CN107988201B

CN107988201B - Preparation method of ethanol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate

Info

Publication number: CN107988201B
Application number: CN201610949314.5A
Authority: CN
Inventors: 邵雷; 陈代杰; 胡晓植; 郑昆; 张骏梁
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2016-10-26
Filing date: 2016-10-26
Publication date: 2021-06-01
Anticipated expiration: 2036-10-26
Also published as: CN107988201A

Abstract

The invention provides a preparation method of an ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate, and particularly relates to a method for preparing the co-crosslinking enzyme aggregate by using supernatant obtained after high-density fermentation, wall breaking and centrifugation as crude enzyme liquid, performing glutaraldehyde crosslinking on the ethanol dehydrogenase and the glucose dehydrogenase in the crude enzyme liquid to prepare the co-crosslinking enzyme aggregate, and increasing the enzyme activity recovery rate of the co-crosslinking enzyme aggregate to 82% by adding Tween 80.

Description

Preparation method of ethanol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate

Technical Field

The invention relates to preparation of an ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate and research on stability and reuse rate of the enzyme aggregate, belonging to the technical field of enzyme immobilization.

Background

On day 3/8/2011, the united states food and drug administration approved crizotinib, produced by the company picrorui, for the treatment of NSCLC with ALK gene rearrangement. The market demand for NSCLC approved for the treatment of ROS1 gene mutation, 3/11/2016, is increasing. During its synthesis, (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol is a key chiral intermediate. At present, (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol is synthesized by a chemical method, but a high-purity chiral compound is not easy to obtain, the synthesis process is complex, and the defects can be overcome by biosynthesis.

The current biosynthesis technology is that the chiral intermediate (S) -1- (2, 6-dichloro-3-fluorophenyl) ketone is synthesized by a fermentation broth of a strain co-expressing ethanol dehydrogenase and glucose dehydrogenase, and a coenzyme cyclic regeneration system of the ethanol dehydrogenase and the glucose dehydrogenase and NADP and NADPH, so as to catalyze 1- (2, 6-dichloro-3-fluorophenyl) ethanone, such as the references (Liuli, Zhang Jun beam, Tan, and the like).

The cross-linked enzyme aggregate is a novel enzyme immobilization technology, can realize the aggregation and immobilization of enzyme, is convenient for recycling, does not need a carrier, and has low preparation cost. Therefore, in order to effectively improve the preparation efficiency of the chiral compound and reduce the preparation cost, the skilled person is devoted to develop the immobilization technology for catalyzing 1- (2, 6-dichloro-3-fluorophenyl) ethanone to synthesize the chiral intermediate (S) -1- (2, 6-dichloro-3-fluorophenyl) enzyme.

Disclosure of Invention

The invention aims to provide a preparation method of an ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate.

In a first aspect of the present invention, there is provided a method for preparing an alcohol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate, the method comprising the steps of:

in the presence of a surfactant, the co-crosslinking reaction of alcohol dehydrogenase and glucose dehydrogenase is carried out by a crosslinking agent.

In another preferred embodiment, the surfactant is selected from the group consisting of: tween 80, and tween 60.

In another preferred example, in the method, the mass ratio of the surfactant to the substrate enzyme (alcohol dehydrogenase and glucose dehydrogenase) in the crosslinking reaction system in which the co-crosslinking reaction is carried out is 0.1 to 1.2: 0.5 to 1; preferably, the mass ratio is 0.2-1: 0.5 to 1; most preferably from about 0.4 to about 0.8: 1.

in another preferred embodiment, the substrate enzyme is defined as the sum of an alcohol dehydrogenase and a glucose dehydrogenase.

In another preferred embodiment, glutaraldehyde is used as the crosslinking agent in the crosslinking reaction.

In another preferred example, the mass ratio of glutaraldehyde to substrate enzymes (alcohol dehydrogenase and glucose dehydrogenase) in the crosslinking reaction system is 0.1-1.2: 0.5 to 1; preferably 0.2-1.2: 1; most preferably about 1: 1.

in another preferred embodiment, the surfactant: a crosslinking agent: the mass ratio of the substrate enzymes (alcohol dehydrogenase and glucose dehydrogenase) is 0.4-0.8: 0.8-1.2: 0.8-1.2.

In another preferred example, the method comprises the steps of:

(1) providing a mixture comprising an alcohol dehydrogenase and a glucose dehydrogenase;

(2) crosslinking reaction

Preparing a crosslinking reaction system, wherein the crosslinking reaction system comprises: and (2) carrying out a crosslinking reaction on the mixture containing the alcohol dehydrogenase and the glucose dehydrogenase, the crosslinking agent and the surfactant provided in the step (1) at the temperature of 0-40 ℃ to prepare the co-crosslinking enzyme aggregate containing the alcohol dehydrogenase and the glucose dehydrogenase.

In another preferred example, the step (1) includes the steps of:

co-expressing alcohol dehydrogenase and glucose dehydrogenase in genetically engineered bacteria to obtain the mixture containing alcohol dehydrogenase and glucose dehydrogenase.

In another preferred example, in the mixture containing alcohol dehydrogenase and glucose dehydrogenase, the mass ratio of alcohol dehydrogenase to glucose dehydrogenase is: 0.5-1: 0.5 to 1.

In another preferred example, the step (1) includes the steps of:

co-expressing alcohol dehydrogenase and glucose dehydrogenase in genetically engineered bacteria to obtain the crude enzyme solution containing the alcohol dehydrogenase and the glucose dehydrogenase; mixing the crude enzyme solution with a precipitator to precipitate the alcohol dehydrogenase and the glucose dehydrogenase so as to obtain the mixture containing the alcohol dehydrogenase and the glucose dehydrogenase; preferably, the precipitating agent is acetone, ethanol, or ammonium sulfate.

In another preferred embodiment, the precipitating agent is 55-100% (v/v) aqueous acetone (preferably 60-100% (v/v); more preferably 80-95% (v/v), e.g., 90%); preferably, the volume of the precipitant and the crude enzyme solution is 10-30: 2-10; preferably 20: 4.

in another preferred example, the step (2) includes the steps of:

adding a cross-linking agent glutaraldehyde aqueous solution to the mixture containing alcohol dehydrogenase and glucose dehydrogenase obtained in step (1), and then adding tween 80.

In another preferred embodiment, the concentration of the glutaraldehyde aqueous solution is 1% to 5% (v/v), preferably 2.5% (v/v).

In another preferred example, the volume ratio of the glutaraldehyde aqueous solution to the mixture containing alcohol dehydrogenase and glucose dehydrogenase is 0.2-1: 15-30; preferably 0.3-0.6: 18 to 25.

In another preferred embodiment, the tween 80 is 30% to 80% (v/v) of tween 80 aqueous solution; preferably, the volume ratio of the tween 80 aqueous solution to the mixture containing the alcohol dehydrogenase and the glucose dehydrogenase is 0.1-0.5: 15-30; preferably 0.2-0.4: 18 to 25.

In a second aspect of the present invention, there is provided a cross-linked enzyme aggregate formed by co-crosslinking alcohol dehydrogenase and glucose dehydrogenase with a cross-linking agent.

In another preferred embodiment, the cross-linked enzyme aggregate is formed by co-crosslinking alcohol dehydrogenase and glucose dehydrogenase with a cross-linking agent, and the cross-linking reaction is carried out in the presence of a surfactant.

In another preferred embodiment, the surfactant is tween 80.

In another preferred embodiment, the alcohol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate is prepared by the method of claim 1.

In a third aspect of the invention, the use of the cross-linking enzyme aggregate according to the second aspect of the invention in catalyzing the synthesis of chiral intermediate (S) -1- (2, 6-dichloro-3-fluorophenyl) from 1- (2, 6-dichloro-3-fluorophenyl) ethanone is provided.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows SDS-PAGE of three lanes, i.e., (A) protein marker (B) crude enzyme solution extracted by E.coli BL21-ADH/GDH fermentation (C) supernatant after 90% acetone precipitation

FIG. 2 shows the recovery rates of enzyme activities in the crude enzyme solutions precipitated by the precipitants at different volume fractions, wherein A represents ADH, B represents GDH, (. diamond-solid.) represents acetone, (■) represents saturated ammonium sulfate, and (. tangle-solidup.) represents ethanol.

FIG. 3 recovery of enzyme activity at different mass ratios of glutaraldehyde and total protein, (. tangle-solidup.) indicating ADH without Tween 80 added at cross-linking; (. diamond.) shows ADH with Tween 80 added at the time of crosslinking; (●) GDH without Tween 80 added during crosslinking; (■) shows GDH to which Tween 80 was added at the time of crosslinking.

FIG. 4 shows recovery curves of enzyme activity at different pHs, wherein A represents ADH, B represents GDH, (. tangle-solidup.) represents aggregate of crosslinked enzyme, and (■) represents free enzyme.

FIG. 5 shows recovery curves of enzyme activity at different temperatures, wherein A represents ADH, B represents GDH, (. tangle-solidup.) represents aggregate of crosslinked enzyme, and (■) represents free enzyme.

Detailed Description

The present inventors have conducted extensive and intensive studies to obtain a method for preparing a co-crosslinking enzyme aggregate of alcohol dehydrogenase and glucose dehydrogenase, wherein genetically engineered bacteria co-expressing alcohol dehydrogenase and glucose dehydrogenase are fermented at high density, the supernatant after wall breaking and centrifugation is used as a crude enzyme solution, the crude enzyme solution is precipitated, the alcohol dehydrogenase and glucose dehydrogenase in the crude enzyme solution are crosslinked by glutaraldehyde to prepare the co-crosslinking enzyme aggregate, and the enzyme activity recovery rate of the co-crosslinking enzyme aggregate is increased to 80% or more by adding tween 80 in the crosslinking reaction.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Cross-linked enzyme aggregates

The cross-linked enzyme aggregate technology (Cross-linked enzyme aggregates, CLEAs) is a novel carrier-free immobilized enzyme technology, which only comprises two steps of precipitation and cross-linking, does not need a fussy and complicated enzyme purification and crystallization process, and has the advantages of simple operation, low cost and high activity per unit volume. The preparation of the cross-linked enzyme aggregate by the cross-linked enzyme aggregate technology is a novel enzyme immobilization technology, has low requirement on the purity of enzyme, can realize the aggregation and immobilization of the enzyme, is convenient for recycling, does not need a carrier, and has low preparation cost.

The invention improves the bioavailability by preparing the ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate. The amino group on the enzyme protein in the crude enzyme solution can have Schiff base reaction with the carbonyl group of the cross-linking agent to form an irreversible chemical bond, thereby preparing the stable water-insoluble enzyme aggregate with the particle size of 1-100 mu m. In the invention, colibacillus BL21 co-expressing ADH/GDH is induced and expressed by IPTG, supernatant obtained by high-density fermentation, wall breaking and centrifugation is used as crude enzyme liquid, ethanol dehydrogenase and glucose dehydrogenase in the crude enzyme liquid are cross-linked by glutaraldehyde to prepare co-cross-linked enzyme aggregates, and the enzyme activity recovery rate of the co-cross-linked enzyme aggregates is improved to 82% by adding Tween 80.

Co-expression of bacteria

In the present invention, co-expressed cells of alcohol dehydrogenase and glucose dehydrogenase are prepared. The plasmid containing ADH and GDH and a vector plasmid pET-28a are subjected to double enzyme digestion and connection by using specific restriction endonucleases NdeI and XhoI to obtain plasmids pET-28a-ADH and pET-28a-GDH containing ampicillin and kanamycin resistance, the two plasmids are introduced into a competent cell E.coli DH5 alpha to obtain a co-expression strain E.coli BL21-ADH/GDH, supernatant obtained by ultrasonic bacteria breaking and centrifugation after fermentation culture is subjected to SDS-page inspection, and as shown in figure 1, the expression of target protein is proved to be successful. Specific methods for the preparation of co-expressed bacteria can be found in "Liuli Dun, Zhang Jun Liang, Tan Jun, et al. ethanol dehydrogenase coupled with glucose dehydrogenase catalyzes the preparation of (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol [ J ]. Industrial microorganisms, 2016,46(3): 8-12", or Chinese patent applications: 201610302564X.

The object of the present invention is to produce co-crosslinked enzyme aggregates of alcohol dehydrogenase and glucose dehydrogenase.

To achieve the above object, the following steps are required: (1) preparing a crude enzyme solution; (2) adding a precipitator to precipitate the enzyme protein in the crude enzyme solution; (3) adding a cross-linking agent to form an enzyme aggregate; (4) and (4) recovering enzyme aggregates.

Wherein the enzyme solution in the step (1) is not obtained by dissolving enzyme protein, but is generated by inducing expression of ADH/GDH co-expression escherichia coli BL 21. E. coli BL21-ADH/GDH is induced and expressed by IPTG, thalli is collected by centrifugation after high-density fermentation, the thalli is washed and then is resuspended in 0.1mol/L triethanolamine buffer solution (pH7.0), the supernatant obtained by ultrasonic wall breaking and centrifugation is crude enzyme solution, and the final concentration of protein is 24.64 mg/ml.

The precipitating agent in the step (2) can be an organic reagent and a high-concentration salt solution, the protein precipitation test is carried out by using ethanol, acetone and ammonium sulfate with different volume fractions, and finally, the highest enzyme activity recovery rate of the alcohol dehydrogenase is almost 98% when 90% acetone is used for precipitating the crude enzyme solution, as shown in figure 2.

Wherein the cross-linking agent in the step (3) is glutaraldehyde, and the final mass ratio of the glutaraldehyde to the protein in the obtained system is 0.5: the enzyme activity recovery rate of the alcohol dehydrogenase in the cross-linked enzyme aggregate reaches 82 percent at most in 1 hour, and the optimal time of cross-linking is 1.5 hours.

The other purpose of the patent is to improve the enzyme activity of the co-crosslinking enzyme aggregate, and to check the pH stability and the temperature stability of the co-crosslinking enzyme aggregate to obtain the co-crosslinking enzyme aggregate with higher enzyme activity and relative stability.

In order to achieve the purpose, the surfactant Tween 80 is added when the enzyme protein molecules are crosslinked with glutaraldehyde. The surface active agent is added to change the hydrophobicity of the surface of the enzyme molecule and increase the affinity between the enzyme molecule and the substrate molecule, so that the enzyme activity of the obtained co-crosslinking enzyme aggregate is improved, the enzyme activity recovery rate of the alcohol dehydrogenase is increased from 59% to 82%, and the enzyme activity recovery rate of the glucose dehydrogenase is increased from 35% to 62%. The pH of the obtained co-crosslinking enzyme aggregate is more stable, as shown in FIG. 4, 80% of relative enzyme activity of the alcohol dehydrogenase and the glucose dehydrogenase is kept at the pH of 4-7, and the enzyme activity recovery rate of the alcohol dehydrogenase is only 40% when the raw enzyme solution is at the pH of 4. Because glucose is continuously converted into gluconic acid by glucose dehydrogenase in the reaction, the pH value of the system is reduced, and the cross-linked enzyme aggregate is more stable under the acidic condition, which is beneficial to the reaction. The temperature stability was also improved relative to the crude enzyme solution as shown in FIG. 5. When the immobilized enzyme is repeatedly used for 7 times, the activity recovery rate of the alcohol dehydrogenase and the glucose dehydrogenase is still 80 percent, so that the repeated use of the immobilized enzyme is realized.

The main advantages of the invention are:

(1) the co-crosslinking enzyme aggregate of the alcohol dehydrogenase and the glucose dehydrogenase is prepared for the first time, so that the repeated use of the immobilized enzyme is realized, and the bioavailability of the enzyme is effectively improved;

(2) provides a method for preparing ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregates, which has higher recovery rate of enzyme activity.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1

Co-expression of alcohol dehydrogenase and glucose dehydrogenase

Plasmids containing ADH and GDH were digested simultaneously with specific restriction enzymes NdeI and XhoI (Thermo, USA), the target fragments were recovered by gel cutting after electrophoresis, and vector plasmid pET-28a was also digested simultaneously with NdeI and XhoI and recovered by gel cutting. The ADH and GDH gene fragments were ligated to the vector fragments, respectively, to obtain plasmids pET-28a-ADH and pET-28a-GDH, both of which had ampicillin and kanamycin resistance. And transferring the two plasmids into a competent cell E.coli DH5 alpha to obtain a co-expression strain E.coli BL21-ADH/GDH of alcohol dehydrogenase and glucose dehydrogenase. The co-expressed strain was inoculated into LB medium containing ampicillin (Amp, 100. mu.g/ml) and kanamycin (Kan, 100. mu.g/ml) and cultured overnight at 37 ℃ on a constant temperature shaker at 230 r/min. Inoculating into shake flask at a volume ratio of 1ml culture solution 100ml LB medium (containing Amp and Kan each 100 μ g/ml), growing in shaker at 37 deg.C (230r/min), and culturing to OD₆₀₀At about 0.8, IPTG was added to a final concentration of 0.2mmol/L, and the mixture was cultured at 25 ℃ for 12 hours (220r/min), and the cells were collected by centrifugation. Resuspending thallus with triethanolamine (0.1mol/L) buffer solution of pH7.0, disrupting cells with ultrasonicator, centrifuging at 3900r/min for 15min to remove disrupted cell tissue to obtain crude enzyme solution containing alcohol dehydrogenase and glucose dehydrogenase (total protein content is 24mg/ml), such as SDS-PAGE electrophoresis chart1, a band with a target protein is obvious, which indicates that the target protein is successfully induced and expressed, and the content ratio of ADH to GDH in the crude enzyme solution is about 1: 1, each representing about 25% to 30% of the total protein mass.

Coli DH5, e.coli BL21(DE3) from CICC23796, plasmids from BioVector plasmid vector strain cell gene collection.

Example 2

Selection of the precipitating agent

The specific implementation steps are as follows:

(1) in this context, 1000. mu.l, 900. mu.l, 800. mu.l, 700. mu.l, 600. mu.l, 500. mu.l of ethanol, acetone and saturated ammonium sulfate solution, respectively, are mixed with 0. mu.l, 100. mu.l, 200. mu.l, 300. mu.l, 400. mu.l, 500. mu.l of 0.1mol/L TEA buffer solution having a pH of 7.0, respectively, to give 100%, 90%, 80%, 70%, 60%, 50% strength by volume of precipitant.

(2) 1ml of precipitant with different concentrations is taken to be put in a 1.5ml EP tube, and is pre-cooled for 5min on an ice water bath, 200 mul of crude enzyme solution is added, and is centrifuged after shaking for 30min on the ice water bath at 150r/min, and the liquid is used for measuring the protein content.

(3) The pellet was resuspended in 600. mu.l TEA buffer for enzyme activity determination. As shown in FIG. 2, when the crude enzyme solution was precipitated with 90% acetone, the recovery rate of the ADH was about 98% at the maximum, and the recovery rate of the GDH was 58%; when 80% ammonium sulfate is used for precipitating the crude enzyme solution, the highest recovery rate of the enzyme activity of GDH is 80%, and the recovery rate of the enzyme activity of ADH is 41%. As ADH is the rate-limiting enzyme in the reaction, 90% acetone is finally selected to precipitate the crude enzyme solution, and the content of the supernatant protein after centrifugation is as shown in a C band in figure 1, so that the ADH and the GDH are obviously absent, and the target protein is precipitated.

Example 3

Preparation of co-crosslinked enzyme aggregates

The specific implementation steps are as follows:

(1) preparing 90% acetone solution from 18ml acetone and 2ml TEA buffer solution, placing in a beaker, sealing, placing in ice water bath, precooling for 5min, stirring, slowly dripping 4ml crude enzyme solution, stirring and precipitating for 30 min.

(2) 480. mu.l of glutaraldehyde solution with a volume fraction of 2.5% was added dropwise, at which point the ratio of glutaraldehyde: the total protein mass ratio was 0.5 and stirred on an ice-water bath for 1.5 hours.

(3) 2ml of the cross-linked immobilized enzyme is taken at 3000r/min, centrifuged at 4 ℃ for 2min to remove supernatant, the enzyme can be washed three times by using equal volume of TEA buffer solution, and the cross-linked enzyme aggregate is resuspended by using 1ml of TEA buffer solution and used for enzyme activity determination.

Example 4

Preparation of Co-Cross-Linked enzyme aggregates with Tween 80

The specific implementation steps are as follows:

(2) 480. mu.l of a 2.5% glutaraldehyde solution was added dropwise, at which point the ratio of glutaraldehyde: and the mass ratio of the total protein is 0.5, 50 percent of Tween 80 is dropwise added after 5min until the mass ratio of the Tween 80 to the total protein is 0.24, and the mixture is stirred for 1.5 hours on an ice-water bath.

(3) Taking 2ml of cross-linked immobilized enzyme, centrifuging at 3000r/min and 4 ℃ for 2min to remove supernatant, washing with equal volume of TEA buffer solution for three times, and resuspending the cross-linked enzyme aggregate with 1ml of TEA buffer solution for enzyme activity determination.

Example 5

Selection of optimum glutaraldehyde concentration for crosslinking

As in example 4, 240. mu.l, 480. mu.l, 960. mu.l, 1200. mu.l, 1680. mu.l of 2.5% glutaraldehyde solution, 1680. mu.l of 5% glutaraldehyde were added dropwise during the preparation thereof so that the ratio of glutaraldehyde: the mass ratio of total protein was 0.25, 0.5, 1.0, 1.25, 1.75, 2.50, and then the obtained cross-linked enzyme aggregate was washed three times and then the enzyme activity was measured, and the results are shown in FIG. 3, with or without Tween 80, glutaraldehyde: when the mass ratio of the total protein is 0.5, the recovery rate of the ADH enzyme activity is the largest, and the recovery rate of the enzyme activity of the co-crosslinking enzyme aggregate added with the Tween 80 is 82 percent and is obviously higher than 59 percent when the enzyme activity is not added.

Example 6

pH stability test of Co-Cross-Linked enzyme aggregates

Preparing co-crosslinking enzyme aggregate according to the fourth step of the specific example, dividing the prepared co-crosslinking enzyme aggregate into 6 parts, each part is 4ml, after centrifuging at 3000r/min, washing three times by using triethanolamine (0.1mol/L) buffer solutions with the same volume of pH respectively being 4,5,6,7,8,9, finally re-suspending by using 2ml buffer solutions with the corresponding pH for measuring the enzyme activity, taking the point with the highest enzyme activity as 100%, and measuring the stability of the crosslinking enzyme aggregate at different pH values.

Diluting 6 parts of 1ml of crude enzyme solution into 2.8ml by using triethanolamine buffer solutions (0.1mol/L) with pH4,5,6,7,8 and 9 respectively, adjusting the pH by using triethanolamine buffer solutions (0.1mol/L) with pH1 and pH12 until the pH of a system is finally 4,5,6,7,8 and 9 respectively, measuring the volume of the system is 3ml, measuring the enzyme activity, taking the point with the highest enzyme activity as 100%, and measuring the stability of free enzyme at different pH values.

Example 7

Temperature stability test of Co-Cross-Linked enzyme aggregates

The co-crosslinked enzyme aggregate was prepared according to the procedure of example 4, and the co-crosslinked enzyme aggregate thus prepared was divided into 5 portions each of 4ml, centrifuged at 3000r/min, washed three times with an equal volume of triethanolamine (0.1mol/L) buffer solution of pH7.0, and finally resuspended in 2ml of buffer solution. And (3) respectively placing the five cross-linked enzyme aggregates in a water bath kettle at the temperature of 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ for heat preservation for 30min, then measuring the enzyme activity, taking the point with the highest enzyme activity as 100%, and measuring the stability of the cross-linked enzyme aggregates at different temperatures.

Diluting 5ml of crude enzyme solution into 15ml by using triethanolamine buffer solution (0.1mol/L) with pH of 7.0, dividing into 5 parts, respectively placing in water bath kettle at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C and 60 deg.C, keeping the temperature for 30min, measuring enzyme activity, taking the point with highest enzyme activity as 100%, and measuring the stability of free enzyme at different temperatures.

Example 8

Enzyme activity measuring method

The method for measuring the enzyme activity of the glucose dehydrogenase comprises the following steps:

200ul of the enzyme reaction system comprises: a.0.1mol/L triethanolamine buffer (pH7.0)110 ul. b.10mmol/L NADP +20 ul. c.1mol/L C6H12O620 ul. d. After 50ul of the enzyme solution was mixed well, the mixture was incubated at 30 ℃ and the time was counted from the time when the GDH crude enzyme solution was added, and the rise of the absorbance at 340nm in 10 seconds was monitored. The amount of NADPH produced by the reaction was calculated from the standard curve. One enzyme activity unit U is defined as: the amount of enzyme required to produce 1. mu. mol of NADPH in 1 minute.

The method for measuring the enzyme activity of the alcohol dehydrogenase comprises the following steps:

200ul of the enzyme reaction system comprises a.0.1mol/L triethanolamine buffer (pH7.0)86 ul. b.10mmol/L NADPH 10 ul. c, 0.1mol/L2, 6-dichloro-3-fluoro acetophenone 4 ul. d. The enzyme solution was 100 ul. Mixing well, keeping temperature at 30 deg.C, adding 100ul enzyme solution, timing, and detecting the decrease of light absorption value at 340nm in 20 s. The amount of NADPH consumed by the reaction was calculated from the standard. 1 enzyme activity unit U is defined as: the amount of enzyme required to consume 1. mu. mol of NADPH in 1 minute.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method for preparing an ethanol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate, comprising the steps of:

wherein the mixture is prepared by the following method: co-expressing alcohol dehydrogenase and glucose dehydrogenase in genetically engineered bacteria to obtain the crude enzyme solution containing the alcohol dehydrogenase and the glucose dehydrogenase; mixing the crude enzyme solution with a precipitator to precipitate the alcohol dehydrogenase and the glucose dehydrogenase so as to obtain the mixture containing the alcohol dehydrogenase and the glucose dehydrogenase; wherein the content of the first and second substances,

the precipitant is 55-100% v/v acetone water solution; and is

The volume of the precipitant and the crude enzyme solution is 10-20: 2-4;

(2) performing crosslinking reaction, namely preparing a crosslinking reaction system, and performing co-crosslinking reaction of the alcohol dehydrogenase and the glucose dehydrogenase provided in the step (1) by using a crosslinking agent in the presence of a surfactant; performing crosslinking reaction at 0-40 ℃ to prepare the co-crosslinking enzyme aggregate of the alcohol dehydrogenase and the glucose dehydrogenase;

wherein the surfactant is tween 80;

glutaraldehyde is used as a cross-linking agent in the cross-linking reaction; and is

The mass ratio of the cross-linking agent to the sum of substrate enzyme alcohol dehydrogenase and glucose dehydrogenase in the cross-linking reaction system is 0.2-1.2: 0.5 to 1.

2. The method according to claim 1, wherein the mass ratio of the cross-linking agent to the sum of the substrate enzymes ethanol dehydrogenase and glucose dehydrogenase in the cross-linking reaction system is 0.2-1.2: 1.

3. the method according to claim 1, wherein the mass ratio of the surfactant to the sum of the substrate enzymes ethanol dehydrogenase and glucose dehydrogenase in the crosslinking reaction system in which the co-crosslinking reaction is carried out is 0.1 to 1.2: 0.5 to 1.

4. The method according to claim 1, wherein the co-crosslinking reaction is carried out in a crosslinking reaction system in which the mass ratio of surfactant to the sum of substrate enzymes, ethanol dehydrogenase and glucose dehydrogenase, is 0.2 to 1: 0.5 to 1.

5. The method according to claim 1, wherein the mass ratio of the cross-linking agent to the sum of the substrate enzymes ethanol dehydrogenase and glucose dehydrogenase in the cross-linking reaction system is 0.5-1.0: 1.

6. the method of claim 1, wherein the surfactant: a crosslinking agent: the mass ratio of the sum of the substrate enzyme ethanol dehydrogenase and the glucose dehydrogenase is 0.4-0.8: 0.8-1.2: 0.8-1.2.

7. The method of claim 1, wherein the method comprises the steps of:

the mass ratio of the cross-linking agent to the sum of the substrate enzyme alcohol dehydrogenase and glucose dehydrogenase in the cross-linking reaction system is 0.5: 1.

8. the method according to claim 1, wherein in the mixture containing the alcohol dehydrogenase and the glucose dehydrogenase, the mass ratio of the alcohol dehydrogenase to the glucose dehydrogenase is: 0.5-1: 0.5 to 1.

9. The method as claimed in claim 7, wherein the step (1) comprises the steps of:

the precipitant is 80-95% acetone water solution.

10. The method of claim 9, wherein the volume of the precipitant and the crude enzyme solution is 20: 4.

11. the method as claimed in claim 1, wherein the step (2) comprises the steps of:

12. The method of claim 11, wherein the aqueous glutaraldehyde solution has a concentration of 1% to 5% v/v.

13. The method of claim 1,

the precipitant is 80-95% v/v acetone water solution;

the mass ratio of the cross-linking agent to the sum of substrate enzyme alcohol dehydrogenase and glucose dehydrogenase in the cross-linking reaction system is 0.5-1.0: 1.

14. the method of claim 13,

the step (2) comprises the following steps:

adding a cross-linking agent glutaraldehyde aqueous solution to the mixture containing alcohol dehydrogenase and glucose dehydrogenase obtained in the step (1), and then adding tween 80; and is

The concentration of the glutaraldehyde water solution is 1-5% v/v.

15. The method of claim 14, wherein the aqueous glutaraldehyde solution has a concentration of 2.5% v/v.

16. A cross-linked enzyme aggregate formed by co-crosslinking alcohol dehydrogenase and glucose dehydrogenase with a cross-linking agent by the method of claim 1.

17. Use of the ethanol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate of claim 16 in catalyzing the synthesis of chiral intermediate (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanone from 1- (2, 6-dichloro-3-fluorophenyl) ethanone.