WO2021108957A1 - 共固定化酶、其制备方法及其应用 - Google Patents

共固定化酶、其制备方法及其应用 Download PDF

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WO2021108957A1
WO2021108957A1 PCT/CN2019/122447 CN2019122447W WO2021108957A1 WO 2021108957 A1 WO2021108957 A1 WO 2021108957A1 CN 2019122447 W CN2019122447 W CN 2019122447W WO 2021108957 A1 WO2021108957 A1 WO 2021108957A1
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enzyme
immobilized
carrier
coenzyme
main
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PCT/CN2019/122447
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English (en)
French (fr)
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洪浩
詹姆斯·盖吉
罗杰斯卡·维亚撒·威廉姆斯
崔瑜霞
张娜
赵佳东
郝明敏
高妍妍
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吉林凯莱英医药化学有限公司
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Priority to EP19955387.6A priority Critical patent/EP4071246A4/en
Priority to PCT/CN2019/122447 priority patent/WO2021108957A1/zh
Priority to JP2022532879A priority patent/JP7420944B2/ja
Priority to US17/781,823 priority patent/US20230025239A1/en
Publication of WO2021108957A1 publication Critical patent/WO2021108957A1/zh

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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/091—Phenol resins; Amino resins
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)

Definitions

  • the present invention relates to the field of immobilized enzymes, in particular to a co-immobilized enzyme, its preparation method and its application.
  • acyltransferase amidase, transaminase, ketoreductase, oxidase, monooxygenase and hydrolase are used in the production of reactions involving antibiotics, herbicides, pharmaceutical intermediates and new era therapeutics .
  • Binay et al (Beilstein J. Org. Chem. 2016, 12, 271-277) reported a highly active immobilized enzyme of FDH from Candida methylica.
  • FDH is covalently immobilized on the epoxy-activated Immobead 150 carrier.
  • the Immobead 150 carrier is first modified with ethylenediamin, and then sequentially activated with glutaraldehyde (FDHIGLU) and functionalized with aldehyde groups (FDHIALD).
  • FDHIGLU glutaraldehyde
  • FDHIALD aldehyde functionalized Immobead 150 was used as the carrier, the highest immobilization yield and active yield were obtained, respectively, 90% and 132%.
  • Jackon et al. reported the immobilization of LDH using glyoxal-agarose. Compared with its soluble counterpart, the thermal stability factor obtained by immobilized LDH is 1600 times larger.
  • thermostable cyclohexanone monooxygenase (TmCHMO) from Thermocrispum municipale and glucose dehydrogenase (GDH) (GDH-Tac) from Thermoplasma acidophilum to amino-functionalized agarose-based carrier (MANA-Sepharose) )on.
  • TmCHMO thermostable cyclohexanone monooxygenase
  • GDH-Tac glucose dehydrogenase
  • MANA-Sepharose amino-functionalized agarose-based carrier
  • the main purpose of the present invention is to provide a co-immobilized enzyme, its preparation method and its application, so as to solve the problem that such enzymes are difficult to recycle in the prior art.
  • a co-immobilized enzyme includes an amino resin carrier and a main enzyme and a coenzyme.
  • the coenzyme is one or more, and the main enzyme and the coenzyme are co-immobilized.
  • 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 of the following enzymes: transaminase, Amino acid dehydrogenase, imine reductase, ketoreductase, ene reductase and monooxygenase.
  • 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 derived from Streptomyces sp or imine reductase of Bacillus cereus; preferably, the ketoreductase is a ketoreductase derived from Sporobolomyces salmonicolor or a ketoreductase derived from Acetobacter sp.CCTCC M209061, more preferably, a ketoreductase derived from Acetobacter sp.CCTCC M209061
  • the enzyme is a mutant with SEO ID NO:1 or SEO ID NO:2 sequence; preferably, the ene reductase is an ene reductase derived from Chry
  • the coenzyme is selected from at least one of the following: lactate dehydrogenase (LDH), ammonium formate dehydrogenase (FDH), glucose dehydrogenase (GDH) and alcohol dehydrogenase; preferably, lactate dehydrogenase is the source D-lactate dehydrogenase from Lactobacillus helveticus; preferably, ammonium formate dehydrogenase is formate dehydrogenase derived from Candida boidinii; preferably, glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10 ;
  • the alcohol dehydrogenase is an alcohol dehydrogenase derived from Thermoanaerobium brockii; preferably, the number of cycles of the co-immobilized enzyme is 4-25 times.
  • the amino resin carrier is an amino resin carrier activated by glutaraldehyde; preferably, the amino resin carrier is an amino resin carrier with a C2 or C4 linking 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.
  • the mass ratio of the main enzyme to the coenzyme is 1-20:1-10; preferably, the sum of the mass of the main enzyme and the coenzyme is denoted as N1, and the mass of the amino resin carrier is denoted as N2, N1/ N2 is 50 to 200 mg: 1 g, and further, 80 to 120 mg: 1 g.
  • 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 is non-covalently immobilized on the amino resin carrier by ion adsorption; preferably, the coenzyme is immobilized on the amino resin carrier by means of ion adsorption. Adsorbed on the amino resin carrier.
  • 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 ion adsorption.
  • a method for preparing any of the above-mentioned co-immobilized enzymes includes: activating an amino resin carrier to obtain an activated amino carrier; and covalently immobilizing the main enzyme on the activated amino resin carrier.
  • the coenzyme corresponding to the main enzyme is covalently and/or non-covalently immobilized on the activated amino carrier to obtain a co-immobilized enzyme; wherein the number of coenzymes corresponding to the main enzyme is one or more.
  • fixing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: mixing the main enzyme and the coenzyme to obtain the first mixed enzyme; and fixing the first mixed enzyme on the activated amino carrier to obtain the co-immobilized enzyme ⁇ zyme.
  • the coenzyme includes the first enzyme
  • fixing 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 the primary immobilized enzyme; The immobilized enzyme is fixed to obtain a co-immobilized enzyme.
  • the coenzyme further includes a second enzyme
  • fixing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: immobilizing the main enzyme and the second enzyme on the activated amino carrier together to obtain the primary immobilized enzyme; The first enzyme is fixed with the initial immobilized enzyme to obtain a co-immobilized enzyme.
  • the first enzyme and the initial immobilized enzyme are immobilized by surface coating with PEI to obtain a co-immobilized enzyme; preferably, PEI is added to the initial immobilized enzyme until the final concentration of PEI is 0.5 w/v % ⁇ 5w/v% to obtain the PEI-primary immobilized enzyme; then the first enzyme is combined with the PEI-primary immobilized enzyme to obtain the co-immobilized enzyme.
  • the first mixed enzyme is immobilized on the activated amino carrier according to a mass ratio of 50-150 mg: 1 g to obtain a co-immobilized enzyme.
  • the mass ratio of the main enzyme to the first enzyme is 1-20:1-10.
  • the mass ratio of the main enzyme and the second enzyme is 1-20:1-10.
  • the main enzyme is transaminase
  • the coenzyme has two cofactors.
  • the two cofactors are LDH (lactate dehydrogenase) and FDH (ammonium formate dehydrogenase), or LDH (lactate dehydrogenase) and GDH (glucose dehydrogenase).
  • Hydrogenase the above preparation method includes any one of the following:
  • the main enzyme is amino acid dehydrogenase
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following:
  • Co-immobilized enzyme is obtained by surface-coating the initially immobilized amino acid dehydrogenase with PEI, and then immobilizing GDH or FDH.
  • the main enzyme is imine reductase
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following:
  • the main enzyme is ketoreductase
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following:
  • ketoreductase, FDH or GDH Mix ketoreductase, FDH or GDH to obtain a ketoreductase mixture; immobilize the ketoreductase mixture on an activated amino carrier to obtain a co-immobilized enzyme;
  • the main enzyme is ene reductase
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following:
  • the main enzyme is cyclohexanone monooxygenase
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following:
  • glutaraldehyde is used to activate the amino resin carrier to obtain an activated amino carrier.
  • the biocatalytic reaction is an intermittent biocatalytic reaction or a continuous biocatalytic reaction; preferably, the co-immobilized enzyme is applied in a continuous fluidized bed or a fixed bed biocatalytic reaction; preferably, co-immobilization
  • the number of cycles of the chemical enzyme in the continuous biocatalytic reaction is 4-25 times.
  • this application realizes the co-immobilization of these main enzymes and their coenzymes by co-immobilizing the above-mentioned main enzymes and their coenzymes on an amino resin carrier, thereby helping to improve enzyme activity and recycling efficiency.
  • PEI Polyethylene imine
  • a co-immobilized enzyme in a typical embodiment of the present application, includes an amino resin carrier and a main enzyme and a coenzyme.
  • the main enzyme and 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 methods; the main enzyme is selected from any of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase , Ketoreductase, ene reductase and monooxygenase.
  • the present application realizes the co-immobilization of these main enzymes and their coenzymes by co-immobilizing the above-mentioned main enzymes and their coenzymes on an amino resin carrier, thereby helping to improve the activity of the enzyme and the efficiency of recycling.
  • 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, imine reduction
  • the enzyme is an imine reductase derived from Streptomyces sp or Bacillus cereus; preferably, the ketoreductase is a ketoreductase derived from Sporobolomyces salmonicolor or a ketoreductase derived from Acetobacter sp.CCTCC M209061, more preferably, derived from Acetobacter sp.
  • the ketoreductase of CCTCC M209061 is a mutant with SEO ID NO:1 or SEO ID NO:2 sequence; preferably, the ene reductase is an ene reductase derived from Chryseobacterium sp.CA49 or Sewanella oneidensis MR-1; preferably , Monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans, or cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 More preferably, the cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1 is a mutant with SEO ID NO: 4 sequence or SEO ID NO: 5 sequence; cyclohexanone monooxygenase derived from Rhodococcus rub
  • the corresponding coenzymes are selected. It is preferable that these coenzymes are coenzymes capable of regenerating NAD(P)+ and NADPH cycles.
  • the coenzyme is selected from at least one of the following: the coenzyme is selected from at least one of the following: lactate dehydrogenase, ammonium formate dehydrogenase, glucose dehydrogenase and alcohol dehydrogenase; preferably, lactate dehydrogenase Hydrogenase is D-lactate dehydrogenase derived from Lactobacillus helveticus; preferably, ammonium formate dehydrogenase is formate dehydrogenase derived from Candida boidinii; preferably, glucose dehydrogenase is glucose 1 derived from Lysinibacillus sphaericus G10 -Dehydrogenase; preferably, the alcohol dehydrogen
  • the coenzymes corresponding to the transaminase are lactate dehydrogenase and ammonium formate dehydrogenase, or lactate dehydrogenase and glucose dehydrogenase.
  • the coenzyme corresponding to amino acid dehydrogenase is ammonium formate dehydrogenase or glucose dehydrogenase.
  • the corresponding coenzyme of imine reductase is ammonium formate dehydrogenase or glucose dehydrogenase.
  • the corresponding coenzyme of ketoreductase is ammonium formate dehydrogenase or glucose dehydrogenase.
  • the coenzyme corresponding to ene reductase is ammonium formate dehydrogenase or glucose dehydrogenase.
  • the coenzyme corresponding to monooxygenase is alcohol dehydrogenase or ammonium formate dehydrogenase or glucose dehydrogenase.
  • lactate dehydrogenase is D-lactate dehydrogenase (abbreviated as LDH) derived from Lactobacillus helveticus; preferably, ammonium formate dehydrogenase is formate dehydrogenase (abbreviated as FDH) derived from Candida boidinii );
  • the glucose dehydrogenase is glucose 1-dehydrogenase (GDH) derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is an alcohol dehydrogenase derived from Thermoanaerobium brocii.
  • the carrier for the co-immobilized enzyme is an amino resin carrier, and various existing amino resin carriers can be used.
  • the amino resin carrier is an amino resin carrier activated by glutaraldehyde; preferably, the amino resin carrier is an amino resin carrier with a C2 or C4 linking arm, more preferably, the amino resin carrier is selected from the following Any one: 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-mentioned main enzyme and coenzyme to be fixed, different amino resin carriers can be selected.
  • the dosage ratio of the main enzyme and the coenzyme varies according to the different substrates catalyzed.
  • the mass ratio of the main enzyme to the coenzyme is 1-20:1-10.
  • the sum of the mass of the main enzyme and the coenzyme is denoted as N1
  • the mass of the amino resin carrier is denoted as N2
  • N1/N2 is 50-200 mg: 1 g, and further, 80-120 mg: 1 g.
  • the mass ratio of the main enzyme to the coenzyme is controlled within the above range, so that all the co-immobilized enzymes of the main enzyme and the coenzyme can catalyze most of the substrates.
  • the mass ratio of the enzyme to the carrier is controlled in the range of 50-200mg:1g, which can realize the co-immobilization of all the above-mentioned main enzymes and coenzymes.
  • the mass ratio of transaminase to its coenzyme lactate dehydrogenase is 5-7:1; the mass ratio of transaminase to its coenzyme ammonium formate dehydrogenase is 5-7:1 to 3; transaminase to its coenzyme glucose
  • the mass ratio of dehydrogenase is 5-7:1-2; the mass ratio of amino acid dehydrogenase and its coenzyme ammonium formate dehydrogenase is 8-10:1; the mass ratio of amino acid dehydrogenase and its coenzyme glucose dehydrogenase is 5 ⁇ 6:1; the mass ratio of imine reductase and its coenzyme ammonium formate dehydrogenase is 4 ⁇ 8; 1; the mass ratio of suban reductase and its coenzyme glucose dehydrogenase is 6 ⁇ 8:1; ketoreductase The mass ratio of its coenzyme ammonium formate de
  • Controlling the mass ratios of the above-mentioned main enzymes and their corresponding different coenzymes within the above-mentioned range enables the main coenzymes to be arranged in stoichiometric ratios as much as possible, thereby improving the catalytic activity and efficiency of the co-immobilized enzymes.
  • both the main enzyme and the coenzyme are immobilized on the carrier, and the specific immobilization method of the two on the carrier is not limited.
  • both the main enzyme and the coenzyme are covalently immobilized on the amino resin carrier.
  • the main enzyme is covalently immobilized on the 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 by PEI.
  • 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 ion adsorption.
  • the first enzyme here refers to an enzyme that is sensitive to certain reagents in the co-immobilization process, such as the cross-linking agent glutaraldehyde, etc., in order to reduce the decrease in activity of the first enzyme during the co-immobilization process, thereby affecting the co-immobilization process
  • the first enzyme is non-covalently immobilized on the amino resin carrier by means of ion adsorption.
  • the transaminase TA-Bt (see Table 1 for details) is co-immobilized with LDH and FDH to the carrier LX1000HA at the optimal ratio, and the maximum number of uses can reach 15 times; the transaminase TA-Bt is co-immobilized with LDH and GDH at the optimal ratio To the carrier LX1000HA, the maximum use times can reach 20-25 times; the monooxygenase CHMO-Rs and GDH are co-immobilized to the carrier LX1000HA at the optimal ratio, and the maximum use times reach 13 times.
  • the monooxygenase CHMO-Rs and GDH ADH was co-immobilized to the carrier LX1000HA at the optimal ratio, and the maximum number of uses was 13-15 times, and it was co-immobilized to the ECR8409 carrier, and the maximum number of uses was 16; the CHMO-Rs mutant V1 and ADH were co-immobilized to the carrier LX1000HA has been used up to 17 times.
  • a method for preparing any of the above-mentioned co-immobilized enzymes includes: activating an amino resin carrier to obtain an activated amino carrier; and covalently immobilizing the main enzyme on Activate the amino carrier, and fix the coenzyme corresponding to the main enzyme on the activated amino carrier in a covalent and/or non-covalent manner to obtain a co-immobilized enzyme.
  • the co-immobilized enzyme can be obtained by first activating the amino resin carrier, and then co-immobilizing any of the above-mentioned main enzymes and coenzymes on the activated amino carrier.
  • immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: mixing the main enzyme and the coenzyme to obtain the first mixed enzyme; immobilizing the first mixed enzyme on the activated amino carrier Above, the co-immobilized enzyme is obtained.
  • the mixing ratio of each is different.
  • the main enzyme and the coenzyme are mixed according to the mass ratio of the aforementioned main enzyme and coenzyme of 1-20:1-10 to obtain the first mixed enzyme; and the first mixed enzyme is fixed according to the aforementioned N1/N2 ratio On the activated amino carrier, a co-immobilized enzyme is obtained.
  • the coenzyme includes the first enzyme
  • fixing 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 the initial immobilized enzyme;
  • the first enzyme is immobilized with the initially immobilized enzyme to obtain a co-immobilized enzyme.
  • the above-mentioned method of fixing together with the main enzyme can be used, or the above-mentioned method of stepwise fixing can be used.
  • a stepwise immobilization method which can retain the activity of the coenzyme to a greater extent, thereby increasing the cycle of the co-immobilized enzyme usage efficiency.
  • the coenzyme further includes a second enzyme.
  • Immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: immobilizing the main enzyme and the second enzyme on the activated amino carrier together to obtain Initially immobilized enzyme; the first enzyme and the initial immobilized enzyme are immobilized to obtain co-immobilized enzyme.
  • the above part can be mixed with the main enzyme and immobilized on the activated amino carrier.
  • the highly sensitive coenzyme is immobilized. On the carrier, this can increase the activity of the sensitive enzyme, thereby improving the overall activity and recycling efficiency of the prepared co-immobilized enzyme.
  • the first enzyme is an enzyme that is relatively 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 separately immobilized.
  • the second immobilization is carried out for the purpose of improving the activity of the coenzyme to be immobilized. Therefore, any improvement in the activity of this type of coenzyme can be achieved, and the activity of the coenzyme can be improved.
  • the fixed methods are applicable to this application.
  • PEI is used to immobilize the first enzyme and the initially immobilized enzyme to obtain a co-immobilized enzyme.
  • PEI is polyethyleneimine, which can be used as a non-solid form of support, which adsorbs the coenzyme to be immobilized on the carrier through ion adsorption.
  • the specific amount of PEI added can be adjusted reasonably according to actual needs. When the final concentration of the addition of PEI reaches 0.5% to 5%, the immobilization of all the aforementioned types of coenzymes can be achieved.
  • the coenzyme 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 fixation method or a stepwise fixation method.
  • the one-step fixation method is to mix two cofactors in different combinations with transaminase to obtain the first enzyme mixture or the second enzyme mixture respectively, and then fix the first enzyme mixture or the second enzyme mixture on the activated amino carrier , To obtain a co-immobilized enzyme.
  • Step-by-step immobilization method mix the transaminase and LDH to obtain the third enzyme mixture; fix the third enzyme mixture on the activated amino carrier to obtain the initially immobilized transaminase; by coating the initially immobilized transaminase with PEI surface, Furthermore, GDH or FDH is immobilized to obtain a co-immobilized enzyme.
  • the preparation method includes any one of the following: mixing amino acid dehydrogenase with FDH or GDH to obtain an amino acid dehydrogenase mixture; fixing the amino acid dehydrogenase mixture On the activated amino carrier, the co-immobilized enzyme is obtained; or, the amino acid dehydrogenase is immobilized on the activated amino carrier to obtain the initially immobilized amino acid dehydrogenase; by PEI surface coating of the initially immobilized amino acid dehydrogenase, Furthermore, GDH or FDH is immobilized to obtain a co-immobilized enzyme.
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following: mixing imine reductase, FDH or GDH to obtain an iminase mixture; fixing the iminase mixture to Activate the amino carrier to obtain a co-immobilized enzyme; or immobilize imine reductase on the activated amino carrier to obtain the initially immobilized imine reductase; by coating the initially immobilized imine reductase with PEI, then Immobilize GDH or FDH to obtain a co-immobilized enzyme.
  • the coenzyme is FDH or GDH
  • the preparation method includes any one of the following: mixing ketoreductase, FDH or GDH to obtain a ketoreductase mixture; fixing the ketoreductase mixture to an activated amino group On the carrier, the co-immobilized enzyme is obtained; or the ketoreductase is immobilized on the activated amino carrier to obtain the initial immobilized ketoreductase; the initial immobilized ketoreductase is coated with PEI, and then GDH or FDH is immobilized , To obtain a co-immobilized enzyme.
  • the preparation method includes any one of the following: mixing ene reductase, FDH or GDH to obtain an ene reductase mixture; fixing the ene reductase mixture to an activated amino group On the carrier, the co-immobilized enzyme is obtained; or, the ene reductase is immobilized on the activated amino carrier to obtain the pre-immobilized ene reductase; the pre-immobilized ene reductase is coated with PEI surface, and then GDH or FDH Immobilized to obtain a co-immobilized enzyme.
  • the preparation method includes any one of the following: mixing cyclohexanone monooxygenase, FDH or GDH to obtain a monooxygenase mixture;
  • the monooxygenase mixture is immobilized on the activated amino carrier to obtain the co-immobilized enzyme; or, the cyclohexanone monooxygenase is immobilized on the activated amino carrier to obtain the initially immobilized cyclohexanone monooxygenase;
  • the method of immobilizing cyclohexanone monooxygenase to coat the surface of PEI, and then immobilizing GDH or FDH to obtain a co-immobilized enzyme.
  • the method of covering the surface of PEI is the same as the aforementioned "adding PEI to the initial immobilized enzyme until the final concentration of PEI is 0.5w/v% ⁇ 5w/v %, the PEI-pre-immobilized enzyme complex is obtained; then the first enzyme is combined with the PEI-pre-immobilized enzyme complex to obtain the co-immobilized enzyme", the operation is the same, and will not be repeated here.
  • an existing activator can be used for activation.
  • glutaraldehyde is used to activate the amino resin carrier to obtain an activated amino carrier.
  • Glutaraldehyde has a wide range of applications and is the most common.
  • the ratio of the total mass of the co-immobilized enzyme to the total mass of the activated amino carrier is also different according to the co-immobilization method and the type and quantity of the co-immobilized coenzyme.
  • the mass ratio of the main enzyme to the coenzyme is also different, and when there are two kinds of coenzymes, the mass ratio of the main enzyme to the first coenzyme and the second coenzyme is also different, which can be adjusted reasonably according to actual needs.
  • the first mixed enzyme is immobilized on the activated amino carrier according to a mass ratio of 50-150 mg: 1 g to obtain a co-immobilized enzyme.
  • the mass ratio of the main enzyme to the first enzyme is 1-20:1-10.
  • the mass ratio of the main enzyme to the second enzyme is 1-20:1-10.
  • the mass ratio of transaminase to its coenzyme lactate dehydrogenase is 5-7:1; the mass ratio of transaminase to its coenzyme ammonium formate dehydrogenase is 5-7:1 to 3; transaminase
  • the mass ratio of amino acid dehydrogenase to coenzyme glucose dehydrogenase is 5 ⁇ 7:1 ⁇ 2; the mass ratio of amino acid dehydrogenase to coenzyme ammonium formate dehydrogenase is 8 ⁇ 10:1; the mass ratio of amino acid dehydrogenase to coenzyme glucose dehydrogenase is 8 ⁇ 10:1.
  • the mass ratio is 4-6:1; the mass ratio of imine reductase and its coenzyme ammonium formate dehydrogenase is 4-6; 1; the mass ratio of suban reductase and its coenzyme glucose dehydrogenase is 5-6:1 ;
  • the mass ratio of ketoreductase to its coenzyme ammonium formate dehydrogenase is 4-6:1; the mass ratio of ketoreductase to its coenzyme glucose dehydrogenase is 1:5 to 10; ene reductase and its coenzyme ammonium formate dehydrogenase
  • the mass ratio of ene reductase to its coenzyme glucose dehydrogenase is 18-20:1; and the mass ratio of monooxygenase to coenzyme alcohol dehydrogenase is 2 ⁇ 3:2 ⁇ 3; The mass ratio of monooxygenase to its coenzyme glucose dehydrogenase is 2
  • Controlling the mass ratios of the above-mentioned main enzymes and their corresponding different coenzymes within the above-mentioned range enables the main coenzymes to be arranged in stoichiometric ratios as much as possible, thereby improving the catalytic activity and efficiency of the co-immobilized enzymes.
  • the application of any one of the above-mentioned co-immobilized enzymes or the preparation method of any one of the above-mentioned co-immobilized enzymes in a biocatalytic reaction is also provided .
  • the biocatalytic reaction is a continuous biocatalytic reaction.
  • PB in the following examples stands for phosphate buffer.
  • a more stable connection can be achieved by reducing the imine double bond with borohydride.
  • 1g of immobilized enzyme was resuspended in 4mL buffer (50mM NaHCO 3 -Na 2 CO 3 , pH 8.0 ⁇ 10.0), and NaBH 4 was added at 5-15°C to make the final concentration of NaBH 4 1mg/mL. After stirring for 1-2 hours at 5-15°C, filter and wash 3 times with 0.1M phosphate buffer (pH 7.5).
  • the co-immobilization activity test of TA and LDH is tested by using free FDH to achieve the regeneration of NADH. Use the following substrate 1 for testing:
  • CHMO The activity of CHMO, ADH, GDH co-immobilized enzyme is detected by reacting with the following substrate 2:
  • CHMO and GDH co-immobilized enzyme The activity of CHMO and GDH co-immobilized enzyme is tested by the following reaction:
  • CHMO and ADH co-immobilized enzyme The activity of CHMO and ADH co-immobilized enzyme is tested by the following reaction:
  • AADH and FDH co-immobilized enzymes The activity of AADH and FDH co-immobilized enzymes is detected by the reaction of the following substrates:
  • the activity detection method of AADH and FDH co-immobilized enzyme is as follows:
  • the activity of the co-immobilized enzyme of KRED and FDH is detected by the reaction of the following substrate 5 or 6:
  • KRED and GDH co-immobilized enzyme The activity of KRED and GDH co-immobilized enzyme is tested by the following reaction:
  • ERED and GDH co-immobilized enzyme The activity of ERED and GDH co-immobilized enzyme is tested by the following reaction:
  • the activity of the co-immobilized enzyme of IRED and FDH uses the following substrate 8 and is tested according to the following method:
  • the transaminase TA-Bt and the coenzymes LDH and FDH were co-immobilized to the carrier LX1000HA, and 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 was 5.9 g.
  • co-immobilized enzyme as in Example 9, 50 g of the co-immobilized enzyme of transaminase TA-Bt and coenzymes LDH and FDH was added to a 200 mL reactor, and 150 mL of phosphate buffer was added.
  • 500g substrate 5, 108mg ammonium chloride dissolve with 4.5L of PB buffer (0.1M, pH8.0), adjust the pH to pH 7.5-8.0 with sodium hydroxide solution, then add 10-50mg NAD + , 80mg ammonium formate , And finally dilute to 5L with PB buffer.
  • PB buffer 0.1M, pH8.0
  • the substrate was continuously added to the continuous stirred tank at a rate of 0.8 mL/min (ie retention time 250 min), and the reaction system was drawn out at the outlet at the same flow rate (a filter was added to the end of the pipeline to prevent the immobilized enzyme from being drawn out). Under these conditions, the conversion rate can reach more than 92%, and continuous operation for 400 hours, the conversion rate is basically not reduced.
  • Table 14 The results are shown in Table 14.
  • Example 1 As in Example 1, after activating the amino carrier, the amount of protein added per gram of carrier was investigated. Taking the co-immobilization of transaminase TA-Bt and its coenzyme lactate dehydrogenase LDH as an example, different amounts of protein were added to detect protein load Quantity and reaction times of repeated use. The results are shown in Table 15.
  • Example 4 taking the co-immobilization of monooxygenase CHMO-Rs and its coenzymes ADH and GDH as an example, different amounts of protein were added to detect the protein load and the number of times of repeated use of the reaction. The results are shown in Table 16. The results show that the range of protein that can be loaded per gram of the selected carrier is 50-200 mg, and the protein loading rate is 50%-100%.
  • Method 2 (two-step method), prepare the co-immobilized enzyme of transaminase TA-Bt and its coenzymes lactate dehydrogenase LDH and ammonium formate dehydrogenase FDH, after the mixed enzyme of TA-Bt and LDH is combined with the carrier , Add different amounts of PEI, and then add the second coenzyme FDH to investigate the concentration range of PEI.
  • Table 17 The results are shown in Table 17.
  • the present application realizes the co-immobilization of the above-mentioned main enzymes and coenzymes on the amino resin carrier. Immobilization is beneficial to improve enzyme activity and recycling efficiency.

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Abstract

本发明提供了一种共固定化酶、其制备方法及其应用。该共固定化酶包括：氨基树脂载体以及主酶和辅酶，主酶和辅酶共固定于氨基树脂载体上，其中，主酶共价固定于氨基树脂载体上，辅酶通过共价和/或非共价方式固定于氨基树脂载体上；主酶选自如下任意一种酶：转氨酶、氨基酸脱氢酶、亚胺还原酶、酮还原酶、烯还原酶及单加氧酶。将主酶及其辅酶共固定于氨基树脂载体上共固定化，提高酶的活性及循环使用效率。

Description

共固定化酶、其制备方法及其应用 技术领域
本发明涉及固定化酶领域，具体而言，涉及一种共固定化酶、其制备方法及其应用。
背景技术
微生物细胞或分离的酶或工程酶的应用使得生物催化取得了重大进展，且使得制造方式发生了转变。许多种类的酶如酰基转移酶、酰胺酶、转氨酶、酮还原酶、氧化酶、单加氧酶及水解酶等被用于生产涉及抗生素、除草剂、医药中间体和新时代治疗剂的反应中。
当游离酶用作生物催化剂时，会有很大的酶浪费，因为回收水溶性酶非常困难。而水不溶性固定化酶，在每个循环后，通过非常简单的过滤就可以容易地回收。
现有技术中已经有报道单一酶的固定化方法，但不同的酶所适合的固定化方法是不同的。比如Bolivar等(Biomacromol.2006,7,669-673)研究了来自假单胞菌SP101的FDH的共价固定化，包括共价固定于改性琼脂糖、CNBr活化的琼脂糖、Sepabeads(葡聚糖)及乙醛琼脂糖的各种载体上。其得出的结论是：固定化于溴化物，聚乙烯亚胺，戊二醛等活化的载体上并不会促进酶在热灭活下的任何稳定作用。然而，高度活化的乙二醛琼脂糖的优化的酶被证明是具有很高的热稳定性、pH稳定性并且在具有增强的稳定性情况下具有超过50％的活性。
Kim et al(J.Mol.Catal B:Enzy 97(2013)209–214)报道了使用交联酶聚集体(CLEA)的方法来固定来自Candida boidinii.的甲酸脱氢酶(formate dehydrogenase，FDH)，并认为葡聚糖多醛(dextran polyaldehyde)作为交联剂代替戊二醛(glutaraldehyde)对于固定化酶更好，经过10次重复使用后残留活性超过95％。此外，葡聚糖多醛形成的交联酶聚集体(Dex-CLEA)的热稳定性比游离酶的热稳定性提高了3.6倍。
Binay et al(Beilstein J.Org.Chem.2016,12,271–277)报道了高活性的来源于Candida methylica的FDH的固定化酶。FDH共价固定到环氧活化Immobead 150载体上，Immobead 150载体先经过乙二胺(ethylenediamin)修饰，然后依次经过戊二醛活化(FDHIGLU)及醛基官能化(FDHIALD)。当使用醛基官能化的Immobead 150作为载体时，分别获得最高的固定化产率和活性产率，分别为90％和132％。在35℃下，游离FDH，FDHI150，FDHIGLU和FDHIALD的半衰期(t1/2)分别计算为10.6、28.9、22.4和38.5小时。FDHI150，FDHIGLU和FDHIALD在10次重复使用后分别保留了其初始活动的69％，38％和51％。
Jackon等(Process Biochem.Vol.1,9,Sep 2016,1248-1255)报道了采用乙二醛-琼脂糖对LDH的固定化。与其可溶性对应物相比，固定化LDH获得的热稳定因子大1600倍。
也有些报道了两种酶的共固定化，比如Valikhani等(Biotech.Bioengg.2018；115:2416–2425)报道了细胞色素P450单加氧酶(色素P450s)与来源于B.megaterium的葡萄糖脱氢酶的共 固定化。Delgrove等人(Appl.Catal.A:Gen.Vol.572,25Feb.2019,134-141)发表了共固定化的Baeyer-Villiger单加氧酶(BVMO)和葡萄糖脱氢酶用于ε-己内酯衍生物的合成。将来自Thermocrispum municipale的热稳定环己酮单加氧酶(TmCHMO)与来自Thermoplasma acidophilum的葡萄糖脱氢酶(GDH)(GDH-Tac)共固定化到氨基官能化琼脂糖基载体(MANA-琼脂糖)上。共固定化被证明是最有效的生物催化剂，在3,3,5-三甲基环己酮的合成中，在15个重复循环中平均转化率为83％。
综上可知，现有技术中仍存在大量的酶还没有有效的固定化酶形式，尤其是某些共同参与同一生物催化反应的酶，这些酶的共固定化以提高酶的可循环利用性便成了亟待解决的问题。
发明内容
本发明的主要目的在于提供一种共固定化酶、其制备方法及其应用，以解决现有技术此类酶难以循环利用的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种共固定化酶，该共固定化酶包括：氨基树脂载体以及主酶和辅酶，辅酶为一种或多种，主酶和辅酶共固定于氨基树脂载体上，其中，主酶共价固定于氨基树脂载体上，辅酶通过共价和/或非共价方式固定于氨基树脂载体上；主酶选自如下任意一种酶：转氨酶、氨基酸脱氢酶、亚胺还原酶、酮还原酶、烯还原酶及单加氧酶。
进一步地，转氨酶为来源于B.thuringiensis或Vibrio fluvialis strain JS17的转氨酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus或Bacillus sphaericus的氨基酸脱氢酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，酮还原酶为来源于Sporobolomyces salmonicolor的酮还原酶或者Acetobacter sp.CCTCC M209061的酮还原酶，更优选地，来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEO ID NO:1或SEO ID NO:2序列的突变体；优选地，烯还原酶为来源于Chryseobacterium sp.CA49或Sewanella oneidensis MR-1的烯还原酶；优选地，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的环己酮单加氧酶；更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEO ID NO：4序列或SEO ID NO：5序列的突变体；来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEO ID NO：7序列或SEO ID NO:8序列的突变体。
进一步地，辅酶选自如下至少一种：乳酸脱氢酶(LDH)、甲酸铵脱氢酶(FDH)、葡萄糖脱氢酶(GDH)及醇脱氢酶；优选地，乳酸脱氢酶为来源于Lactobacillus helveticus的D-乳酸脱氢酶；优选地，甲酸铵脱氢酶为来源于Candida boidinii的甲酸脱氢酶；优选地，葡萄糖脱氢酶为来源于Lysinibacillus sphaericus G10的葡萄糖1-脱氢酶；优选地，醇脱氢酶为来源于Thermoanaerobium brockii的醇脱氢酶；优选地，共固定化酶的循环利用次数为4～25次。
进一步地，氨基树脂载体为戊二醛活化的氨基树脂载体；优选地，氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，更优选地，氨基树脂载体选自如下任意一种：
Figure PCTCN2019122447-appb-000001
LX1000EA、LX1000HA、LX1000NH、HFA、LX1000EPN、HM100D、
Figure PCTCN2019122447-appb-000002
Lifetech TM ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、
Figure PCTCN2019122447-appb-000003
ESR-1、ESR-3、ESR-5及ESR-8。
进一步地，共固定化酶中，主酶与辅酶的质量比为1～20：1～10；优选地，主酶和辅酶的质量和记为N1，氨基树脂载体的质量记为N2，N1/N2为50～200mg:1g，进一步地，为80～120mg：1g。
进一步地，主酶和辅酶均共价固定于氨基树脂载体上；或者主酶共价固定于氨基树脂载体上，辅酶以离子吸附的方式非共价固定于氨基树脂载体上；优选通过PEI将辅酶吸附于氨基树脂载体上。
进一步地，辅酶包括第一酶和第二酶，主酶与第二酶共价固定于氨基树脂载体上，第一酶以离子吸附的方式固定于氨基树脂载体上。
在本申请的第二个方面，提供了一种上述任一种共固定化酶的制备方法，该制备方法包括：将氨基树脂载体进行活化，得到活化氨基载体；将主酶共价固定于活化氨基载体上，并将主酶对应的辅酶通过共价和/或非共价的方式固定于活化氨基载体上，得到共固定化酶；其中，主酶对应的辅酶的数量为一个或多个。
进一步地，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶与辅酶混合，得到第一混合酶；将第一混合酶固定于活化氨基载体上，得到共固定化酶。
进一步地，辅酶包括第一酶，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶固定于活化氨基载体上，得到初固定化酶；将第一酶与初固定化酶进行固定，得到共固定化酶。
进一步地，辅酶还包括第二酶，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶和第二酶同固定于活化氨基载体上，得到初固定化酶；将第一酶与初固定化酶进行固定，得到共固定化酶。
进一步地，通过表面包覆PEI的方式，将第一酶与初固定化酶进行固定，得到共固定化酶；优选地，向初固定化酶中添加PEI至PEI的终浓度为0.5w/v％～5w/v％，得到PEI-初固定化酶；然后将第一酶于PEI-初固定化酶结合，得到共固定化酶。
进一步地，按照质量比为50～150mg:1g的质量比将第一混合酶固定于活化氨基载体上，得到共固定化酶。
进一步地，主酶与第一酶的质量比为1～20：1～10。
进一步地，主酶与第二酶的质量比为1～20：1～10。
进一步地，主酶为转氨酶，辅酶具有两种辅因子，两种辅因子为LDH(乳酸脱氢酶)与FDH(甲酸铵脱氢酶)，或者LDH(乳酸脱氢酶)与GDH(葡萄糖脱氢酶)，上述制备方法包括如下任意一种：
(1)将转氨酶、LDH与FDH进行混合，得到第一酶混物；将第一酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将转氨酶、LDH与GDH进行混合，得到第二酶混物；将第二酶混物固定到活化氨基载体上，得到共固定化酶；
(3)将转氨酶与LDH进行混合，得到第三酶混物；将第三酶混物固定到活化氨基载体上，得到初固定转氨酶；通过对初固定转氨酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，主酶为氨基酸脱氢酶，辅酶为FDH或GDH，制备方法包括如下任意一种：
(1)将氨基酸脱氢酶与FDH或GDH混合，得到氨基酸脱氢酶混物；将氨基酸脱氢酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将氨基酸脱氢酶固定到活化氨基载体上，得到初固定氨基酸脱氢酶；
通过对初固定氨基酸脱氢酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，主酶为亚胺还原酶，辅酶为FDH或GDH，制备方法包括如下任意一种：
(1)将亚胺还原酶、FDH或GDH进行混合，得到亚胺酶混物；将亚胺酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将亚胺还原酶固定到活化氨基载体上，得到初固定亚胺还原酶；通过对初固定亚胺还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，主酶为酮还原酶，辅酶为FDH或GDH，制备方法包括如下任意一种：
(1)将酮还原酶、FDH或GDH进行混合，得到酮还原酶混物；将酮还原酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将酮还原酶固定到活化氨基载体上，得到初固定酮还原酶；通过对初固定酮还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，主酶为烯还原酶，辅酶为FDH或GDH，制备方法包括如下任意一种：
(1)将烯还原酶、FDH或GDH进行混合，得到烯还原酶混物；将烯还原酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将烯还原酶固定到活化氨基载体上，得到初固定烯还原酶；通过对初固定烯还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，主酶为环己酮单加氧酶，辅酶为FDH或GDH，制备方法包括如下任意一种：
(1)将环己酮单加氧酶、FDH或GDH进行混合，得到单加氧酶混物；将单加氧酶混物固定到活化氨基载体上，得到共固定化酶；
(2)将环己酮单加氧酶固定到活化氨基载体上，得到初固定环己酮单加氧酶；通过对初固定环己酮单加氧酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
进一步地，采用戊二醛对氨基树脂载体进行活化，得到活化氨基载体。
根据本申请的第三个方面，提供了上述任一种共固定化酶，或者上述任一种共固定化酶的制备方法所制备的共固定化酶在生物催化反应中的应用。
进一步地，生物催化反应为间歇性的生物催化反应或连续化的生物催化反应；优选地，共固定化酶应用于连续的流化床中或固定床的生物催化反应中；优选地，共固定化酶在连续化的生物催化反应中的循环利用次数为4～25次。
应用本发明的技术方案，本申请通过将上述主酶及其辅酶共固定于氨基树脂载体上，实现了这些主酶及其辅酶的共固定化，从而有利于提高酶的活性及循环使用效率。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
Polyethylene imine，简称PEI，聚乙烯亚胺。
在本申请一种典型的实施方式中，提供了一种共固定化酶，该共固定化酶包括：氨基树脂载体以及主酶和辅酶，主酶和辅酶共固定于氨基树脂载体上，其中，主酶共价固定于氨基树脂载体上，辅酶通过共价和/或非共价方式固定于氨基树脂载体上；主酶选自如下任意一种酶：转氨酶、氨基酸脱氢酶、亚胺还原酶、酮还原酶、烯还原酶及单加氧酶。
本申请通过将上述主酶及其辅酶共固定于氨基树脂载体上，实现了这些主酶及其辅酶的共固定化，从而有利于提高酶的活性及循环使用效率。
上述各种主酶根据来源的不同，具体的种类和活性也有所不同。在一次优选的实施例中，转氨酶为来源于B.thuringiensis或Vibrio fluvialis strain JS17的转氨酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus或Bacillus sphaericus的氨基酸脱氢酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，酮还原酶为来源于Sporobolomyces  salmonicolor的酮还原酶或者Acetobacter sp.CCTCC M209061的酮还原酶，更优选地，来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEO ID NO:1或SEO ID NO:2序列的突变体；优选地，烯还原酶为来源于Chryseobacterium sp.CA49或Sewanella oneidensis MR-1的烯还原酶；优选地，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的环己酮单加氧酶；更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEO ID NO：4序列或SEO ID NO：5序列的突变体；来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEO ID NO：7序列或SEO ID NO:8序列的突变体。
根据上述主酶种类的不同，选择各自对应的辅酶，优选这些辅酶是能进行NAD(P)+和NADPH循环再生的辅酶。在一次优选的实施例中，辅酶选自如下至少一种：辅酶选自如下至少一种：乳酸脱氢酶、甲酸铵脱氢酶、葡萄糖脱氢酶及醇脱氢酶；优选地，乳酸脱氢酶为来源于Lactobacillus helveticus的D-乳酸脱氢酶；优选地，甲酸铵脱氢酶为来源于Candida boidinii的甲酸脱氢酶；优选地，葡萄糖脱氢酶为来源于Lysinibacillus sphaericus G10的葡萄糖1-脱氢酶；优选地，醇脱氢酶为来源于Thermoanaerobium brockii的醇脱氢酶。在另一优选的实施例中，共固定化酶的循环利用次数为4～25次。
上述优选实施例中，转氨酶对应的辅酶是乳酸脱氢酶和甲酸铵脱氢酶，或者是乳酸脱氢酶和葡萄糖脱氢酶。氨基酸脱氢酶对应的辅酶是甲酸铵脱氢酶或葡萄糖脱氢酶。亚胺还原酶对应的辅酶是甲酸铵脱氢酶或葡萄糖脱氢酶。酮还原酶对应的辅酶是甲酸铵脱氢酶或葡萄糖脱氢酶。烯还原酶对应的辅酶是甲酸铵脱氢酶或葡萄糖脱氢酶。单加氧酶对应的辅酶是醇脱氢酶或甲酸铵脱氢酶或葡萄糖脱氢酶。
在一次优选的实施例中，乳酸脱氢酶为来源于Lactobacillus helveticus的D-乳酸脱氢酶(简称LDH)；优选地，甲酸铵脱氢酶为来源于Candida boidinii的甲酸脱氢酶(简称FDH)；优选地，葡萄糖脱氢酶为来源于Lysinibacillus sphaericus G10的葡萄糖1-脱氢酶(简称GDH)；优选地，醇脱氢酶为来源于Thermoanaerobium brockii的醇脱氢酶。
现有技术中都没有对上述优选实施例中各来源的主酶及辅酶的共固定化酶，将这些主酶和辅酶共固定化，有助于提高这些酶的循环利用效率。
上述共固定化酶的载体为氨基树脂载体，可以采用现有的各种氨基树脂载体。在一次优选的实施例中，氨基树脂载体为戊二醛活化的氨基树脂载体；优选地，氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，更优选地，氨基树脂载体选自如下任意一种：
Figure PCTCN2019122447-appb-000004
LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HFA、HM100D、
Figure PCTCN2019122447-appb-000005
Lifetech TM ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、
Figure PCTCN2019122447-appb-000006
ESR-1、ESR-3、ESR-5及ESR-8。上述根据所固定的主酶和辅酶的不同，可以选择不同的氨基树脂载体。
上述共固定化酶中，主酶和辅酶的用量比根据所催化底物的不同而有所不同。在一次优选的实施例中，上述共固定化酶中，主酶与辅酶的质量比为1～20：1～10。优选地，主酶和辅 酶的质量和记为N1，氨基树脂载体的质量记为N2，N1/N2为50～200mg:1g，进一步地，为80～120mg：1g。
将主酶与辅酶的质量比控制在上述范围内，使得上述所有主酶与辅酶的共固定化酶能够催化绝大部分的底物。且酶与载体的质量比控制在50～200mg:1g的范围内，能够实现对上述所有主酶与辅酶的共固定化。
在一些更优选的实施例中，转氨酶与其辅酶乳酸脱氢酶的质量比为5～7：1；转氨酶与其辅酶甲酸铵脱氢酶的质量比为5～7：1～3；转氨酶与其辅酶葡萄糖脱氢酶的质量比为5～7：1～2；氨基酸脱氢酶与其辅酶甲酸铵脱氢酶的质量比为8～10：1；氨基酸脱氢酶与其辅酶葡萄糖脱氢酶的质量比为5～6：1；亚胺还原酶与其辅酶甲酸铵脱氢酶的质量比为4～8；1；亚安还原酶与其辅酶葡萄糖脱氢酶的质量比为6～8：1；酮还原酶与其辅酶甲酸铵脱氢酶的质量比为4～8：1；同还原酶与其辅酶葡萄糖脱氢酶的质量比为1：3～10；烯还原酶与其辅酶甲酸铵脱氢酶的质量比为6～10：1；烯还原酶与其辅酶葡萄糖脱氢酶的质量比为12～20：1；及单加氧酶与其辅酶醇脱氢酶的质量比为甲酸铵脱氢酶的质量比为2～3：2～3；单加氧酶与其辅酶葡萄糖脱氢酶的质量比为2～3：1。将上述各主酶与其对应的不同辅酶的质量比控制在上述范围内，能够使得主辅酶的尽可能按照化学计量比进行配置，从而提高共固定化酶的催化活性及效率。
上述共固定化酶中，主酶与辅酶都固定在载体上，对于两者在载体上的具体固定方式不限。在一次优选的实施例中，主酶和辅酶均共价固定于氨基树脂载体上。在另一优选的实施例中，主酶共价固定于氨基树脂载体上，辅酶以离子吸附的方式非共价固定于氨基树脂载体上；优选通过PEI将辅酶吸附于氨基树脂载体上。上述两种不同的共固定化方式可以采用不同的共固定化方法得到。
上述共固定化酶中，有些主酶只有一种辅酶，有些主酶有两种辅酶。两者的共固定化方式同上，可以相同，也可以有所不同，根据具体的辅酶的性质特点而定。在一次优选的实施例中，辅酶包括第一酶和第二酶，主酶与第二酶共价固定于氨基树脂载体上，第一酶以离子吸附的方式固定于氨基树脂载体上。此处第一酶是指对共固定化过程中的某些试剂，比如交联剂戊二醛等比较敏感的酶，为了减少第一酶在共固定化过程中活性降低，从而影响共固定化酶的活性及后续的循环利用效率，上述优选实施例中通过离子吸附的方式将第一酶非共价固定于氨基树脂载体上。
转氨酶TA-Bt(具体见表1)与LDH及FDH在最优比例下共固定化至载体LX1000HA，最高使用次数可达15次；转氨酶TA-Bt与LDH及GDH在最优比例下共固定化至载体LX1000HA，最高使用次数可达20～25次；单加氧酶CHMO-Rs与GDH在最优比例下共固定化至载体LX1000HA，最高使用次数达13次，单加氧酶CHMO-Rs与ADH在最优比例下共固定化至载体LX1000HA，最高使用次数达13～15次，共固定化至ECR8409载体，最高使用次数达16次；CHMO-Rs的突变体V1与ADH共固定化至载体LX1000HA，最高使用次数达17次，突变体V2与ADH共固定化至载体LX1000HA，最高使用次数达17次；CHMO-Bp与ADH或GDH共固定化至LX1000HA载体，使用次数达14-15次；CHMO-Bp突变体V1 与ADH共固定化至LX1000HA载体，使用次数达19次；CHMO-Bp突变体V1与ADH共固定化至LX1000HA载体，使用次数达21次；AADH-Bc与FDH最优比例下共固定化至载体LX1000HA，最高使用次数达12次；AADH-Bs与GDH最优比例下共固定化至载体LX1000HA，最高使用次数达15次；KRED-Ac与FDH共固定化至载体ECR8409，最高使用次数达18次；KRED-Ac-V1与FDH共固定化至载体ECR8409，最高使用次数达24次；KRED-Ac与GDH共固定化至载体ECR8409，最高使用次数达14次；ERED-Sc与FDH最优比例下固定化至载体LX1000HA，最高使用次数达17次；ERED-Sc与GDH最优比例下固定化至载体LX1000HA，最高使用次数达21次；ERED-Chr最优比例下固定化至载体LX1000EPN，最高使用次数达21次；IRED-Str与FDH最优比例下固定化至载体LX1000EPN，最高使用次数达17次；IRED-Bc与GDH最优比例下固定化至载体LX1000EPN，最高使用次数达19次。
在本申请第二种典型的实施方式中，提供了上述任一种共固定化酶的制备方法，该制备方法包括：将氨基树脂载体进行活化，得到活化氨基载体；将主酶共价固定于活化氨基载体上，并将主酶对应的辅酶通过共价和/或非共价的方式固定于活化氨基载体上，得到共固定化酶。
上述共固定化酶的方法，通过先对氨基树脂载体进行活化，再将上述任一种主酶及其辅酶共固定于活化氨基载体上即可得共固定化酶。
对上述主酶与辅酶共固定于活化的氨基载体上的具体方法，可以根据辅酶的种类及性质不同而选择合适的步骤进行。
在一次优选的实施例中，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶与辅酶混合，得到第一混合酶；将第一混合酶固定于活化氨基载体上，得到共固定化酶。
该优选方法中，不论辅酶有几种，都可以通过与主酶一起混合后，固定至载体上。根据不同的主酶及辅酶，各自混合的比例有所不同。优选地，按照前述主酶与辅酶的质量比1～20：1～10的比例，将主酶与辅酶混合，得到第一混合酶；并按照前述N1/N2的比例，将第一混合酶固定于活化氨基载体上，得到共固定化酶。
在一次优选的实施例中，辅酶包括第一酶，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶固定于活化氨基载体上，得到初固定化酶；将第一酶与初固定化酶进行固定，得到共固定化酶。
当辅酶只有一种的时候，可以采用上述与主酶混合后一起固定的方法，也可以采用上述分步固定的方法。对于某些对活化氨基载体步骤中的戊二醛敏感的辅酶的共固定化来说，优选采用分步固定的方法，这样能够更大程度上保留辅酶的活性，从而提高共固定化酶的循环利用效率。
在一次优选的实施例中，辅酶还包括第二酶，将主酶及辅酶固定于活化氨基载体上，得到共固定化酶包括：将主酶和第二酶同固定于活化氨基载体上，得到初固定化酶；将第一酶与初固定化酶进行固定，得到共固定化酶。
当辅酶不仅只有一种时，根据辅酶与戊二醛等活化剂的敏感性程度，可以采用上述部分与主酶一起混合后固定于活化氨基载体上，第二步再将敏感性高的辅酶固定到载体上，这样能够提高敏感性酶的活性，从而提高所制备的共固定化酶的整体活性及循环回收效率。上述优选实施例中，第一酶为对戊二醛比较敏感的酶，第二酶的敏感性相对低，因而将第二酶与主酶混合固定，再将第一酶进行单独固定。
在上述分步固定化的步骤中，第二次固定化均是出于提高所欲固定的辅酶的活性考虑而进行的，因次，任何能够实现提高该类辅酶的活性，且能实现对其固定的方式均适用于本申请。在一次优选的实施例中，利用PEI将第一酶与初固定化酶进行固定，得到共固定化酶。在另一些优选的实施例中，向初固定化酶中添加PEI至PEI的终浓度(质量体积浓度)为0.5％～5％，得到PEI-初固定化酶；向PEI-初固定化酶中添加第一酶进行孵育，得到共固定化酶。
PEI为聚乙烯亚胺，可以作为一种非固体形式的支撑物，其通过离子吸附的方式将待固定的辅酶吸附于载体上，具体所添加的PEI的量可以根据实际需要进行合理调整。将PEI的添加的终浓度达到0.5％～5％时，能够实现上述所有种类的辅酶的固定化。
当主酶为转氨酶时，辅酶具有两种辅因子LDH与FDH，或者为LDH与GDH，其制备方法包括一步固定法或分步固定法。一步固定法是通过将不同组合的两种辅因子与转氨酶混合，分别得到第一酶混物或第二酶混物，然后将第一酶混物或第二酶混物固定到活化氨基载体上，得到共固定化酶。分步固定法：将转氨酶与LDH进行混合，得到第三酶混物；将第三酶混物固定到活化氨基载体上，得到初固定转氨酶；通过对初固定转氨酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
当主酶为氨基酸脱氢酶时，辅酶为FDH或GDH，制备方法包括如下任意一种：将氨基酸脱氢酶与FDH或GDH混合，得到氨基酸脱氢酶混物；将氨基酸脱氢酶混物固定到活化氨基载体上，得到共固定化酶；或者，将氨基酸脱氢酶固定到活化氨基载体上，得到初固定氨基酸脱氢酶；通过对初固定氨基酸脱氢酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
当主酶为亚胺还原酶时，辅酶为FDH或GDH，制备方法包括如下任意一种：将亚胺还原酶、FDH或GDH进行混合，得到亚胺酶混物；将亚胺酶混物固定到活化氨基载体上，得到共固定化酶；或者，将亚胺还原酶固定到活化氨基载体上，得到初固定亚胺还原酶；通过对初固定亚胺还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
当主酶为酮还原酶时，辅酶为FDH或GDH，制备方法包括如下任意一种：将酮还原酶、FDH或GDH进行混合，得到酮还原酶混物；将酮还原酶混物固定到活化氨基载体上，得到共固定化酶；或者将酮还原酶固定到活化氨基载体上，得到初固定酮还原酶；通过对初固定酮还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
当主酶为烯还原酶时，辅酶为FDH或GDH，制备方法包括如下任意一种：将烯还原酶、FDH或GDH进行混合，得到烯还原酶混物；将烯还原酶混物固定到活化氨基载体上，得到共固定化酶；或者，将烯还原酶固定到活化氨基载体上，得到初固定烯还原酶；通过对初固定烯还原酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
当主酶为环己酮单加氧酶时，辅酶为FDH或GDH，制备方法包括如下任意一种：将环己酮单加氧酶、FDH或GDH进行混合，得到单加氧酶混物；将单加氧酶混物固定到活化氨基载体上，得到共固定化酶；或者，将环己酮单加氧酶固定到活化氨基载体上，得到初固定环己酮单加氧酶；通过对初固定环己酮单加氧酶进行PEI表面包覆的方式，进而将GDH或FDH进行固定，得到共固定化酶。
上述各种主酶及其辅酶的共固定化的制备方法中，PEI包面包覆的方式与前述向“初固定化酶中添加PEI至PEI的终浓度为0.5w/v％～5w/v％，得到PEI-初固定化酶复合物；然后将第一酶与PEI-初固定化酶复合物结合，得到共固定化酶”的操作相同，此处不再赘述。
上述对氨基载体进行活化的步骤中，可以采用现有的活化剂进行活化。在一次优选的实施例中，采用戊二醛对氨基树脂载体进行活化，得到活化氨基载体。戊二醛的应用范围广且最常见。
上述共固定化的步骤中，根据共固定化方法及共固定化的辅酶的种类及数量的不同，共固定化的酶的总质量与活化氨基载体的总质量之比也有所不同。相应地，主酶与辅酶的质量比也有所不同，且当辅酶有两种时，主酶与第一种辅酶和第二种辅酶的质量比也有所不同，可以根据实际需要进行合理调整。
在一次优选的实施例中，按照质量比为50～150mg:1g的质量比将第一混合酶固定于活化氨基载体上，得到共固定化酶。
在一次优选的实施例中，主酶与第一酶的质量比为1～20：1～10。
在一次优选的实施例中，主酶与第二酶的质量比为1～20：1～10。
优选地，在上述共固定化的步骤中，转氨酶与其辅酶乳酸脱氢酶的质量比为5～7：1；转氨酶与其辅酶甲酸铵脱氢酶的质量比为5～7：1～3；转氨酶与其辅酶葡萄糖脱氢酶的质量比为5～7：1～2；氨基酸脱氢酶与其辅酶甲酸铵脱氢酶的质量比为8～10：1；氨基酸脱氢酶与其辅酶葡萄糖脱氢酶的质量比为4～6：1；亚胺还原酶与其辅酶甲酸铵脱氢酶.的质量比为4～6；1；亚安还原酶与其辅酶葡萄糖脱氢酶的质量比为5～6：1；酮还原酶与其辅酶甲酸铵脱氢酶的质量比为4～6：1；酮还原酶与其辅酶葡萄糖脱氢酶的质量比为1：5～10；烯还原酶与其辅酶甲酸 铵脱氢酶的质量比为6～8：1；烯还原酶与其辅酶葡萄糖脱氢酶的质量比为18～20：1；及单加氧酶与其辅酶醇脱氢酶的质量比为2～3：2～3；单加氧酶与其辅酶葡萄糖脱氢酶的质量比为2～3：1。
将上述各主酶与其对应的不同辅酶的质量比控制在上述范围内，能够使得主辅酶的尽可能按照化学计量比进行配置，从而提高共固定化酶的催化活性及效率。
在本申请第三种典型的实施方式中，还提供了上述任一种共固定化酶，或者上述任一种共固定化酶的制备方法所制备的共固定化酶在生物催化反应中的应用。优选地，生物催化反应为连续化的生物催化反应。
下面将结合具体的实施例来进一步说明本申请的有益效果。
以下实施例中用到的酶及其来源见下表1，表2至表4显示的是部分酶的序列。
表1：
Figure PCTCN2019122447-appb-000007
表2：
Figure PCTCN2019122447-appb-000008
Figure PCTCN2019122447-appb-000009
表3：
Figure PCTCN2019122447-appb-000010
表4：
Figure PCTCN2019122447-appb-000011
Figure PCTCN2019122447-appb-000012
下列实施例中的PB代表磷酸缓冲液的意思。
实施例1
用4～5mL 0.1M磷酸缓冲液(pH 7.5)洗涤1g氨基树脂，用4mL 0.1M磷酸缓冲液(pH7.5)重悬，滴加25％～50％(w/v)戊二醛水溶液，使戊二醛终浓度为2％(w/v)，在20℃温和振荡下孵育1小时，过滤并用0.1M磷酸缓冲液(pH 7.5)洗涤3次。
将含有100～120mg蛋白质的4mL酶溶液(调节适当比例的TA和LDH)加入到戊二醛活化树脂中，在20-25℃温和振荡下孵育后，用0.1M磷酸缓冲液(pH 7.5)过滤并洗涤3次。
通过用硼氢化物还原亚胺双键可以实现更稳定的连接。将1g固定化酶用4mL缓冲液(50mM NaHCO 3-Na  2CO 3，pH 8.0～10.0)重悬，并在5-15℃下加入NaBH 4，使NaBH 4终浓度为1mg/mL。在5-15℃搅拌1-2小时后，过滤并用0.1M磷酸缓冲液(pH 7.5)洗涤3次。
TA和LDH的共固定化活性测试，通过利用游离的FDH来实现NADH的再生进行测试。使用以下底物1进行测试：
Figure PCTCN2019122447-appb-000013
将5mL 0.1M磷酸缓冲液(pH 8.0)加入10mL反应器中，然后加入100mg上述底物1，80mg甲酸铵(FDH)，5mg PLP，调节pH至pH 7.5-8.0，然后加入5mg NAD+和30mg FDH游离酶，100mg共固定酶(湿酶，含50～80％的水)。在30℃下反应16-20小时，通过HPLC检测转化率，结果见下表。
表5：
Figure PCTCN2019122447-appb-000014
Figure PCTCN2019122447-appb-000015
实施例2 TA-Bt、LDH及FDH的共固定化
方法1：一步共固定化
用1-2mL 0.1M PB(PH 7.5)洗涤1g氨基树脂，用4mL 0.1M PB(PH 7.5)重悬，将浓度为25(w/v)％～50(w/v)％的戊二醛水溶液滴加入重悬液中使戊二醛的终浓度为2(w/v)％。在20℃温和振荡下孵育1小时，随后过滤并用0.1M PB(PH 7.5)洗涤3次。将含有100-120mg蛋白质的4mL酶溶液(调节适当比例的TA和LDH和FDH)加入到戊二醛活化树脂中，在20-25℃温和振荡下孵育后，过滤并用0.1M PB(pH 7.5)洗涤3次。
方法2：两步共固定化
为提高FDH的活性和稳定性，1g共固定化的TA和LDH由0.1M的PB(pH为7.0-7.5)重悬，并加入PEI溶液(终浓度2％)，随后加入20mg的FDH，在20-25℃下温和振荡孵育后，过滤并用0.1M PB(pH7.5)洗涤3次。
共固定化的TA，LDH及FDH的活性通过以下反应进行检测：
5mL的0.1M PB(pH 8.0)装入10ml的反应器中，随后通过加入100mg上述底物1、80mg甲酸铵及5mg PLP，调节pH至pH 7.5-8.0，然后加入5mg NAD+和100mg共固定化酶(湿的，含有50～80％的水)。在30℃下反应16-20小时，测试转化率。测试结果见下表。
表6:
Figure PCTCN2019122447-appb-000016
Figure PCTCN2019122447-appb-000017
实施例3 TA-Bt、LDH及GDH的共固定化
方法1和方法2，除了用GDH替代FDH外，其余步骤都与实施例2相同。
共固定化的TA，LDH及GDH的活性通过以下反应进行检测：
5mL的0.1M PB(pH 8.0)装入10ml的反应器中，随后通过加入100mg上述底物1、120mg葡萄糖及5mg PLP，调节pH至pH 7.5-8.0，然后加入5mg NAD+和100mg共固定化酶(湿的，含有50～80％的水)。在30℃下反应16-20小时，测试转化率。测试结果见表7。
表7:
Figure PCTCN2019122447-appb-000018
Figure PCTCN2019122447-appb-000019
实施例4 CHMO与ADH及GDH的共固定化
采用实施例2中的方法1和方法2，除了将主酶和辅酶替换为CHMO与ADH、GDH外，其余步骤都相同。
CHMO与ADH、GDH共固定化酶的活性通过利用以下底物2进行反应来检测：
Figure PCTCN2019122447-appb-000020
CHMO与GDH共固定化酶的活性通过以下反应来检测：
3mL的0.1M PB(pH 8.0)装入10ml的反应瓶中，随后通过加入50毫克底物2、100mg葡萄糖及5mg NADP+，然后加入200-300mg的CHMO与GDH的共固定化酶(湿的，含有50～80％的水)。在30℃下反应16-20小时，测试转化率。
CHMO与ADH共固定化酶的活性通过以下反应来检测：
0.3mL的异丙醇装入10ml的反应瓶中，随后加入500mg的底物2，然后加入3mL含有5mg NADP+的0.1M PB(pH 8.0)及100-200mg的CHMO与ADH的共固定化酶(湿的，含有50～80％的水)。在30℃下反应16-20小时，测试转化率。测试结果见表8。
表8:
Figure PCTCN2019122447-appb-000021
Figure PCTCN2019122447-appb-000022
实施例5 AADH与FDH/GDH的共固定化
方法1：
除了酶不同外，其余步骤都与实施例2中的方法1相同。
方法2：
用1-2mL 0.1M PB(PH 7.5)洗涤1g氨基树脂，用4mL 0.1M PB(PH 7.5)重悬，将质量浓度为25％～50％的戊二醛水溶液滴加入重悬液中使戊二醛的终浓度为2％。在20℃温和振荡下孵育1小时，随后过滤并用0.1M PB(PH 7.5)洗涤3次。
将含有50-100mg蛋白质(仅AADH)的4mL酶溶液加入到戊二醛活化树脂中，在20-25℃温和振荡下孵育后，过滤并用0.1M PB(pH 7.5)洗涤3次。用0.1M的PB(pH为7.0-7.5)重悬后，加入PEI溶液(终浓度2％)，随后加入20-50mg的GDH/FDH，在20-25℃下温和振荡孵育后，过滤并用0.1M PB(pH7.5)洗涤3次。
AADH及FDH共固定化酶的活性通过以下底物的反应进行检测：
Figure PCTCN2019122447-appb-000023
将5mL 0.1M Tris-Cl缓冲液(pH 8.0-9.0)加入10mL反应器中，然后加入100mg底物3或4，或1，108mg氯化铵，调节pH至pH 7.5-8.0，然后加入10-50mg NAD +，80mg甲酸铵和100mg共固定化酶。在30℃下反应16-20小时后，进行转化试验。
AADH及FDH共固定化酶的活性检测方法如下：
将5mL 0.1M Tris-Cl缓冲液(pH 8.0-9.0)加入10mL反应器中，然后加入100mg底物5或6，或7，108mg氯化铵，调节pH至pH 7.5-8.0，然后加入10-50mg NAD+，150mg葡萄糖和100mg共固定化酶。在30℃下反应16-20小时后，进行转化试验。检测结果见表9。
Figure PCTCN2019122447-appb-000024
表9：
Figure PCTCN2019122447-appb-000025
实施例6 KRED与FDH/GDH的共固定化
除酶不同外，其余的方法1和2的步骤均与实施例5相同。
KRED与FDH共固定化酶的活性通过以下底物5或6的反应进行检测：
3mL的0.1M PB(pH7.0-8.0)装入10ml的反应器中，随后通过加入100mg的底物5或6，接着加入10-50mg NAD(P)+，80mg甲酸铵及100mg的共固定化酶。在30℃下反应16-20小时，测试转化率。
KRED与GDH共固定化酶的活性通过以下反应来检测：
3mL的0.1M PB(pH7.0-8.0)装入10ml的反应器中，随后通过加入100mg的底物5或6，接着加入10-50mg NAD(P)+，120mg葡萄糖及100mg的共固定化酶。在30℃下反应16-20小时，测试转化率。
测试结果见表10。
Figure PCTCN2019122447-appb-000026
Figure PCTCN2019122447-appb-000027
实施例7 ERED与FDH/GDH的共固定化
除酶不同外，其余的方法1和2的步骤均与实施例5相同。
ERED与FDH共固定化酶的活性通过与底物7的反应进行检测：
3mL的0.1M PB(pH7.0-8.0)装入10ml的反应器中，随后通过加入100mg的底物7，接着加入10-50mg NAD(P)+，80mg甲酸铵及100mg的共固定化酶。在30℃下反应16-20小时，测试转化率。
ERED与GDH共固定化酶的活性通过以下反应来检测：
3mL的0.1M PB(pH7.0-8.0)装入10ml的反应器中，随后通过加入100mg的底物7，接着加入10-50mg NAD(P)+，120mg葡萄糖及100mg的共固定化酶。在30℃下反应16-20小时，测试转化率。
测试结果见表11。
Figure PCTCN2019122447-appb-000028
Figure PCTCN2019122447-appb-000029
实施例8 IRED与FDH/GDH的共固定化
除酶不同外，其余的方法1和2的步骤均与实施例5相同。
IRED和FDH共固定化酶的活性采用以下底物8，并按以下方法测试：
Figure PCTCN2019122447-appb-000030
将2mL 0.1M PB缓冲液(pH 7.0-8.0)加入10mL反应器中，然后加入100mg上述底物，然后加入10-50mg NAD(P)+，60mg甲酸铵和100mg共固定酶。在30℃下反应16-20小时后，进行转化试验。
IRED和GDH共固定化酶的活性以下方法测试：
3mL的0.1M PB缓冲液(pH 7.0-8.0)加入10mL反应器中，随后加入100mg底物，再加入10-50mg NAD(P)+，100mg葡萄糖和100mg共固定化酶。在30℃下反应16-20小时后，进行转化试验。
测试结果见表12。
Figure PCTCN2019122447-appb-000031
实施例9 共固定化酶在填充床连续反应中的应用
按实施例2中的方法2，转氨酶TA-Bt与辅酶LDH及FDH共固定化至载体LX1000HA，所得共固定化酶填充于10mL柱体积的柱状反应器中，固定化酶用量5.9g。
500g底物5，108mg氯化铵，用4.5L的PB缓冲液(0.1M,pH8.0)溶解，氢氧化钠溶液调节pH至pH 7.5-8.0，然后加入10-50mg NAD+，80mg甲酸铵，最后用PB缓冲液定容至5L。
设置流速0.1mL/min，即保留时间100min，进行连续化反应，出口端流出液检测转化率，转化率＞98％，持续运行300h，转化率无降低，运行348h，转化率降低值88.4％。具体见表13。
表13.TA-Bt+LDH+FDH共固定化酶在填充床连续反应中的反应结果
Figure PCTCN2019122447-appb-000032
实施例10 共固定化酶在连续搅拌罐反应中的应用
使用同实施例9的共固定化酶，200mL反应器中加入50g转氨酶TA-Bt与辅酶LDH,FDH的共固定化酶，加入150mL磷酸缓冲液。
500g底物5，108mg氯化铵，用4.5L的PB缓冲液(0.1M,pH8.0)溶解，氢氧化钠溶液调节pH至pH 7.5-8.0，然后加入10-50mg NAD +，80mg甲酸铵，最后用PB缓冲液定容至5L。
以0.8mL/min的速度向连续搅拌罐中连续添加底物(即保留时间250min)，同时以同样的流速在出口抽出反应体系(管道末端加过滤头，防止将固定化酶抽出)。在该条件下，转化率可达92％以上，且连续运行400h，转化率基本无降低。结果如表14所示。
表14.TA-Bt+LDH+FDH共固定化酶在连续搅拌罐连续反应中的反应结果
Figure PCTCN2019122447-appb-000033
实施例11 每克载体负载蛋白量的调查
同实施例1，活化氨基载体后，对每克载体中加入蛋白的量进行考察，以转氨酶TA-Bt与其辅酶乳酸脱氢酶LDH的共固定化为例，加入不同的蛋白量，检测蛋白负载量和反应重复使用次数。结果见表15。
同实施例4，以单加氧酶CHMO-Rs与其辅酶ADH和GDH的共固定化为例，加入不同的蛋白量，检测蛋白负载量和反应重复使用次数，结果见表16。结果显示，每克所选载体可负载的蛋白范围50～200mg，蛋白负载率在50％～100％。
表15.TA-Bt与LDH混和酶在不同载体上负载蛋白量的考察
Figure PCTCN2019122447-appb-000034
表16.CHMO-Rs和ADH混和酶在不同载体上蛋白负载量的考察
Figure PCTCN2019122447-appb-000035
从表15，16的数据可见，针对绝大多数载体，蛋白上样量为50-100mg时，蛋白负载率在90％以上。在相同蛋白负载率的情况下，不同载体所形成的共固定化酶的重复使用次数不同。从该表中，可以看出，LX1000HA，LX1000EPN和ECR8409这几种载体所体现的固定化酶活性和稳定性较好。
实施例12 PEI终浓度的调查
同实施例2，方法2(两步法)，制备转氨酶TA-Bt与其辅酶乳酸脱氢酶LDH和甲酸铵脱氢酶FDH的共固定化酶，TA-Bt与LDH的混和酶与载体结合后，加入不同量的PEI，再加入第二辅酶FDH，考察PEI的浓度范围，结果见表17。
表17.TA-Bt与其辅酶LDH及FDH两步固定法中PEI用量的考察
载体 PEI的终浓度 重复使用次数
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
从表17的数据可以看出，PEI的浓度在1％～5％的范围内效果基本一致，超出这个范围，固定化酶的稳定性有所下降。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本申请通过将上述主酶及其辅酶共固定于氨基树脂载体上，实现了这些主酶及其辅酶的共固定化，从而有利于提高酶的活性及循环使用效率。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (24)

  1. 一种共固定化酶，其特征在于，所述共固定化酶包括：
    氨基树脂载体，以及
    主酶和辅酶，所述辅酶为一种或多种，所述主酶和所述辅酶共固定于所述氨基树脂载体上，其中，所述主酶共价固定于所述氨基树脂载体上，所述辅酶通过共价和/或非共价方式固定于所述氨基树脂载体上；
    所述主酶选自如下任意一种酶：转氨酶、氨基酸脱氢酶、亚胺还原酶、酮还原酶、烯还原酶及单加氧酶。
  2. 根据权利要求1所述的共固定化酶，其特征在于，所述转氨酶为来源于B.thuringiensis或Vibrio fluvialis strain JS17的转氨酶；
    优选地，所述氨基酸脱氢酶为来源于Bacillus cereus或Bacillus sphaericus的氨基酸脱氢酶；
    优选地，所述亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；
    优选地，所述酮还原酶为来源于Sporobolomyces salmonicolor的酮还原酶或者Acetobacter sp.CCTCC M209061的酮还原酶，更优选地，所述来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEO ID NO:1或SEO ID NO:2序列的突变体；
    优选地，所述烯还原酶为来源于Chryseobacterium sp.CA49或Sewanella oneidensis MR-1的烯还原酶；
    优选地，所述单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的环己酮单加氧酶；更优选地，所述来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEO ID NO：4序列或SEO ID NO：5序列的突变体；所述来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEO ID NO：7序列或SEO ID NO:8序列的突变体。
  3. 根据权利要求1所述的共固定化酶，其特征在于，所述辅酶选自如下至少一种：乳酸脱氢酶、甲酸铵脱氢酶、葡萄糖脱氢酶及醇脱氢酶；
    优选地，所述乳酸脱氢酶为来源于Lactobacillus helveticus的D-乳酸脱氢酶；
    优选地，所述甲酸铵脱氢酶为来源于Candida boidinii的甲酸脱氢酶；
    优选地，所述葡萄糖脱氢酶为来源于Lysinibacillus sphaericus G10的葡萄糖1-脱氢酶；
    优选地，所述醇脱氢酶为来源于Thermoanaerobium brockii的醇脱氢酶；
    优选地，所述共固定化酶的循环利用次数为4～25次。
  4. 根据权利要求1至3中任一项所述的共固定化酶，其特征在于，所述氨基树脂载体为戊二 醛活化的氨基树脂载体；
    优选地，所述氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，更优选地，所述氨基树脂载体选自如下任意一种：
    Figure PCTCN2019122447-appb-100001
    LX1000HA、LX1000NH、HFA、LX1000EPN、HM100D、
    Figure PCTCN2019122447-appb-100002
    Lifetech TMECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、
    Figure PCTCN2019122447-appb-100003
    ESR-3、ESR-5及ESR-8。
  5. 根据权利要求1所述的共固定化酶，其特征在于，所述共固定化酶中，所述主酶与所述辅酶的质量比为1～20：1～10；
    优选地，所述主酶和所述辅酶的质量和记为N1，所述氨基树脂载体的质量记为N2，N1/N2为50～200mg:1g，进一步地，为80～120mg：1g。
  6. 根据权利要求1或5所述的共固定化酶，其特征在于，
    所述主酶和所述辅酶均共价固定于所述氨基树脂载体上；或者
    所述主酶共价固定于所述氨基树脂载体上，所述辅酶以离子吸附的方式非共价固定于所述氨基树脂载体上；优选通过PEI将所述辅酶吸附于所述氨基树脂载体上。
  7. 根据权利要求6所述的共固定化酶，其特征在于，所述辅酶包括第一酶和第二酶，所述主酶与所述第二酶共价固定于所述氨基树脂载体上，所述第一酶以离子吸附的方式固定于所述氨基树脂载体上。
  8. 权利要求1至7中任一项所述的共固定化酶的制备方法，其特征在于，所述制备方法包括：
    将氨基树脂载体进行活化，得到活化氨基载体；
    将主酶共价固定于所述活化氨基载体上，并将所述所述主酶对应的辅酶通过共价和/或非共价的方式固定于所述活化氨基载体上，得到所述共固定化酶；
    其中，所述主酶对应的所述辅酶的数量为一个或多个。
  9. 根据权利要求8所述的制备方法，其特征在于，将所述主酶及所述辅酶固定于所述活化氨基载体上，得到所述共固定化酶包括：
    按照权利要求5所述的共固定化酶中的所述主酶与所述辅酶的质量比，将所述主酶与所述辅酶混合，得到第一混合酶；
    按照权利要求5所述的共固定化酶中的所述N1/N2的比例，将所述第一混合酶固定于所述活化氨基载体上，得到所述共固定化酶。
  10. 根据权利要求8所述的制备方法，其特征在于，所述辅酶包括第一酶，将所述主酶及所述辅酶固定于所述活化氨基载体上，得到所述共固定化酶包括：
    将所述主酶固定于所述活化氨基载体上，得到初固定化酶；
    将所述第一酶与所述初固定化酶进行固定，得到所述共固定化酶。
  11. 根据权利要求10所述的制备方法，其特征在于，所述辅酶还包括第二酶，将所述主酶及所述辅酶固定于所述活化氨基载体上，得到所述共固定化酶包括：
    将所述主酶和所述第二酶同固定于所述活化氨基载体上，得到所述初固定化酶；
    将所述第一酶与所述初固定化酶进行固定，得到所述共固定化酶。
  12. 根据权利要求11所述的制备方法，其特征在于，通过表面包覆PEI的方式，将所述第一酶与所述初固定化酶进行固定，得到所述共固定化酶；
    优选地，向所述初固定化酶中添加PEI至所述PEI的终浓度为0.5w/v％～5w/v％，得到PEI-初固定化酶复合物；
    然后将所述第一酶与所述PEI-初固定化酶复合物结合，得到所述共固定化酶。
  13. 根据权利要求10所述的制备方法，其特征在于，按照质量比为50～150mg:1g的质量比将所述第一混合酶固定于所述活化氨基载体上，得到所述共固定化酶。
  14. 根据权利要求11所述的制备方法，其特征在于，所述主酶与所述第一酶的质量比为1～20：1～10。
  15. 根据权利要求11所述的制备方法，其特征在于，所述主酶与所述第二酶的质量比为1～20：1～10。
  16. 根据权利要求8所述的制备方法，其特征在于，所述主酶为转氨酶，所述辅酶具有两种辅因子，两种所述辅因子为LDH与FDH，或者为LDH与GDH，所述制备方法包括如下任意一种：
    (1)将所述转氨酶、所述LDH与FDH进行混合，得到第一酶混物；将所述第一酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    (2)将所述转氨酶、所述LDH与GDH进行混合，得到第二酶混物；将所述第二酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    (3)将所述转氨酶与所述LDH进行混合，得到第三酶混物；将所述第三酶混物固定到所述活化氨基载体上，得到初固定转氨酶；通过对所述初固定转氨酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  17. 根据权利要求8所述的制备方法，其特征在于，所述主酶为氨基酸脱氢酶，所述辅酶为FDH或GDH，所述制备方法包括如下任意一种：
    将氨基酸脱氢酶与FDH或GDH混合，得到氨基酸脱氢酶混物；将所述氨基酸脱氢酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    或者
    将所述氨基酸脱氢酶固定到所述活化氨基载体上，得到所述初固定氨基酸脱氢酶；
    通过对所述初固定氨基酸脱氢酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  18. 根据权利要求8所述的制备方法，其特征在于，所述主酶为亚胺还原酶，所述辅酶为FDH或GDH，所述制备方法包括如下任意一种：
    将所述亚胺还原酶、所述FDH或所述GDH进行混合，得到亚胺酶混物；
    将所述亚胺酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    或者
    将所述亚胺还原酶固定到所述活化氨基载体上，得到初固定亚胺还原酶；
    通过对所述初固定亚胺还原酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  19. 根据权利要求8所述的制备方法，其特征在于，所述主酶为酮还原酶，所述辅酶为FDH或GDH，所述制备方法包括如下任意一种：
    将所述酮还原酶、所述FDH或所述GDH进行混合，得到酮还原酶混物；
    将所述酮还原酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    或者
    将所述酮还原酶固定到所述活化氨基载体上，得到初固定酮还原酶；
    通过对所述初固定酮还原酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  20. 根据权利要求8所述的制备方法，其特征在于，所述主酶为烯还原酶，所述辅酶为FDH或GDH，所述制备方法包括如下任意一种：
    将所述烯还原酶、所述FDH或所述GDH进行混合，得到烯还原酶混物；
    将所述烯还原酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    或者
    将所述烯还原酶固定到所述活化氨基载体上，得到初固定烯还原酶；
    通过对所述初固定烯还原酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  21. 根据权利要求8所述的制备方法，其特征在于，所述主酶为环己酮单加氧酶，所述辅酶为FDH或GDH，所述制备方法包括如下任意一种：
    将所述环己酮单加氧酶、所述FDH或所述GDH进行混合，得到单加氧酶混物；
    将所述单加氧酶混物固定到所述活化氨基载体上，得到所述共固定化酶；
    或者
    将所述环己酮单加氧酶固定到所述活化氨基载体上，得到初固定环己酮单加氧酶；
    通过对所述初固定环己酮单加氧酶进行PEI表面包覆的方式，进而将所述GDH或所述FDH进行固定，得到所述共固定化酶。
  22. 根据权利要求8所述的制备方法，其特征在于，采用戊二醛对所述氨基树脂载体进行活化，得到所述活化氨基载体。
  23. 权利要求1至7中任一项所述的共固定化酶，或者权利要求8至22中任一项所述的共固定化酶的制备方法所制备的共固定化酶在生物催化反应中的应用。
  24. 根据权利要求23所述的应用，其特征在于，所述生物催化反应为间歇性的生物催化反应或连续化的生物催化反应；
    优选地，所述共固定化酶应用于连续的流化床中或固定床的生物催化反应中；
    优选地，所述共固定化酶在所述连续化的生物催化反应中的循环利用次数为4～25次。
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