and 40 mg of a PVA-CFP membrane immobilized enzyme containing a mixed enzyme of alkene reductase and GDH were added. The reaction was carried out at 30°C for 16 hours to detect the conversion rate, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. The test results are shown in Table 15.

[0137] [Table 15]

[0138] Example 13 Immobilization of imine reductase using PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to imine reductase IRED-Str or IRED-Bc, and could also be a mixed enzyme of alkene reductase and its coenzyme glucose dehydrogenase GDH or ammonium formate dehydrogenase FDH, with the ratio of the two enzymes being ERED:GDH (or FDH) = 4:1. Activity and stability studies: Reaction models employed in the conversion study: [ka] In the above reaction scheme, R1 and R2 are each independently selected from H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclyl group, and a substituted or unsubstituted heterocycloalkyl group; or R1 and R2 form a fused ring system with the heterocycle or aromatic ring to which they are attached. The activity of the PVA-CFP membrane-immobilized enzyme of IRED was detected using the following substrate 8 in the following manner. [ka] Add 2 mL of 0.1 M PB buffer (pH 7.0-8.0) to a 10 mL reaction flask, followed by 100 mg of the substrate 8, 10 mg of NAD(P) + A PVA-CFP membrane immobilized enzyme containing 60 mg of ammonium formate, 10 mg of FDH, and 40 mg of IRED was added. After reacting at 30°C for 16 hours, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times of repeated use was examined. Substrate 8 was used to detect the activity of the PVA-CFP membrane-immobilized enzymes IRED and FDH in the following manner. Add 2 mL of 0.1 M PB buffer (pH 7.0-8.0) to a 10 mL reaction flask, and add 100 mg of the substrate 8, followed by 10 mg of NAD(P). +60 mg of ammonium formate was added, followed by the addition of 50 mg of the PVA-CFP membrane-immobilized enzyme containing a mixed enzyme of IRED and FDH. After 16 hours of reaction at 30°C, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. The activities of the PVA-CFP membrane-immobilized enzymes IRED and GDH were detected by the following method. Add 3 mL of 0.1 M PB buffer (pH 7.0-8.0) to a 10 mL reaction flask, followed by 100 mg of substrate 8, and then 10 mg of NAD(P). + 100 mg of glucose and 50 mg of the PVA-CFP membrane immobilized enzyme containing the mixed enzymes of IRED and GDH were added. After reacting at 30°C for 16 hours, a conversion test was performed. The test results are shown in Table 16.

[0139] [Table 16]

[0140] Example 14 Immobilization of nitrile hydrolase on PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to a nitrile hydrolase NIT-An or NIT-Nc enzyme solution (enzyme concentration 0.1 g / mL) or a suspension of NIT-An or NIT-Nc crosslinked enzyme aggregates (0.5 g / mL). Activity and stability studies: Reaction models employed in the conversion study: [ka] The activity of the PVA-CFP membrane-immobilized enzyme of nitrile hydrolase was detected using the following substrate 9 by the following method. [ka] 2 mL of 0.1 M PB buffer (pH 7.0-8.0) was added to a 10 mL reaction flask, and 100 mg of the substrate 9 was added, followed by the addition of 20 mg of the PVA-CFP membrane immobilized enzyme containing NIT. After reacting at 30°C for 16 hours, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and repeatedly used in the next reaction, and the number of times of repeated use was examined. The test results are shown in Table 17.

[0141] [Table 17]

[0142] Example 15 Immobilization of ammonia decomposition enzyme using PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to the ammonia decomposition enzyme PAL-An or PAL-Ss. Activity and stability studies: Reaction models employed in the conversion study: [ka] The activity of the ammonia decomposition enzyme PAL immobilized on a PVA-CFP membrane was detected using the following substrate 10 by the following method. [ka] 8 mL of 4 M ammonium aminoformate aqueous solution (pH 9.0-9.5) was added to a 10 mL reaction flask, and 100 mg of the substrate 10 was added, followed by the addition of 40 mg of the PVA-CFP membrane immobilized enzyme containing NIT. After reacting at 30°C for 16 hours, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and repeatedly used in the next reaction, and the number of times of repeated use was examined. The test results are shown in Table 18.

[0143] [Table 18] (Example 16) Immobilization of amino acid dehydrogenase on PVA-CFP membrane: The immobilization method was the same as in Example 10, except that the PVA-encapsulated enzyme was changed to amino acid dehydrogenase AADH-Bc or AADH-Bs, or it could be a mixed enzyme of amino acid dehydrogenase and its coenzyme glucose dehydrogenase GDH or ammonium formate dehydrogenase FDH, with the ratio of the two enzymes being AADH:GDH (or FDH) = 4:1. Activity and stability studies: Reaction models employed in the conversion study: [ka] R is a substituted or unsubstituted aryl group. The activity of the PVA-CFP membrane-immobilized AADH enzyme was detected using the following substrates 11 and 12 in the following manner. [ka] Add 5 mL of 0.1 M Tris-Cl buffer (pH 8.0-9.0) to a 10 mL reaction flask, then add 100 mg of substrate 11 or 12, 108 mg of ammonium chloride, adjust the pH to 7.5-8.0, and then add 10 mg of NAD. + Then, 150 mg of glucose and 10 mg of GDH were added, and finally, 20 mg of the PVA-CFP membrane immobilized enzyme embedded with AADH was added. After reacting for 16 hours at 30°C, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times of repeated use was examined. The method for detecting the activity of the co-immobilized enzymes AADH and FDH was as follows. Add 5 mL of 0.1 M Tris-Cl buffer (pH 8.0-9.0) to a 10 mL reaction flask, then add 100 mg of substrate 11 or 12, 108 mg of ammonium chloride, adjust the pH to 7.5-8.0, and then add 10 mg of NAD. +, 80 mg of ammonium formate, and 50 mg of the PVA-CFP membrane-immobilized enzyme containing a mixed enzyme of AADH and FDH were added. After reacting for 16 hours at 30°C, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times it could be used repeatedly was examined. The method for detecting the activity of the PVA-CFP membrane-immobilized enzymes AADH and GDH was as follows. Add 5 mL of 0.1 M Tris-Cl buffer (pH 8.0-9.0) to a 10 mL reaction flask, then add 100 mg of substrate 11 or 12, 108 mg of ammonium chloride, adjust the pH to 7.5-8.0, and then add 10 mg of NAD. + 150 mg of glucose and 50 mg of a PVA-CFP membrane immobilized enzyme containing a mixed enzyme of AADH and GDH were added. After reacting for 16 hours at 30°C, the conversion rate was detected, and the immobilized enzyme was separated after one reaction and used repeatedly in the next reaction, and the number of times it was used repeatedly was examined. The test results are shown in Table 19.

[0144] [Table 19]

[0145] As can be seen from the above description, the embodiments described in the present invention achieve the following technical advantages. The present application uses a PVA porous membrane as a carrier to immobilize the enzyme by embedding, the process of embedding and immobilizing is simple, the conditions are mild, and it has a good immobilization effect on both purified and crude enzymes, and the enzyme is relatively stable when embedded and immobilized in the PVA porous membrane, and is not easily leached during use, and the porous structure of the PVA porous membrane used can better deliver reactants and products, making it suitable for use in continuous flow biocatalysis. The embedding and immobilization is a mechanical fixation, so it has a wide range of compatibility with enzymes. The PVA porous membrane is structured three-dimensionally to provide a three-dimensional structure, and has a larger surface area, which provides more embedding sites, and increases the enzyme loading capacity while ensuring high enzyme activity and stability.

[0146] The above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. that do not deviate from the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. a PVA porous membrane and an enzyme embedded in the PVA porous membrane, the PVA porous membrane being a three-dimensionally structured PVA porous membrane, the enzyme being selected from any one of aminotransferase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, alkene reductase, nitrile hydrolase, ammonia decomposition enzyme, amino acid dehydrogenase, imine reductase, alcohol dehydrogenase, ammonium formate dehydrogenase, glucose 1-dehydrogenase and mutants thereof; The three-dimensionally structured PVA porous membrane has a three-dimensional structure formed by protrusions or recesses, The PVA porous membrane further contains polyethylene glycol and / or polyethyleneimine, The molecular weight of the polyethylene glycol is PEG 400 to PEG 6000; The molecular weight of the polyethyleneimine is 3 KDa to 70 KDa. A PVA membrane-immobilized enzyme.

2. 2. The PVA membrane-immobilized enzyme according to claim 1, wherein the enzyme is a free enzyme or a cross-linked enzyme aggregate.

3. the aminotransferase is an aminotransferase derived from Chromobacterium violaceum DSM30191, an aminotransferase derived from Arthrobacter citreus, or an aminotransferase derived from Bacillus thuringiensis; The ketoreductase is a ketoreductase derived from Acetobacter sp. CCTCC M209061, The cyclohexanone monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans, or cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1; The ammonia decomposition enzyme is an ammonia decomposition enzyme derived from Solenostemon scutellarioides, The alkene reductase is an alkene reductase derived from Saccharomyces cerevisiae, the imine reductase is an imine reductase derived from Streptomyces sp. and an imine reductase derived from Bacillus cereus; the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus and a phenylalanine dehydrogenase derived from Bacillus sphaericus; The Chromobacterium violaceum DSM30191 DSM30191) has the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the mutant of the aminotransferase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, in which threonine at position 7 is mutated to cysteine, serine at position 47 is mutated to cysteine, lysine at position 90 is mutated to glycine, alanine at position 95 is mutated to proline, isoleucine at position 297 is mutated to leucine, lysine at position 304 is mutated to aspartic acid, glutamine at position 380 is mutated to leucine, arginine at position 405 is mutated to glutamic acid, and arginine at position 416 is mutated to threonine at one or more mutation sites selected from positions 7, 47, 90, 95, 297, 304, 380, 405, and 416, The aminoacid transferase derived from Arthrobacter citreus has the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the aminoacid transferase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 2, in which leucine at position 3 is mutated to serine, valine at position 5 is mutated to serine, cysteine ​​at position 60 is mutated to tyrosine, and phenylalanine at position 164 is mutated to leucine at one or more mutation sites selected from positions 3, 5, 60, 164, 171, 178, 180, 186, 187, 252, 370, 384, 389, 404, 411, 423, and 424. , glutamic acid at position 171 mutates to aspartic acid, alanine at position 178 mutates to leucine, isoleucine at position 180 mutates to valine, serine at position 186 mutates to glycine, serine at position 187 mutates to alanine, valine at position 252 mutates to isoleucine, leucine at position 370 mutates to alanine, tyrosine at position 384 mutates to phenylalanine, isoleucine at position 389 mutates to phenylalanine, leucine at position 404 mutates to glutamine, glycine at position 411 mutates to aspartic acid, methionine at position 423 mutates to lysine, and glutamic acid at position 424 mutates to glutamine, The ketoreductase derived from Acetobacter sp. CCTCC M209061 has the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by causing mutations in the amino acid sequence shown in SEQ ID NO: 3, in which alanine at position 94 is mutated to asparagine, glutamic acid at position 144 is mutated to serine, and asparagine at position 156 is mutated to threonine or valine at one or more mutation sites selected from positions 94, 144, and 156, The cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 has the amino acid sequence shown in SEQ ID NO: 4, and the amino acid sequence of the mutant of the cyclohexanone monooxygenase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 4, in which at one or more mutation sites selected from positions 280, 435, 436, 438, 441, 508, and 510, phenylalanine at position 280 is mutated to tyrosine, phenylalanine at position 435 is mutated to asparagine, phenylalanine at position 436 is mutated to serine, leucine at position 438 is mutated to alanine, serine at position 441 is mutated to valine, and leucine at position 510 is mutated to valine, 3. The PVA membrane-immobilized enzyme according to claim 1 or 2, wherein the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 has the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of the mutant cyclohexanone monooxygenase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO:5, and wherein at one or more mutation sites selected from positions 45, 190, 249, 257, 393, 504, and 559, methionine at position 45 is mutated to threonine, proline at position 190 is mutated to leucine, cysteine ​​at position 249 is mutated to valine, cysteine ​​at position 257 is mutated to alanine, cysteine ​​at position 393 is mutated to valine, proline at position 504 is mutated to valine, and tyrosine at position 559 is mutated to methionine.

4. The PVA membrane-immobilized enzyme according to claim 1, further comprising a coenzyme and a cofactor for each enzyme, the coenzyme and the cofactor being embedded in the PVA porous membrane.

5. The PVA membrane-immobilized enzyme according to any one of claims 1 to 4, characterized in that a mass ratio of the polyethylene glycol to the PVA porous membrane is 5:4 to 75:4, and a mass ratio of the polyethyleneimine to the PVA porous membrane is 1:12 to 1:

240.

6. 2. The PVA membrane-immobilized enzyme according to claim 1, wherein the enzyme is a crude enzyme.

7. The enzyme loading capacity is 0.05 to 0.4 g of free enzyme / cm ２ Membrane or 0.03-0.06 g of dried cross-linked enzyme aggregates / cm ２ 7. The PVA membrane-immobilized enzyme according to claim 1 or 6, which is a membrane.

8. A method for producing the PVA membrane-immobilized enzyme according to any one of claims 1 to 7, comprising the steps of: Step S1: mixing raw materials including an enzyme and a PVA solution for a predetermined time to obtain a mixed system; Step S2: adding the mixture to a mold and drying the mixture to obtain a membrane-embedded enzyme, the mold being a three-dimensionally structured mold for forming a three-dimensionally structured PVA porous membrane; and a step S3 of immersing the membrane-embedded enzyme in a phosphate buffer solution to wash the enzyme, and then obtaining the PVA membrane-immobilized enzyme. The step S1 is mixing the PVA aqueous solution and the modifier solution for a first predetermined time to form a second mixture system; mixing the second mixture with the enzyme system for a second predetermined period of time to form the mixture; the modifying agent solution comprises an aqueous solution of polyethylene glycol in which a cofactor is dispersed and / or an aqueous solution of polyethyleneimine in which a cofactor is dispersed, The molecular weight of the polyethylene glycol is PEG 400 to PEG 6000; The concentration of the polyethylene glycol in the mixed system is 3 to 10 g / 100 mL; The molecular weight of the polyethyleneimine is 3 KDa to 70 KDa. A manufacturing method comprising the steps of:

9. The step S1 is preparing an enzyme suspension in which the enzyme is cross-linked enzyme aggregates or an enzyme solution in which the enzyme is decellularized free enzyme; The method according to claim 8, further comprising mixing the suspension or the enzyme solution with the PVA solution for a predetermined period of time to obtain the mixed system.

10. 10. The method according to claim 9, wherein the predetermined time is 10 to 60 minutes, the molecular weight of PVA in the PVA solution is 20 KDa to 200 KDa, the content of PVA in the PVA solution is 10 to 50 g / 100 mL, acetic acid, methanol and sulfuric acid are dispersed in the PVA solution, the pH of the PVA solution is 5.5 to 6.5, the ratio of the enzyme to the PVA solution is 1 to 50 g / 100 mL, the suspension or the enzyme solution further comprises a phosphate buffer, a cofactor and a coenzyme, and the weight ratio of the coenzyme to the enzyme is 10:1 to 1:

10.

11. The step S1 The method of claim 8, further comprising mixing an aqueous PVA solution and cross-linked enzyme particles to form the mixed system.

12. 12. The method of claim 11, wherein the ratio of the cross-linked enzyme particles to the PVA aqueous solution is 1-50 g / 100 mL, the predetermined time is 10-60 minutes, and the cross-linked enzyme particles contain an enzyme, a cofactor and a coenzyme.

13. 9. The method according to claim 8, wherein the concentration of the PVA aqueous solution is 5 to 30 g / 100 mL, the molecular weight of the polyethyleneimine is 3 KDa to 50 KDa, the concentration of the polyethyleneimine in the mixed system is 0.1 to 1 g / 100 mL, the enzyme system comprises an enzyme, a cofactor, a coenzyme, and a phosphate buffer, the enzyme is a free enzyme from which cells have been removed or a cross-linked enzyme aggregate, the concentration of the cofactor in the enzyme system is 1 to 20 mg / mL, the weight ratio of the coenzyme to the enzyme in the enzyme system is 10:1 to 1:10, and the ratio of the enzyme to the PVA solution is 1 to 50 g / 100 mL.

14. The step S2 is The manufacturing method according to any one of claims 8 to 13, further comprising the steps of: placing the mixture in a mold and leaving it for a third predetermined period of time; and then adding a dehydration promoter, which is any one or more selected from the group consisting of acetonitrile, ethanol, and acetone, to the mold to perform a drying treatment.

15. The method according to claim 14, characterized in that a volume ratio of the dehydration accelerator to the mixed system is 1:10 to 5:1, the third predetermined time is 2 to 4 hours, the mold is a three-dimensionally structured mold, and the three-dimensionally structured mold has protrusions or grooves.

16. The step S3 is The method according to any one of claims 8 to 13, further comprising immersing the membrane-embedded enzyme in the phosphate buffer for 2 to 16 hours, and then washing the membrane-embedded enzyme with fresh phosphate buffer to obtain the PVA membrane-immobilized enzyme.

Images