CN109652474A - The method of biocatalysis acrylonitrile hydration reaction, the ablation method for producing nitrile bacterial strain - Google Patents
The method of biocatalysis acrylonitrile hydration reaction, the ablation method for producing nitrile bacterial strain Download PDFInfo
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Abstract
A kind of method that the present invention discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps: Step 1: carrying out tolerance training to Nocard's bacillus；Step 2: then fermented culture, screening, obtains qualified fermentation liquid；Step 3: qualified fermentation liquid removes supernatant, cleans to obtain bacterium solution after being centrifuged；Step 4: bacterium solution carries out inactivation treatment；Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.Invention additionally discloses a kind of ablation methods for producing nitrile bacterial strain.The present invention has the advantages that reach the production quantity for reducing by-product acrylic acid.
Description
Technical field
The method reacted the present invention relates to acrylamide preparation technical field more particularly to biocatalysis acrylonitrile hydration,
Produce the ablation method of nitrile bacterial strain.
Background technique
It is that acrylonitrile, raw water and immobilized biocatalyst are deployed into hydration solution that bioanalysis, which produces acrylamide,
Isolating dead catalyst just after catalysis reaction can be obtained acrylamide product.This method compares other prior arts such as sulfuric acid water
There are following advantages for legal, catalytic hydration: safe operation, without side reaction, at low cost, with high purity.
Nitrile hydratase is the catalyst of Production of Acrylamide by Microbial Method, but in the life of bioanalysis production acrylamide
Nitrile hydratase and nitrilase can be generated during production simultaneously.
Wherein, nitrile hydratase is a kind of constitutive enzyme, is only determined by the inhereditary material of microorganism itself.And nitrilase is more
Tendency is a kind of induced enzyme, during microbial catalyst catalysis hydration reacts and generates acrylamide, with acrylamide
The promotion of concentration, stimulation of the microorganism by substrate acrylamide start to synthesize more nitrilases, nitrilase in vivo
Meeting catalysed partial acrylamide is hydrolyzed into acrylic acid, and the presence of this by-product of acrylic acid will affect acrylamide solution product
Quality and storage stability, while will also result in the consumption of raw material propylene nitrile, finally affect the quality for generating liquid product and
The stability of post storage.
Summary of the invention
In view of the above technical problems, present invention aims to first carry out inactivation treatment to production bacterium by inactivator, after processing
The biocatalyst catalysis acrylonitrile hydration reaction being prepared into, to reach the technology for reducing the production quantity of by-product acrylic acid
Effect.
The present invention is realized by following technological means solves a kind of above-mentioned technical problem: biocatalysis acrylonitrile hydration
The method of reaction, comprising the following steps:
Step 1: carrying out tolerance training to Nocard's bacillus；
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；
Step 3: qualified fermentation liquid removes supernatant, cleans to obtain bacterium solution after being centrifuged；
Step 4: bacterium solution carries out inactivation treatment；
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.
Preferably, the tolerance training of the step 1 is to impregnate strain with the acrylamide aqueous solution of 55~60wt%
Then 16~40h is isolated and purified, rejuvenation culture.
The plate that the present invention utilizes solid medium to prepare, is reached with the method for dilution butteron on plate spread plate culture of the prior art
To the purpose isolated and purified.Bacterium solution is added dropwise in planar surface, then with sterile glass painting stick that bacterium solution is evenly dispersed to entire
Planar surface, the picking single bacterium colony after culture 4 days.It is trained using the rejuvenation that reaches for Tube propagation 12 days that solid medium is prepared
Feeding purpose.
Preferably, the temperature of fermentation tank culture is 23.5~27.5 DEG C in fermented and cultured in the step 2, is passed through compression
Air mass flow is 210~240m3/ h, holding pressure inside the tank are 0.05~0.07Mpa, and incubation time is 55~60h.
Preferably, the screening in the step 2 specifically comprises the processes of: enzyme activity detection is done in sampling, is examined with gas chromatography
Enzyme activity is surveyed, enzyme activity reaches 1600~2000 μ g/mLh for qualified fermentation liquid.
Preferably, solid medium includes the raw material of following mass fraction: 0.5~1 part of glucose, soybean meal hydrolysate 0.1
~0.3 part, 0.05~0.1 part of sodium chloride, 0.01~0.03 part of potassium phosphate, 0.01~0.03 part of magnesium sulfate, agar powder 1~3
Part, 90~95 parts of water.
Preferably, fermentation medium includes the raw material of following mass fraction: 2~3.5 parts of glucose, soybean meal hydrolysate 0.1
~0.3 part, 0.8~1.0 part of urea, 0.03~0.05 part of potassium phosphate, 0.03~0.05 part of sulfate of ammoniac, sodium glutamate 0.05~
0.075 part, 0.0001~0.0005 part of cobalt chloride, 94~95 parts of water
Preferably, the revolving speed being centrifuged in the step 3 is 4000r/min, centrifugation time 15min.
Preferably, inactivation treatment in the step 4 specifically comprises the processes of: the inactivation of bacterium solution total volume 0.5~1% is added
Agent formaldehyde carries out inactivation treatment to bacterium solution, 30~37 DEG C of temperature is controlled when inactivation, inactivation time is controlled in 3~5h；Wherein institute
Inactivator formaldehyde is to analyze pure AR.
Preferably, the catalyst in the step 5, pure water, acrylonitrile three mass ratio be 1:240:75.Reaction
21~23 DEG C of process control temp, the hydration reaction time is 15~16h.
Invention additionally discloses a kind of ablation methods for producing nitrile bacterial strain, comprising the following steps:
Step 1: carrying out tolerance training to Nocard's bacillus；
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；
Step 3: qualified fermentation liquid removes supernatant, cleans to obtain bacterium solution after being centrifuged；
Step 4: bacterium solution carries out inactivation treatment.
The present invention has the advantages that inactivation is added before thallus is as the reaction of biocatalyst catalysis hydration in the present invention
Agent pretreatment, the thallus after inactivation reach reduction catalysis reaction again as biocatalyst catalysis acrylonitrile hydration reaction
The production quantity of middle by-product acrylic acid, reduces the consumption of raw material propylene nitrile, and the reduction of by-product acrylic acid content can also make
The conductivity of acrylamide aqueous solution product is low, is conducive to the quality of product.Method of the invention can generate catalysis reaction
Acrylic acid content in liquid reduces by 70% or more, is conducive to product quality and storage stability.
Inactivate for thallus 30~37 DEG C of control temperature of the present invention, control time are that 3~5h is to realize spy of the invention
Parameter area is determined, if technical effect of the invention is not achieved in technical effect not in this parameter area.It is gone out with inactivator
The temperature controlled when processing living is excessively high, then will have a direct impact on the activity of nitrile hydratase, so that enzymatic activity reduces, because of nitrile water
Synthase is a kind of protein, it has certain optimum temperature the experiment has found that 30~37 DEG C of control temperature of thallus inactivation, and
The control temperature of the optimum temperature of the nitrile hydratase, inactivation is lower than this range, then thallus cannot be by thorough enzyme activity, the present invention
It is that formaldehyde enters thallus and directly destroys its inhereditary material with the principle of formalin-inactivated, thallus is made to lose genetic function, in this way inactivation
Thallus cannot largely synthesize nitrilase again afterwards, but not destroy the activity of nitrile hydratase, and important point of the invention is just
It is this inactivation characteristic of the inactivator formaldehyde to the promise cassette bacterium of discovery and the specific temperature with the formalin-inactivated strain.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, implement below in conjunction with the present invention
Example, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention
A part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, shall fall within the protection scope of the present invention.
It should be noted that it can directly on the other element when element is referred to as " being fixed on " another element
Or there may also be elements placed in the middle.When an element is considered as " connection " another element, it, which can be, is directly connected to
To another element or it may be simultaneously present centering elements.
Nocard's bacillus strain of the invention is the prior art, derives from Shanghai Pesticide Research Institute, and preservation condition is 3~4
DEG C, strain number 0918C018F3.
Embodiment 1
A kind of method that the present embodiment discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 16h to the Nocard's bacillus acrylamide aqueous solution of 55wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 90 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 27.5 DEG C, being passed through compressed air require is 210m3/ h, holding pressure inside the tank are 0.05Mpa, incubation time 55h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
1600 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: the inactivator formaldehyde of bacterium solution total volume 0.5% is added, inactivation treatment is carried out to bacterium solution, when inactivation controls
30 DEG C of temperature, inactivation time is controlled in 3h；Inactivator formaldehyde used in it is to analyze pure AR.
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.Catalyst, pure water, propylene
The mass ratio of nitrile three is 1:240:75.Reaction process controls 21 DEG C of temperature, and the hydration reaction time is 15h.
Embodiment 2
A kind of method that the present embodiment discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 16h to the Nocard's bacillus acrylamide aqueous solution of 60wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 95 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 27.5 DEG C, being passed through compressed air require is 210m3/ h, holding pressure inside the tank are 0.05Mpa, incubation time 55h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
1600 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: the inactivator formaldehyde of bacterium solution total volume 0.5% is added, inactivation treatment is carried out to bacterium solution, when inactivation controls
35 DEG C of temperature, inactivation time is controlled in 3h；Inactivator formaldehyde used in it is to analyze pure AR.
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.Catalyst, pure water, propylene
The mass ratio of nitrile three is 1:240:75.Reaction process controls 21 DEG C of temperature, and the hydration reaction time is 15h.
Embodiment 3
A kind of method that the present embodiment discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 40h to the Nocard's bacillus acrylamide aqueous solution of 55wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 95 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 26.5 DEG C, being passed through compressed air require is 240m3/ h, holding pressure inside the tank are 0.07Mpa, incubation time 60h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
2000 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: the inactivator formaldehyde of bacterium solution total volume 1% is added, inactivation treatment is carried out to bacterium solution, control temperature when inactivation
32 DEG C of degree, inactivation time are controlled in 5h；Inactivator formaldehyde used in it is to analyze pure AR.
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.Catalyst, pure water, propylene
The mass ratio of nitrile three is 1:240:75.Reaction process controls 23 DEG C of temperature, and the hydration reaction time is 16h.
Embodiment 4
A kind of method that the present embodiment discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 25h to the Nocard's bacillus acrylamide aqueous solution of 55wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 95 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 25.5 DEG C, being passed through compressed air require is 230m3/ h, holding pressure inside the tank are 0.06Mpa, incubation time 58h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
1800 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: the inactivator formaldehyde of bacterium solution total volume 0.6% is added, inactivation treatment is carried out to bacterium solution, when inactivation controls
37 DEG C of temperature, inactivation time is controlled in 3h；Inactivator formaldehyde used in it is to analyze pure AR.
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.Catalyst, pure water, propylene
The mass ratio of nitrile three is 1:240:75.Reaction process controls 22 DEG C of temperature, and the hydration reaction time is 15h.
Comparative example 1
A kind of method that this comparative example discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 16h to the Nocard's bacillus acrylamide aqueous solution of 60wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 95 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 26.5 DEG C, being passed through compressed air require is 210m3/ h, holding pressure inside the tank are 0.05Mpa, incubation time 55h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
1600 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: being catalyzed acrylonitrile hydration reaction using the bacterium solution in step 3 as catalyst.Catalyst, pure water, third
The mass ratio of alkene nitrile three is 1:240:75.Reaction process controls 21 DEG C of temperature, and the hydration reaction time is 15h.
Comparative example 2
A kind of method that this comparative example discloses biocatalysis acrylonitrile hydration reaction, comprising the following steps:
Step 1: impregnating 25h to the Nocard's bacillus acrylamide aqueous solution of 55wt%, then isolate and purify, rejuvenation training
It supports；The plate prepared using solid medium is reached with the method for dilution butteron on plate spread plate culture of the prior art and to be isolated and purified
Purpose achievees the purpose that rejuvenation culture using the Tube propagation that solid medium is prepared.Solid medium includes following mass parts
Several raw materials: 0.5 part of glucose, 0.1 part of soybean meal hydrolysate, 0.05 part of sodium chloride, 0.01 part of potassium phosphate, 0.01 part of magnesium sulfate,
1 part of agar powder, 95 parts of water.
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；The temperature of fermentation tank culture in fermented and cultured
It is 25.5 DEG C, being passed through compressed air require is 230m3/ h, holding pressure inside the tank are 0.06Mpa, incubation time 58h.Fermentation
Culture medium includes the raw material of following mass fraction: 2 parts of glucose, 0.3 part of soybean meal hydrolysate, 0.5 part of urea, potassium phosphate 0.03
Part, 0.03 part of sulfate of ammoniac, 0.05 part of sodium glutamate, 0.0005 part of cobalt chloride, 95 parts of water.
Enzyme activity is detected with gas chromatography, enzyme activity detection is done in sampling, detects enzyme activity with gas chromatography, enzyme activity reaches
1800 μ g/mLh are qualified fermentation liquid.
Step 3: qualified fermentation liquid obtains clean bacterium solution through pure water displacement, centrifugation removal supernatant；Centrifugation
Revolving speed is 4000r/min, centrifugation time 15min.
Step 4: being catalyzed acrylonitrile hydration reaction using the bacterium solution of step 3 as catalyst.Catalyst, pure water, propylene
The mass ratio of nitrile three is 1:240:75.Reaction process controls 22 DEG C of temperature, and the hydration reaction time is 15h.
Embodiment 2, embodiment 4, comparative example 1,2 catalysis hydration of comparative example is measured respectively to react third in the generation liquid obtained
Olefin(e) acid content, as shown in table 1:
Table 1
It is analyzed by the data to table 1, it is known that, catalytic water is participated in using biocatalyst prepared by method of the invention
The acrylic acid that the acrylic acid content that reaction obtains is reacted much larger than the biocatalyst participation catalysis hydration not inactivated is closed to contain
Amount.Inactivator pretreatment, the thallus after inactivation is added before thallus is as the reaction of biocatalyst catalysis hydration in the present invention
Again as biocatalyst catalysis acrylonitrile hydration reaction, and then reach the generation for reducing by-product acrylic acid in catalysis reaction
Amount reduces the consumption of raw material propylene nitrile, and generating acrylic acid content in liquid may be up to 210ppm, and yield is non-inactivation treatment
6 times.
Herein, such as first and second or the like relational terms are used merely to an entity or behaviour if it exists
Make to distinguish with another entity or operation, without necessarily requiring or implying between these entities or operation, there are any
This actual relationship or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment for including a series of elements not only includes those elements, and
It and further include other elements that are not explicitly listed, or further include solid by this process, method, article or equipment
Some elements.In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that wrapping
Include in the process, method, article or equipment of the element that there is also other identical elements.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (10)
1. a kind of method of biocatalysis acrylonitrile hydration reaction, which comprises the following steps:
Step 1: carrying out tolerance training to Nocard's bacillus；
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；
Step 3: qualified fermentation liquid removes supernatant, cleans to obtain bacterium solution after being centrifuged；
Step 4: bacterium solution carries out inactivation treatment；
Step 5: being catalyzed acrylonitrile hydration reaction using the bacterium solution after inactivation as catalyst.
2. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that the step 1
Tolerance training is to impregnate 16~40h of strain with the acrylamide aqueous solution of 55~60wt%, is then isolated and purified, rejuvenation training
It supports.
3. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that in the step 2
The temperature of fermentation tank culture is 23.5~27.5 DEG C in fermented and cultured, and being passed through compressed air require is 210~240m3/ h is kept
Pressure inside the tank is 0.05~0.07Mpa, and incubation time is 55~60h.
4. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that in the step 2
Screening specifically comprises the processes of: enzyme activity detection is done in sampling, and enzyme activity reaches 1600~2000 μ g/mLh for qualified fermentation liquid.
5. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that in the step 3
The revolving speed of centrifugation is 4000r/min, centrifugation time 15min.
6. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that in the step 4
Inactivation treatment specifically comprises the processes of: the inactivator formaldehyde of bacterium solution total volume 0.5~1% is added, inactivation treatment is carried out to bacterium solution, is gone out
30~37 DEG C of temperature are controlled when living, inactivation time is controlled in 3~5h.
7. the method for biocatalysis acrylonitrile hydration reaction according to claim 1, which is characterized in that in the step 5
Catalyst, pure water, acrylonitrile three mass ratio be 1:240:75.
8. a kind of ablation method for producing nitrile bacterial strain, it is characterised in that: the following steps are included:
Step 1: carrying out tolerance training to Nocard's bacillus；
Step 2: then fermented culture, screening, obtains qualified fermentation liquid；
Step 3: qualified fermentation liquid removes supernatant, cleans to obtain bacterium solution after being centrifuged；
Step 4: bacterium solution carries out inactivation treatment.
9. the ablation method according to claim 8 for producing nitrile bacterial strain, it is characterised in that: the tolerance training of the step 1
It is to impregnate 16~40h of strain with the acrylamide aqueous solution of 55~60wt%, then isolates and purifies, rejuvenation culture.
10. the ablation method according to claim 8 for producing nitrile bacterial strain, it is characterised in that: inactivation treatment in the step 4
Specifically comprises the processes of: the inactivator formaldehyde of bacterium solution total volume 0.5~1% is added, inactivation treatment is carried out to bacterium solution, when inactivation controls
30~37 DEG C of temperature, inactivation time is controlled in 3~5h.
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