CN107988201A - A kind of preparation method of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate - Google Patents
A kind of preparation method of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate Download PDFInfo
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Abstract
The present invention provides the preparation method of a kind of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate, supernatant after the specific present invention is centrifuged by high density fermentation broken wall is used as crude enzyme liquid, alcohol dehydrogenase in crude enzyme liquid and glucose dehydrogenase are prepared into co-crosslinking enzyme aggregate through glutaraldehyde cross-linking, and the enzyme activity rate of recovery of co-crosslinking enzyme aggregate is brought up to 82% by adding Tween 80.
Description
Technical field
Preparation and the enzyme the present invention relates to a kind of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate gather
The stability of collective and the research of repeat usage, belong to technical field of enzyme immobilization.
Background technology
On August 3rd, 2011, U.S. Food and Drug Administration's approval are used to control by gram azoles of Pfizer's production for Buddhist nun
Treat the NSCLC that ALK gene is reset.On March 11st, 2016, and it is approved for the NSCLC for the treatment of ROS1 gene mutations, its market
Demand is continuously increased.In its building-up process, (S) -1- (2,6- bis- chloro- 3- fluorophenyls) ethanol is crucial chiral intermediate.
It is now more that (S) -1- (2,6- bis- chloro- 3- fluorophenyls) ethanol is synthesized by chemical method, but it is not readily available the chiral compound of high-purity
Thing, and building-up process is complicated, biosynthesis can overcome these drawbacks.
Existing newest biosynthesis technology is the hair that bacterial strain is co-expressed by alcohol dehydrogenase and glucose dehydrogenase
Zymotic fluid, 1- (2,6- is catalyzed by alcohol dehydrogenase and glucose dehydrogenase and the coenzyme circular regeneration system of NADP and NADPH
Two chloro- 3- fluorophenyls) ethyl ketone synthesis of chiral intermediate (S) -1- (the chloro- 3- fluorophenyls of 2,6- bis-) such as bibliography (Liu Liqin,
Fine horse beam, Tan Jun, waits alcohol dehydrogenases and coupling catalysed preparation (S) -1- (the chloro- 3- fluorophenyls of the 2,6- bis-) second of glucose dehydrogenase
Alcohol [J] industrial microorganisms, 2016,46 (3):8-12.).
Cross-linked enzyme aggregate is a kind of new enzyme immobilization technology, the aggregation and immobilization of enzyme can be realized, easy to recycle
Utilize, and carrier is not required, manufacturing cost is low.Therefore, in order to effectively improve the preparation efficiency of chipal compounds, system is reduced
Standby cost, those skilled in the art are directed to exploitation catalysis 1- (2,6- bis- chloro- 3- fluorophenyls) ethyl ketone synthesis of chiral intermediate
(S) -1- (the chloro- 3- fluorophenyls of 2,6- bis-) enzyme immobilization technology.
The content of the invention
It is an object of the invention to provide the preparation of a kind of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate
Method.
A kind of the first aspect of the present invention, there is provided the system of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate
Preparation Method, the method include step：
In the presence of surfactants, alcohol dehydrogenase and the co-crosslinking of glucose dehydrogenase are carried out for crosslinked dose
Reaction.
In another preference, the surfactant is selected from the group：Tween 80 and polysorbate60.
In another preference, in the method, the cross-linking reaction system inner surface activity of the co-crosslinking reaction is carried out
The mass ratio of agent and substrates enzymes (alcohol dehydrogenase and glucose dehydrogenase) is 0.1~1.2：0.5~1；Preferably mass ratio is
0.2~1：0.5~1；Most preferably about 0.4~0.8：1.
In another preference, it is the sum of alcohol dehydrogenase and glucose dehydrogenase to define the substrates enzymes.
In another preference, glutaraldehyde is used in the cross-linking reaction as crosslinking agent.
In another preference, (alcohol dehydrogenase and glucose take off for glutaraldehyde and substrates enzymes in the cross-linking reaction system
Hydrogen enzyme) mass ratio be 0.1~1.2：0.5~1；Preferably 0.2~1.2：1；Most preferably about 1：1.
In another preference, surfactant in cross-linking reaction：Crosslinking agent：Substrates enzymes (alcohol dehydrogenase and glucose
Dehydrogenase) mass ratio be 0.4-0.8：0.8-1.2：0.8-1.2.
In another preference, the method includes the steps：
(1) mixture containing alcohol dehydrogenase and glucose dehydrogenase is provided；
(2) cross-linking reaction
Cross-linking reaction system is prepared, the cross-linking reaction system includes：Step (1) provide containing alcohol dehydrogenase and
Mixture, crosslinking agent and the surfactant of glucose dehydrogenase, carry out cross-linking reaction under the conditions of 0 DEG C~40 DEG C, so as to be made
The alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate.
In another preference, in the step (1), including step：
Alcohol dehydrogenase and glucose dehydrogenase are co-expressed in genetic engineering bacterium, so as to contain alcohol dehydrogenase described in obtaining
The mixture of enzyme and glucose dehydrogenase.
In another preference, in the mixture containing alcohol dehydrogenase and glucose dehydrogenase, alcohol dehydrogenase
Mass ratio with glucose dehydrogenase is：0.5~1：0.5~1.
In another preference, in the step (1), including step：
Alcohol dehydrogenase and glucose dehydrogenase are co-expressed in genetic engineering bacterium, so as to contain alcohol dehydrogenase described in obtaining
The crude enzyme liquid of enzyme and glucose dehydrogenase；Take the crude enzyme liquid and precipitant mix so that the alcohol dehydrogenase and glucose
Dehydrogenase precipitates, so as to obtain the mixture containing alcohol dehydrogenase and glucose dehydrogenase；Preferably, it is described heavy
Shallow lake agent is acetone, ethanol or ammonium sulfate.
In another preference, the precipitating reagent be 55%~100% (v/v) aqueous acetone solution (be preferably 60%~
100% (v/v)；More preferably 80%~95% (v/v), such as 90%)；Preferably, the body of the precipitating reagent and the crude enzyme liquid
Product is 10~30：2~10；Preferably 20：4.
In another preference, in the step (2), including step：
Crosslinking agent is added in the mixture containing alcohol dehydrogenase and glucose dehydrogenase obtained into step (1)
Glutaraldehyde water solution, then adds Tween 80.
In another preference, the concentration of the glutaraldehyde water solution is 1%~5% (v/v), is preferably 2.5% (v/
v)。
In another preference, the glutaraldehyde water solution and described mixed containing alcohol dehydrogenase and glucose dehydrogenase
The volume ratio of compound is 0.2~1：15~30；Preferably 0.3~0.6：18~25.
In another preference, the Tween 80 is the Tween 80 aqueous solution of 30%~80% (v/v)；Spat described in preferable
The volume ratio of warm 80 aqueous solutions and the mixture containing alcohol dehydrogenase and glucose dehydrogenase is 0.1~0.5：15~
30；Preferably 0.2~0.4：18~25.
The second aspect of the present invention, there is provided a kind of cross-linked enzyme aggregate, the cross-linked enzyme aggregate is by alcohol dehydrogenase
Formed with crosslinked dose of co-crosslinking of glucose dehydrogenase.
In another preference, the cross-linked enzyme aggregate is handed over for crosslinked dose altogether by alcohol dehydrogenase and glucose dehydrogenase
Connection is formed, and in the presence of surfactants, carries out cross-linking reaction.
In another preference, the surfactant is Tween 80.
In another preference, surfactant in cross-linking reaction：Crosslinking agent：Substrates enzymes (alcohol dehydrogenase and glucose
Dehydrogenase) mass ratio be 0.4-0.8：0.8-1.2：0.8-1.2.
In another preference, the alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate pass through claim
It is prepared by the method described in 1.
The third aspect of the present invention, there is provided cross-linked enzyme aggregate as described in respect of the second aspect of the invention catalysis 1- (2,
The chloro- 3- fluorophenyls of 6- bis-) purposes in ethyl ketone synthesis of chiral intermediate (S) -1- (the chloro- 3- fluorophenyls of 2,6- bis-).
It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical solution.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Tri- swimming lanes of Fig. 1 .SDS-PAGE are (A) albumen Marker (B) E.coli BL21-ADH/GDH fermentation extractions respectively
90% acetone precipitation of crude enzyme liquid (C) after supernatant
Fig. 2 different volumes fractions precipitating reagent precipitates the enzyme activity rate of recovery of crude enzyme liquid, and A represents that ADH, B represent GDH, (◆) table
Show acetone, (■) represents saturated ammonium sulfate, and (▲) represents ethanol.
Fig. 3 differences glutaraldehydes and total protein quality than when the enzyme activity rate of recovery, (▲) represents to be not added with Tween 80 during crosslinking
ADH；(◆) represents the ADH for adding Tween 80 during crosslinking；(●) represents to be not added with the GDH of Tween 80 during crosslinking；When (■) represents crosslinking
Add the GDH of Tween 80.
Enzyme activity recycling curve during Fig. 4 difference pH, A represent that ADH, B represent GDH, and (▲) represents cross-linked enzyme aggregate,
(■) represents resolvase.
Enzyme activity recycling curve during Fig. 5 different temperatures, A represent that ADH, B represent GDH, and (▲) represents cross-linked enzyme aggregate,
(■) represents resolvase.
Embodiment
The present inventor and in-depth study, obtains a kind of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme by extensive
The preparation method of aggregation, in the method, alcohol dehydrogenase and the base of glucose dehydrogenase is co-expressed by high density fermentation
Because of engineering bacteria, supernatant after being centrifuged using broken wall as crude enzyme liquid, crude enzyme liquid after precipitation, by the alcohol dehydrogenase in crude enzyme liquid and
Glucose dehydrogenase prepares co-crosslinking enzyme aggregate through glutaraldehyde cross-linking, and will altogether by adding Tween 80 in cross-linking reaction
The enzyme activity rate of recovery of cross-linked enzyme aggregate brings up to more than 80%.
Before describing the present invention, it should be understood that the invention is not restricted to the specific method and experiment condition, because this
Class method and condition can change.It should also be understood that its purpose of term used herein is only that description specific embodiment, and
And it is not intended to be restricted, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as fields of the present invention with scientific terminology
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in the numerical value specifically enumerated is mentioned
" about " mean that the value can be changed from the value enumerated and be not more than 1%.For example, as used herein, " about 100 " include 99 Hes for statement
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although it can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention
And material, herein place enumerate preferable method and material.
Cross-linked enzyme aggregate
A kind of cross-linked enzyme aggregate technology (cross-linked enzyme aggregates, CLEAs) new carrier-free
Enzyme immobilization technology, it is only formed by precipitating and being crosslinked two steps, it is not necessary to the enzyme purification crystallization process of very complicated, operation letter
It is single, of low cost, activity per unit volume is big.It is a kind of new enzyme to prepare cross-linked enzyme aggregate by cross-linked enzyme aggregate technology
Immobilization technology, the purity requirement to enzyme is not high, can realize the aggregation and immobilization of enzyme, easy to recycle, and need not
Carrier, manufacturing cost are low.
The present invention improves bioavailability by preparing alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate.
Schiff reaction can occur with the carbonyl of crosslinking agent for the amino in crude enzyme liquid on zymoprotein, form irreversible chemical bond,
So as to which the insoluble in water enzyme aggregate that stable granular size is 1-100 μm be made.The preparation of existing cross-linked enzyme aggregate is logical
The molten solution of zymoprotein weight is crossed to be crosslinked, is led in the present invention with coexpression ADH/GDH e. coli bl21s through IPTG induced expressions
Supernatant after the centrifugation of overpopulation breaking wall by fermentation passes through the alcohol dehydrogenase in crude enzyme liquid and glucose dehydrogenase as crude enzyme liquid
Glutaraldehyde cross-linking prepares co-crosslinking enzyme aggregate, and is put forward the enzyme activity rate of recovery of co-crosslinking enzyme aggregate by adding Tween 80
It is high to 82%.
Co-express thalline
Alcohol dehydrogenase and the coexpression thalline of glucose dehydrogenase are prepared in the present invention.The present invention is with specificity limitation
Enzyme cutting NdeI and XhoI carries out double digestion and connection to the plasmid containing ADH and GDH and vector plasmid pET-28a, is contained
The plasmid pET-28a-adh and pET-28a-gdh of ampicillin and kalamycin resistance, and the two plasmids import competence
Cell E.coli DH5 α, obtain coexpression bacterial strain E.coli BL21-ADH/GDH, carrying out ultrasonic bacteria breaking centrifuges after fermented culture
The supernatant arrived, is examined by SDS-page, as shown in Figure 1, it was demonstrated that target protein is expressed successfully.It is specific to prepare coexpression thalline
Method may be referred to document " Liu Liqin, Zhang Junliang, Tan Jun, wait alcohol dehydrogenases and the coupling catalysed preparation of glucose dehydrogenase
(S) -1- (the chloro- 3- fluorophenyls of 2,6- bis-) ethanol [J] industrial microorganisms, 2016,46 (3):8-12. " or Chinese patent application：
201610302564X。
The purpose of the present invention is prepare the co-crosslinking enzyme aggregate of alcohol dehydrogenase and glucose dehydrogenase.
To achieve the above object, it is necessary to follow the steps below：(1) preparation of crude enzyme liquid；(2) adding precipitating reagent makes crude enzyme liquid
In zymoprotein precipitate；(3) add crosslinking agent and form enzyme aggregate；(4) recycling of enzyme aggregate.
Enzyme liquid wherein in above-mentioned steps (1) is not dissolved to obtain by zymoprotein, but co-expresses large intestine by ADH/GDH
Bacillus BL21 induced expressions produce.E.coli BL21-ADH/GDH are after IPTG induced expressions, by being centrifuged after high density fermentation
Thalline is collected, thalline is once purged to be resuspended in 0.1mol/L Triethanolamine buffers (pH7.0), what ultrasonication centrifuged
Supernatant is crude enzyme liquid, and the final concentration of albumen is 24.64mg/ml.
Precipitating reagent wherein in above-mentioned steps (2) can be organic reagent, high concentration salt solutions, and this patent is by using difference
Ethanol, acetone, the ammonium sulfate of volume fraction carry out albumen precipitation experiment, are finally taken off with ethanol during 90% acetone precipitation crude enzyme liquid
The enzyme activity rate of recovery highest of hydrogen enzyme has nearly reached 98%, as shown in Figure 2.
Crosslinking agent wherein in above-mentioned steps (3) is glutaraldehyde, and obtaining the whole mass ratio of glutaraldehyde and albumen in system is
0.5：The enzyme activity rate of recovery of alcohol dehydrogenase has been up to 82% in cross-linked enzyme aggregate when 1, and crosslinked optimum time is 1.5
Hour.
Another goal of the invention of this patent is to improve the enzyme activity of co-crosslinking enzyme aggregate, and examines its pH stability, temperature
Stability, obtains that enzyme activity is higher and metastable co-crosslinking enzyme aggregate.
To achieve the above object, this patent adds surface active agent tween 80 in zymoprotein molecule and glutaraldehyde cross-linking.
The hydrophobicity on enzyme molecule surface can be changed by adding surfactant, increase its affinity between substrate molecule, so as to get
The enzyme activity of co-crosslinking enzyme aggregate improve, the enzyme activity rate of recovery of alcohol dehydrogenase increases to 82% by original 59%, grape
The enzyme activity rate of recovery of glucocorticoid dehydrogenase increases to 62% by original 35%.And obtained co-crosslinking enzyme aggregate pH is more stable, such as
In pH4-7, alcohol dehydrogenase and glucose dehydrogenase all remain 80% opposite enzyme activity shown in Fig. 4, and original enzyme liquid is in pH4
When alcohol dehydrogenase the enzyme activity rate of recovery there was only 40%.Since glucose is constantly converted into grape by glucose dehydrogenase in reaction
Saccharic acid, declines the pH of system, and cross-linked enzyme aggregate is more stable in acid condition, this is conducive to the progress of reaction.Temperature is steady
Qualitative also relatively former crude enzyme liquid makes moderate progress as shown in Figure 5.When reusing 7 times, alcohol dehydrogenase and glucose dehydrogenase
Activity recovery still have 80%, to realize repeatedly using for immobilised enzymes.
Main advantages of the present invention are：
(1) alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate are prepared for first, realize immobilised enzymes
Repeatedly use, be effectively improved the bioavailability of enzyme；
(2) provide and come a kind of method for preparing alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate, the side
Method has higher activity recovery.
With reference to specific embodiment, the further old present invention in detail.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002)
Described in condition, or according to the condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
Calculate.Experiment material and reagent used can be obtained from commercially available channel unless otherwise instructed in following embodiments.
Embodiment 1
Alcohol dehydrogenase and glucose dehydrogenase coexpression
Double enzymes are carried out to the plasmid containing ADH and GDH with specificity limitation restriction endonuclease NdeI and XhoI (U.S. Thermo)
Cut, run electrophoresis after to target segment carry out gel extraction, to vector plasmid pET-28a also with NdeI and XhoI carry out double digestion with
Gel extraction.ADH and GDH genetic fragments are attached with carrier segment respectively, obtain plasmid pET-28a-adh and pET-
28a-gdh, two plasmids have ampicillin and kalamycin resistance.Two plasmids are transferred to competent cell E.coli
DH5 α, obtain alcohol dehydrogenase and the coexpression bacterial strain E.coli BL21-ADH/GDH of glucose dehydrogenase.Bacterial strain will be co-expressed
It is inoculated into the LB culture mediums containing ampicillin (Amp, 100 μ g/ml) and kanamycins (Kan, 100 μ g/ml), 37 DEG C of perseverances
Warm shaking table 230r/min is incubated overnight.By the volume ratio of 1ml nutrient solution 100mlLB culture mediums (each 100 μ g/ml containing Amp and Kan)
It is inoculated into shaking flask, is grown in 37 DEG C of (230r/min) shaking tables, OD is arrived in culture600When about 0.8, IPTG is added, makes its final concentration
For 0.2mmol/L, (220r/min) cultivates 12h at 25 DEG C, and thalline is collected by centrifugation.With the triethanolamine (0.1mol/ of pH7.0
L) thalline is resuspended in buffer solution, and using Ultrasonic Cell Disruptor smudge cells, 3900r/min centrifugations 15min removes smudge cells tissue,
The crude enzyme liquid (in terms of total protein, total protein content 24mg/ml) containing alcohol dehydrogenase and glucose dehydrogenase is obtained, such as
Shown in the B swimming bands of SDS-PAGE electrophoresis 1, there is obvious target protein band, illustrate the success of target protein induced expression, thick enzyme
The content ratio of ADH and GDH is about 1 in liquid：1, respectively account for about the 25%~30% of total protein quality.
E.coli DH5, E.coli BL21 (DE3) are purchased from CICC23796, and plasmid is purchased from BioVector plasmid vector bacterium
Kind cytogene collection.
Embodiment 2
The selection of precipitating reagent
Specific implementation step is as follows：
(1) ethanol, acetone and saturated ammonium sulfate solution 1000 μ l, 900 μ l, 800 μ l, 700 μ l, 600 μ l are taken respectively herein,
500 μ l, by its respectively with the μ of 0 μ l, 100 μ l, 200 l, 300 μ l, 400 μ l, the TEA buffer solutions of the pH7.0 of 500 μ l0.1mol/L
Mixing, is made 100%, 90%, 80%, 70%, 60%, the precipitating reagent of 50% volumetric concentration.
(2) 1ml various concentrations precipitating reagent is taken in 1.5mlEP pipes, is placed on precooling 5min on ice-water bath, and it is thick to add 200 μ l
Enzyme liquid, on ice-water bath 150r/min shake 30min after centrifuge, above liquid be used for measure protein content.
(3) precipitation is resuspended with 600 μ lTEA buffer solutions again, for measuring enzyme activity.The results are shown in Figure 2, when with 90%
Acetone precipitation crude enzyme liquid when, the enzyme activity rate of recovery a maximum of about of 98%, the GDH enzyme activity rate of recovery of ADH is 58%；When with 80% sulfuric acid
When ammonium precipitates crude enzyme liquid, the enzyme activity rate of recovery of GDH is up to that 80%, the ADH enzyme activity rate of recovery is 41%.Due to ADH in this reaction
For rate-limiting enzyme, thus the acetone precipitation crude enzyme liquid of final choice 90%, the C bands in supernatant protein content such as Fig. 1 after centrifugation,
It can be seen that apparently without ADH and GDH, illustrate that target protein all is deposited.
Embodiment 3
The preparation of co-crosslinking enzyme aggregate
Specific implementation step is as follows：
(1) 18ml acetone and the acetone soln of 2mlTEA buffer preparations 90% is taken to seal in beaker and be placed on ice
In water-bath, start to stir after precooling 5min, 4ml crude enzyme liquids, stirring precipitation 30min is slowly added dropwise.
(2) the 480 μ l of glutaraldehyde solution that volume fraction is 2.5% are added dropwise, at this time glutaraldehyde in solution：Total protein mass ratio
For 0.5, when stirring 1.5 is small on ice-water bath.
(3) take the immobilised enzymes after 2ml crosslinkings to centrifuge 2min at 3000r/min, 4 DEG C and remove supernatant, isometric TEA can be used
Cache solution cleaning three times, cross-linked enzyme aggregate is resuspended with 1mlTEA buffer solutions, for enzyme activity determination.
Embodiment 4
Add the preparation of the co-crosslinking enzyme aggregate of Tween 80
Specific implementation step is as follows：
(1) 18ml acetone and the acetone soln of 2mlTEA buffer preparations 90% is taken to seal in beaker and be placed on ice
In water-bath, start to stir after precooling 5min, 4ml crude enzyme liquids, stirring precipitation 30min is slowly added dropwise.
(2) 2.5% 480 μ l of glutaraldehyde solution are added dropwise, at this time glutaraldehyde in solution：Total protein mass ratio is 0.5,5min
50% Tween 80 is added dropwise afterwards until the mass ratio of Tween 80 and total protein is 0.24, when stirring 1.5 is small on ice-water bath.
(3) take the immobilised enzymes after 2ml crosslinkings to centrifuge 2min at 3000r/min, 4 DEG C and remove supernatant, isometric TEA can be used
Cache solution cleaning three times, cross-linked enzyme aggregate is resuspended with 1ml TEA buffer solutions, for enzyme activity determination.
Embodiment 5
The selection of optimal glutaraldehyde concentration during crosslinking
The step of by embodiment 4, be added dropwise 2.5% glutaraldehyde solution 240 μ l, 480 μ l, 960 μ l in its preparation process,
1200 μ l, 1680 μ l, 5% 1680 μ l of glutaraldehyde, make glutaraldehyde in solution：Total protein mass ratio is 0.25,0.5,1.0,
1.25th, 1.75,2.50, the cleaning of the cross-linked enzyme aggregate that obtains afterwards measures enzyme activity afterwards three times, and the results are shown in Figure 3, add or
It is not added with Tween 80, glutaraldehyde：The ADH enzyme activity rate of recovery is maximum when total protein mass ratio is 0.5, and adds the co-crosslinking enzyme of Tween 80
The enzyme activity rate of recovery of aggregation is 82% apparently higher than the 59% of the not added-time.
Embodiment 6
The pH stability tests of co-crosslinking enzyme aggregate
Co-crosslinking enzyme aggregate is prepared according to the step of specific embodiment four, obtained co-crosslinking enzyme aggregate is divided equally into
6 parts, be respectively that 4,5,6,7,8,9 triethanolamines (0.1mol/L) are slow with isometric pH after 3000r/min centrifugations per portion 4ml
Rush solution washing three times, the buffer solution resuspension of pH is finally corresponded to 2ml, for the measure of enzyme activity, take the highest point of enzyme activity to be
100%, stability of the measure cross-linked enzyme aggregate in different pH.
6 parts of 1ml crude enzyme liquids are diluted to the triethanolamine buffer solution (0.1mol/L) of pH4,5,6,7,8,9 respectively
2.8ml, pH is adjusted with the 0.1mol/L Triethanolamine buffers of pH1 and pH12, until system pH is respectively finally 4,5,6,7,
8,9, and volume is 3ml, measures enzyme activity, it is 100% to take the highest point of enzyme activity, stability of the measure resolvase in different pH.
Embodiment 7
The temperature stability test of co-crosslinking enzyme aggregate
Co-crosslinking enzyme aggregate is prepared according to the step of specific embodiment 4, obtained co-crosslinking enzyme aggregate is divided into 5
Part, per portion 4ml, after 3000r/min centrifugations, three are washed with triethanolamine (0.1mol/L) buffer solution of isometric pH7.0
It is secondary, finally it is resuspended with 2ml buffer solutions.Five parts of cross-linked enzyme aggregates are individually placed to 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C of water
30min is kept the temperature in bath, measures enzyme activity afterwards, it is 100% to take the highest point of enzyme activity, and measure cross-linked enzyme aggregate is in different temperatures
When stability.
5ml crude enzyme liquids are diluted to 15ml with the triethanolamine buffer solution (0.1mol/L) of pH7.0, are divided equally into 5 parts, point
It is not placed on 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, keeps the temperature 30min in 60 DEG C of water-baths, measure enzyme activity afterwards, takes the highest point of enzyme activity to be
100%, stability of the measure resolvase in different temperatures.
Embodiment 8
Enzyme activity determination method
Glucose dehydrogenase enzyme activity determination method：
Enzyme reaction system 200ul includes：A.0.1mol/L Triethanolamine buffer (pH7.0) 110ul.b.10mmol/L
NADP+20ul.c.1mol/L C6H12O620ul.D. enzyme liquid 50ul puts 30 DEG C of insulations after mixing, from addition GDH crude enzyme liquids
That time start timing, in monitoring 10s at 340nm light absorption value rising.Calculate reaction generation NADPH's according to mark song
Amount.One enzyme-activity unit U is defined as：1 μm of required enzyme amount of ol NADPH is produced in 1 minute.
Alcohol dehydrogenase enzyme activity assay method：
Enzyme reaction system 200ul includes:A.0.1mol/L Triethanolamine buffer (pH7.0) 86ul.b.10mmol/L
NADPH 10ul.C.0.1mol/L the chloro- 3- fluoro acetophenones 4ul of 2,6- bis-.D. enzyme liquid 100ul.30 DEG C of guarantors are put after mixing
Temperature, starts timing, the reduction of the interior light absorption values at 340nm of detection 20s after adding 100ul enzyme liquids.Reaction is calculated according to mark song to disappear
Consume the amount of NADPH.1 enzyme-activity unit U is defined as：1 μm of required enzyme amount of ol NADPH is consumed in 1 minute.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
Enclose.
Claims (10)
- A kind of 1. preparation method of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate, it is characterised in that the side Method includes step：In the presence of surfactants, crosslinked dose of progress alcohol dehydrogenase and the co-crosslinking of glucose dehydrogenase are anti- Should.
- 2. the method as described in claim 1, it is characterised in that the surfactant is selected from the group：Tween 80 and tween 60。
- 3. the method as described in claim 1, it is characterised in that in the method, the crosslinking for carrying out the co-crosslinking reaction is anti- It is 0.1~1.2 to answer the mass ratio of system internal surfactants and substrates enzymes (alcohol dehydrogenase and glucose dehydrogenase)：0.5~ 1；Preferably mass ratio is 0.2~1：0.5~1；Most preferably about 0.4~0.8：1.
- 4. the method as described in claim 1, it is characterised in that glutaraldehyde is used in the cross-linking reaction as crosslinking agent.
- 5. the method as described in claim 1, it is characterised in that the cross-linking reaction system internal crosslinker and substrates enzymes (ethanol Dehydrogenase and glucose dehydrogenase) mass ratio be 0.1~1.2：0.5~1；Preferably 0.2~1.2：1；Most preferably about 1： 1。
- 6. the method as described in claim 1, it is characterised in that surfactant in cross-linking reaction：Crosslinking agent：Substrates enzymes (second Alcohol dehydrogenase and glucose dehydrogenase) mass ratio be 0.4-0.8：0.8-1.2：0.8-1.2.
- 7. the method as described in claim 1, it is characterised in that the method includes the steps：(1) mixture containing alcohol dehydrogenase and glucose dehydrogenase is provided；(2) cross-linking reactionCross-linking reaction system is prepared, the cross-linking reaction system includes：What step (1) provided contains alcohol dehydrogenase and grape Mixture, crosslinking agent and the surfactant of glucocorticoid dehydrogenase, carry out cross-linking reaction under the conditions of 0 DEG C~40 DEG C, so as to be made described Alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate.
- 8. a kind of cross-linked enzyme aggregate, it is characterised in that the cross-linked enzyme aggregate is by alcohol dehydrogenase and glucose dehydrogenase Crosslinked dose of co-crosslinking is formed.
- 9. cross-linked enzyme aggregate as claimed in claim 8, it is characterised in that the cross-linked enzyme aggregate by alcohol dehydrogenase and Crosslinked dose of co-crosslinking of glucose dehydrogenase is formed；Also, in the presence of surfactants, carry out cross-linking reaction.
- 10. alcohol dehydrogenase as claimed in claim 2 and glucose dehydrogenase co-crosslinking enzyme aggregate are in catalysis 1- (2,6- bis- Chloro- 3- fluorophenyls) purposes in ethyl ketone synthesis of chiral intermediate (S) -1- (the chloro- 3- fluorophenyls of 2,6- bis-).
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| CN110643556A (en) * | 2019-08-23 | 2020-01-03 | 浙江工业大学 | Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof |
| CN115011574A (en) * | 2022-06-24 | 2022-09-06 | 杭州师范大学 | Preparation method and application of site-controllable and ordered cross-linked double-enzyme aggregate |
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| CN109536479A (en) * | 2018-12-05 | 2019-03-29 | 清华大学 | A kind of crosslinking immobilized bi-enzyme-surfactant complex and preparation method thereof |
| WO2020113717A1 (en) * | 2018-12-05 | 2020-06-11 | 清华大学 | Crosslinking immobilized bienzyme-surfactant coordination complex, and preparation method therefor |
| CN110643556A (en) * | 2019-08-23 | 2020-01-03 | 浙江工业大学 | Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof |
| CN115011574A (en) * | 2022-06-24 | 2022-09-06 | 杭州师范大学 | Preparation method and application of site-controllable and ordered cross-linked double-enzyme aggregate |
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