WO2021142617A1

WO2021142617A1 - Immobilized enzyme, and preparation method therefor and use thereof

Info

Publication number: WO2021142617A1
Application number: PCT/CN2020/072008
Authority: WO
Inventors: 洪浩; 詹姆斯•盖吉; 肖毅; 威廉姆斯罗杰斯卡•维亚撒•; 崔瑜霞; 张娜; 赵佳东; 高妍妍
Original assignee: 吉林凯莱英医药化学有限公司
Priority date: 2020-01-14
Filing date: 2020-01-14
Publication date: 2021-07-22

Abstract

Disclosed are an immobilized enzyme, and a preparation method therefor and the use thereof. Specifically, the preparation method comprises the following steps: using polyethylene glycol to modify a crosslinker to obtain a polyethylene-glycol-modified crosslinker; precipitating a free enzyme; and then adding the polyethylene-glycol-modified crosslinker thereto for cross-linking immobilization to obtain an immobilized enzyme. The enzyme is immobilized by means of using the polyethylene-glycol-modified crosslinker, and upon immobilization, the carrier-free immobilized enzyme has a better stable structure and a stronger mechanical stability.

Description

Immobilized enzyme, its preparation method and application

Technical field

The present invention relates to the technical field of biocatalysis, in particular to an immobilized enzyme, its preparation method and application.

Background technique

Biocatalysis has become one of the most promising and advantageous technologies for the green synthesis of important drug parts, structural units and intermediates, especially providing a unique alternative route for chiral synthesis. The increasing interest of synthetic chemists in continuous flow synthesis is now drawing attention to biotransformation. The increasing use of enzymes as catalysts in industrial processes has led to an increased demand for enzymes in immobilized form because it provides unique process and cost advantages. Immobilized enzymes commonly referred to as "biocatalysts" are widely used in industrial organic synthesis and biotransformation.

Biocatalysts can be produced using whole live cells, dead cells, crude enzymes, or purified enzymes, depending on the type and application of the enzyme. The expandable production of enzymes and advances in protein engineering make it possible for biocatalysts to achieve commercial applications and have good economic value.

The emergence of a new immobilization platform makes immobilized enzymes perfectly integrated in continuous flow biocatalysis. The discovery and evolution of new high-efficiency enzymes, new reverse synthesis methods that emphasize biocatalysis, reduced recombinant protein costs and enzyme immobilization strategies are all good foundations for the application of biocatalysis in continuous synthesis.

Conceptually, the immobilization used in the preparation of immobilized enzymes mainly includes physical adsorption, carrier binding, embedding and cross-linking methods. Among them, physical adsorption depends on the affinity between the enzyme and the carrier. Although simple, the interaction is very weak, and the enzyme is easily desorbed and lost; carrier binding involves connecting the enzyme to the water-insoluble carrier through ionic or covalent bonds. This method Having the disadvantage of partially inactivating enzyme molecules due to strong chemical bonds, the target enzyme also requires a more purified form, which ultimately increases the cost of catalyst production.

In recent years, among various immobilization technologies, the immobilization of self-crosslinked enzymes without a carrier has become more and more important. In the past few decades, carrier-free cross-linking enzyme technology has become an attractive method to enhance enzyme stability [Roy et al, 2004]. In this technology, the enzyme is cross-linked by a bifunctional cross-linking agent to form a stable heterogeneous biocatalyst, which is significantly better than conventional immobilized enzymes. Generally, immobilized enzymes using various supports are "support-bound biocatalysts", and the presence of most non-catalytic supports (about 90-99% of the total mass) leads to the dilution of their volumetric activity. The cross-linking enzyme called "unsupported biocatalyst" exhibits very high catalytic activity per unit volume, thereby maximizing volumetric productivity and space-time yield [Sheldon et al. 2005].

At present, there have been reports about carrier-free methods for immobilizing lipase, penicillin acylase, protease and aminoacylase, etc. However, in other sensitive enzymes or newly emerging enzymes such as monooxygenase, ammonia lyase, ene There are few reports on the application of reductase, imine reductase, transaminase, ketoreductase, amino acid dehydrogenase and nitrilase into immobilized enzymes.

In addition, the disadvantages of current carrier-free immobilized enzymes include: without solid support, carrier-free immobilized enzymes cannot be fully expanded to mass production because they exhibit low mechanical stability to shear stress and harsh stirring conditions. And may also cause filtering problems. When carrying out biotransformation in batch stirred reaction, STY (space-time yield) is quite low, which will lead to relatively low productivity, while continuous flow reaction can significantly improve STY, resulting in higher productivity and controllable Sexuality and environmental friendliness.

Therefore, continue to develop a carrier-free immobilized enzyme with strong mechanical stability.

Summary of the invention

The present invention aims to provide an immobilized enzyme, its preparation method and application, so as to improve the mechanical stability of the immobilized enzyme.

In order to achieve the above objective, according to one aspect of the present invention, a method for preparing an immobilized enzyme is provided. The preparation method includes the following steps: using polyethylene glycol to modify the cross-linking agent to obtain a polyethylene glycol-modified cross-linking agent; co-precipitating free enzymes and adding the polyethylene glycol-modified cross-linking agent for cross-linking and fixing Obtain immobilized enzyme.

Further, the step of using polyethylene glycol to modify the cross-linking agent includes: mixing the cross-linking agent with polyethylene glycol to obtain a polyethylene glycol-modified cross-linking agent; preferably, the polyethylene glycol is PEG 2000 to PEG 6000 ; Preferably, the mass ratio of polyethylene glycol to the cross-linking agent is 1:5 to 5:1, more preferably, the mass ratio is 1:2 to 3:1; preferably, the cross-linking agent is glutaric acid Aldehydes or aldehyde-based dextran; preferably, mixing at room temperature for 3-20 hours.

Further, the enzyme is ω-transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine dehydrogenase or nitrilase.

Further, precipitating the free enzyme includes: dripping the precipitant into the solution containing the free enzyme, or dripping the solution containing the free enzyme into the precipitating agent; preferably, the operation of precipitating the free enzyme includes dropping the precipitant Add to the free enzyme-containing solution, or drop the free enzyme-containing solution into the precipitation agent and stir.

Further, the precipitating agent is one or more selected from the group consisting of ammonium sulfate, ethanol, n-propanol, isopropanol, acetonitrile, PEG 2000-6000 and acetone; wherein, as the precipitating agent, the concentration of ammonium sulfate 20～70g/100mL, preferably 30～60g/100mL; the concentration of ethanol is 60～90g/100mL, preferably 70～90g/100mL; the concentration of n-propanol is 50～90g/100mL, preferably 60～80g /100mL; the concentration of acetonitrile is 50～90g/100mL, preferably 60～70g/100mL; the concentration of acetone is 50～90g/100mL, preferably 60～80g/100mL; the concentration of PEG 2000-6000 is 10～60g/ 100mL, preferably 20-40g/100mL.

Further, the concentration of the polyethylene glycol-modified cross-linking agent in the cross-linking reaction system of the enzyme precipitation and the polyethylene glycol-modified cross-linking agent is 20 mM to 300 mM, more preferably 50 to 250 mM; preferably, the reaction The temperature of the system is 2-30°C; preferably, the reaction system is stirred for 0.5-20 hours and then suction filtered, and the immobilized enzyme obtained is washed with a buffer solution. Preferably, the buffer solution is a phosphate buffer solution.

According to another aspect of the present invention, an immobilized enzyme is provided. The immobilized enzyme is prepared by any one of the above-mentioned preparation methods.

Further, the immobilized enzyme is an immobilized enzyme of ω-transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine dehydrogenase or nitrilase .

Further, the ω-transaminase is a transaminase derived from Chromobacterium violaceum DSM30191 or a transaminase derived from Arthrobacter citreus, or a transaminase derived from B. thuringiensis; preferably, the ketoreductase is a ketoreductase derived from Acetobacter sp.CCTCC M209061, Or a ketoreductase derived from Candida macedoniensis AKU4588; preferably, the monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans;

Preferably, the ammonia lyase is an ammonia lyase derived from Aspergillus niger CBS 513.88 and an ammonia lyase derived from Solenostemon scutellarioides; preferably, the ene reductase is an ene reductase derived from Saccharomyces cerevisiae and an ene reductase derived from Chryseobacterium sp.CA49 Preferably, the imine reductase is an imine reductase derived from Streptomyces sp or Bacillus cereus; preferably, the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus or a leucine dehydrogenase derived from Bacillus sphaericus Phenylalanine dehydrogenase; preferably, the nitrilase is a nitrilase derived from Aspergillus niger CBS 513.88 and a nitrilase derived from Neurospora crassa OR74A. Preferably, the transaminase derived from Chromobacterium violaceum DSM30191 is a mutant with SEO ID NO.2 sequence or SEO ID NO.3; the transaminase derived from Arthrobacter citreus has SEO ID NO.5 sequence or SEO ID NO.6 sequence The ketoreductase derived from Acetobacter sp.CCTCC M209061 is a mutant with the SEO ID NO.8 sequence or the SEO ID NO.9 sequence; the cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1 has SEO ID NO.11 sequence or SEO ID NO.12 sequence mutant; Cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 is a mutant with SEO ID NO.14 sequence or SEO ID NO.15 sequence.

According to another aspect of the present invention, there is provided an application of the above-mentioned immobilized enzyme in an aqueous buffer reaction system or an organic solvent reaction system.

Further, the aqueous buffer reaction system or the organic solvent reaction system is used in a packed bed reactor or a continuous stirred tank reactor.

By applying the technical scheme of the present invention, a polyethylene glycol-modified crosslinking agent is used to immobilize the enzyme, and after immobilization, it has a better stable structure and stronger mechanical stability as a carrier-free immobilized enzyme.

Detailed ways

It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other if there is no conflict. The present invention will be described in detail below in conjunction with embodiments.

Noun explanation and abbreviations:

SCE: Self-Cross Linked Enzyme, self-crosslinking enzyme.

GA: Glutaraldehyde, glutaraldehyde.

DA: Dextran aldehyde, aldehyde dextran

PEG: Polyethylene Glycol, polyethylene glycol.

PVA: Polyvinyl Alchohol, polyvinyl alcohol.

CSTR: Continuous stirred tank reactor, continuous stirred tank reactor.

PBR: Packed bed reactor, packed bed reactor.

According to a typical embodiment of the present invention, a method for preparing an immobilized enzyme is provided. The preparation method of the immobilized enzyme includes the following steps: using polyethylene glycol to modify the crosslinking agent to obtain the polyethylene glycol modified crosslinking agent; co-precipitating the free enzyme and adding the polyethylene glycol modified crosslinking agent at the same time The immobilized enzyme is obtained by cross-linking and immobilization.

In a typical embodiment of the present invention, the step of using polyethylene glycol to modify the crosslinking agent includes: diluting the crosslinking agent with water, adding polyethylene glycol and mixing at room temperature to obtain a polyethylene glycol modified crosslinking agent . After such modification, the cross-linking process is also improved, has a better buffer, and can achieve higher activity recovery and stability. Preferably, the concentration of the crosslinking agent after dilution with water is 2-10% w/v; the polyethylene glycol is PEG 2000 to PEG 6000.

Typically, the crosslinking agent is glutaraldehyde or aldodextran. It is well known that glutaraldehyde (GA), which is widely used as a linker, can cause enzyme denaturation. In the present invention, the cross-linking process is also significantly improved, achieving higher activity recovery and stability. GA is used together with PEG or PEI or used for enzyme activity protection to improve enzyme activity and stability. The aldehyde group of glutaraldehyde is covalently bonded to the hydroxyl group of PEG or the amino group of PEI, and finally forms a network structure dispersed with aldehyde groups and amino groups/hydroxyl groups. Each functional group in this network structure is covalently connected to the enzyme protein. It is combined in various ways, such as interaction, hydrogen bonding, ionic interaction, and hydrophobic interaction, instead of just covalent bonding like glutaraldehyde, which avoids only covalent bonding that destroys enzyme activity. Preferably, it is mixed at room temperature for 3-20 hours.

According to a typical embodiment of the present invention, the enzyme is ω-transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine dehydrogenase or nitrile hydrolysis Enzyme. The immobilized transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine dehydrogenase or nitrilase produced by the method of the present invention are used as independent enzymes The catalyst or as a co-immobilized fusion enzyme catalyst exhibits higher activity and mechanical stability, and is repeatedly used for >10 cycles in each model reaction, showing enhanced operational stability. When the immobilized enzymes of these enzymes are used for substrate conversion, under the best fixed strip, that is, polyethylene glycol or ammonium sulfate is used as a precipitant, and polyethylene glycol-modified glutaraldehyde is used as a cross-linking agent. The ratio of ethylene glycol to glutaraldehyde is PEG6000:GA=2:1, and the concentration of the polyethylene glycol modified crosslinker in the reaction system of enzyme precipitation and polyethylene glycol modified crosslinker for crosslinking is 200mM, the temperature of the reaction system was 20°C; after stirring the reaction system for 60 minutes, the immobilized enzyme was filtered with suction and washed with a buffer solution to obtain an activity of more than 30%, and there was no obvious activity drop after 10 cycles of use. Among them, the mutant of the transaminase TA-Cv uses acetonitrile as a precipitant, and PEG-modified glutaraldehyde (PEG6000:GA=2:1) is used as a cross-linking agent. The cross-linking agent is used in enzyme precipitation and polyethylene glycol-modified cross-linking. The concentration of the reagent in the cross-linking reaction system is 200 mM, and the cross-linking is 60 minutes. The immobilized enzyme obtained can be reused 21 times in the aqueous phase reaction and 18 times in the organic phase reaction. During the precipitation, the precipitation agent is added dropwise to the enzyme solution, or the enzyme solution is added dropwise to the precipitation agent acetonitrile, both of which can achieve the desired effect.

According to a typical embodiment of the present invention, precipitating the free enzyme includes: adding the precipitant dropwise to the solution containing the free enzyme, or adding the solution containing the free enzyme dropwise to the precipitating agent; preferably, adding the free enzyme The precipitation operation is carried out at 2～10℃; under this condition, the free enzyme can be effectively precipitated and cross-linked. More preferably, the free enzyme precipitation operation includes dropping the precipitation agent into the solution containing the free enzyme, or adding the solution containing the free enzyme dropwise to the precipitation agent and stirring for 1 to 2 hours.

Typically, the precipitating agent is one or more selected from the group consisting of ammonium sulfate, ethanol, n-propanol, isopropanol, acetonitrile, PEG 2000-6000 and acetone; among them, as the precipitating agent, the concentration of ammonium sulfate 20～70g/100mL, preferably 30～60g/100mL; the concentration of ethanol is 60～90g/100mL, preferably 70～90g/100mL; the concentration of n-propanol is 50～90g/100mL, preferably 60～80g /100mL; the concentration of acetonitrile is 50～90g/100mL, preferably 60～70g/100mL; the concentration of acetone is 50～90g/100mL, preferably 60～80g/100mL; the concentration of PEG 2000-6000 is 10～60g/ 100mL, preferably 20-40g/100mL. That is to say, various precipitating agents in this application can be used alone or in combination with other co-solvents and co-precipitating agents. When PEG is used as a precipitant, the enzymes listed above all have enhanced activity and stability. Preferably, the concentration of the polyethylene glycol-modified cross-linking agent in the reaction system where the enzyme precipitation and the polyethylene glycol-modified cross-linking agent are cross-linked is 20 mM to 200 mM; more preferably, the temperature of the reaction system is 2 to 200 mM. 20°C; after stirring the reaction system for 30-60 minutes, the immobilized enzyme is filtered with suction and washed with a buffer solution. Preferably, the buffer solution is a phosphate buffer solution.

According to a typical embodiment of the present invention, an immobilized enzyme is provided. The immobilized enzyme is prepared by any of the above-mentioned preparation methods. The immobilized enzyme can be the immobilization of ω-transaminase, lipase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine dehydrogenase or nitrilase Enzymes, etc., preferably, the ω-transaminase is D-amino acid transaminase derived from B. thuringiensis or pyruvate transaminase derived from Vibrio fluvialis strain JS17, preferably, the ketoreductase is a ketoreductase derived from Sporobolomyces salmonicolor, or a source The ketoreductase of Candida macedoniensis AKU4588; preferably, the monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans; preferably, ammonia The lyase is an ammonia lyase derived from Aspergillus niger CBS 513.88 and an ammonia lyase derived from Solenostemon scutellarioides; preferably, the ene reductase is an ene reductase derived from Bacillus cereus and an ene reductase derived from Bacillus sphaericus; preferably , Imine reductase is imine reductase derived from Streptomyces sp or Bacillus cereus; preferably, the amino acid dehydrogenase is leucine dehydrogenase derived from Bacillus cereus or phenylalanine dehydrogenase derived from Bacillus sphaericus Enzyme; Preferably, the nitrilase is a nitrilase derived from Aspergillus niger CBS 513.88 and a nitrilase derived from Neurospora crassa OR74A.

According to a typical embodiment of the present invention, the application of the above-mentioned immobilized enzyme in an aqueous buffer reaction system or an organic solvent reaction system is provided. Typically, the aqueous buffer reaction system or the organic solvent reaction system is used in a packed bed reactor or a continuous stirred tank reactor. Due to good mechanical stability and operational stability, these fixed Huamei can be used in PBR (packed bed reactor) without high back pressure, and can also be applied to CSTR (continuous stirred tank reactor) without filtration problems.

According to a typical embodiment of the present invention, a method for preparing an immobilized enzyme is provided. The preparation method includes the following steps: precipitating the free enzyme, and then adding a cross-linking agent for cross-linking and fixing to obtain an immobilized enzyme. The enzymes are ω-transaminase, lipase, ketoreductase, cyclohexanone monooxygenase, and ammonia cleavage. Enzymes, ene reductase, imine reductase, leucine dehydrogenase or nitrilase immobilized enzymes. The present invention further provides an immobilized form of the above-mentioned enzyme. In a typical embodiment of the present invention, precipitating the free enzyme includes: dropping the precipitant into the solution containing the free enzyme, or dropping the solution containing the free enzyme into the precipitating agent; preferably, adding the free enzyme The precipitation operation is carried out at 2-10°C; preferably, the free enzyme precipitation operation includes dropping the precipitation agent into the solution containing the free enzyme, or adding the solution containing the free enzyme dropwise to the precipitation agent and stirring for 1 to 2 hours Preferably, the precipitating agent is one or more selected from the group consisting of ammonium sulfate, ethanol, n-propanol, isopropanol, acetonitrile, PEG 2000-6000 and acetone.

According to a typical embodiment of the present invention, an immobilized enzyme is provided, and the immobilized enzyme is prepared by the above preparation method.

The following will further illustrate the beneficial effects of the present application in conjunction with examples and comparative examples.

The enzymes used in the following examples and their sources are shown in Tables 1-1 to 1-5 below (mutants are shown in the lower part of Tables 1-2 to 1-5, the rest are wild-type, and the specific sequences are shown in GeneBank).

Table 1-1:

Table 1-2:

Mutant sequence

Table 1-3:

Table 1-3:

Table 1-4:

Table 1-5:

The chemical process of the reaction involved in the above enzymes is briefly described as follows:

_{R, R 1} and R ₂ in the above reaction formula may be independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aralkyl, substituted or unsubstituted The heterocyclic group, substituted or unsubstituted heterocycloalkyl group, or R1 and the heterocyclic ring to which it is connected form a condensed ring system.

Example 1

Enzyme molecule self-crosslinking covalent immobilization (SCE)

Dissolve 3mg/mL PLP (pyridoxal 5'-phosphate) into 10mL transaminase TA-Cv enzyme solution, slowly add 20mL acetonitrile to the enzyme solution under ice-water bath stirring, or slowly drop the enzyme solution into 20mL acetonitrile, After the addition, after stirring for 60 minutes, add 25% or 50% glutaraldehyde solution (to make the final concentration 200mM), stir in an ice-water bath for 30-40 minutes, centrifuge or filter, wash the precipitate 3 times with phosphate buffer, and store at 4°C , Can be directly applied to the water phase reaction. Alternatively, the cross-linked enzyme aggregates are freeze-dried, and the freeze-dried powder of the cross-linked enzyme aggregates obtained after freeze-drying can be used in the reaction between the aqueous phase and the organic phase.

Example 2

Optimized self-crosslinking and covalent immobilization of enzyme molecules (SCE)

Preparation of PEG-modified GA (PEG-GA): Dilute glutaraldehyde (GA) with water to a final concentration of 20% (w/v, 20g/100mL), and add PEG 2000 or PEG 6000, which is PEG and GA The mass ratio is 2:1, mixed at room temperature for 1 to 3 hours, and kept until use.

3mg/mL PLP (pyridoxal 5'-phosphate) was dissolved in 10mL enzyme solution, acetonitrile was added to the enzyme solution under ice-water bath stirring, after the addition, after stirring for 1-2h, then add PEG-modified GA (GA final Concentration 200mM), stirred in an ice water bath for 30-40 minutes, centrifuged or filtered, washed the precipitate 3 times with phosphate buffer, stored at 4°C, and can be directly applied to the water phase reaction. Or freeze-dry the cross-linked enzyme aggregates, and the freeze-dried powder of the cross-linked enzyme aggregates obtained after freeze-drying can be used in the reaction between the aqueous phase and the organic phase.

Example 3

Aldehydated dextran (DA) is used as cross-linking agent to prepare immobilized enzyme

In 10mL ketoreductase KRED-Ac enzyme solution, add about 5g solid ammonium sulfate under ice bath conditions, after addition, stir for 60min, then add aldehyde dextran (to make the final concentration 100mM), stir for 30-40min in ice water bath After centrifugation or filtration, the precipitate is washed 3 times with phosphate buffer and stored at 4°C. It can be directly applied to the water phase reaction. Alternatively, the cross-linked enzyme aggregates are freeze-dried, and the freeze-dried powder of the cross-linked enzyme aggregates obtained after freeze-drying can be used in the reaction between the aqueous phase and the organic phase.

Example 4

Optimized Aldehydated Dextran Crosslinker for Preparation of Immobilized Enzyme

Preparation of PEG-modified DA (PEG-DA): Dilute glutaraldehyde (GA) with water to a final concentration of 20% w/v, and add PEG 2000 or PEG 6000, so that the mass ratio of PEG to DA is 3: 1. Mix at room temperature for 1 to 3 hours, keep it until use.

3mg/mL PLP (pyridoxal 5'-phosphate) is dissolved in 10mL enzyme solution, acetonitrile is added to the enzyme solution under ice-water bath stirring, after the addition, after stirring for 1-2h, add PEG-modified DA (DA final Concentration 100mM), stirred in an ice water bath for 30-40 minutes, centrifuged or filtered, washed the precipitate 3 times with phosphate buffer, and stored at 4°C. It can be directly applied to the water phase reaction. Or freeze-dry the cross-linked enzyme poly, and the freeze-dried powder of cross-linked enzyme aggregates obtained after freeze-drying can be used in the reaction of the aqueous phase and the organic phase.

Example 5

The temperature and time of the PEG modified crosslinking agent were investigated, and the specific parameters are shown in Table 2.

Table 2

Example 6

Transaminase self-crosslinking covalently immobilized enzyme water phase reaction test

In a 10mL reaction flask, add 0.3mL MeOH, dissolve 0.1g of the above main raw material 1 or main raw material 2, and add 4eq isopropylamine hydrochloride and 1.0mg PLP (5'-pyridoxal phosphate), plus 0.1M PB 7.0 until the final volume of the reaction solution is 1 mL, then add 5 mg of enzyme powder or cross-linked enzyme aggregate wet enzyme or cross-linked enzyme aggregate lyophilized powder prepared from 10 mg of enzyme powder, and stir at 30°C for 16-20 hours. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 3, Table 4):

Table 3. Overview of water phase reaction of transaminase TA-Cv self-crosslinking immobilized enzyme

Table 4. Overview of water phase reaction of transaminase TA-Ac self-crosslinking immobilized enzyme

Table 5. TA-Bt cross-linked enzyme aggregates water phase reaction test results

Example 7

Self-crosslinked covalently immobilized enzyme organic phase reaction test

In a 10mL reaction flask, add 1mL of water-saturated methyl tert-butyl ether, then add 100mg of the main raw material 1 and 4eq of isopropylamine, and then add 20mg of enzyme or a cross-linked enzyme aggregate lyophilized powder prepared from 20mg of enzyme, at 30℃ Stir for 16h. The conversion rate of the system was detected by HPLC, and the reaction data is as follows (Table 6)

Table 6. Overview of the organic phase reaction of TA-Cv self-crosslinked immobilized enzyme

Example 8

Self-crosslinking covalently immobilized enzyme water phase-organic phase two-phase reaction test

In a 10 mL reaction flask, add 1 mL of water-saturated methyl tert-butyl ether, then add 100 mg of the main raw material and 4 eq of isopropylamine hydrochloride, then add 5 mg of enzyme or cross-linking enzyme prepared from 20 mg of enzyme, and stir at 30°C for 16 hours. The conversion rate of the system was detected by HPLC, and the reaction data is as follows (Table 7)

Table 7. Overview of the two-phase reaction of TA-Ac self-crosslinked immobilized enzyme aqueous phase-organic phase

Example 9

Water-phase reaction test of ketoreductase self-crosslinked immobilized enzyme aggregates

The preparation method of the cross-linking enzyme is the same as in Examples 1 to 4

In a 10mL reaction flask, add 0.5mL of isopropanol (IPA), dissolve 0.1g of the main raw material 3 or 4, and add 0.5mL 0.1M PB 7.0 and 1mg NAD+, then add 5mg enzyme or immobilized prepared from 20mg enzyme Enzyme, stir at 30°C for 16-20h. The conversion rate of the system was tested by GC, and the conversion rate of free enzyme was >99%. The reaction data of immobilized enzyme is shown in Table 8.

Table 8. Overview of water phase reaction of ketoreductase covalently immobilized enzyme

Example 10

Organic phase reaction of ketoreductase self-crosslinking immobilized enzyme

In a 10 mL reaction flask, add 1 mL of water-saturated methyl tert-butyl ether, then add 100 mg of the main raw material 3 and 0.1 mL of isopropanol, and then add 100 mg of free enzyme freeze-dried powder or 100 mg of enzyme powder to prepare cross-linked enzyme aggregates Lyophilized powder (PEG6000 as precipitant, PEG6000:GA=2:1 as crosslinking agent), stirred at 30°C for 16h. The conversion rate of the system was detected by HPLC, and the results are shown in Table 9.

Table 9

Example 11

Aqueous Reaction Activity Test of Amino Acid Dehydrogenase GP Covalently Immobilized Enzyme Aggregate

Add 5mL 0.1M Tris-Cl buffer (pH 8.0-9.0) into a 10mL reaction flask, then add 100mg of the main raw material 5, 6, or 7,108mg of ammonium chloride, adjust the pH to pH 7.5 to 8.0, and then add 10 The raw material 50mg NAD+, and 5mg GDH, and 10mg AADH or immobilized AADH made of 30mg free enzyme. After reacting at 30°C for 16-20 hours, a conversion test was performed. The conversion rate of free enzyme is 99%.

See Table 10 for an overview of the water phase reaction of amino acid dehydrogenase GP covalently immobilized enzyme.

Table 10

Example 12

Enzyme self-crosslinking covalently immobilized enzyme water phase reaction test

In a 10 mL reaction flask, add 50 mg of the above-mentioned main raw material 8, and add 80 mg of ammonium formate, 10 mg of FDH, and ^{0.6 mg of NAD +} and NADP ⁺ . Add 0.1M PB 7.5 to the final volume of the reaction solution to 2.4 mL, then add 10 mg Enzyme or cross-linked enzyme aggregate prepared from 30 mg of enzyme, wet enzyme or cross-linked enzyme aggregate, stirred at 30°C for 16-20h. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 11):

Table 11. Overview of water phase reaction of ene reductase self-crosslinking immobilized enzyme

Example 13

Aqueous reaction test of ammonia lyase self-crosslinking covalently immobilized enzyme

In a 20mL reaction flask, add 0.1g of the above-mentioned main raw materials and 2mL of purified water and mix well, then add 4mL of 4M ammonium carbamate, and then add 10mg of enzyme or cross-linked enzyme aggregate prepared from 30mg of enzyme. Wet enzyme or cross-linked enzyme aggregates Body, stirring at 30°C for 40-48h. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 12):

Table 12. Aqueous reaction overview of ammonia lyase self-crosslinking immobilized enzyme

Example 14

Aqueous reaction test of imine reductase self-crosslinking covalently immobilized enzyme

In a 10mL reaction flask, add 0.1g of the above-mentioned main raw materials, ^{add 0.36g glucose, NAD +} 3mg, NADP ⁺ 3mg, and GDH 5mg, add 0.1MPB 7.5 until the final volume of the reaction solution is 5mL, and add 10mg enzyme or Wet enzyme or cross-linked enzyme aggregate prepared by 30mg enzyme, stir at 30°C for 16-20h. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 13):

Table 13. Overview of water phase reaction of imine reductase self-crosslinking immobilized enzyme

Example 15

Nitrilase self-crosslinking covalently immobilized enzyme water phase reaction test

In a 10mL reaction flask, add 0.1g of the above-mentioned main raw material, and 0.1MPB 7.5 3mL, and add 10mg enzyme powder or cross-linked enzyme aggregate prepared from 30mg enzyme powder, wet enzyme or cross-linked enzyme aggregate, and stir at 30°C 16-20h. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 14):

Table 14. Overview of water phase reaction of nitrilase self-crosslinking immobilized enzyme

Example 16

Monooxygenase self-crosslinking covalently immobilized enzyme water phase reaction test

In a 10mL reaction flask, add 0.1g of the above-mentioned main raw materials, add 0.3mL of isopropanol, NADP ⁺ 10mg, and 2mg of ADH enzyme powder, add 0.1M KPB 8.0 to the final volume of the reaction solution to 4mL, and then add 20mg of enzyme Or the wet enzyme or cross-linked enzyme aggregate prepared from 40mg enzyme, stirred at 30°C for 16-20h. The conversion rate of the system was detected by HPLC, and the reaction data are as follows (Table 15):

Table 15. Overview of water phase reaction of monooxygenase self-crosslinking immobilized enzyme

Example 17

Application of Transaminase Cross-linked Enzyme Aggregate in Continuous Packed Bed Reaction

In Example 6, the transaminase TA-Cv-V1 used acetonitrile as the precipitant and PEG-modified glutaraldehyde (PEG2000:GA=3:1) as the cross-linking agent, and the obtained cross-linking enzyme was filled into a column reactor with a column volume of 100 mL In, the amount of immobilized enzyme is 60g.

500g substrate 1, dissolve with 1.5L methanol, and add 4eq of isopropylamine hydrochloride (1.8L of 6M isopropylamine hydrochloride aqueous solution) and 5g PLP, without PB buffer (0.1M, pH8.0) Set the volume to 5L.

Set the flow rate to 0.8mL/min, that is, the retention time is 125min, to carry out the continuous reaction, and the conversion rate of the effluent at the outlet end is measured. The conversion rate is >97%. Continuous operation for 424h has no reduction in the conversion rate. After 436h, the conversion rate is reduced to 90%. See Table 16.

Table 16

Example 18

Application of immobilized transaminase in continuous stirred tank reaction

TA-Ac-V2 uses ammonium sulfate as precipitant and PEG-modified glutaraldehyde (PEG6000:GA=2:1) as cross-linking agent. Add 50 g of transaminase TA-Ac-V2 cross-linking enzyme aggregates to a 200 mL reactor, and add 150 mL of phosphate buffer.

The substrate solution was continuously added to the reaction flask at a rate of 0.5 mL/min (ie retention time was 400 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 480h, the conversion rate basically does not decrease. The results are shown in Table 17.

Table 17

Example 19

Application of Ammonia Lyase Immobilized Enzyme in Continuous Stirred Tank Reaction

The ammonia lyase PAL-Ss uses ammonium sulfate as the precipitant and PEG-modified glutaraldehyde (PEG6000:GA=2:1) as the cross-linking agent, and 6 g of the obtained immobilized enzyme is filled into a 10 mL column reactor.

500g of substrate 9 was dissolved with 4.5L of ammonium carbamate aqueous solution (4M, pH 9.0-9.5).

Set the flow rate to 0.03mL/min, that is, the retention time is 330min, and the continuous reaction is carried out. The conversion rate of the effluent at the outlet end is detected. The conversion rate is 80%. Continuous operation for 400h has no reduction in the conversion rate. After 412h, the conversion rate is reduced to 76%. See Table 18.

Table 18

Example 20

Application of ketoreductase cross-linking enzyme in continuous reaction stirred tank

The ketoreductase KRED-Ac uses PEG6000 as a precipitant, and PEG-modified aldehyde-based dextran (PEG6000:DA=3:1) is used as a cross-linking agent, and 6.2 g of the obtained immobilized enzyme is filled into a 10 mL column reactor. .

100g of substrate 3 was dissolved with 0.3L of isopropanol, and 0.7L of PB buffer (0.1M, pH7.0) was added to dissolve, and then 0.1g of NAD+ was added.

Set the flow rate to 0.05mL/min, that is, the retention time is 200min, and the continuous reaction is carried out. The conversion rate of the effluent at the outlet end is measured. The conversion rate is >90%. Continuous operation for 180h has no reduction in conversion rate. After 200h operation, the conversion rate is reduced to 84%. See Table 19.

Table 19. Reaction results of KRED-Ac-V1 cross-linked enzyme aggregates in a packed bed continuous reaction

Example 21

Precipitant and precipitant concentration screening

The preparation of immobilized enzyme is the same as in Example 2. Preparation of PEG-modified GA (PEG-GA): Dilute glutaraldehyde (GA) with water to a final concentration of 20% (w/v, 20g/100mL), and add PEG 2000 or PEG 6000, which is PEG and GA The mass ratio is 2:1, mixed at room temperature for 1 to 3 hours, and kept until use.

3mg/mL PLP (pyridoxal 5'-phosphate) was dissolved in 10mL enzyme solution, and the precipitant was added to the enzyme solution under ice-water bath stirring. After the addition, after stirring for 1-2h, add PEG-modified GA (GA The final concentration is 200mM), stirred in an ice-water bath for 30-40min, then centrifuged or filtered, the precipitate is washed 3 times with phosphate buffer, stored at 4°C, and directly applied to the water phase reaction. See Table 20 for the influence of the type of precipitant and the concentration of each precipitant on the yield, enzyme activity and use times. Too low concentration of precipitant will affect the yield, and the number of repeated use will be slightly worse. Excessive concentration of organic solvent precipitant will also affect the number of repeated uses of the enzyme. When the concentration of ammonium sulfate and PEG is higher than a certain value, the continuous increase will not have much influence on the yield, enzyme activity and the number of repeated use. Considering the cost, there is no need to further increase the concentration of the precipitant.

Table 20. Influence of precipitant and concentration of precipitant on yield, enzyme activity and stability of repeated use

The above are only preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

A preparation method of immobilized enzyme is characterized in that it comprises the following steps:

Using polyethylene glycol to modify the cross-linking agent to obtain a polyethylene glycol-modified cross-linking agent;

The free enzyme is co-precipitated, and the polyethylene glycol modified cross-linking agent is added for cross-linking and fixing to obtain the immobilized enzyme.
The preparation method according to claim 1, wherein the step of using polyethylene glycol to modify the cross-linking agent comprises: mixing the cross-linking agent with polyethylene glycol to obtain the polyethylene glycol-modified cross-linking agent Agent

Preferably, the polyethylene glycol is PEG 2000 to PEG 6000;

Preferably, the mass ratio of the polyethylene glycol to the crosslinking agent is 1:5 to 5:1, and more preferably, the mass ratio is 1:2 to 3:1;

Preferably, the crosslinking agent is glutaraldehyde or aldehyde dextran

Preferably, it is mixed at room temperature for 3-20 hours.
The preparation method according to claim 1, wherein the enzyme is ω-transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, leucine Dehydrogenase or nitrilase.
The preparation method according to claim 1, wherein the precipitating the free enzyme comprises:

Drop the precipitation agent into the solution containing free enzyme, or drop the solution containing free enzyme into the precipitation agent;

Preferably, the operation of precipitating the free enzyme includes dripping the precipitating agent into the solution containing the free enzyme, or dripping the solution containing the free enzyme into the precipitating agent and stirring.
The preparation method according to claim 1, wherein the precipitating agent is one selected from the group consisting of ammonium sulfate, ethanol, n-propanol, isopropanol, acetonitrile, PEG 2000-6000 and acetone. Many; among them, as a precipitant, the concentration of ammonium sulfate is 20～70g/100mL, preferably 30～60g/100mL; the concentration of ethanol is 60～90g/100mL, preferably 70～90g/100mL; n-propanol The concentration is 50~90g/100mL, preferably 60~80g/100mL; the concentration of acetonitrile is 50~90g/100mL, preferably 60~70g/100mL; the concentration of acetone is 50~90g/100mL, preferably 60~80g/ 100mL; the concentration of PEG 2000-6000 is 10-60g/100mL, preferably 20-40g/100mL.
The preparation method according to claim 1, wherein the concentration of the polyethylene glycol-modified cross-linking agent in the reaction system of the enzyme precipitation and the polyethylene glycol-modified cross-linking agent for cross-linking is 20mM~ 300mM, more preferably 50-250mM;

Preferably, the temperature of the reaction system is 2-30°C;

Preferably, the reaction system is stirred for 0.5-20 hours and then filtered with suction, and the immobilized enzyme is washed with a buffer solution. Preferably, the buffer solution is a phosphate buffer solution.
An immobilized enzyme, characterized in that the immobilized enzyme is prepared by the preparation method according to any one of claims 1 to 6.
The immobilized enzyme according to claim 7, wherein the immobilized enzyme is ω-transaminase, ketoreductase, cyclohexanone monooxygenase, ammonia lyase, ene reductase, imine reductase, An immobilized enzyme of leucine dehydrogenase or nitrilase.
The immobilized enzyme according to claim 8, wherein the ω-transaminase is a transaminase derived from Chromobacterium violaceum DSM30191 or a transaminase derived from Arthrobacter citreus, or a transaminase derived from B. thuringiensis;

Preferably, the ketoreductase is a ketoreductase derived from Acetobacter sp.CCTCC M209061, or a ketoreductase derived from Candida macedoniensis AKU4588;

Preferably, the monooxygenase is cyclohexanone monooxygenase derived from Rhodococcus sp.Phi1, or cyclohexanone monooxygenase derived from Brachymonas petroleovorans;

Preferably, the ammonia lyase is an ammonia lyase derived from Aspergillus niger CBS 513.88 and an ammonia lyase derived from Solenostemon scutellarioides;

Preferably, the ene reductase is an ene reductase derived from Saccharomyces cerevisiae and an ene reductase derived from Chryseobacterium sp.CA49;

Preferably, the imine reductase is an imine reductase derived from Streptomyces sp or Bacillus cereus;

Preferably, the leucine dehydrogenase is leucine dehydrogenase derived from Bacillus cereus or phenylalanine dehydrogenase derived from Bacillus sphaericus;

Preferably, the nitrilase is a nitrilase derived from Aspergillus niger CBS 513.88 and a nitrilase derived from Neurospora crassa OR74A;

Preferably, the transaminase derived from Chromobacterium violaceum DSM30191 is a mutant with SEO ID NO.2 sequence or SEO ID NO.3; said transaminase derived from Arthrobacter citreus has SEO ID NO.5 sequence or SEO ID A mutant of the NO.6 sequence; the ketoreductase derived from Acetobacter sp.CCTCC M209061 is a mutant with the SEO ID NO.8 sequence or the SEO ID NO.9 sequence; the loop derived from Rhodococcus sp.Phi1 The hexanone monooxygenase is a mutant with the SEO ID NO.11 sequence or the SEO ID NO.12 sequence; the cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1 has the SEO ID NO.14 sequence or SEO ID NO.15 sequence mutant.
The application of the immobilized enzyme according to claim 7 in an aqueous buffer reaction system or an organic solvent reaction system.
The application according to claim 10, wherein the aqueous buffer reaction system or the organic solvent reaction system is used in a packed bed reactor or a continuous stirred tank reactor.