Co-immobilized enzyme, its preparation method and its application

Abstract

The present invention provides a co-immobilized enzyme, a preparation method thereof and an application thereof. The co-immobilized enzyme includes: an amino resin carrier, a main enzyme and a coenzyme, wherein the main enzyme and the coenzyme are co-immobilized on the amino resin carrier, the main enzyme is covalently immobilized on the amino resin carrier, and there is one kind of coenzyme which is immobilized on the amino resin carrier by a non-covalent method; the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketone reductase, alkene reductase and monooxygenase. Co-immobilizing the main enzyme and its coenzyme on the amino resin carrier can improve the enzyme activity and the recycling efficiency.

Description

Technical Field

[0001] The present invention relates to the field of immobilized enzymes, and more particularly, to a co-immobilized enzyme, a preparation method thereof, and an application thereof. Background Art

[0002] The application of microbial cells or isolated enzymes or engineered enzymes has led to significant progress in biocatalysis and a transformation in manufacturing methods. Many types of enzymes such as acyltransferases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, and hydrolases are used in reactions involving antibiotics, herbicides, pharmaceutical intermediates, and new era therapeutic agents.

[0003] When free enzymes are used as biocatalysts, there is a significant waste of enzymes because it is very difficult to recover water-soluble enzymes. In contrast, water-insoluble immobilized enzymes can be easily recovered by very simple filtration after each cycle.

[0004] There have been reports in the prior art on the immobilization methods of single enzymes, but different enzymes are suitable for different immobilization methods. For example, Bolivar et al (Biomacromol. 2006, 7, 669-673) studied the covalent immobilization of FDH from Pseudomonas sp. SP101 on various carriers including modified agarose, CNBr-activated agarose, Sepabeads (dextran), and glyoxal agarose. The conclusion they reached was that immobilization on carriers activated with bromide, polyethyleneimine, glutaraldehyde, etc. did not promote any stabilization of the enzyme under thermal inactivation. However, the optimized enzyme immobilized on highly activated glyoxal agarose was shown to have high thermal stability, pH stability, and more than 50% activity with enhanced stability.

[0005] Kim et al (J. Mol. Catal B: Enzy 97 (2013) 209–214) reported a method of using cross-linked enzyme aggregates (CLEAs) to immobilize formate dehydrogenase (FDH) from Candida boidinii, and considered that dextran polyaldehyde as a cross-linking agent instead of glutaraldehyde was better for immobilized enzymes. After 10 repeated uses, the residual activity exceeded 95%. In addition, the thermal stability of the cross-linked enzyme aggregates (Dex-CLEA) formed by dextran polyaldehyde was 3.6 times higher than that of the free enzyme.

[0006] Binay et al (Beilstein J. Org. Chem. 2016, 12, 271–277) reported the immobilized enzyme of highly active FDH from Candida methylica. FDH was covalently immobilized on the epoxy-activated Immobead 150 support, which was first modified with ethylenediamin and then sequentially activated with glutaraldehyde (FDHIGLU) and aldehyde-functionalized (FDHIALD). When using the aldehyde-functionalized Immobead 150 as the support, the highest immobilization yield and activity yield were obtained, which were 90% and 132% respectively. At 35 °C, the half-lives (t1 / 2) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated to be 10.6, 28.9, 22.4 and 38.5 hours respectively. FDHI150, FDHIGLU and FDHIALD retained 69%, 38% and 51% of their initial activities respectively after 10 repeated uses.

[0007] Jackon et al (Process Biochem. Vol. 1, 9, Sep 2016, 1248-1255) reported the immobilization of LDH using glyoxal-agarose. The thermal stability factor obtained for the immobilized LDH was 1600 times greater than that of its soluble counterpart.

[0008] There are also some reports on the co-immobilization of two enzymes. For example, Valikhani et al (Biotech. Bioengg. 2018; 115:2416–2425) reported the co-immobilization of cytochrome P450 monooxygenase (P450s) and glucose dehydrogenase from B. megaterium. Delgrove et al (Appl. Catal. A: Gen. Vol. 572, 25 Feb. 2019, 134-141) published the co-immobilization of Baeyer-Villiger monooxygenase (BVMO) and glucose dehydrogenase for the synthesis of ε-caprolactone derivatives. The thermostable cyclohexanone monooxygenase (TmCHMO) from Thermocrispum municipale and glucose dehydrogenase (GDH) from Thermoplasma acidophilum (GDH-Tac) were co-immobilized on an amino-functionalized agarose-based support (MANA-agarose). The co-immobilization was proven to be the most effective biocatalyst, with an average conversion rate of 83% in 15 repeated cycles for the synthesis of 3,3,5-trimethylcyclohexanone.

[0009] In summary, there are still a large number of enzymes in the prior art that do not have an effective immobilized enzyme form. In particular, some enzymes that jointly participate in the same biocatalytic reaction, and the co-immobilization of these enzymes to improve the recyclability of the enzymes has become an urgent problem to be solved. Summary of the Invention

[0010] The main object of the present invention is to provide a co-immobilized enzyme, a preparation method thereof and an application thereof, so as to solve the problem that such enzymes in the prior art are difficult to recycle.

[0011] To achieve the above object, according to one aspect of the present invention, there is provided a co-immobilized enzyme, which comprises: an amino resin carrier, a main enzyme and a coenzyme. The coenzyme is one or more. The main enzyme and the coenzyme are co-immobilized on the amino resin carrier. Among them, the main enzyme is covalently immobilized on the amino resin carrier, and the coenzyme is immobilized on the amino resin carrier by covalent and / or non-covalent means; the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketone reductase, alkene reductase and monooxygenase.

[0012] Furthermore, the transaminase is a transaminase derived from B. thuringiensis or Vibrio fluvialis strain JS17; preferably, the amino acid dehydrogenase is an amino acid dehydrogenase derived from Bacillus cereus or Bacillus sphaericus; preferably, the imine reductase is an imine reductase derived from Streptomyces sp or Bacillus cereus; preferably, the ketone reductase is a ketone reductase derived from Sporobolomyces salmonicolor or the ketone reductase of Acetobacter sp. CCTCC M209061, and more preferably, the ketone reductase derived from Acetobacter sp. CCTCC M209061 is a mutant having the sequence of SEO ID NO: 1 or SEO ID NO: 2; preferably, the enol reductase is an enol reductase derived from Chryseobacterium sp. CA49 or Shewanella oneidensis MR-1; preferably, the monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans, or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1; more preferably, the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having the sequence of SEO ID NO: 4 or SEO ID NO: 5; the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant having the sequence of SEO ID NO: 7 or SEO ID NO: 8.

[0013] Furthermore, the coenzyme is selected from at least one of the following: lactate dehydrogenase (LDH), formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and alcohol dehydrogenase; preferably, the lactate dehydrogenase is D-lactate dehydrogenase derived from Lactobacillus helveticus; preferably, the formate dehydrogenase is formate dehydrogenase derived from Candida boidinii; preferably, the glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is alcohol dehydrogenase derived from Thermoanaerobium brockii; preferably, the number of recycling times of the co-immobilized enzyme is 4 to 25 times.

[0014] Further, the amino resin carrier is a glutaraldehyde-activated amino resin carrier; preferably, the amino resin carrier is an amino resin carrier with a C2 or C4 linker arm, and more preferably, the amino resin carrier is selected from any one of the following: LX1000EA, LX1000HA, LX1000NH, HFA, LX1000EPN, HM100D, Lifetech TM ECR8309, ECR8409, ECR8305, ECR8404, ECR8315, ECR8415, ESR-1, ESR-3, ESR-5 and ESR-8.

[0015] Further, in the co-immobilized enzyme, the mass ratio of the main enzyme to the coenzyme is 1-20:1-10; preferably, the sum of the masses of the main enzyme and the coenzyme is denoted as N1, and the mass of the amino resin carrier is denoted as N2, and N1 / N2 is 50-200 mg:1 g, and further, it is 80-120 mg:1 g.

[0016] Further, both the main enzyme and the coenzyme are covalently immobilized on the amino resin carrier; or the main enzyme is covalently immobilized on the amino resin carrier, and the coenzyme of the main enzyme is non-covalently immobilized on the amino resin carrier by ionic adsorption; preferably, the coenzyme is adsorbed on the amino resin carrier through PEI.

[0017] Further, the coenzyme includes a first enzyme and a second enzyme, the main enzyme and the second enzyme are covalently immobilized on the amino resin carrier, and the first enzyme is immobilized on the amino resin carrier by ionic adsorption.

[0018] In the second aspect of the present application, a preparation method of any of the above co-immobilized enzymes is provided, and the preparation method includes: activating the amino resin carrier to obtain an activated amino carrier; covalently immobilizing the main enzyme on the activated amino carrier, and immobilizing the coenzyme corresponding to the main enzyme on the activated amino carrier by covalent and / or non-covalent methods to obtain a co-immobilized enzyme; wherein, the number of coenzymes corresponding to the main enzyme is one or more.

[0019] Further, immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain a co-immobilized enzyme includes: mixing the main enzyme and the coenzyme to obtain a first mixed enzyme; immobilizing the first mixed enzyme on the activated amino carrier to obtain a co-immobilized enzyme.

[0020] Further, the coenzyme includes a first enzyme, and immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain a co-immobilized enzyme includes: immobilizing the main enzyme on the activated amino carrier to obtain a preliminarily immobilized enzyme; immobilizing the first enzyme and the preliminarily immobilized enzyme to obtain a co-immobilized enzyme.

[0021] Further, the coenzyme further includes a second enzyme. Fixing the main enzyme and the coenzyme to an activated amino carrier to obtain a co-immobilized enzyme includes: co-fixing the main enzyme and the second enzyme to the activated amino carrier to obtain a primary immobilized enzyme; fixing the first enzyme to the primary immobilized enzyme to obtain a co-immobilized enzyme.

[0022] Further, the first enzyme is fixed to the primary immobilized enzyme by surface coating with PEI to obtain a co-immobilized enzyme; preferably, PEI is added to the primary immobilized enzyme to a final concentration of 0.5 w / v% to 5 w / v% to obtain a PEI-primary immobilized enzyme; then the first enzyme is combined with the PEI-primary immobilized enzyme to obtain a co-immobilized enzyme.

[0023] Further, the first mixed enzyme is fixed to the activated amino carrier at a mass ratio of 50 to 150 mg:1 g to obtain a co-immobilized enzyme.

[0024] Further, the mass ratio of the main enzyme to the first enzyme is 1 to 20:1 to 10.

[0025] Further, the mass ratio of the main enzyme to the second enzyme is 1 to 20:1 to 10.

[0026] Further, the main enzyme is a transaminase, and the coenzyme has two cofactors, and the two cofactors are LDH (lactate dehydrogenase) and FDH (formate dehydrogenase), or LDH (lactate dehydrogenase) and GDH (glucose dehydrogenase). The above preparation method includes any one of the following:

[0027] (1) Mix the transaminase, LDH and FDH to obtain a first enzyme mixture; fix the first enzyme mixture to an activated amino carrier to obtain a co-immobilized enzyme;

[0028] (2) Mix the transaminase, LDH and GDH to obtain a second enzyme mixture; fix the second enzyme mixture to an activated amino carrier to obtain a co-immobilized enzyme;

[0029] (3) Mix the transaminase and LDH to obtain a third enzyme mixture; fix the third enzyme mixture to an activated amino carrier to obtain a primary immobilized transaminase; by means of PEI surface coating of the primary immobilized transaminase, further fix GDH or FDH to obtain a co-immobilized enzyme.

[0030] Further, the main enzyme is an amino acid dehydrogenase, and the coenzyme is FDH or GDH. The preparation method includes any one of the following:

[0031] (1) Mix the amino acid dehydrogenase with FDH or GDH to obtain an amino acid dehydrogenase mixture; fix the amino acid dehydrogenase mixture to an activated amino carrier to obtain a co-immobilized enzyme;

[0032] (2) Immobilize the amino acid dehydrogenase on the activated amino carrier to obtain the initially immobilized amino acid dehydrogenase;

[0033] Fix GDH or FDH by means of PEI surface coating of the initially immobilized amino acid dehydrogenase to obtain the co-immobilized enzyme.

[0034] Furthermore, the main enzyme is imine reductase, and the coenzyme is FDH or GDH. The preparation method includes any one of the following:

[0035] (1) Mix the imine reductase, FDH or GDH to obtain an imine enzyme mixture; immobilize the imine enzyme mixture on the activated amino carrier to obtain the co-immobilized enzyme;

[0036] (2) Immobilize the imine reductase on the activated amino carrier to obtain the initially immobilized imine reductase; fix GDH or FDH by means of PEI surface coating of the initially immobilized imine reductase to obtain the co-immobilized enzyme.

[0037] Furthermore, the main enzyme is ketone reductase, and the coenzyme is FDH or GDH. The preparation method includes any one of the following:

[0038] (1) Mix the ketone reductase, FDH or GDH to obtain a ketone reductase mixture; immobilize the ketone reductase mixture on the activated amino carrier to obtain the co-immobilized enzyme;

[0039] (2) Immobilize the ketone reductase on the activated amino carrier to obtain the initially immobilized ketone reductase; fix GDH or FDH by means of PEI surface coating of the initially immobilized ketone reductase to obtain the co-immobilized enzyme.

[0040] Furthermore, the main enzyme is alkene reductase, and the coenzyme is FDH or GDH. The preparation method includes any one of the following:

[0041] (1) Mix the alkene reductase, FDH or GDH to obtain an alkene reductase mixture; immobilize the alkene reductase mixture on the activated amino carrier to obtain the co-immobilized enzyme;

[0042] (2) Immobilize the alkene reductase on the activated amino carrier to obtain the initially immobilized alkene reductase; fix GDH or FDH by means of PEI surface coating of the initially immobilized alkene reductase to obtain the co-immobilized enzyme.

[0043] Furthermore, the main enzyme is cyclohexanone monooxygenase, and the coenzyme is FDH or GDH. The preparation method includes any one of the following:

[0044] (1) Mix the cyclohexanone monooxygenase, FDH or GDH to obtain a monooxygenase mixture; immobilize the monooxygenase mixture on the activated amino carrier to obtain the co-immobilized enzyme;

[0045] (2) Fix cyclohexanone monooxygenase onto an activated amino carrier to obtain a preliminarily fixed cyclohexanone monooxygenase; then fix GDH or FDH by means of PEI surface coating of the preliminarily fixed cyclohexanone monooxygenase to obtain a co-immobilized enzyme.

[0046] Further, activate the amino resin carrier with glutaraldehyde to obtain an activated amino carrier.

[0047] According to the third aspect of the present application, there is provided the use of any of the above co-immobilized enzymes, or the co-immobilized enzymes prepared by the preparation method of any of the above co-immobilized enzymes, in biocatalytic reactions.

[0048] Further, the biocatalytic reaction is an intermittent biocatalytic reaction or a continuous biocatalytic reaction; preferably, the co-immobilized enzyme is applied to a continuous fluidized bed or a biocatalytic reaction in a fixed bed; preferably, the number of recycling times of the co-immobilized enzyme in the continuous biocatalytic reaction is 4 to 25 times.

[0049] By applying the technical solution of the present invention, the present application co-immobilizes the above-mentioned main enzyme and its coenzyme on an amino resin carrier, realizing the co-immobilization of these main enzyme and its coenzyme, thereby being beneficial to improving the activity and recycling efficiency of the enzyme. Detailed Embodiments

[0050] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0051] Polyethylene imine, abbreviated as PEI, is polyethyleneimine.

[0052] In a typical embodiment of the present application, there is provided a co-immobilized enzyme, which includes: an amino resin carrier, a main enzyme and a coenzyme, the main enzyme and the coenzyme are co-immobilized on the amino resin carrier, wherein the main enzyme is covalently immobilized on the amino resin carrier, and the coenzyme is immobilized on the amino resin carrier by covalent and / or non-covalent means; the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketone reductase, alkene reductase and monooxygenase.

[0053] The present application co-immobilizes the above-mentioned main enzyme and its coenzyme on an amino resin carrier, realizing the co-immobilization of these main enzyme and its coenzyme, thereby being beneficial to improving the activity and recycling efficiency of the enzyme.

[0054] The specific types and activities of the above various main enzymes vary depending on their sources. In a preferred embodiment, the transaminase is a transaminase derived from B. thuringiensis or Vibrio fluvialis strain JS17; preferably, the amino acid dehydrogenase is an amino acid dehydrogenase derived from Bacillus cereus or Bacillus sphaericus; preferably, the imine reductase is an imine reductase derived from Streptomyces sp or Bacillus cereus; preferably, the ketone reductase is a ketone reductase derived from Sporobolomyces salmonicolor or the ketone reductase of Acetobacter sp. CCTCC M209061. More preferably, the ketone reductase derived from Acetobacter sp. CCTCC M209061 is a mutant having the sequence of SEO ID NO:1 or SEO ID NO:2; preferably, the enol reductase is an enol reductase derived from Chryseobacterium sp. CA49 or Shewanella oneidensis MR-1; preferably, the monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or a cyclohexanone monooxygenase derived from Brachymonas petroleovorans, or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1; more preferably, the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having the sequence of SEO ID NO:4 or SEO ID NO:5; the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant having the sequence of SEO ID NO:7 or SEO ID NO:8.

[0055] According to the types of the above-mentioned main enzymes, their corresponding coenzymes are selected. Preferably, these coenzymes are coenzymes capable of recycling NAD(P)+ and NADPH. In a preferred embodiment, the coenzyme is selected from at least one of the following: the coenzyme is selected from at least one of the following: lactate dehydrogenase, formate dehydrogenase, glucose dehydrogenase and alcohol dehydrogenase; preferably, the lactate dehydrogenase is D-lactate dehydrogenase derived from Lactobacillus helveticus; preferably, the formate dehydrogenase is formate dehydrogenase derived from Candida boidinii; preferably, the glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is alcohol dehydrogenase derived from Thermoanaerobium brockii. In another preferred embodiment, the number of times of recycling the co-immobilized enzyme is 4 to 25 times.

[0056] In the above preferred embodiment, the coenzymes corresponding to the transaminase are lactate dehydrogenase and formate dehydrogenase, or lactate dehydrogenase and glucose dehydrogenase. The coenzymes corresponding to the amino acid dehydrogenase are formate dehydrogenase or glucose dehydrogenase. The coenzymes corresponding to the imine reductase are formate dehydrogenase or glucose dehydrogenase. The coenzymes corresponding to the ketone reductase are formate dehydrogenase or glucose dehydrogenase. The coenzymes corresponding to the enol reductase are formate dehydrogenase or glucose dehydrogenase. The coenzymes corresponding to the monooxygenase are alcohol dehydrogenase or formate dehydrogenase or glucose dehydrogenase.

[0057] In a preferred embodiment, the lactate dehydrogenase is D-lactate dehydrogenase (abbreviated as LDH) derived from Lactobacillus helveticus; preferably, the formate dehydrogenase is formate dehydrogenase (abbreviated as FDH) derived from Candida boidinii; preferably, the glucose dehydrogenase is glucose 1-dehydrogenase (abbreviated as GDH) derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is alcohol dehydrogenase derived from Thermoanaerobium brockii.

[0058] In the prior art, there is no co-immobilized enzyme of the main enzymes and coenzymes from the above preferred embodiments. Co-immobilizing these main enzymes and coenzymes helps to improve the recycling efficiency of these enzymes.

[0059] The carrier of the co-immobilized enzyme is an amino resin carrier, and various existing amino resin carriers can be used. In a preferred embodiment, the amino resin carrier is a glutaraldehyde-activated amino resin carrier; preferably, the amino resin carrier is an amino resin carrier with a C2 or C4 linker arm. More preferably, the amino resin carrier is selected from any one of the following: LX1000EA, LX1000HA, LX1000NH, LX1000EPN, HFA, HM100D, Lifetech TM ECR8309, ECR8409, ECR8305, ECR8404, ECR8315, ECR8415, ESR-1, ESR-3, ESR-5 and ESR-8. According to the above different immobilized main enzymes and coenzymes, different amino resin carriers can be selected.

[0060] In the above co-immobilized enzymes, the dosage ratio of the main enzyme and the coenzyme varies according to the different substrates to be catalyzed. In a preferred embodiment, in the above co-immobilized enzymes, the mass ratio of the main enzyme to the coenzyme is 1-20:1-10. Preferably, the sum of the masses of the main enzyme and the coenzyme is denoted as N1, and the mass of the amino resin carrier is denoted as N2, and N1 / N2 is 50-200 mg:1 g, and further, 80-120 mg:1 g.

[0061] Controlling the mass ratio of the main enzyme to the coenzyme within the above range enables all the above co-immobilized enzymes of the main enzyme and the coenzyme to catalyze most of the substrates. And controlling the mass ratio of the enzyme to the carrier within the range of 50-200 mg:1 g can achieve the co-immobilization of all the above main enzymes and coenzymes.

[0062] In some more preferred embodiments, the mass ratio of the transaminase to its coenzyme lactate dehydrogenase is 5-7:1; the mass ratio of the transaminase to its coenzyme formate dehydrogenase is 5-7:1-3; the mass ratio of the transaminase to its coenzyme glucose dehydrogenase is 5-7:1-2; the mass ratio of the amino acid dehydrogenase to its coenzyme formate dehydrogenase is 8-10:1; the mass ratio of the amino acid dehydrogenase to its coenzyme glucose dehydrogenase is 5-6:1; the mass ratio of the imine reductase to its coenzyme formate dehydrogenase is 4-8:1; the mass ratio of the imine reductase to its coenzyme glucose dehydrogenase is 6-8:1; the mass ratio of the ketone reductase to its coenzyme formate dehydrogenase is 4-8:1; the mass ratio of the ketone reductase to its coenzyme glucose dehydrogenase is 1:3-10; the mass ratio of the enol reductase to its coenzyme formate dehydrogenase is 6-10:1; the mass ratio of the enol reductase to its coenzyme glucose dehydrogenase is 12-20:1; and the mass ratio of the monooxygenase to its coenzyme alcohol dehydrogenase to the formate dehydrogenase is 2-3:2-3; the mass ratio of the monooxygenase to its coenzyme glucose dehydrogenase is 2-3:1. Controlling the mass ratio of the above main enzymes to their corresponding different coenzymes within the above range can enable the main coenzymes to be configured as much as possible according to the stoichiometric ratio, thereby improving the catalytic activity and efficiency of the co-immobilized enzymes.

[0063] In the above co-immobilized enzyme, both the main enzyme and the coenzyme are immobilized on the carrier, and there is no limitation on the specific immobilization method of the two on the carrier. In a preferred embodiment, both the main enzyme and the coenzyme are covalently immobilized on an amino resin carrier. In another preferred embodiment, the main enzyme is covalently immobilized on an amino resin carrier, and the coenzyme is non-covalently immobilized on the amino resin carrier by ion adsorption; preferably, the coenzyme is adsorbed on the amino resin carrier through PEI. The above two different co-immobilization methods can be obtained by different co-immobilization methods.

[0064] Among the above co-immobilized enzymes, some main enzymes have only one coenzyme, and some main enzymes have two coenzymes. The co-immobilization methods of the two are the same as above, which can be the same or different, depending on the specific properties of the coenzyme. In a preferred embodiment, the coenzyme includes a first enzyme and a second enzyme. The main enzyme and the second enzyme are covalently immobilized on an amino resin carrier, and the first enzyme is immobilized on the amino resin carrier by ion adsorption. Here, the first enzyme refers to an enzyme that is sensitive to certain reagents in the co-immobilization process, such as cross-linking agent glutaraldehyde, etc. In order to reduce the decrease in the activity of the first enzyme during the co-immobilization process, thereby affecting the activity of the co-immobilized enzyme and the subsequent recycling efficiency, in the above preferred embodiment, the first enzyme is non-covalently immobilized on the amino resin carrier by ion adsorption.

[0065] Transaminase TA-Bt (see Table 1 for details) was co-immobilized with LDH and FDH to carrier LX1000HA at an optimal ratio, and the maximum number of uses could reach 15 times; transaminase TA-Bt was co-immobilized with LDH and GDH to carrier LX1000HA at an optimal ratio, and the maximum number of uses could reach 20 - 25 times; monooxygenase CHMO-Rs was co-immobilized with GDH to carrier LX1000HA at an optimal ratio, and the maximum number of uses reached 13 times. Monooxygenase CHMO-Rs was co-immobilized with ADH to carrier LX1000HA at an optimal ratio, and the maximum number of uses reached 13 - 15 times. When co-immobilized to carrier ECR8409, the maximum number of uses reached 16 times; mutant V1 of CHMO-Rs was co-immobilized with ADH to carrier LX1000HA, and the maximum number of uses reached 17 times. Mutant V2 was co-immobilized with ADH to carrier LX1000HA, and the maximum number of uses reached 17 times; CHMO-Bp was co-immobilized with ADH or GDH to carrier LX1000HA, and the number of uses reached 14 - 15 times; mutant V1 of CHMO-Bp was co-immobilized with ADH to carrier LX1000HA, and the number of uses reached 19 times; mutant V1 of CHMO-Bp was co-immobilized with ADH to carrier LX1000HA, and the number of uses reached 21 times; AADH-Bc was co-immobilized with FDH to carrier LX1000HA at an optimal ratio, and the maximum number of uses reached 12 times; AADH-Bs was co-immobilized with GDH to carrier LX1000HA at an optimal ratio, and the maximum number of uses reached 15 times; KRED-Ac was co-immobilized with FDH to carrier ECR8409, and the maximum number of uses reached 18 times; KRED-Ac-V1 was co-immobilized with FDH to carrier ECR8409, and the maximum number of uses reached 24 times; KRED-Ac was co-immobilized with GDH to carrier ECR8409, and the maximum number of uses reached 14 times; ERED-Sc was immobilized to carrier LX1000HA at an optimal ratio with FDH, and the maximum number of uses reached 17 times; ERED-Sc was immobilized to carrier LX1000HA at an optimal ratio with GDH, and the maximum number of uses reached 21 times; ERED-Chr was immobilized to carrier LX1000EPN at an optimal ratio, and the maximum number of uses reached 21 times; IRED-Str was immobilized to carrier LX1000EPN at an optimal ratio with FDH, and the maximum number of uses reached 17 times; IRED-Bc was immobilized to carrier LX1000EPN at an optimal ratio with GDH, and the maximum number of uses reached 19 times.

[0066] In the second typical embodiment of the present application, a preparation method for any of the above co-immobilized enzymes is provided. The preparation method includes: activating an amino resin carrier to obtain an activated amino carrier; covalently immobilizing a main enzyme on the activated amino carrier, and covalently and / or non-covalently immobilizing the coenzyme corresponding to the main enzyme on the activated amino carrier to obtain a co-immobilized enzyme.

[0067] The above method for co - immobilizing enzymes can obtain co - immobilized enzymes by first activating the amino resin carrier and then co - immobilizing any one of the above - mentioned main enzymes and its coenzyme on the activated amino carrier.

[0068] For the specific method of co - immobilizing the above - mentioned main enzyme and coenzyme on the activated amino carrier, appropriate steps can be selected according to the type and properties of the coenzyme.

[0069] In a preferred embodiment, immobilizing the main enzyme and coenzyme on the activated amino carrier to obtain co - immobilized enzymes includes: mixing the main enzyme and coenzyme to obtain a first mixed enzyme; immobilizing the first mixed enzyme on the activated amino carrier to obtain co - immobilized enzymes.

[0070] In this preferred method, regardless of the number of coenzymes, they can be mixed with the main enzyme and then immobilized on the carrier. According to different main enzymes and coenzymes, the mixing ratios are different. Preferably, the main enzyme and coenzyme are mixed according to the mass ratio of the main enzyme to the coenzyme of 1 - 20:1 - 10 mentioned above to obtain a first mixed enzyme; and the first mixed enzyme is immobilized on the activated amino carrier according to the ratio of N1 / N2 mentioned above to obtain co - immobilized enzymes.

[0071] In a preferred embodiment, the coenzyme includes a first enzyme. Immobilizing the main enzyme and coenzyme on the activated amino carrier to obtain co - immobilized enzymes includes: immobilizing the main enzyme on the activated amino carrier to obtain a preliminarily immobilized enzyme; immobilizing the first enzyme and the preliminarily immobilized enzyme to obtain co - immobilized enzymes.

[0072] When there is only one coenzyme, the method of mixing with the main enzyme and then immobilizing together can be used, or the step - by - step immobilization method can also be used. For the co - immobilization of some coenzymes that are sensitive to glutaraldehyde in the step of activating the amino carrier, the step - by - step immobilization method is preferably used, which can retain the activity of the coenzyme to a greater extent, thereby improving the recycling efficiency of the co - immobilized enzymes.

[0073] In a preferred embodiment, the coenzyme further includes a second enzyme. Immobilizing the main enzyme and coenzyme on the activated amino carrier to obtain co - immobilized enzymes includes: co - immobilizing the main enzyme and the second enzyme on the activated amino carrier to obtain a preliminarily immobilized enzyme; immobilizing the first enzyme and the preliminarily immobilized enzyme to obtain co - immobilized enzymes.

[0074] When there is more than one coenzyme, according to the sensitivity of the coenzyme to activators such as glutaraldehyde, the above-mentioned part can be mixed with the main enzyme and then immobilized on the activated amino carrier. In the second step, the coenzyme with high sensitivity is immobilized on the carrier, which can improve the activity of the sensitive enzyme, thereby improving the overall activity and recycling efficiency of the prepared co-immobilized enzyme. In the above preferred embodiment, the first enzyme is sensitive to glutaraldehyde, and the sensitivity of the second enzyme is relatively low. Therefore, the second enzyme is mixed and immobilized with the main enzyme, and then the first enzyme is immobilized separately.

[0075] In the above stepwise immobilization steps, the second immobilization is carried out in consideration of improving the activity of the coenzyme to be immobilized. Therefore, any method that can improve the activity of this type of coenzyme and can achieve its immobilization is applicable to this application. In a preferred embodiment, the first enzyme and the initially immobilized enzyme are immobilized using PEI to obtain a co-immobilized enzyme. In other preferred embodiments, PEI is added to the initially immobilized enzyme until the final concentration (mass-volume concentration) of PEI is 0.5% - 5% to obtain PEI-initially immobilized enzyme; the first enzyme is added to the PEI-initially immobilized enzyme for incubation to obtain a co-immobilized enzyme.

[0076] PEI is polyethyleneimine, which can be used as a support in a non-solid form. It adsorbs the coenzyme to be immobilized on the carrier by ionic adsorption, and the specific amount of PEI added can be reasonably adjusted according to actual needs. When the final concentration of the added PEI reaches 0.5% - 5%, the immobilization of all the above types of coenzymes can be achieved.

[0077] When the main enzyme is transaminase, the coenzyme has two cofactors, LDH and FDH, or LDH and GDH, and its preparation method includes a one-step immobilization method or a stepwise immobilization method. The one-step immobilization method is to mix two cofactors in different combinations with transaminase to obtain a first enzyme mixture or a second enzyme mixture respectively, and then immobilize the first enzyme mixture or the second enzyme mixture on the activated amino carrier to obtain a co-immobilized enzyme. Stepwise immobilization method: Transaminase and LDH are mixed to obtain a third enzyme mixture; the third enzyme mixture is immobilized on the activated amino carrier to obtain initially immobilized transaminase; by means of PEI surface coating of the initially immobilized transaminase, GDH or FDH is further immobilized to obtain a co-immobilized enzyme.

[0078] When the main enzyme is amino acid dehydrogenase, the coenzyme is FDH or GDH, and the preparation method includes any one of the following: mixing amino acid dehydrogenase with FDH or GDH to obtain an amino acid dehydrogenase mixture; immobilizing the amino acid dehydrogenase mixture on an activated amino carrier to obtain a co-immobilized enzyme; or immobilizing amino acid dehydrogenase on an activated amino carrier to obtain a preliminarily immobilized amino acid dehydrogenase; and then immobilizing GDH or FDH by means of PEI surface coating of the preliminarily immobilized amino acid dehydrogenase to obtain a co-immobilized enzyme.

[0079] When the main enzyme is imine reductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the following: mixing imine reductase, FDH or GDH to obtain an imine enzyme mixture; immobilizing the imine enzyme mixture on an activated amino carrier to obtain a co-immobilized enzyme; or immobilizing imine reductase on an activated amino carrier to obtain a preliminarily immobilized imine reductase; and then immobilizing GDH or FDH by means of PEI surface coating of the preliminarily immobilized imine reductase to obtain a co-immobilized enzyme.

[0080] When the main enzyme is ketone reductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the following: mixing ketone reductase, FDH or GDH to obtain a ketone reductase mixture; immobilizing the ketone reductase mixture on an activated amino carrier to obtain a co-immobilized enzyme; or immobilizing ketone reductase on an activated amino carrier to obtain a preliminarily immobilized ketone reductase; and then immobilizing GDH or FDH by means of PEI surface coating of the preliminarily immobilized ketone reductase to obtain a co-immobilized enzyme.

[0081] When the main enzyme is alkene reductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the following: mixing alkene reductase, FDH or GDH to obtain an alkene reductase mixture; immobilizing the alkene reductase mixture on an activated amino carrier to obtain a co-immobilized enzyme; or immobilizing alkene reductase on an activated amino carrier to obtain a preliminarily immobilized alkene reductase; and then immobilizing GDH or FDH by means of PEI surface coating of the preliminarily immobilized alkene reductase to obtain a co-immobilized enzyme.

[0082] When the main enzyme is cyclohexanone monooxygenase, the coenzyme is FDH or GDH, and the preparation method includes any one of the following: mixing cyclohexanone monooxygenase, FDH or GDH to obtain a monooxygenase mixture; immobilizing the monooxygenase mixture on an activated amino carrier to obtain a co-immobilized enzyme; or immobilizing cyclohexanone monooxygenase on an activated amino carrier to obtain a preliminarily immobilized cyclohexanone monooxygenase; and then immobilizing GDH or FDH by means of PEI surface coating of the preliminarily immobilized cyclohexanone monooxygenase to obtain a co-immobilized enzyme.

[0083] In the preparation methods for the co-immobilization of the above various main enzymes and their coenzymes, the method of coating with PEI is the same as the aforementioned operation of "adding PEI to the initially immobilized enzyme until the final concentration of PEI is 0.5 w / v% to 5 w / v% to obtain a PEI-initially immobilized enzyme complex; then binding the first enzyme to the PEI-initially immobilized enzyme complex to obtain the co-immobilized enzyme", which will not be elaborated here.

[0084] In the above step of activating the amino group carrier, existing activators can be used for activation. In a preferred embodiment, glutaraldehyde is used to activate the amino resin carrier to obtain the activated amino group carrier. Glutaraldehyde has a wide range of applications and is the most common.

[0085] In the above step of co-immobilization, according to the different co-immobilization methods and the types and amounts of coenzymes for co-immobilization, the ratio of the total mass of the co-immobilized enzymes to the total mass of the activated amino group carrier also varies. Correspondingly, the mass ratio of the main enzyme to the coenzyme also varies, and when there are two coenzymes, the mass ratios of the main enzyme to the first coenzyme and the second coenzyme also vary, which can be reasonably adjusted according to actual needs.

[0086] In a preferred embodiment, the first mixed enzyme is immobilized on the activated amino group carrier at a mass ratio of 50 - 150 mg:1 g to obtain the co-immobilized enzyme.

[0087] In a preferred embodiment, the mass ratio of the main enzyme to the first enzyme is 1 - 20:1 - 10.

[0088] In a preferred embodiment, the mass ratio of the main enzyme to the second enzyme is 1 - 20:1 - 10.

[0089] Preferably, in the above step of co-immobilization, the mass ratio of the transaminase to its coenzyme lactate dehydrogenase is 5 - 7:1; the mass ratio of the transaminase to its coenzyme formate dehydrogenase is 5 - 7:1 - 3; the mass ratio of the transaminase to its coenzyme glucose dehydrogenase is 5 - 7:1 - 2; the mass ratio of the amino acid dehydrogenase to its coenzyme formate dehydrogenase is 8 - 10:1; the mass ratio of the amino acid dehydrogenase to its coenzyme glucose dehydrogenase is 4 - 6:1; the mass ratio of the imine reductase to its coenzyme formate dehydrogenase is 4 - 6:1; the mass ratio of the imine reductase to its coenzyme glucose dehydrogenase is 5 - 6:1; the mass ratio of the ketone reductase to its coenzyme formate dehydrogenase is 4 - 6:1; the mass ratio of the ketone reductase to its coenzyme glucose dehydrogenase is 1:5 - 10; the mass ratio of the alkene reductase to its coenzyme formate dehydrogenase is 6 - 8:1; the mass ratio of the alkene reductase to its coenzyme glucose dehydrogenase is 18 - 20:1; and the mass ratio of the monooxygenase to its coenzyme alcohol dehydrogenase is 2 - 3:2 - 3; the mass ratio of the monooxygenase to its coenzyme glucose dehydrogenase is 2 - 3:1.

[0090] Controlling the mass ratio of each of the above main enzymes to their corresponding different coenzymes within the above range can enable the main coenzymes to be configured as much as possible according to the stoichiometric ratio, thereby improving the catalytic activity and efficiency of the co-immobilized enzyme.

[0091] In the third typical embodiment of the present application, there is also provided the use of any of the above co-immobilized enzymes, or the co-immobilized enzyme prepared by the preparation method of any of the above co-immobilized enzymes in a biocatalytic reaction. Preferably, the biocatalytic reaction is a continuous biocatalytic reaction.

[0092] The beneficial effects of the present application will be further illustrated below with specific examples.

[0093] The enzymes used in the following examples and their sources are shown in Table 1 below. Tables 2 to 4 show the sequences of some enzymes.

[0094] Table 1:

[0095]

[0096] Table 2:

[0097]

[0098]

[0099] Table 3:

[0100]

[0101] Table 4:

[0102]

[0103]

[0104] In the following examples, PB represents phosphate buffer.

[0105] Example 1

[0106] Wash 1 g of amino resin with 4 - 5 mL of 0.1 M phosphate buffer (pH 7.5), resuspend with 4 mL of 0.1 M phosphate buffer (pH 7.5), add dropwise 25% - 50% (w / v) aqueous glutaraldehyde solution to make the final concentration of glutaraldehyde 2% (w / v), incubate at 20°C with gentle shaking for 1 hour, filter and wash 3 times with 0.1 M phosphate buffer (pH 7.5).

[0107] Add 4 mL of an enzyme solution containing 100 - 120 mg of protein (adjusting the appropriate ratio of TA and LDH) to glutaraldehyde-activated resin. After incubation with gentle shaking at 20 - 25 °C, filter and wash 3 times with 0.1 M phosphate buffer (pH 7.5).

[0108] More stable linkage can be achieved by reducing the imine double bond with borohydride. Resuspend 1 g of immobilized enzyme with 4 mL of buffer (50 mM NaHCO 3 -Na 2 CO 3 , pH 8.0 - 10.0), and add NaBH 4 at 5 - 15 °C to make the final concentration of NaBH 4 1 mg / mL. After stirring at 5 - 15 °C for 1 - 2 hours, filter and wash 3 times with 0.1 M phosphate buffer (pH 7.5).

[0109] The co-immobilization activity test of TA and LDH is carried out by using free FDH to regenerate NADH for testing. The following substrate 1 is used for testing:

[0110]

[0111] Add 5 mL of 0.1 M phosphate buffer (pH 8.0) to a 10 mL reactor, then add 100 mg of the above substrate 1, 80 mg of ammonium formate (FDH), 5 mg of PLP, adjust the pH to pH 7.5 - 8.0, then add 5 mg of NAD+ and 30 mg of free FDH enzyme, 100 mg of co-immobilized enzyme (wet enzyme, containing 50 - 80% water). React at 30 °C for 16 - 20 hours, and detect the conversion rate by HPLC. The results are shown in the following table.

[0112] Table 5:

[0113]

[0114]

[0115] Example 2 Co-immobilization of TA - Bt, LDH and FDH

[0116] Method 1: One-step co-immobilization

[0117] Wash 1 g of amino resin with 1 - 2 mL of 0.1 M PB (pH 7.5), resuspend it with 4 mL of 0.1 M PB (pH 7.5), and add an aqueous solution of glutaraldehyde with a concentration of 25 (w / v)% - 50 (w / v)% dropwise into the resuspension to make the final concentration of glutaraldehyde 2 (w / v)%. Incubate for 1 hour with gentle shaking at 20 °C, then filter and wash 3 times with 0.1 M PB (pH 7.5). Add 4 mL of enzyme solution (adjusting the appropriate ratio of TA, LDH, and FDH) containing 100 - 120 mg of protein to the glutaraldehyde-activated resin, incubate with gentle shaking at 20 - 25 °C, then filter and wash 3 times with 0.1 M PB (pH 7.5).

[0118] Method 2: Two-step co-immobilization

[0119] To improve the activity and stability of FDH, 1 g of co-immobilized TA and LDH was resuspended with 0.1 M PB (pH 7.0 - 7.5), and PEI solution (final concentration 2%) was added. Subsequently, 20 mg of FDH was added, incubated with gentle shaking at 20 - 25 °C, then filtered and washed 3 times with 0.1 M PB (pH 7.5).

[0120] The activities of co-immobilized TA, LDH, and FDH were detected by the following reaction:

[0121] Load 5 mL of 0.1 M PB (pH 8.0) into a 10 mL reactor, then adjust the pH to pH 7.5 - 8.0 by adding 100 mg of the above substrate 1, 80 mg of ammonium formate, and 5 mg of PLP. Then add 5 mg of NAD+ and 100 mg of co-immobilized enzyme (wet, containing 50 - 80% water). React at 30 °C for 16 - 20 hours and test the conversion rate. The test results are shown in the following table.

[0122] Table 6:

[0123]

[0124]

[0125] Example 3 Co-immobilization of TA-Bt, LDH, and GDH

[0126] For Method 1 and Method 2, except that GDH was used instead of FDH, the remaining steps were the same as in Example 2.

[0127] The activities of co-immobilized TA, LDH, and GDH were detected by the following reaction:

[0128] 5 mL of 0.1 M PB (pH 8.0) was loaded into a 10 ml reactor. Subsequently, by adding 100 mg of the above-mentioned substrate 1, 120 mg of glucose, and 5 mg of PLP, the pH was adjusted to pH 7.5 - 8.0, and then 5 mg of NAD+ and 100 mg of co-immobilized enzyme (wet, containing 50 - 80% water) were added. The reaction was carried out at 30 °C for 16 - 20 hours, and the conversion rate was tested. The test results are shown in Table 7.

[0129] Table 7:

[0130]

[0131]

[0132] Example 4 Co-immobilization of CHMO with ADH and GDH

[0133] The methods 1 and 2 in Example 2 were adopted, and except that the main enzyme and coenzyme were replaced with CHMO, ADH, and GDH, the remaining steps were the same.

[0134] The activity of the co-immobilized enzyme of CHMO with ADH and GDH was detected by reacting with the following substrate 2:

[0135]

[0136] The activity of the co-immobilized enzyme of CHMO with GDH was detected by the following reaction:

[0137] 3 mL of 0.1 M PB (pH 8.0) was loaded into a 10 ml reaction flask. Subsequently, by adding 50 mg of substrate 2, 100 mg of glucose, and 5 mg of NADP+, and then 200 - 300 mg of the co-immobilized enzyme of CHMO with GDH (wet, containing 50 - 80% water) was added. The reaction was carried out at 30 °C for 16 - 20 hours, and the conversion rate was tested.

[0138] The activity of the co-immobilized enzyme of CHMO with ADH was detected by the following reaction:

[0139] 0.3 mL of isopropanol was loaded into a 10 ml reaction flask. Subsequently, 500 mg of substrate 2 was added, and then 3 mL of 0.1 M PB (pH 8.0) containing 5 mg of NADP+ and 100 - 200 mg of the co-immobilized enzyme of CHMO with ADH (wet, containing 50 - 80% water) was added. The reaction was carried out at 30 °C for 16 - 20 hours, and the conversion rate was tested. The test results are shown in Table 8.

[0140] Table 8:

[0141]

[0142]

[0143] Example 5 Co - immobilization of AADH and FDH / GDH

[0144] Method 1:

[0145] Except for the different enzymes, the remaining steps are the same as Method 1 in Example 2.

[0146] Method 2:

[0147] Wash 1 g of amino resin with 1 - 2 mL of 0.1 M PB (pH 7.5), resuspend with 4 mL of 0.1 M PB (pH 7.5), and add an aqueous solution of glutaraldehyde with a mass concentration of 25% - 50% dropwise to the resuspended solution so that the final concentration of glutaraldehyde is 2%. Incubate for 1 hour with gentle shaking at 20 °C, then filter and wash 3 times with 0.1 M PB (pH 7.5).

[0148] Add 4 mL of enzyme solution containing 50 - 100 mg of protein (only AADH) to the glutaraldehyde - activated resin, incubate with gentle shaking at 20 - 25 °C, then filter and wash 3 times with 0.1 M PB (pH 7.5). After resuspending with 0.1 M PB (pH 7.0 - 7.5), add PEI solution (final concentration 2%), then add 20 - 50 mg of GDH / FDH, incubate with gentle shaking at 20 - 25 °C, then filter and wash 3 times with 0.1 M PB (pH 7.5).

[0149] The activity of the co - immobilized enzyme of AADH and FDH is detected by the reaction of the following substrates:

[0150]

[0151] Add 5 mL of 0.1 M Tris - Cl buffer (pH 8.0 - 9.0) to a 10 mL reactor, then add 100 mg of substrate 3 or 4, or 1,108 mg of ammonium chloride, adjust the pH to pH 7.5 - 8.0, then add 10 - 50 mg of NAD + , 80 mg of ammonium formate and 100 mg of the co - immobilized enzyme. After reacting at 30 °C for 16 - 20 hours, a conversion test is carried out.

[0152] The method for detecting the activity of the co - immobilized enzyme of AADH and FDH is as follows:

[0153] Add 5 mL of 0.1 M Tris-Cl buffer (pH 8.0 - 9.0) to a 10 mL reactor, then add 100 mg of substrate 5 or 6, or 7, 108 mg of ammonium chloride, adjust the pH to pH 7.5 - 8.0, and then add 10 - 50 mg of NAD⁺, 150 mg of glucose, and 100 mg of co-immobilized enzyme. After reacting at 30 °C for 16 - 20 hours, a conversion test is carried out. The test results are shown in Table 9.

[0154]

[0155] Table 9:

[0156]

[0157] Example 6 Co-immobilization of KRED and FDH / GDH

[0158] Except for the different enzymes, the steps of Methods 1 and 2 are the same as those in Example 5.

[0159] The activity of the co-immobilized enzyme of KRED and FDH is detected by the reaction of the following substrate 5 or 6:

[0160] Load 3 mL of 0.1 M PB (pH 7.0 - 8.0) into a 10 mL reactor, then add 100 mg of substrate 5 or 6, followed by adding 10 - 50 mg of NAD(P)⁺, 80 mg of ammonium formate, and 100 mg of the co-immobilized enzyme. React at 30 °C for 16 - 20 hours and test the conversion rate.

[0161] The activity of the co-immobilized enzyme of KRED and GDH is detected by the following reaction:

[0162] Load 3 mL of 0.1 M PB (pH 7.0 - 8.0) into a 10 mL reactor, then add 100 mg of substrate 5 or 6, followed by adding 10 - 50 mg of NAD(P)⁺, 120 mg of glucose, and 100 mg of the co-immobilized enzyme. React at 30 °C for 16 - 20 hours and test the conversion rate.

[0163] The test results are shown in Table 10.

[0164]

[0165]

[0166] Example 7 Co-immobilization of ERED and FDH / GDH

[0167] Except for the different enzymes, the steps of Methods 1 and 2 are the same as those in Example 5.

[0168] The activity of the co-immobilized enzyme of ERED and FDH was detected by reacting with substrate 7:

[0169] 3 mL of 0.1 M PB (pH 7.0 - 8.0) was loaded into a 10 mL reactor, followed by adding 100 mg of substrate 7, then adding 10 - 50 mg of NAD(P)+, 80 mg of ammonium formate and 100 mg of the co-immobilized enzyme. The reaction was carried out at 30 °C for 16 - 20 hours, and the conversion rate was tested.

[0170] The activity of the co-immobilized enzyme of ERED and GDH was detected by the following reaction:

[0171] 3 mL of 0.1 M PB (pH 7.0 - 8.0) was loaded into a 10 mL reactor, followed by adding 100 mg of substrate 7, then adding 10 - 50 mg of NAD(P)+, 120 mg of glucose and 100 mg of the co-immobilized enzyme. The reaction was carried out at 30 °C for 16 - 20 hours, and the conversion rate was tested.

[0172] The test results are shown in Table 11.

[0173]

[0174]

[0175] Example 8 Co-immobilization of IRED with FDH / GDH

[0176] Except for the different enzymes, the steps of Methods 1 and 2 were the same as those in Example 5.

[0177] The activity of the co-immobilized enzyme of IRED and FDH was tested using the following substrate 8 and by the following method:

[0178]

[0179] 2 mL of 0.1 M PB buffer (pH 7.0 - 8.0) was added to a 10 mL reactor, then 100 mg of the above substrate was added, then 10 - 50 mg of NAD(P)+, 60 mg of ammonium formate and 100 mg of the co-immobilized enzyme were added. After reacting at 30 °C for 16 - 20 hours, a conversion test was carried out.

[0180] The activity of the co-immobilized enzyme of IRED and GDH was tested by the following method:

[0181] 3 mL of 0.1 M PB buffer (pH 7.0 - 8.0) was added to a 10 mL reactor, followed by adding 100 mg of the substrate, then adding 10 - 50 mg of NAD(P)+, 100 mg of glucose and 100 mg of the co-immobilized enzyme. After reacting at 30 °C for 16 - 20 hours, a conversion test was carried out.

[0182] The test results are shown in Table 12.

[0183]

[0184] Application of the co-immobilized enzyme in Example 9 in continuous reaction in a packed bed

[0185] According to Method 2 in Example 2, the transaminase TA-Bt, coenzyme LDH and FDH were co-immobilized onto the carrier LX1000HA. The obtained co-immobilized enzyme was filled in a column reactor with a column volume of 10 mL, and the amount of the immobilized enzyme used was 5.9 g.

[0186] 500 g of substrate 5, 108 mg of ammonium chloride were dissolved in 4.5 L of PB buffer (0.1 M, pH 8.0), and the pH was adjusted to pH 7.5 - 8.0 with sodium hydroxide solution. Then 10 - 50 mg of NAD+ and 80 mg of ammonium formate were added, and finally the volume was made up to 5 L with PB buffer.

[0187] The flow rate was set at 0.1 mL / min, i.e., the retention time was 100 min, and continuous reaction was carried out. The conversion rate was detected at the outlet end of the effluent. The conversion rate > 98%. The reaction was continuously run for 300 h, and the conversion rate did not decrease. After running for 348 h, the decrease value of the conversion rate was 88.4%. See Table 13 for details.

[0188] Table 13. Reaction results of the co-immobilized enzyme TA-Bt + LDH + FDH in continuous reaction in a packed bed

[0189]

[0190] Application of the co-immobilized enzyme in Example 10 in continuous stirred tank reaction

[0191] The same co-immobilized enzyme as in Example 9 was used. 50 g of the co-immobilized enzyme of transaminase TA-Bt and coenzymes LDH and FDH were added to a 200 mL reactor, and 150 mL of phosphate buffer was added.

[0192] 500 g of substrate 5, 108 mg of ammonium chloride were dissolved in 4.5 L of PB buffer (0.1 M, pH 8.0), and the pH was adjusted to pH 7.5 - 8.0 with sodium hydroxide solution. Then 10 - 50 mg of NAD + , 80 mg of ammonium formate were added, and finally the volume was made up to 5 L with PB buffer.

[0193] The substrate was continuously added to the continuously stirred tank at a rate of 0.8 mL / min (i.e., the retention time was 250 min), and at the same time, the reaction system was withdrawn at the outlet at the same flow rate (a filter head was added at the end of the pipeline to prevent the immobilized enzyme from being withdrawn). Under this condition, the conversion rate could reach over 92%, and after continuous operation for 400 h, the conversion rate basically did not decrease. The results are shown in Table 14.

[0194] Table 14. Reaction results of the co-immobilized enzyme of TA-Bt + LDH + FDH in continuous reaction in a continuously stirred tank

[0195]

[0196] Investigation of the protein loading amount per gram of carrier in Example 11

[0197] Same as Example 1, after activating the amino carrier, the amount of protein added per gram of carrier was investigated. Taking the co-immobilization of the transaminase TA-Bt and its coenzyme lactate dehydrogenase LDH as an example, different amounts of protein were added, and the protein loading amount and the number of reaction reuse times were detected. The results are shown in Table 15.

[0198] Same as Example 4, taking the co-immobilization of the monooxygenase CHMO-Rs and its coenzymes ADH and GDH as an example, different amounts of protein were added, and the protein loading amount and the number of reaction reuse times were detected. The results are shown in Table 16. The results showed that the range of protein that could be loaded per gram of the selected carrier was 50 - 200 mg, and the protein loading rate was 50% - 100%.

[0199] Table 15. Investigation of the protein loading amount of the mixed enzyme of TA-Bt and LDH on different carriers

[0200]

[0201] Table 16. Investigation of the protein loading amount of the mixed enzyme of CHMO-Rs and ADH on different carriers

[0202]

[0203] It can be seen from the data in Tables 15 and 16 that for the vast majority of carriers, when the protein loading amount was 50 - 100 mg, the protein loading rate was over 90%. Under the condition of the same protein loading rate, the number of reuse times of the co-immobilized enzyme formed by different carriers was different. From this table, it can be seen that several carriers such as LX1000HA, LX1000EPN, and ECR8409 showed better immobilized enzyme activity and stability.

[0204] Investigation of the final concentration of PEI in Example 12

[0205] Same as Example 2, Method 2 (two-step method), to prepare the co-immobilized enzyme of transaminase TA-Bt and its coenzymes lactate dehydrogenase LDH and formate dehydrogenase FDH. After the mixed enzyme of TA-Bt and LDH was combined with the carrier, different amounts of PEI were added, and then the second coenzyme FDH was added. The concentration range of PEI was investigated, and the results are shown in Table 17.

[0206] Table 17. Investigation of the amount of PEI used in the two-step immobilization method of TA-Bt and its coenzymes LDH and FDH

[0207] carrier Final concentration of PEI Number of reuse times LX1000HA 0.3％ 10 LX1000HA 0.5％ 12 LX1000HA 1％ 12 LX1000HA 3％ 12 LX1000HA 5％ 12 HFA 1％ 9 HFA 3％ 9 HFA 5％ 9 HFA 7％ 7 ECR8409 2％ 13 ECR8409 5％ 13 ECR8409 7％ 13 ESR-1 0.5％ 10 ESR-1 2％ 11 ESR-1 5％ 11

[0208] It can be seen from the data in Table 17 that the effects are basically the same when the concentration of PEI is in the range of 1% - 5%. Beyond this range, the stability of the immobilized enzyme decreases.

[0209] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By co-immobilizing the above main enzyme and its coenzymes on the amino resin carrier, the co-immobilization of these main enzyme and its coenzymes is realized, which is beneficial to improving the enzyme activity and recycling efficiency.

[0210] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Jilin Caelux Pharmaceutical Chemistry Co., Ltd. <120> Co-immobilized Enzyme, Its Preparation Method and Application <130> PN187140KLY <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 253 <212> PRT <213> Ketoreductase (Acetobacter sp.) <220> <221> VARIANT <222> (1)..(253) <223> KRED-Ac-V2 <220> <221> MUTAGEN <222> (1)..(253) <223> E144S+A94T+N156T <400> 1 Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 1 5 10 15 Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 20 25 30 Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 35 40 45 Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 50 55 60 Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 65 70 75 80 Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Thr Tyr Ser 85 90 95 Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 100 105 110 Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 115 120 125 Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser 130 135 140 Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Thr Ala Ser Lys Gly 145 150 155 160 Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 165 170 175 Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 180 185 190 Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 195 200 205 Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 210 215 220 Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 225 230 235 240 Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 245 250 <210> 2 <211> 253 <212> PRT <213> Ketoreductase (Acetobacter sp.) <220> <221> VARIANT <222> (1)..(253) <223> KRED-Ac-V1 <220> <221> MUTAGEN <222> (1)..(253) <223> E144S+A94N+N156V <400> 2 Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 1 5 10 15 Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 20 25 30 Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 35 40 45 Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 50 55 60 Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 65 70 75 80 Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Asn Tyr Ser 85 90 95 Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 100 105 110 Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 115 120 125 Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser 130 135 140 Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Val Ala Ser Lys Gly 145 150 155 160 Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 165 170 175 Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 180 185 190 Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 195 200 205 Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 210 215 220 Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 225 230 235 240 Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 245 250 <210> 3 <211> 253 <212> PRT <213> Ketoreductase (Acetobacter sp.) <220> <221> SITE <222> (1)..(253) <223> KRED-Ac-mother <400> 3 Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 1 5 10 15 Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 20 25 30 Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 35 40 45 Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 50 55 60 Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 65 70 75 80 Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Ala Tyr Ser 85 90 95 Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 100 105 110 Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 115 120 125 Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Glu 130 135 140 Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Asn Ala Ser Lys Gly 145 150 155 160 Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 165 170 175 Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 180 185 190 Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 195 200 205 Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 210 215 220 Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 225 230 235 240 Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 245 250 <210> 4 <211> 541 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus sp. Phi1) <220> <221> VARIANT <222> (1)..(541) <223> CHMO-Rs-V2 <220> <221> MUTAGEN <222> (1)..(541) <223> F508Y+F435N+L438A+T436S+F280V+S441V+L510V <400> 4 Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 1 5 10 15 Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 20 25 30 Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 35 40 45 Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 50 55 60 Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 65 70 75 80 Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 85 90 95 Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 100 105 110 Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 115 120 125 Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 130 135 140 Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 145 150 155 160 Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 165 170 175 Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 180 185 190 Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 195 200 205 Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 210 215 220 Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 225 230 235 240 Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 245 250 255 Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 260 265 270 Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly 275 280 285 Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 290 295 300 Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 305 310 315 320 Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 325 330 335 Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 340 345 350 Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 355 360 365 Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 370 375 380 Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 385 390 395 400 Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 405 410 415 Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 420 425 430 Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp 435 440 445 Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 450 455 460 Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 465 470 475 480 Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 485 490 495 Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Val Gly Gly 500 505 510 Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 515 520 525 Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 530 535 540 <210> 5 <211> 541 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus sp. Phi1) <220> <221> VARIANT <222> (1)..(541) <223> CHMO-Rs-V1 <220> <221> MUTAGEN <222> (1)..(541) <223> F508Y+F435N+L438A+T436S+F280V+S441V <400> 5 Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 1 5 10 15 Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 20 25 30 Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 35 40 45 Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 50 55 60 Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 65 70 75 80 Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 85 90 95 Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 100 105 110 Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 115 120 125 Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 130 135 140 Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 145 150 155 160 Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 165 170 175 Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 180 185 190 Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 195 200 205 Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 210 215 220 Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 225 230 235 240 Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 245 250 255 Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 260 265 270 Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly 275 280 285 Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 290 295 300 Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 305 310 315 320 Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 325 330 335 Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 340 345 350 Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 355 360 365 Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 370 375 380 Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 385 390 395 400 Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 405 410 415 Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 420 425 430 Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp 435 440 445 Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 450 455 460 Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 465 470 475 480 Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 485 490 495 Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Leu Gly Gly 500 505 510 Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 515 520 525 Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 530 535 540 <210> 6 <211> 541 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus sp. Phi1) <220> <221> SITE <222> (1)..(541) <223> CHMO-Rs-mother <400> 6 Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 1 5 10 15 Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 20 25 30 Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 35 40 45 Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 50 55 60 Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 65 70 75 80 Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 85 90 95 Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 100 105 110 Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 115 120 125 Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 130 135 140 Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 145 150 155 160 Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 165 170 175 Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 180 185 190 Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 195 200 205 Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 210 215 220 Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 225 230 235 240 Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 245 250 255 Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 260 265 270 Ala Trp Asp His Gly Gly Gly Phe Arg Phe Met Phe Gly Thr Phe Gly 275 280 285 Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 290 295 300 Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 305 310 315 320 Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 325 330 335 Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 340 345 350 Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 355 360 365 Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 370 375 380 Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 385 390 395 400 Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 405 410 415 Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 420 425 430 Gly Pro Phe Thr Asn Leu Pro Pro Ser Ile Glu Thr Gln Val Glu Trp 435 440 445 Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 450 455 460 Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 465 470 475 480 Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 485 490 495 Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Phe Tyr Leu Gly Gly 500 505 510 Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 515 520 525 Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 530 535 540 <210> 7 <211> 603 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus ruber - SD1) <220> <221> VARIANT <222> (1)..(603) <223> CHMO - Rr - V2 <220> <221> MUTAGEN <222> (1)..(603) <223> Y559M+P190L+P504V <400> 7 Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 1 5 10 15 Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 20 25 30 Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr 35 40 45 Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 50 55 60 Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 65 70 75 80 Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 85 90 95 Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 100 105 110 Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 115 120 125 Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 130 135 140 Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 145 150 155 160 Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 165 170 175 Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His 180 185 190 Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 195 200 205 Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 210 215 220 Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 225 230 235 240 Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr 245 250 255 Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 260 265 270 Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 275 280 285 Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 290 295 300 Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 305 310 315 320 Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 325 330 335 Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 340 345 350 Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 355 360 365 Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 370 375 380 Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro 385 390 395 400 Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 405 410 415 Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 420 425 430 Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 435 440 445 Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 450 455 460 Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 465 470 475 480 His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 485 490 495 Ala Ala Leu Gly Ser Asn Ile Val His Asn Phe Val Glu Ala Ala Arg 500 505 510 Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 515 520 525 Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 530 535 540 Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr 545 550 555 560 Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 565 570 575 Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 580 585 590 Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 595 600 <210> 8 <211> 603 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus ruber - SD1) <220> <221> VARIANT <222> (1)..(603) <223> CHMO - Rr - V1 <220> <221> MUTAGEN <222> (1)..(603) <223> P190L + Y559M + C249V + C393V + C257A + M45T <400> 8 Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 1 5 10 15 Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 20 25 30 Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Thr Pro Ile Thr 35 40 45 Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 50 55 60 Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 65 70 75 80 Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 85 90 95 Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 100 105 110 Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 115 120 125 Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 130 135 140 Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 145 150 155 160 Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 165 170 175 Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His 180 185 190 Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 195 200 205 Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 210 215 220 Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 225 230 235 240 Gly Thr Gly Ala Thr Ala Val Gln Val Val Pro Glu Leu Ala Lys Tyr 245 250 255 Ala Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 260 265 270 Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 275 280 285 Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 290 295 300 Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 305 310 315 320 Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 325 330 335 Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 340 345 350 Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 355 360 365 Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 370 375 380 Phe Arg Gln Met Cys Lys Arg Pro Val Phe His Asp Asp Tyr Leu Pro 385 390 395 400 Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 405 410 415 Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 420 425 430 Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 435 440 445 Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 450 455 460 Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 465 470 475 480 His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 485 490 495 Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg 500 505 510 Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 515 520 525 Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 530 535 540 Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr 545 550 555 560 Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 565 570 575 Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 580 585 590 Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 595 600 <210> 9 <211> 603 <212> PRT <213> Cyclohexanone monooxygenase (Rhodococcus ruber - SD1) <220> <221> SITE <222> (1)..(603) <223> CHMO - Rr - parent <400> 9 Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 1 5 10 15 Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 20 25 30 Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr 35 40 45 Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 50 55 60 Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 65 70 75 80 Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 85 90 95 Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 100 105 110 Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 115 120 125 Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 130 135 140 Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 145 150 155 160 Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 165 170 175 Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Pro Leu His 180 185 190 Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 195 200 205 Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 210 215 220 Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 225 230 235 240 Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr 245 250 255 Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 260 265 270 Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 275 280 285 Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 290 295 300 Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 305 310 315 320 Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 325 330 335 Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 340 345 350 Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 355 360 365 Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 370 375 380 Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro 385 390 395 400 Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 405 410 415 Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 420 425 430 Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 435 440 445 Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 450 455 460 Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 465 470 475 480 His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 485 490 495 Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg 500 505 510 Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 515 520 525 Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 530 535 540 Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Tyr Tyr 545 550 555 560 Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 565 570 575 Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 580 585 590 Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 595 600

Claims

1. A co-immobilized enzyme, characterized in that, the co-immobilized enzyme comprises: an amino resin carrier, and a main enzyme and a coenzyme, the main enzyme is covalently immobilized on the amino resin carrier, and the coenzyme is immobilized on the amino resin carrier by a non-covalent method; the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketone reductase, alkene reductase, and monooxygenase; the non-covalent immobilization is by adsorbing on the amino resin carrier by PEI in an ion adsorption manner; the coenzyme is selected from any one of the following: lactate dehydrogenase, formate dehydrogenase, glucose dehydrogenase, and alcohol dehydrogenase; the amino resin carrier is selected from any one of the following: LX1000EA, LX1000HA, LX1000NH, HFA, LX1000EPN, HM100D, ECR8309, ECR8409, ECR8305, ECR8404, ECR8315, ECR8415, ESR-1, ESR-3, ESR-5, and ESR-8.

2. The co-immobilized enzyme according to claim 1, characterized in that, the transaminase is a transaminase derived from B.thuringiensis or Vibrio fluvialis strain JS17; the amino acid dehydrogenase is an amino acid dehydrogenase derived from Bacillus cereus or Bacillus sphaericus; the imine reductase is an imine reductase derived from Streptomyces sp or Bacillus cereus; the ketone reductase is a ketone reductase derived from Sporobolomyces salmonicolor or Acetobactersp. CCTCC M209061 ketone reductase; the alkene reductase is an alkene reductase derived from Chryseobacterium sp.CA49 or Sewanella oneidensis MR-1; the monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans, or cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1.

3. The co-immobilized enzyme according to claim 2, characterized in that, the ketone reductase derived from Acetobactersp.CCTCCM209061 is a mutant having the sequence of SEO ID NO:1 or SEO ID NO:

2.

4. The co-immobilized enzyme according to claim 2, characterized in that, The cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having the sequence of SEO ID NO: 4 or SEO ID NO: 5; the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant having the sequence of SEO ID NO: 7 or SEO ID NO:

8.

5. The co-immobilized enzyme according to claim 4, wherein, the lactate dehydrogenase is D-lactate dehydrogenase derived from Lactobacillus helveticus; the formate dehydrogenase is formate dehydrogenase derived from Candida boidinii; the glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10; the alcohol dehydrogenase is alcohol dehydrogenase derived from Thermoanaerobium brockii.

6. The co-immobilized enzyme according to claim 4, wherein, the number of recycling times of the co-immobilized enzyme is 4 to 25 times.

7. The co-immobilized enzyme according to claim 1, wherein, in the co-immobilized enzyme, the mass ratio of the main enzyme to the coenzyme is 1 to 20: 1 to 10.

8. The co-immobilized enzyme according to claim 7, wherein, the sum of the masses of the main enzyme and the coenzyme is denoted as N1, the mass of the amino resin carrier is denoted as N2, and N1 / N2 is 50 to 200 mg: 1 g.

9. The co-immobilized enzyme according to claim 8, wherein, N1 / N2 is 80 to 120 mg: 1 g.

10. The preparation method of the co-immobilized enzyme according to any one of claims 1 to 9, wherein, the preparation method includes: activating the amino resin carrier to obtain an activated amino carrier; covalently immobilizing the main enzyme on the activated amino carrier, non-covalently immobilizing the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme; the non-covalent immobilization on the activated carrier means adsorbing on the activated carrier by PEI in an ion adsorption manner.

11. The preparation method according to claim 10, wherein, the immobilization of the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: immobilizing the main enzyme on the activated amino carrier to obtain a preliminarily immobilized enzyme; coating a PEI layer on the surface of the preliminarily immobilized enzyme and then adsorbing the coenzyme to obtain the co-immobilized enzyme.

12. The preparation method according to claim 11, wherein, adding PEI to the preliminarily immobilized enzyme until the final concentration of PEI is 0.5 w / v% to 5 w / v% to obtain a PEI-preliminarily immobilized enzyme complex; then binding the coenzyme to the PEI-preliminarily immobilized enzyme complex to obtain the co-immobilized enzyme.

13. The preparation method according to claim 12, wherein, the mass ratio of the main enzyme to the coenzyme is 1 to 20: 1 to 10.

14. The preparation method according to claim 10, characterized in that, the main enzyme is amino acid dehydrogenase, the coenzyme is FDH or GDH, and the preparation method includes: fixing the amino acid dehydrogenase to the activated amino carrier to obtain the initially fixed amino acid dehydrogenase; fixing the GDH or the FDH by means of PEI surface coating of the initially fixed amino acid dehydrogenase to obtain the co-immobilized enzyme.

15. The preparation method according to claim 10, characterized in that, the main enzyme is imine reductase, the coenzyme is FDH or GDH, and the preparation method includes: fixing the imine reductase to the activated amino carrier to obtain the initially fixed imine reductase; fixing the GDH or the FDH by means of PEI surface coating of the initially fixed imine reductase to obtain the co-immobilized enzyme.

16. The preparation method according to claim 10, characterized in that, the main enzyme is ketone reductase, the coenzyme is FDH or GDH, and the preparation method includes: fixing the ketone reductase to the activated amino carrier to obtain the initially fixed ketone reductase; fixing the GDH or the FDH by means of PEI surface coating of the initially fixed ketone reductase to obtain the co-immobilized enzyme.

17. The preparation method according to claim 10, characterized in that, the main enzyme is alkene reductase, the coenzyme is FDH or GDH, and the preparation method includes: fixing the alkene reductase to the activated amino carrier to obtain the initially fixed alkene reductase; fixing the GDH or the FDH by means of PEI surface coating of the initially fixed alkene reductase to obtain the co-immobilized enzyme.

18. The preparation method according to claim 10, characterized in that, the main enzyme is cyclohexanone monooxygenase, the coenzyme is FDH or GDH, and the preparation method includes: fixing the cyclohexanone monooxygenase to the activated amino carrier to obtain the initially fixed cyclohexanone monooxygenase; fixing the GDH or the FDH by means of PEI surface coating of the initially fixed cyclohexanone monooxygenase to obtain the co-immobilized enzyme.

19. The preparation method according to claim 10, characterized in that, the amino resin carrier is activated with glutaraldehyde to obtain the activated amino carrier.

20. Application of the co-immobilized enzyme according to any one of claims 1 to 9, or the co-immobilized enzyme prepared by the preparation method of the co-immobilized enzyme according to any one of claims 10 to 19 in a biocatalytic reaction.

21. The application according to claim 20, characterized in that, the biocatalytic reaction is an intermittent biocatalytic reaction or a continuous biocatalytic reaction.

22. The application according to claim 21, characterized in that, the co-immobilized enzyme is applied in a continuous fluidized bed or a fixed bed biocatalytic reaction.

23. The application according to claim 22, characterized in that, The recycling times of the co-immobilized enzyme in the continuous biocatalytic reaction are 4 to 25 times.

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