CN106191002B - A method of improving pyrethroids class degrading enzyme expression quantity - Google Patents
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 29
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 28
- 230000014509 gene expression Effects 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000000593 degrading effect Effects 0.000 title claims abstract description 14
- VEMKTZHHVJILDY-UXHICEINSA-N bioresmethrin Chemical class CC1(C)[C@H](C=C(C)C)[C@H]1C(=O)OCC1=COC(CC=2C=CC=CC=2)=C1 VEMKTZHHVJILDY-UXHICEINSA-N 0.000 title claims abstract description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 72
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 18
- 230000006698 induction Effects 0.000 claims abstract description 15
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 14
- 238000011218 seed culture Methods 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 239000006052 feed supplement Substances 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000000725 suspension Substances 0.000 claims abstract description 9
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 8
- 239000008101 lactose Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims abstract description 5
- 230000001939 inductive effect Effects 0.000 claims abstract description 5
- 239000002953 phosphate buffered saline Substances 0.000 claims abstract description 5
- 238000002525 ultrasonication Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- -1 isopropylthio Chemical group 0.000 claims abstract 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims abstract 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims 1
- 229910052731 fluorine Inorganic materials 0.000 claims 1
- 239000011737 fluorine Substances 0.000 claims 1
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 5
- 238000004108 freeze drying Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 12
- 229930027917 kanamycin Natural products 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 229910052927 chalcanthite Inorganic materials 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 229910052564 epsomite Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000012805 post-processing Methods 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000003519 ventilatory effect Effects 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 239000011686 zinc sulphate Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- VQXSOUPNOZTNAI-UHFFFAOYSA-N Pyrethrin I Natural products CC(=CC1CC1C(=O)OC2CC(=O)C(=C2C)CC=C/C=C)C VQXSOUPNOZTNAI-UHFFFAOYSA-N 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000005935 Sulfuryl fluoride Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 150000002597 lactoses Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- HYJYGLGUBUDSLJ-UHFFFAOYSA-N pyrethrin Natural products CCC(=O)OC1CC(=C)C2CC3OC3(C)C2C2OC(=O)C(=C)C12 HYJYGLGUBUDSLJ-UHFFFAOYSA-N 0.000 description 1
- VJFUPGQZSXIULQ-XIGJTORUSA-N pyrethrin II Chemical compound CC1(C)[C@H](/C=C(\C)C(=O)OC)[C@H]1C(=O)O[C@@H]1C(C)=C(C\C=C/C=C)C(=O)C1 VJFUPGQZSXIULQ-XIGJTORUSA-N 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- OBTWBSRJZRCYQV-UHFFFAOYSA-N sulfuryl difluoride Chemical compound FS(F)(=O)=O OBTWBSRJZRCYQV-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
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Abstract
The present invention provides a kind of method for improving pyrethroids class degrading enzyme expression quantity.Pass through preparation production strain E. coli BLR (DE3)/pET-28a-est825；Seed culture；Fermented and cultured: 30-35 DEG C is cooled to using the mixing inducing solution (molar concentration rate 7:3) of lactose and isopropylthio galactolipin (IPTG) and starts to induce, the mixing feed supplement liquid of Induction Process relaying afterflow glycerol adding and yeast powder, the concentration for maintaining glycerol in fermentation liquid is 1-2g/L, and the time of induction continues 6-7h；Finally by thallus with the phosphate buffered saline of the pH6.5-7.0 containing 0.005mol/L phenylmethylsulfonyl fluoride at the bacteria suspension of mass concentration 15%-20%, up to high enzyme activity degrading enzyme enzyme powder after ultrasonication centrifugation freeze-drying.
Description
[technical field]
The present invention relates to field of microbial fermentation, and in particular to prokaryotic expression system is a kind of to improve pyrethroids class degrading enzyme table
Up to the method for amount.
[background technique]
Prokaryotic expression system is one of current most widely used heterologous gene expression system, and there are commonly Escherichia coli
Expression system, bacillus subtilis expression system, Expression System of streptomyces etc., wherein again the most normal with escherichia expression system
See.Escherichia expression system advantage is that gene expression product can be obtained within a short period of time and required cost is opposite
It is cheaper, it has been applied to the expression of pyrethroids class degrading enzyme.BLR (DE3) is a kind of protease-deficient Type B bacterial strain, is
The derivative bacterium of BL21recA of A.Roca (University of Wisconsin) building can stablize certain with repetitive sequence
Target gene.
Pyrethrin pesticide is a kind of widely used insecticide, has been widely applied in modern agriculture, due to they
Extensive use produces certain influence to ecological environment, this environmental problem of pesticide residue attracts attention in recent years, utilizes biology
Biodegrading process can solve Pesticides Residues, become the research hotspot of environmental science.The scholars such as existing Zhongshan University, the country are to it
The screening of degrading enzyme and the clone of degrading enzyme gene have done a series of researchs.If CN 201010547206.8 has recombinated est825,
E. coli BL21 (the DE3)/pET-28a-est825 for constructing recombinant expression is used to prepare degrading enzyme.Huang Hao etc.
People uses lactose fermentation in CN 201210412214.0, improves the yield of degrading enzyme, right above-mentioned enzyme powder yield still compared with
It is low.
[summary of the invention]
The purpose of the present invention is in view of the shortcomings of the prior art, developing a kind of side for improving pyrethroids class degrading enzyme expression quantity
Method.
In order to achieve the above objectives, including following the present invention provides a kind of method for improving pyrethroids class degrading enzyme expression quantity
Step:
1) preparation production strain E. coli BLR (DE3)/pET-28a-est825；
The sequence of est825 is referred to patent CN 201210412214.0, and genetic engineering constructs the operation of host cell
It is referred to patent CN 201010547206.8.
2) seed culture is carried out；
3) fermented and cultured is carried out: as the OD of fed-batch fermentation culture to thallus600nmIt is disposable to add when value reaches 120-140
Lactose+isopropylthio galactolipin (IPTG) mixing inducing solution (molar concentration rate 7:3), makes above-mentioned mixing in fermentation liquid
The concentration of induction is 9-13g/L, is down to temperature and starts to induce to 30-35 DEG C, and Induction Process relays afterflow glycerol adding and yeast powder
Mixing feed supplement liquid, maintaining the concentration of glycerol in fermentation liquid is 1-2g/L, and the time of induction continues 6-7h；
In some embodiments, the substrate cultivation base of fermentation can be NZCYM culture medium or LB culture medium；At other
In embodiment, the pH of the fermentation medium is 6.8, and the fermentation medium includes following components: glycerol 5-8g/L, albumen
Peptone 3-10g/L, yeast powder 3-8g/L, (NH4)2SO4 5-10g/L, MgSO47H2O 1-3g/L, KH2PO4 3.4-6.85g/
L, K2HPO4·12H2O 5.68-11.35g/L, liquid microelement 1-2mL/L, kanamycins 50-100mg/L.
4) after fermentation, 4-8 DEG C at a temperature of thalline were collected by centrifugation, by thallus use contain 0.005mol/L benzyl
The phosphate buffered saline of the pH6.5-7.0 of sulfuryl fluoride at mass concentration 15%-20% bacteria suspension, by bacteria suspension in 4-8
20KHz ultrasonication at a temperature of DEG C is crushed liquid and is centrifuged obtained supernatant, is lyophilized up to enzyme powder.
The research of pyrethroids class biodegradable enzyme is also relatively fewer, by the numerous studies of the present inventor, the choosing of host cell
It selects, for expressing this biodegradable enzyme, than using expression quantity under BL21 (DE3) same case to be higher by 20% or more；Use conjunction
The lactose of suitable ratio and the mixed solution of lactose analog carry out adding a small amount of phenylmethylsulfonyl fluoride in induction and disruption solution
Higher enzyme powder output can be help to obtain.Preparation method through the invention can obtain the high activity not less than 200g/L
The enzyme powder amount of pyrethroids class degrading enzyme.
Specific embodiment
As described below is the preferred embodiment of the present invention, and what the present invention was protected is not limited to following preferred implementation side
Formula.It should be pointed out that for those skilled in the art on the basis of the inventive concept, several deformations for making and
It improves, belongs to protection scope of the present invention.
The measuring method of the glycerol of following example, cell concentration and enzyme activity is referring to patent CN 201210412214.0.
Embodiment 1
1., will with glycerol tube using recombination bacillus coli E.coli BLR (DE3)/pET-28a-est825 as production strain
Production strain -86 DEG C at a temperature of save.
2. seed culture: the production strain in glycerol tube being accessed shake-flask seed culture medium by 1.5% inoculum concentration, to turn
Speed is 220r/min, temperature is 37 DEG C, condition of culture cultivates 10h in constant-temperature table；The pH of the shake-flask seed culture medium is
7.0, the shake-flask seed culture medium is the LB culture medium of 50mg/L containing kanamycin.
3. fermented and cultured: cultured seed liquor is equipped with to the fermentation of 5.5L fermentation medium with 4% inoculum concentration access
Through row fermented and cultured in tank, the time of fermented and cultured is 26h, maintains oxygen dissolving value 20%, ventilatory capacity 3vvm, speed of agitator
1000r/min, 37 DEG C of fermentation temperature, the pH that fermentation liquid is controlled using 25% ammonium hydroxide and the hydrochloric acid solution of 3mol/L is 6.8；Hair
The pH of ferment culture medium is 6.8, this experiment fermentation medium includes: glycerol 5g/L, peptone 10g/L, yeast powder 8g/L, (NH4)2SO45g/L, MgSO4·7H2O 3g/L, KH2PO43.4g/L, K2HPO4·12H2O 11.35g/L, liquid microelement 1mL/L,
Kanamycins 50mg/L.Above-mentioned liquid microelement includes: CaCl22.0g/L, ZnSO4·7H2O 5.0g/L, MnSO4·H2O
0.5g/L, Na2- EDTA 18.0g/L, FeSO4·7H2O 10.0g/L, CuSO4·5H2O 2.5g/L, CoCl2·6H2O0.2g/
L, (NH4)6Mo7O24·4H2O 0.1g/L。
4. fed-batch fermentation culture: after fermented and cultured 4h, flowing the mixing feed supplement liquid of glycerol adding and yeast powder, control the ratio of thallus
Growth rate is 0.1-0.2h-1, fed-batch fermentation cultivation stage duration is 7h；The mixing feed supplement of the glycerol and yeast powder
Liquid includes following components: glycerol 500g/L, yeast powder 100g/L, kanamycins 50mg/L；
5. carrying out fermented and cultured: as the OD of fed-batch fermentation culture to thallus600nmIt is disposable to add when value reaches 130 or so
The mixing inducing solution (molar concentration rate 7:3) of lactose+IPTG makes the concentration 9/L of above-mentioned mixing induction in fermentation liquid, drop
Start to induce to temperature to 30 DEG C, Induction Process relays the mixing feed supplement liquid of afterflow glycerol adding and yeast powder, maintains in fermentation liquid
The concentration of glycerol is 1g/L, and the time of induction continues 7h.
6. post-processing: after fermentation, 4-8 DEG C at a temperature of thalline were collected by centrifugation, by thallus with containing 0.005mol/
The phosphate buffered saline of the pH6.5 of L phenylmethylsulfonyl fluoride at mass concentration 20% bacteria suspension, by bacteria suspension at 4 DEG C
At a temperature of, 20KHz ultrasonication, be crushed liquid and be centrifuged supernatant is made, be lyophilized up to enzyme powder, enzyme powder yield is 267g/L.Enzyme activity
For 70000U/g.
Embodiment 2
1., will with glycerol tube using recombination bacillus coli E.coli BLR (DE3)/pET-28a-est825 as production strain
Production strain -86 DEG C at a temperature of save.
2. seed culture: the production strain in glycerol tube being accessed shake-flask seed culture medium by 1.5% inoculum concentration, to turn
Speed is 220r/min, temperature is 37 DEG C, condition of culture cultivates 10h in constant-temperature table；The pH of the shake-flask seed culture medium is
7.0, the shake-flask seed culture medium is the LB culture medium of 50mg/L containing kanamycin.
3. fermented and cultured: cultured seed liquor is equipped with to the fermentation of 5.5L fermentation medium with 4% inoculum concentration access
Through row fermented and cultured in tank, the time of fermented and cultured is 26h, maintains oxygen dissolving value 20%, ventilatory capacity 3vvm, speed of agitator
1000r/min, 37 DEG C of fermentation temperature, the pH that fermentation liquid is controlled using 25% ammonium hydroxide and the hydrochloric acid solution of 3mol/L is 6.8；Hair
The pH of ferment culture medium is 6.8, this experiment fermentation medium includes: glycerol 8g/L, peptone 3g/L, yeast powder 3g/L, (NH4)2SO410g/L, MgSO4·7H2O 1g/L, KH2PO46.85g/L K2HPO4·12H2O 5.68g/L, liquid microelement 1mL/
L, kanamycins 50mg/L.Above-mentioned liquid microelement includes: CaCl22.0g/L, ZnSO4·7H2O 5.0g/L, MnSO4·H2O
0.5g/L, Na2- EDTA 18.0g/L, FeSO4·7H2O 10.0g/L, CuSO4·5H2O 2.5g/L, CoCl2·6H2O0.2g/
L, (NH4)6Mo7O24·4H2O 0.1g/L；
4. fed-batch fermentation culture: after fermented and cultured 4h, flowing the mixing feed supplement liquid of glycerol adding and yeast powder, control the ratio of thallus
Growth rate is 0.1-0.2h-1, fed-batch fermentation cultivation stage duration is 9h；The mixing feed supplement of the glycerol and yeast powder
Liquid includes following components: glycerol 500g/L, yeast powder 100g/L, kanamycins 50mg/L；
5. carrying out fermented and cultured: as the OD of fed-batch fermentation culture to thallus600nmIt is disposable to add when value reaches 130 or so
The mixing inducing solution (molar concentration rate 7:3) of lactose+IPTG makes the concentration 9/L of above-mentioned mixing induction in fermentation liquid, drop
Start to induce to temperature to 30 DEG C, Induction Process relays the mixing feed supplement liquid of afterflow glycerol adding and yeast powder, maintains in fermentation liquid
The concentration of glycerol is 1g/L, and the time of induction continues 7h.
6. post-processing: after fermentation, 4-8 DEG C at a temperature of thalline were collected by centrifugation, by thallus with containing 0.005mol/
The phosphate buffered saline of the pH6.5 of L phenylmethylsulfonyl fluoride at mass concentration 20% bacteria suspension, by bacteria suspension at 4 DEG C
At a temperature of, 20KHz ultrasonication, be crushed liquid and be centrifuged supernatant is made, be lyophilized up to enzyme powder, enzyme powder yield is 315g/L.Enzyme activity
For 72000U/g.
Above description is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, it is all the present invention suggested by technical spirit under completed same changes or modifications change, should all belong to
In the covered the scope of the patents of the present invention.
Claims (1)
1. a kind of method for improving pyrethroids class degrading enzyme expression quantity, comprising the following steps:
1) preparation production strain E. coli BLR (DE3)/pET-28a-est825；
2) seed culture is carried out；
3) fermented and cultured is carried out: as the OD of fed-batch fermentation culture to thallus600nmWhen value reaches 120-140, disposably add lactose+
The mixing inducing solution of isopropylthio galactolipin (IPTG) makes the concentration 9-13g/L of above-mentioned mixing induction liquid in fermentation liquid,
The molar concentration rate of the lactose and isopropylthio galactolipin is 7:3, and temperature is down to 30-35 DEG C and starts to induce, Induction Process
The mixing feed supplement liquid of afterflow glycerol adding and yeast powder is relayed, maintaining the concentration of glycerol in fermentation liquid is 1-2g/L, the time of induction
Continue 6-7h；
4) after fermentation, 4-8 DEG C at a temperature of thalline were collected by centrifugation, by thallus use contain 0.005mol/L benzyl sulphonyl
The phosphate buffered saline of the pH6.5-7.0 of fluorine at mass concentration 15%-20% bacteria suspension, by bacteria suspension at 4-8 DEG C
At a temperature of 20KHz ultrasonication, be crushed liquid and be centrifuged supernatant is made, be lyophilized up to enzyme powder.
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