Patent Application
20230025239
The present invention relates to the field of immobilized enzymes, in particular to a co-immobilized enzyme, a preparation method and use thereof.
The applications of microbial cells or separated or engineered enzymes make great progress in biocatalysis, and manufacturing modes are transformed. Many types of enzymes such as an acyltransferase, an amidase, a transaminase, a ketoreductase, an oxidase, a monooxygenase and a hydrolase are used in production of reactions involving antibiotics, herbicides, pharmaceutical intermediates and new age therapeutic agents.
While a free enzyme is used as a biocatalyst, there is a great waste of the enzyme because it is very difficult to recover a water-soluble enzyme. However, a water-insoluble immobilized enzyme may be easily recovered by very simple filtration after each cycle.
Immobilization methods for single enzymes are already reported in existing technologies, but the immobilization methods suitable for the different enzymes are different. For example, Bolivar et al. (Biomacromol. 2006, 7, 669-673) research the covalent immobilization of a formate dehydrogenase (FDH) from pseudomonas SP101, including the covalent immobilization on various carriers such as a modified agarose, a CNBr-activated agarose, Sepabeads (dextran) and an acetaldehyde agarose. It is concluded that the immobilization on activated carriers such as a bromide, a polyethyleneimine, and a glutaraldehyde do not promote any stabilization effects of the enzyme under heat inactivation. However, an optimized enzyme of a highly activated glyoxal agarose is proved to have high heat stability and pH stability, and have more than 50% of the activity in the case of enhanced stability.
Kim et al (J. Mol. Catal B: Enzy 97(2013) 209-214) report that a method of a cross-linked enzyme aggregate (CLEA) is used to immobilize FDH from Candida boidinii, and it is believed that a dextran polyaldehyde as a cross-linking agent instead of a glutaraldehyde is better for immobilizing the enzyme, and the residual activity exceeds 95% after 10 times of repeated uses. In addition, the heat stability of a cross-linked enzyme aggregate (Dex-CLEA) formed by the dextran polyaldehyde is 3.6 times higher than that of the free enzyme.
Binay et al (Beilstein J. Org. Chem. 2016, 12, 271-277) report a highly active immobilized enzyme of FDH derived from Candida methylica. FDH is covalently immobilized on an epoxy-activated Immobead 150 carrier. The Immobead 150 carrier is firstly modified with an ethylenediamin, then activated with glutaraldehyde (FDHIGLU) and functionalized with an aldehyde group (FDHIALD). The highest immobilization and activity yields are obtained while the aldehyde-functionalized Immobead 150 is used as a carrier, which is 90% and 132%, respectively. At 35° C., the half-lives (t1/2) of free FDH, FDHI150, FDHIGLU and FDHIALD are respectively calculated to be 10.6, 28.9, 22.4 and 38.5 hours. FDHI150, FDHIGLU and FDHIALD retain 69%, 38%, and 51% of the initial activity thereof respectively after 10 times of repeated uses.
Jackon et al. (Process Biochem. Vol. 1, 9, Sep. 2016, 1248-1255) report immobilization of a lactate dehydrogenase (LDH) using glyoxal-agarose. Compared to a soluble counterpart thereof, a heat stability factor obtained by the immobilized LDH is 1600 times greater.
Co-immobilization of two enzymes is also reported, such as Valikhani et al. (Biotech. Bioeng. 2018; 115:2416-2425) report co-immobilization of a cytochrome P450 monooxygenase (pigments P450s) and a glucose dehydrogenase derived from B. megaterium. Delgrove et al. (Appl. Catal. A: Gen. Vol. 572, 25 Feb. 2019, 134-141) publish that co-immobilized Baeyer-Villiger monooxygenase (BVMO) and glucose dehydrogenase are used for synthesis of a ε-caprolactone derivative. A thermostable cyclohexanone monooxygenase (TmCHMO) from Thermocrispum municipale and a glucose dehydrogenase (GDH) (GDH-Tac) from Thermoplasma acidophilum are co-immobilized on an amino-functionalized agarose-based carrier (MANA-agarose). The co-immobilization is proved to be the most efficient biocatalyst. In the synthesis of 3,3,5-trimethylcyclohexanone, the average conversion rate is 83% in 15 repeated cycles.
In conclusion, there are still a large number of enzymes in the existing technologies that do not have effective immobilized enzyme forms, especially some enzymes that jointly participate in the same biocatalytic reaction. The co-immobilization of these enzymes to improve the recyclability of the enzymes becomes an urgent problem to be solved.
A main purpose of the present invention is to provide a co-immobilized enzyme, a preparation method therefor and an application thereof, as to solve the problem that such enzymes are difficult to recycle in the prior art.
In order to achieve the above purpose, according to one aspect of the present invention, a co-immobilized enzyme is provided, the co-immobilized enzyme includes: an amino resin carrier, a main enzyme, and a coenzyme, there is one or more coenzymes, and the main enzyme and the coenzyme are co-immobilized on the amino resin carrier, herein the main enzyme is covalent-immobilized on the amino resin carrier, and the coenzyme is immobilized on the amino resin carrier by a mode of covalent and/or non-covalent; and the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketoreductase, enoyl reductase, and monooxygenase.
Further, the transaminase is derived from B. thuringiensis or Vibrio fluvialis strain JS17; preferably, the amino acid dehydrogenase is derived from Bacillus cereus or from Bacillus sphaericus; preferably, the imine reductase is derived from Streptomyces sp or from Bacillus cereus; preferably, the ketoreductase is derived from Sporobolomyces salmonicloror fromAcetobacter sp. CCTCC M209061; more preferably, the ketoreductase derived from Acetobacter sp. CCTCC M209061 is a mutant having the sequence of SEQ ID NO:1 or SEQ ID NO:2; preferably, the enoyl reductase is derived from Chryseobacterium sp. CA49 or Sewanella oneidensis MR-1; preferably, the monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or from Brachymonas petroleovorans, or from Rhodococcus ruber-SD1; more preferably, the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having the sequence of SEQ ID NO:4 or SEQ ID NO:5, the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant having the sequence of SEQ ID NO:7 or SEQ ID NO:8.
Further, the coenzyme is selected from at least one of the followings: lactate dehydrogenase (LDH), formate dehydrogenase (FDH), glucose dehydrogenase (GDH) and alcohol dehydrogenase (ADH); preferably, the lactate dehydrogenase is a D-lactate dehydrogenase derived from Lactobacillus helveticus; preferably, the formate dehydrogenase is derived from Candida boidinii; preferably, the glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is derived from Thermoanaerobium brockii; and preferably, the co-immobilized enzyme is re-used for 4-25 cycles.
Further, 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 linker arm, more preferably, the amino resin carrier is selected from at least one of the followings: SEPLITE® LX1000EA, LX1000HA, LX1000NH, HFA, LX1000EPN, HM100D, Purolite® Lifetech™ ECR8309, ECR8409, ECR8305, ECR8404, ECR8315, ECR8415, HECHENG® ESR-1, ESR-3, ESR-5 and ESR-8.
Further, in the co-immobilized enzyme, a mass ratio of the main enzyme and the coenzyme is 1-20:1-10; preferably, a sum of masses of the main enzyme and the coenzyme is marked as N1, a mass of the amino resin carrier is marked as N2, N1:N2 is 50-200 mg:1 g, and preferably, 80-120 mg:1 g.
Further, either the main enzyme and the coenzyme are covalently immobilized on the amino resin carrier; or only the main enzyme is covalent-immobilized on the amino resin carrier and the coenzyme is non-covalently immobilized on the amino resin carrier by an ionic adsorption mode; preferably, the coenzyme is adsorbed on the amino resin carrier by polyethyleneimine (PEI).
Further, the coenzyme includes a first enzyme and a second enzyme, the main enzyme and the second enzyme are covalent-immobilized on the amino resin carrier, and the first enzyme is immobilized on the amino resin carrier by the ionic adsorption mode.
In a second aspect of the present application, a preparation method for any one of the above co-immobilized enzymes is provided, and the preparation method includes: activating an amino resin carrier to obtain an activated amino carrier; and covalently immobilizing a main enzyme on the activated amino carrier, and covalently and/or non-covalently immobilizing a coenzyme corresponding to the main enzyme on the activated amino carrier, to obtain the co-immobilized enzyme; herein the number of the coenzyme corresponding to the main enzyme is one or more.
Further, immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: mixing the main enzyme with the coenzyme, to obtain a first mixed enzyme; and immobilizing the first mixed enzyme on the activated amino carrier, to obtain the co-immobilized enzyme.
Further, the coenzyme includes a first enzyme, and immobilizing 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 primary immobilized enzyme; and immobilizing the first enzyme and the primary immobilized enzyme, to obtain the co-immobilized enzyme.
Further, the coenzyme further includes a second enzyme, and 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, to obtain a primary immobilized enzyme; and immobilizing the first enzyme and the primary immobilized enzyme, to obtain the co-immobilized enzyme.
Further, immobilizing the first enzyme and the primary immobilized enzyme by surface coating of PEI, to obtain the co-immobilized enzyme; preferably, adding PEI to the primary immobilized enzyme until a final concentration of the PEI ranges from 0.5 w/v % to 5 w/v %, to obtain a PEI-primary immobilized enzyme; and binding the first enzyme to the PEI-primary immobilized enzyme, to obtain the co-immobilized enzyme.
Further, immobilizing the first mixed enzyme in a mass ratio of 50˜150 mg:1 g on the activated amino carrier, to obtain the co-immobilized enzyme.
Further, a mass ratio of the main enzyme and the first enzyme is 1˜20:1˜10.
Further, a mass ratio of the main enzyme and the second enzyme is 1˜20:1˜10.
Further, the main enzyme is a transaminase, and the coenzyme has two co-factors, and the two factors are LDH and FDH, or LDH and GDH, and the above preparation method includes any one of the followings:
(1) mixing the transaminase with both LDH and FDH, to obtain a first enzyme mixture, and immobilizing the first enzyme mixture on an activated amino carrier, to obtain the co-immobilized enzyme;
(2) mixing the transaminase with both LDH and GDH, to obtain a second enzyme mixture, and immobilizing the second enzyme mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(3) mixing the transaminase with LDH, to obtain a third enzyme mixture; immobilizing the third enzyme mixture on the activated amino carrier, to obtain a primary immobilized transaminase; and then performing surface coating of PEI on the primary immobilized transaminase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further, the main enzyme is an amino acid dehydrogenase, the coenzyme is FDH or GDH, and the preparation method includes any one of the followings:
(1) mixing the amino acid dehydrogenase with either FDH or GDH, to obtain an amino acid dehydrogenase mixture; and immobilizing the amino acid dehydrogenase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(2) immobilizing the amino acid dehydrogenase on the activated amino carrier, to obtain a primary immobilized amino acid dehydrogenase; and then performing surface coating of PEI on the primary immobilized amino acid dehydrogenase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further, the main enzyme is an imine reductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the followings:
(1) mixing the imine reductase with either FDH or GDH, to obtain an imine reductase mixture; and immobilizing the imine reductase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(2) immobilizing the imine reductase on the activated amino carrier, to obtain a primary immobilized imine reductase; and then performing surface coating of PEI on the primary immobilized imine reductase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further, the main enzyme is a ketoreductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the followings:
(1) mixing the ketoreductase with either FDH or GDH, to obtain a ketoreductase mixture; and immobilizing the ketoreductase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(2) immobilizing the ketoreductase on the activated amino carrier, to obtain a primary immobilized ketoreductase; and then performing surface coating of PEI on the primary immobilized ketoreductase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further, the main enzyme is an enoyl reductase, the coenzyme is FDH or GDH, and the preparation method includes any one of the followings:
(1) mixing the enoyl reductase with either FDH or GDH, to obtain a enoyl reductase mixture; and immobilizing the enoyl reductase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(2) immobilizing the enoyl reductase on the activated amino carrier, to obtain a primary immobilized enoyl reductase; and then performing surface coating of PEI on the primary immobilized enoyl reductase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further, the main enzyme is a cyclohexanone monoxygenase, the coenzyme is FDH or GDH, and the preparation method includes any one of the followings:
(1) mixing the cyclohexanone monoxygenase with either FDH or GDH, to obtain a monoxygenase mixture; and immobilizing the monoxygenase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; and
(2) immobilizing the cyclohexanone monoxygenase on the activated amino carrier, to obtain a primary immobilized cyclohexanone monoxygenase; and then performing surface coating of PEI on the primary immobilized cyclohexanone monoxygenase, and immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
Further the amino resin carrier is activated by glutaraldehyde, to obtain the activated amino carrier.
According to a third aspect of the present application, an application of any one of the above co-immobilized enzymes or the co-immobilized enzyme prepared by any one of the preparation methods for the co-immobilized enzymes in a biocatalytic reaction is provided.
Further, 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 fixed bed biocatalytic reaction; and preferably, the co-immobilized enzyme is re-used for 4-25 cycles in the continuous biocatalytic reaction.
By applying a technical scheme of the present invention, the present application achieves the co-immobilization of these main enzymes and the coenzymes thereof by co-immobilizing the above main enzymes and the coenzymes thereof on the amino resin carrier, thereby it is beneficial to improve the activity and recycling efficiency of the enzyme.
It should be noted that embodiments in the present application and features of the embodiments may be combined with each other in the case without conflicting. The present invention is described in detail below with reference to the embodiments.
Polyethylene imine, referred to as PEI.
In a typical embodiment of the present application, a co-immobilized enzyme is provided, and the co-immobilized enzyme 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, herein the main enzyme is covalent-immobilized on the amino resin carrier, and the coenzyme is immobilized on the amino resin carrier by a mode of covalent and/or non-covalent; and the main enzyme is selected from any one of the following enzymes: transaminase, amino acid dehydrogenase, imine reductase, ketoreductase, enoyl reductase, and monooxygenase.
The present application achieves the co-immobilization of these main enzymes and the coenzymes thereof by co-immobilizing the above main enzymes and the coenzymes thereof on the amino resin carrier, thereby it is beneficial to improve the activity and recycling efficiency of the enzyme.
The above various main enzymes have different specific types and activities according to different sources. In a preferred embodiment, the transaminase is derived from B. thuringiensis or Vibrio fluvialis strain JS17; preferably, the amino acid dehydrogenase is derived from Bacillus cereus or from Bacillus sphaericus; preferably, the imine reductase is derived from Streptomyces sp or from Bacillus cereus; preferably, the ketoreductase is derived from Sporobolomyces salmonicolor or from Acetobacter sp. CCTCC M209061; more preferably, the ketoreductase derived from Acetobacter sp. CCTCC M209061 is a mutant having the sequence of SEQ ID NO:1 or SEQ ID NO:2; preferably, the enoyl reductase is derived from Chryseobacterium sp. CA49 or Sewanella oneidensis MR-1; preferably, the monooxygenase is a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, or from Brachymonas petroleovorans, or from Rhodococcus ruber-SD1; more preferably, the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having the sequence of SEQ ID NO:4 or SEQ ID NO:5, the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant having the sequence of SEQ ID NO:7 or SEQ ID NO:8.
According to the different types of the above main enzymes, the 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 followings: lactate dehydrogenase, formate dehydrogenase, glucose dehydrogenase and alcohol dehydrogenase; preferably, the lactate dehydrogenase is a D-lactate dehydrogenase derived from Lactobacillus helveticus; preferably, the formate dehydrogenase is derived from Candida boidinii; preferably, the glucose dehydrogenase is glucose 1-dehydrogenase derived from Lysinibacillus sphaericus G10; preferably, the alcohol dehydrogenase is derived from Thermoanaerobium brockii. In another preferred embodiment, the co-immobilized enzyme is re-used for 4-25 cycles.
In the above preferred embodiment, the coenzyme corresponding to the transaminase is lactate dehydrogenase and ammonium formate dehydrogenase, or lactate dehydrogenase and glucose dehydrogenase. The coenzyme corresponding to the amino acid dehydrogenase is ammonium formate dehydrogenase or glucose dehydrogenase. The coenzyme corresponding to the imine reductase is ammonium formate dehydrogenase or glucose dehydrogenase. The coenzyme corresponding to the ketoreductase is ammonium formate dehydrogenase or glucose dehydrogenase. The coenzyme corresponding to the enoyl reductase is ammonium formate dehydrogenase or glucose dehydrogenase. The coenzyme corresponding to the monooxygenase is alcohol dehydrogenase or ammonium formate dehydrogenase or glucose dehydrogenase.
In a preferred embodiment, the lactate dehydrogenase is a D-lactate dehydrogenase (LDH for short) derived from Lactobacillus helveticus; preferably, the ammonium formate dehydrogenase is a formate dehydrogenase (FDH for short) derived from Candida boidinii; preferably, the glucose dehydrogenase is a glucose 1-dehydrogenase (GDH for short) derived from Lysinibacillus sphaericus G10; and preferably, the alcohol dehydrogenase is an alcohol dehydrogenase derived from Thermoanaerobium brockii.
There is no co-immobilized enzyme for the main enzymes and co-enzymes from each source in the above preferred embodiment in the prior art, and the co-immobilization of these main enzymes and co-enzymes is helpful to improve the recycling efficiency of these enzymes.
The carrier of the co-immobilized enzyme is an amino resin carrier, and various existing amino resin carriers may be used. In a preferred embodiment, 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 linker arm, more preferably, the amino resin carrier is selected from at least one of the followings: SEPLITE® LX1000EA, LX1000HA, LX1000NH, HFA, LX1000EPN, HFA, HM100D, Purolite® Lifetech™ ECR8309, ECR8409, ECR8305, ECR8404, ECR8315, ECR8415, HECHENG® ESR-1, ESR-3, ESR-6 and ESR-8. According to the difference of the main enzyme and coenzyme to be immobilized, the different amino resin carriers may be selected.
In the above co-immobilized enzyme, the amount ratios of the main enzyme and the co-enzyme are different according to different substrates to be catalyzed. In a preferred embodiment, in the above co-immobilized enzyme, a mass ratio of the main enzyme and the coenzyme is 1-20:1-10. Preferably, a sum of masses of the main enzyme and the coenzyme is marked as N1, a mass of the amino resin carrier is marked as N2, N1:N2 is 50-200 mg:1 g, and preferably, 80-120 mg:1 g.
The mass ratio of the main enzyme and the coenzyme is controlled within the above range, so that all the above co-immobilized enzymes of the main enzyme and the coenzyme may catalyze most of the substrates. In addition, the mass ratio of the enzyme to the carrier is controlled in the range of 50-200 mg:1 g, it may achieve the co-immobilization of all the above main enzymes and coenzymes.
In some more preferred embodiments, the mass ratio of the transaminase to the coenzyme lactate dehydrogenase thereof is 5˜7:1; the mass ratio of the transaminase to the coenzyme ammonium formate dehydrogenase thereof is 5˜7:1˜3; the mass ratio of the transaminase to the coenzyme glucose dehydrogenase thereof is 5˜7:1˜2; the mass ratio of the amino acid dehydrogenase to the coenzyme ammonium formate dehydrogenase thereof is 8˜10:1; the mass ratio of the amino acid dehydrogenase to the coenzyme glucose dehydrogenase thereof is 5˜6:1; the mass ratio of the imine reductase to the coenzyme ammonium formate dehydrogenase thereof is 4˜8; 1; the mass ratio of the imine reductase to the coenzyme glucose dehydrogenase thereof is 6˜8:1; the mass ratio of the ketoreductase to the coenzyme ammonium formate dehydrogenase is 4˜8:1; the mass ratio of the ketoreductase to the coenzyme glucose dehydrogenase thereof is 1:3˜10; the mass ratio of the enoyl reductase to the coenzyme ammonium formate dehydrogenase thereof is 6˜10:1; the mass ratio of the enoyl reductase to the coenzyme glucose dehydrogenase thereof is 12˜20:1; the mass ratio of the monooxygenase to the coenzyme alcohol dehydrogenase thereof is 2˜3:2˜3; and the mass ratio of the monooxygenase to the coenzyme glucose dehydrogenase thereof is 2˜3:1. The mass ratios of the above main enzymes and the corresponding different coenzymes thereof are controlled within the above range, so that the main enzymes and the coenzymes may be configured according to the stoichiometric ratio as much as possible, thereby the catalytic activity and efficiency of the co-immobilized enzyme are improved.
In the above co-immobilized enzyme, both the main enzyme and the coenzyme are immobilized on the carrier, and a specific immobilization mode of the two on the carrier is not limited. In a preferred embodiment, both the main enzyme and the coenzyme are covalently immobilized on the amino resin carrier. In another preferred embodiment, the main enzyme is covalently immobilized on the amino resin carrier, and the coenzyme is non-covalently immobilized on the amino resin carrier by a mode of ionic adsorption; preferably, the coenzyme is adsorbed on the amino resin carrier by PEI. The above two different co-immobilization modes may be obtained by adopting different co-immobilization methods.
In the above co-immobilized enzymes, some main enzymes have only one coenzyme, and some main enzymes have two coenzymes. The co-immobilization modes of the two are the same as above, it may be the same or different, and determined according to the properties and characteristics of the specific coenzymes. 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 the amino resin carrier, and the first enzyme is immobilized on the amino resin carrier by a mode of ionic adsorption. Here, the first enzyme refers to an enzyme that is sensitive to some reagents in a co-immobilization process, such as a cross-linking agent glutaraldehyde. In order to reduce the decrease of the activity of the first enzyme in the co-immobilization process, and reduce the influence on the activity of the co-immobilized enzyme and the subsequent recycling efficiency, the first enzyme is non-covalently immobilized on the amino resin carrier by the mode of ionic adsorption in the above preferred embodiment.
Transaminase TA-Bt (see Table 1 for details) and LDH and FDH are co-immobilized to a carrier LX1000HA in the optimal ratio, and the maximum number of uses may reach 15 times; the transaminase TA-Bt and LDH and GDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses may reach 20-25 times; monooxygenase CHMO-Rs and GDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 13 times, the monooxygenase CHMO-Rs and ADH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 13-15 times, they are co-immobilized to a carrier ECR8409, and the maximum number of uses reaches 16 times; mutant V1 of CHMO-Rs and ADH are co-immobilized to the carrier LX1000HA, and the maximum number of uses reaches 17 times, mutant V2 and ADH are co-immobilized to the carrier LX1000HA, and the maximum number of uses reaches 17 times; CHMO-Bp and ADH or GDH are co-immobilized to the carrier LX1000HA, and the number of uses reaches 14-15 times; CHMO-Bp mutant V1 and ADH are co-immobilized to the carrier LX1000HA, and the number of uses reaches 19 times; the CHMO-Bp mutant V1 and ADH are co-immobilized to the carrier LX1000HA, and the number of uses reaches 21 times; AADH-Bc and FDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 12 times; AADH-Bs and GDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 15 times; KRED-Ac and FDH are co-immobilized to the carrier ECR8409, and the maximum number of uses reaches 18 times; KRED-Ac-V1 and FDH are co-immobilized to the carrier ECR8409, and the maximum number of uses reaches 24 times; KRED-Ac and GDH are co-immobilized to the carrier ECR8409, and the maximum number of uses reaches 14 times; ERED-Sc and FDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 17 times; ERED-Sc and GDH are co-immobilized to the carrier LX1000HA in the optimal ratio, and the maximum number of uses reaches 21 times; ERED-Chr is immobilized to a carrier LX1000EPN in the optimal ratio, and the maximum number of uses reaches 21 times; IRED-Str and FDH are co-immobilized to the carrier LX1000EPN in the optimal ratio, and the maximum number of uses reaches 17 times; and IRED-Bc and GDH are co-immobilized to the carrier LX1000EPN in the optimal ratio, and the maximum number of uses reaches 19 times.
In a second typical embodiment of the present application, a preparation method for any one 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 immobilizing a coenzyme corresponding to the main enzyme on the activated amino carrier in a mode of covalent and/or non-covalent, to obtain the co-immobilized enzyme.
In the above method for the co-immobilized enzyme, the co-immobilized enzyme may be obtained by firstly activating the amino resin carrier, and then co-immobilizing any one of the above main enzymes and the coenzymes thereof on the activated amino carrier.
For the specific method of co-immobilizing the above main enzymes and coenzymes on the activated amino carrier, appropriate steps may be selected according to the different types and properties of the coenzymes.
In a preferred embodiment, immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: mixing the main enzyme with the coenzyme, to obtain a first mixed enzyme; and immobilizing the first mixed enzyme on the activated amino carrier, to obtain the co-immobilized enzyme.
In this preferred method, regardless of the number of the coenzymes, the coenzyme may be immobilized on the carrier after being mixed with the main enzyme. According to the different main enzymes and coenzymes, the mixing ratios are different. Preferably, according to the previous mass ratio 1˜20:1˜10 of the main enzyme and the coenzyme, the main enzyme is mixed with the coenzyme, to obtain the first mixed enzyme; and according to the previous ratio of N1/N2, the first mixed enzyme is immobilized on the activated amino carrier, to obtain the co-immobilized enzyme.
In a preferred embodiment, the coenzyme includes a first enzyme, and immobilizing 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 primary immobilized enzyme; and immobilizing the first enzyme and the primary immobilized enzyme, to obtain the co-immobilized enzyme.
While there is only one coenzyme, the above method of immobilizing together with the main enzyme after mixing may be adopted, or the above method of step-by-step immobilizing may be adopted. For the co-immobilization of some coenzymes sensitive to the glutaraldehyde in the step of activating the amino carrier, a step-by-step immobilization method is preferentially adopted, this may retain the activity of the coenzyme to a greater extent, thereby the recycling efficiency of the co-immobilized enzyme is improved.
In a preferred embodiment, the coenzyme further includes a second enzyme, and immobilizing the main enzyme and the coenzyme on the activated amino carrier to obtain the co-immobilized enzyme includes: co-immobilizing the main enzyme and the second enzyme on the activated amino carrier, to obtain a primary immobilized enzyme; and immobilizing the first enzyme and the primary immobilized enzyme, to obtain the co-immobilized enzyme.
While there is more than one coenzyme, according to the sensitivities of the coenzyme and glutaraldehyde and other activators, the above part may be used to mix with the main enzyme and then immobilized on the activated amino carrier. In the second step, the highly sensitive coenzyme is immobilized on the carrier, thus the activity of the highly sensitive enzyme may be improved, thereby the overall activity and recycling efficiency of the prepared co-immobilized enzyme are improved. In the above preferred embodiment, the first enzyme is an enzyme that is relatively sensitive to the glutaraldehyde, and the sensitivity of the second enzyme is relatively low, so the second enzyme and the main enzyme are mixed and immobilized, and then the first enzyme is separately immobilized.
In the above step-by-step immobilization step, the second time of immobilization is performed for the purpose of improving the activity of the coenzyme to be immobilized. Therefore, any modes that may improve the activity of this type of the coenzymes and may achieve the immobilization to it are applicable to the present application. In a preferred embodiment, the first enzyme is immobilized with the primary immobilized enzyme through PEI, to obtain the co-immobilized enzyme. In other preferred embodiments, PEI is added to the primary immobilized enzyme until a final concentration (mass volume concentration) of PEI is 0.5%˜5%, to obtain a PEI-primary immobilized enzyme; and the first enzyme is added to the PEI-primary immobilized enzyme for incubation, to obtain the co-immobilized enzyme.
PEI is polyethylene imine, it may be used as a support in the non-solid form, and it adsorbs the coenzyme to be immobilized on the carrier by the mode of ionic adsorption. The specific amount of PEI added may be reasonably adjusted according to actual needs. While the final concentration of PEI added is 0.5%˜5%, the immobilization of all types of the coenzymes described above may be achieved.
While the main enzyme is the transaminase, the coenzyme has two cofactors, LDH and FDH, or LDH and GDH, and the preparation method thereof includes a one-step immobilization method or a step-by-step immobilization method. The one-step immobilization method is to obtain a first enzyme mixture or a second enzyme mixture by mixing the two cofactors in different combinations with the transaminase respectively, and then immobilize the first enzyme mixture or the second enzyme mixture on the activated amino carrier, to obtain the co-immobilized enzyme. Step-by-step immobilization method: mixing the transaminase with LDH, to obtain a third enzyme mixture; immobilizing the third enzyme mixture on the activated amino carrier, to obtain a primary immobilized transaminase; and through a mode of performing surface coating of PEI on the primary immobilized transaminase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
While the main enzyme is the amino acid dehydrogenase, and the coenzyme is FDH or GDH, the preparation method includes any one of the followings: mixing the amino acid dehydrogenase with FDH or GDH, to obtain an amino acid dehydrogenase mixture; immobilizing the amino acid dehydrogenase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; or, immobilizing the amino acid dehydrogenase on the activated amino carrier, to obtain a primary immobilized amino acid dehydrogenase; through a mode of performing surface coating of PEI on the primary immobilized amino acid dehydrogenase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
While the main enzyme is the imine reductase, and the coenzyme is FDH or GDH, the preparation method includes any one of the followings: mixing the imine reductase, and FDH or GDH, to obtain an imine enzyme mixture; immobilizing the imine enzyme mixture on the activated amino carrier, to obtain the co-immobilized enzyme; or, immobilizing the imine reductase on the activated amino carrier, to obtain a primary immobilized imine reductase; and through a mode of performing surface coating of PEI on the primary immobilized imine reductase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
While the main enzyme is the ketoreductase, and the coenzyme is FDH or GDH, the preparation method includes any one of the followings: mixing the ketoreductase, and FDH or GDH, to obtain a ketoreductase mixture; immobilizing the ketoreductase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; or immobilizing the ketoreductase on the activated amino carrier, to obtain a primary immobilized ketoreductase; and through a mode of performing surface coating of PEI on the primary immobilized ketoreductase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
While the main enzyme is the enoyl reductase, and the coenzyme is FDH or GDH, the preparation method includes any one of the followings: mixing the enoyl reductase, and FDH or GDH, to obtain an enoyl reductase mixture; immobilizing the enoyl reductase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; or, immobilizing the enoyl reductase on the activated amino carrier, to obtain a primary immobilized enoyl reductase; and through a mode of performing surface coating of PEI on the primary immobilized enoyl reductase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
While the main enzyme is the cyclohexanone monooxygenase, and the coenzyme is FDH or GDH, the preparation method includes any one of the followings: mixing the cyclohexanone monooxygenase, and FDH or GDH, to obtain a monooxygenase mixture; immobilizing the monooxygenase mixture on the activated amino carrier, to obtain the co-immobilized enzyme; or, immobilizing the cyclohexanone monooxygenase on the activated amino carrier, to obtain a primary immobilized cyclohexanone monooxygenase; and through a mode of performing surface coating of PEI on the primary immobilized cyclohexanone monooxygenase, immobilizing GDH or FDH, to obtain the co-immobilized enzyme.
In the above preparation methods for the co-immobilization of various main enzymes and coenzymes thereof, the PEI surface coating method is the same as the previous operation of “adding PEI to the primary immobilized enzyme until the final concentration of PEI is 0.5 w/v %˜5 w/v %, to obtain the PEI-primary immobilized enzyme complex; and combining the first enzyme with the PEI-primary immobilized enzyme complex, to obtain the co-immobilized enzyme”, it is not repeatedly described here.
In the above step of activating the amino carrier, an existing activator may be used for activation. In a preferred embodiment, the glutaraldehyde is used to activate the amino resin carrier, to obtain the activated amino carrier. The glutaraldehyde has a wide range of applications and is the most common.
In the above co-immobilization step, according to the different co-immobilization methods and the different types and numbers of the co-immobilized coenzymes, the ratios of the total mass of the co-immobilized coenzymes to the total mass of the activated amino carriers are also different. Correspondingly, the mass ratios of the main enzymes to the coenzymes are also different, and while there are two types of the coenzymes, the mass ratios of the main enzymes to the first coenzyme and the second coenzyme are also different, and it may be reasonably adjusted according to actual needs.
In a preferred embodiment, the first mixed enzyme is immobilized on the activated amino carrier at the mass ratio of 50˜150 mg:1 g, to obtain the co-immobilized enzyme.
In a preferred embodiment, the mass ratio of the main enzyme to the first enzyme is 1˜20:1˜10.
In a preferred embodiment, the mass ratio of the main enzyme to the second enzyme is 1˜20:1˜10.
Preferably, in the above co-immobilization step, the mass ratio of the transaminase to the coenzyme lactate dehydrogenase thereof is 5˜7:1; the mass ratio of the transaminase to the coenzyme ammonium formate dehydrogenase thereof is 5˜7:1˜3; the mass ratio of the transaminase to the coenzyme glucose dehydrogenase thereof is 5˜7:1˜2; the mass ratio of the amino acid dehydrogenase to the coenzyme ammonium formate dehydrogenase thereof is 8˜10:1; the mass ratio of the amino acid dehydrogenase to the coenzyme glucose dehydrogenase thereof is 4˜6:1; the mass ratio of the imine reductase to the coenzyme ammonium formate dehydrogenase thereof is 4˜6; 1; the mass ratio of the imine reductase to the coenzyme glucose dehydrogenase thereof is 5˜6:1; the mass ratio of the ketoreductase to the coenzyme ammonium formate dehydrogenase is 4˜6:1; the mass ratio of the ketoreductase to the coenzyme glucose dehydrogenase thereof is 1:5˜10; the mass ratio of the enoyl reductase to the coenzyme ammonium formate dehydrogenase thereof is 6˜8:1; the mass ratio of the enoyl reductase to the coenzyme glucose dehydrogenase thereof is 18˜20:1; the mass ratio of the monooxygenase to the coenzyme alcohol dehydrogenase thereof is 2˜3:2˜3; and the mass ratio of the monooxygenase to the coenzyme glucose dehydrogenase thereof is 2˜3:1.
The mass ratios of the above main enzymes and the corresponding different coenzymes thereof are controlled within the above range, so that the main enzymes and the coenzymes may be configured according to the stoichiometric ratio as much as possible, thereby the catalytic activity and efficiency of the co-immobilized enzyme are improved.
In a third typical embodiment of the present application, an application of any one of the above co-immobilized enzymes or the co-immobilized enzyme prepared by any one of the preparation methods for the co-immobilized enzymes in a biocatalytic reaction is provided. Preferably, the biocatalytic reaction is a continuous biocatalytic reaction.
The beneficial effects of the present application are further described below with reference to the specific embodiments.
The enzymes used in the following embodiments and sources thereof are shown in Table 1 below, and Tables 2 to 4 show sequences of some enzymes.
B. thuringiensis
Vibrio fluvialis strain JS17
Sporobolomyces salmonicolor
Acetobacter sp. CCTCC M209061
Thermoanaerobium brockii
Lactobacillus helveticus
Candida boidinii
Lysinibacillus sphaericus G10
Rhodococcus sp. Phil
Brachymonas petroleovorans
Rhodococcus ruber-SDI
Saccharomyces cerevisiae
Chryseobacterium sp. CA49
Streptomyces sp.
Bacillus cereus
Bacillus cereus
Baccillus sphaericus
PB in the following embodiments represents a phosphate buffer.
4˜5 mL of 0.1 M phosphate buffer (pH 7.5) is used to wash 1 g of an amino resin, 4 mL of 0.1M phosphate buffer (pH 7.5) is used to resuspend, 25%˜50% (w/v) of glutaraldehyde aqueous solution is dropwise added, so that a final concentration of glutaraldehyde is 2% (w/v), and it is incubated for 1 hour at 20° C. with gentle shaking, filtered and washed for 3 times with 0.1 M phosphate buffer (pH 7.5).
4 mL of enzyme solution (the appropriate ratio of TA and LDH is adjusted) containing 100˜120 mg of a protein is added to the glutaraldehyde-activated resin, and after being incubated at 20˜25° C. with gentle shaking, it is filtered and washed with 0.1 M phosphate buffer (pH 7.5) for 3 times.
The more stable linkage may be achieved by reducing an imine double-bond with a borohydride. 1 g of an immobilized enzyme is resuspended with 4 mL of a buffer (50 mM NaHCO3—Na2CO3, pH 8.0-10.0), and NaBH4 is added at 5˜15° C., so that a final concentration of NaBH4 is 1 mg/mL. After being stirred for 1-2 hours at 5-15° C., it is filtered and washed with 0.1 M phosphate buffer (pH 7.5) for 3 times.
The co-immobilization activity of TA and LDH is tested with free FDH to achieve regeneration of NADH. It is tested with the following substrate 1:
5 mL of 0.1 M phosphate buffer (pH 8.0) is added to a 10 mL reactor, and then 100 mg of the above substrate 1, 80 mg of an ammonium format. (FDH), and 5 mg of PLP are added, pH is adjusted to 7.5-8.0, then 5 mg of NAD+ and 30 mg of FDH free enzyme are added, to obtain 100 mg of the co-immobilized enzyme (wet enzyme, containing 50-80 of water). It is reacted at 30° C. for 16-20 hours, and the conversion rate is detected by a High Performance Liquid Chromatography (HPLC), and results are shown in the following table.
Method 1: One-Step Co-Immobilization
1-2 mL of 0.1 M PB (PH 7.5) is used to wash 1 g of an amino resin, 4 mL of 0.1 M PB (PH 7.5) is used to resuspend, and glutaraldehyde aqueous solution with a concentration of 25 (w/v) %˜50 (w/v) % is dropwise added to resuspension so that a final concentration of glutaraldehyde is 2 (w/v) %. It is incubated for 1 hour at 20° C. with gentle shaking, then filtered and washed with 0.1 M PB (pH 7.5) for 3 times. 4 mL of enzyme solution (the appropriate ratio of TA and LDH and FDH is adjusted) containing 100˜120 mg of a protein is added to the glutaraldehyde-activated resin, and after being incubated at 20˜25° C. with gentle shaking, it is filtered and washed with 0.1 M phosphate buffer (pH 7.5) for 3 times.
Method 2: Two-Step Co-Immobilization
In order to improve the activity and stability of FDH, 1 g of co-immobilized TA and LDH is resuspended with 0.1 M PB (pH 7.0-7.5), and PEI solution (final concentration 2%) is added, and then 20 mg of FDH is added. After being incubated at 20-25° C. with gentle shaking, it is filtered and washed with 0.1 M PB (pH 7.5) for 3 times.
The activities of co-immobilized TA, LDH and FDH are detected by the following reactions:
5 mL of 0.1 M phosphate buffer (pH 8.0) is added to a 10 mL reactor, and then 100 mg of the above substrate 1, 80 mg of FDH, and 5 mg of PLP are added, pH is adjusted to 7.5-8.0, then 5 mg of NAD+ and 100 mg of the co-immobilized enzyme (wet, containing 50-80% of water) are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested. Test results are shown in the following table.
Method 1 and Method 2 are the same as Embodiment 2 except that GDH is used instead of FDH.
The activities of co-immobilized TA, LDH and GDH are detected by the following reactions:
5 mL of 0.1 M phosphate buffer (pH 8.0) is added to a 10 mL reactor, and then 100 mg of the above substrate 1, 120 mg or glucose and 5 mg of PLP are added, pH is adjusted to 7.5-8.0, then 5 mg of NAD+ and 100 mg of the co-immobilized enzyme (wet, containing 50-80% of water) are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested. Test results are shown in Table 7.
Method 1 and Method 2 in Embodiment 2 are used, and the remaining steps are the same except that the main enzyme and coenzyme are replaced by CHMO, ADH, and GDH.
The activity of CHMO, ADH and GDH co-immobilized enzyme is detected by reacting with the following substrate 2:
The activity of CHMO and GDH co-immobilized enzyme is detected by the following reaction:
3 mL of 0.1 M PB (pH 8.0) is loaded in a 10 ml reaction flask, and then, 50 mg of the substrate 2, 100 mg of glucose and 5 mg of NADP+ are added, and 200,300 mg of CHMO and GDH co-immobilized enzyme (wet, containing 50˜80% of water) is added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested.
The activity of CHMO and ADH co-immobilized enzyme is detected by the following reaction:
0.3 mL of isopropanol is loaded in a 10 mL reaction flask, and then 500 mg of the substrate 2 is added, and 3 mL of 0.1 M PB (pH 8.0), containing 5 mg of NADP+ and 100-200 mg of CHMO and ADH co-immobilized enzyme (wet, containing 50430% of water) are added. It is reacted at 30° C. for 16.20 hours, and the conversion rate is tested. Test results are shown in Table 8.
Method 1:
The remaining steps are the same as Method 1 in Embodiment 2, except that the enzymes are different.
Method 2:
1-2 mL of 0.1 M PB (PH 7.5) is used to wash 1 g of an amino resin, and 4 mL of 0.1 M PB (PH 7.5) is used to resuspend; and glutaraldehyde aqueous solution with a concentration of 25%˜50% is dropwise added to resuspension so that a final concentration of glutaraldehyde is 2%. It is incubated for 1 hour at 20′C with gentle shaking, then filtered and washed with 0.1 M PB (pH 7.5) for 3 times.
4 mL of enzyme solution containing 50˜100 mg of a protein (only AADH) is added to the glutaraldehyde-activated resin, and after being incubated at 20˜25′C with gentle shaking, it is filtered and washed with 0.1 M phosphate buffer (pH 7.5) for 3 times. After being resuspended with 0.1 M PB (pH 7.0-7.5). PEI solution (final concentration 2%) is added, and 20-50 mg of GDH/FDH is added, after being incubated at 20-25′C with gentle shaking, it is filtered and washed with 0.1 M PB (pH 7.5) for 3 times.
The activity of AADH and FDH co-immobilized enzyme is detected by reactions of the following substrates:
5 mL of 0.1 M Tris-Cl buffer (pH 8.0-9.0) is added to a 10 mL reactor, and then 100 mg of the substrate 3 or 4 or 1, and 108 mg of an ammonium chloride are added, pH is adjusted to 7.5-8.0, and then 10-50 mg of NAD+, 80 mg of an ammonium formate and 100 mg of the co-immobilized enzyme are added. After it is reacted at 30° C. for 16-20 hours, a conversion experiment is performed.
An activity detection method of AADH and FDH co-immobilized enzyme is as follows:
5 mL of 0.1 M Tris-Cl buffer (pH 8.0-9.0) is added to a 10 mL reactor, and then 100 mg of the substrate 5 or H or 7, and 108 mg of an ammonium chloride are added, pH is adjusted to 7.5-8.0, and then 10-50 mg of NAD. 150 mg of glucose and 100 mg of the co-immobilized enzyme are added. After it is reacted at 30° C. for 16-20 hours, a conversion experiment is performed. Detection results are shown in Table 9.
Except that the enzymes are different, the remaining steps of Methods 1 and 2 are the same as Embodiment 5.
The activity of KRED and FDH co-immobilized enzyme is detected by reactions of the following substrate 5 or 6:
3 mL of 0.1 M PB (pH 7.0-8.0) is loaded in a 10 ml reactor, and then 100 mg of the substrate 5 or 6 is added, and 10-50 mg of NAD(P)+, 80 mg of an ammonium formate and 100 mg of the co-immobilization enzyme are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested.
The activity of KRED and GDH co-immobilized enzyme is detected by the following reaction:
3 mL of 0.1 M PB (pH 7.0-8.0) is loaded in a 10 mL reactor, and then 100 mg of the substrate 5 or 6 is added, and 10-50 mg of NAD(P)+. 120 mg of glucose and 100 mg of the co-immobilized enzyme are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested.
Test results are shown in Table 10.
Except that the enzymes are different, the remaining steps of Methods 1 and 2 are the same as Embodiment 5.
The activity of ERED and FDH co-immobilized enzyme is detected by reactions of the following substrate 7:
3 mL of 0.1 M PB (pH 7.0-8.0) is loaded in a 10 mL reactor, and then 100 mg of the substrate 7 is added, and 10-50 mg of NAD(P)+, 80 mg of an ammonium formate and 100 mg of the co-immobilization enzyme are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested.
The activity of ERED and GDH co-immobilized enzyme is detected by the following reaction:
3 mL of 0.1 M PB (pH 7.0-8.0) is loaded in a 10 ml reactor, and then 100 mg of the substrate 7 is added, and 10-50 mg of NAD(P)+, 120 mg of glucose and 100 mg of the co-immobilized enzyme are added. It is reacted at 30° C. for 16-20 hours, and the conversion rate is tested.
Test results are shown in Table 11.
Except that the enzymes are different, the remaining steps of Methods 1 and 2 are the same as Embodiment 5.
The activity of IRED and FDH co-immobilized enzyme is tested by using the following substrate 8 and according to the following method:
2 mL of 0.1 M PB (pH 7.0-0) is loaded in a 10 mL reactor, and then 100 mg of the above substrate is added, and 10-50 mg of NAD(P)+, 60 mg of an ammonium formate and 100 mg of the co-immobilization enzyme are added. It is reacted at 3° C. for 16-20 hours, and the conversion rate is tested.
The activity of IRED and GDH co-immobilized enzyme is tested by the following method:
3 mL of 0.1 M PB (pH 7.0-8.0) is loaded in a 10 mL reactor, and then 100 mg of the substrate is added, and 10.50 mg of NAD(P)+, 100 mg of glucose and 100 mg of the co-immobilized enzyme are added. After itis reacted at 30° C. for 16-20 hours, a conversion experiment is performed.
Test results are shown in Table 12
According to Method 2 in Embodiment 2, transaminase TA-Bt, coenzyme LDH and FDH are co-immobilized on a carrier LX1000HA, and the obtained co-immobilized enzyme is filled in a column-shaped reactor with a column volume of 10 mL, and the amount of the immobilized enzyme is 5.9 g.
500 g of the substrate 5, and 108 mg of an ammonium chloride are dissolved with 4.5 L of PB buffer (0.1 M, pH 8.0), pH is adjusted to 7.5-8.0 with sodium hydroxide solution, then 10-50 mg of NAD+, and 80 mg of an ammonium formate are added, and finally, the volume is fixed to 5 L with the PB buffer.
The flow rate is set to 0.1 mL/min, namely the retention time is 100 min, and a continuous reaction is performed. Effluent liquid at an outlet is detected for the conversion rate, and the conversion rate is greater than 98%. After 300 h of continuous operation, the conversion rate is not decreased. After 348 h of operation, the conversion rate is decreased by 88.4%. See Table 13 for details.
The co-immobilized enzyme same as Embodiment 9 is used. 50 g of the co-immobilized enzyme of transaminase TA-Bt and coenzyme LDH and FDH is added to a 200 mL reactor, and 150 mL of a phosphate buffer is added.
500 g of the substrate 5, and 108 mg of an ammonium chloride are dissolved with 4.5 L of the PB buffer (0.1 M, pH 8.0), pH is adjusted to 7.5-8.0 with sodium hydroxide solution, then 10-50 mg of NAD+, and 80 mg of an ammonium formate are added, and finally the volume is fixed to 5 L with the PB buffer.
The substrate is continuously added to a continuous stirred tank at a rate of 0.8 mL/min (namely the retention time is 250 min), and a reaction system is extracted at an outlet at the same flow rate (a filter head is added at a tail end of a pipeline, as to prevent the immobilized enzyme from being extracted). Under this condition, the conversion rate may reach more than 92%, and the conversion rate is basically not reduced after continuous operation for 400 h. Results are shown in Table 14.
Same as Embodiment 1, after the amino carrier is activated, the amount of a protein added per gram of the carrier is Investigated, the co-immobilization of transaminase TA-Bt and coenzyme lactate dehydrogenase LDH thereof is taken as an example, the different amounts of the protein is added, as to detect a protein load amount and reaction reuse times. Results are shown in Table 15.
Same as Embodiment 4, the co-immobilization of monooxygenase CHMO-Rs and coenzymes ADH and GDH thereof is taken as an example, the different amounts of the protein is added, as to detect a protein load amount and reaction reuse times, and results are shown in Table 16. The results show that the range of the protein loaded per gram of the selected carrier is 50˜200 mg, and the protein loading rate is 50%˜100%.
It may be seen from data in Tables 15 and 16 that for most of the carriers, while the protein loading amount is 50-100 mg, the protein loading rate is above 90%. In the case of the same protein loading rate, the reuse times of the co-immobilized enzymes formed by the different carriers are different. It may be seen from this table that these carriers LX1000HA, LX1000EPN and ECR8409 have the better immobilized enzyme activity and stability.
Same as Embodiment 2, and Method 2 (two-step method), the co-immobilized enzyme of transaminase TA-Mt and coenzyme lactate dehydrogenase LDH and ammonium formate dehydrogenase FDH thereof is prepared, after the mixed enzyme of TA-Bt and LDH is combined to with the carrier, the different amounts of PEI is added, and then the second coenzyme FDH is added, to investigate the concentration range of PEI. Results are shown in Table 17.
It may be seen from data in Table 17 that the effect of the concentration of PEI is basically the same in the range of 1%˜5%, and beyond this range, the stability of the immobilized enzyme is decreased.
It may be seen from the above description that the above embodiments of the present invention achieve the following technical effects: the present application achieves the co-immobilization of these main enzymes and the coenzymes thereof by co-immobilizing the above main enzymes and the coenzymes thereof on the amino resin carrier, thereby it is beneficial to improve the activity of the enzyme and the recycling efficiency.
The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvement and the like made within the spirit and principle of the present invention shall be included within a scope of protection of the present invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/122447 | 12/2/2019 | WO |
