CN106191002A - A kind of method improving chrysanthemum esters digestive enzyme expression - Google Patents
A kind of method improving chrysanthemum esters digestive enzyme expression Download PDFInfo
- Publication number
- CN106191002A CN106191002A CN201610531523.8A CN201610531523A CN106191002A CN 106191002 A CN106191002 A CN 106191002A CN 201610531523 A CN201610531523 A CN 201610531523A CN 106191002 A CN106191002 A CN 106191002A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- induction
- liquid
- concentration
- glycerol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 15
- 102000038379 digestive enzymes Human genes 0.000 title claims abstract description 13
- 108091007734 digestive enzymes Proteins 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims abstract description 13
- 235000007516 Chrysanthemum Nutrition 0.000 title claims abstract description 10
- 150000002148 esters Chemical class 0.000 title claims abstract description 10
- 244000189548 Chrysanthemum x morifolium Species 0.000 title claims abstract 3
- 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 49
- 230000004151 fermentation Effects 0.000 claims abstract description 49
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 24
- 230000006698 induction Effects 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 14
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims abstract description 11
- 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
- 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
- YNAGLKOBLOKHEO-VGRMVHKJSA-N (4r,5s,6s,7r)-4,5,6,7,8-pentahydroxy-2-methyloctane-3-thione Chemical compound CC(C)C(=S)[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO YNAGLKOBLOKHEO-VGRMVHKJSA-N 0.000 claims abstract description 6
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 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
- 239000006228 supernatant Substances 0.000 claims description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims 1
- -1 benzyl sulphonyl Chemical group 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
- 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 abstract 1
- 239000001963 growth medium Substances 0.000 description 12
- 241000723353 Chrysanthemum Species 0.000 description 7
- 229930027917 kanamycin Natural products 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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
- 229960000318 kanamycin Drugs 0.000 description 7
- 229930182823 kanamycin A Natural products 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 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
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229910021529 ammonia Inorganic materials 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
- 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
- 238000011160 research Methods 0.000 description 2
- 238000009423 ventilation Methods 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
- 241001198387 Escherichia coli BL21(DE3) 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
- 238000003556 assay Methods 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
- 230000000593 degrading effect Effects 0.000 description 1
- 230000000694 effects 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
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 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
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
-
- 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)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides a kind of method improving chrysanthemum esters digestive enzyme expression.I.e. produce strain E. coli BLR (DE3)/pET 28a est825 by preparation；Seed culture；Fermentation culture: use the mixing inducing solution (molar concentration rate is 7:3) of lactose and isopropylthio galactose (IPTG) to be cooled to 30 35 DEG C and start induction, Induction Process relaying afterflow glycerol adding and the mixing feed supplement liquid of yeast powder, maintaining the concentration of glycerol in fermentation liquid is 1 2g/L, and the time of induction continues 6 7h；Thalline finally becomes the bacteria suspension of mass concentration 15% 20% with the phosphate buffered saline of the pH6.5 7.0 containing 0.005mol/L Phenylmethanesulfonyl fluoride, and ultrasonication i.e. obtains high enzyme and lives digestive enzyme enzyme powder after being centrifuged lyophilizing.
Description
[technical field]
The present invention relates to field of microbial fermentation, be specifically related to prokaryotic expression system one and can improve chrysanthemum esters digestive enzyme table
The method of the amount of reaching.
[background technology]
Prokaryotic expression system, is one of current most widely used heterologous gene expression system, and conventional has escherichia coli
Expression system, bacillus subtilis expression system, Expression System of streptomyces etc., the most normal with escherichia expression system the most again
See.Escherichia expression system advantage is can to obtain gene expression product within a short period of time and required cost is relative
Ratio is less expensive, has been applied to the expression of chrysanthemum esters digestive enzyme.BLR (DE3) is a kind of protease-deficient Type B bacterial strain, for
The BL21recA that A.Roca (University of Wisconsin) builds derives bacterium, can stablize some with repetitive sequence
Genes of interest.
Pyrethrin pesticide is a widely used insecticide of class, has been widely applied in modern agriculture, due to they
Extensively application creates certain impact to ecological environment, and this environmental problem of pesticide residues receives publicity in recent years, utilizes biology
Biodegrading process can solve Pesticides Residues, becomes the study hotspot of environmental science.The scholars such as domestic existing Zhongshan University are to it
The screening of digestive enzyme and the clone of degrading enzyme gene have done a series of research.As CN 201010547206.8 has recombinated est825,
Construct recombinant expressed E. coli BL21 (DE3)/pET-28a-est825 to be used for preparing digestive enzyme.Huang Hao etc.
People uses lactose fermentation in CN 201210412214.0, improves the yield of digestive enzyme, and right above-mentioned enzyme powder yield is the most relatively
Low.
[summary of the invention]
It is an object of the invention to for prior art not enough, develop a kind of side improving chrysanthemum esters digestive enzyme expression
Method.
For reaching above-mentioned purpose, the invention provides a kind of method improving chrysanthemum esters digestive enzyme expression, including following
Step:
1) preparation produces strain E. coli BLR (DE3)/pET-28a-est825；
The sequence of est825 is referred to patent CN 201210412214.0, and genetic engineering builds the operation of host cell
It is referred to patent CN 201010547206.8.
2) seed culture is carried out；
3) fermentation culture is carried out: when fed-batch fermentation cultivates the OD to thalline600nmWhen value reaches 120-140, disposably add
The mixing inducing solution (molar concentration rate is 7:3) of lactose+isopropylthio galactose (IPTG), makes above-mentioned mixing in fermentation liquid
The concentration of induction is 9-13g/L, is down to temperature and starts induction, Induction Process relaying afterflow glycerol adding and yeast powder to 30-35 DEG C
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 described fermentation medium is 6.8, and described fermentation medium includes following components: glycerol 5-8g/L, albumen
Peptone 3-10g/L, yeast powder 3-8g/L, (NH4)2SO4 5-10g/L, MgSO4 7H2O 1-3g/L, KH2PO4 3.4-6.85g/
L, K2HPO4·12H2O 5.68-11.35g/L, liquid microelement 1-2mL/L, kanamycin 50-100mg/L.
4), after fermentation ends, centrifugal collection thalline at a temperature of 4-8 DEG C, by thalline with containing 0.005mol/L benzyl
The phosphate buffered saline of the pH6.5-7.0 of sulfuryl fluoride becomes the bacteria suspension of mass concentration 15%-20%, by bacteria suspension at 4-8
20KHz ultrasonication at a temperature of DEG C, broken liquid is centrifugal prepares supernatant, and lyophilizing i.e. obtains enzyme powder.
The research of chrysanthemum esters biodegradable enzyme is the most relatively fewer, through the numerous studies of the present inventor, the choosing of host cell
Select, for expressing this biodegradable enzyme, exceed more than 20% than using expression under BL21 (DE3) same case；Use and close
The suitable lactose of ratio and the mixed solution of lactose analog carry out adding a small amount of Phenylmethanesulfonyl fluoride in induction and disruption solution
Can beneficially obtain higher enzyme powder volume of production.By the preparation method of the present invention, can obtain being not less than the high activity of 200g/L
The enzyme powder amount of chrysanthemum esters digestive enzyme.
Detailed description of the invention
The following stated is the preferred embodiment of the present invention, and what the present invention was protected is not limited to the following side of being preferable to carry out
Formula.It should be pointed out that, for a person skilled in the art these innovation and creation conceive on the basis of, the some deformation made and
Improve, broadly fall into protection scope of the present invention.
The assay method that the glycerol of example below, cell concentration and enzyme are lived is with reference to patent CN 201210412214.0.
Embodiment 1
1. using recombination bacillus coli E.coli BLR (DE3)/pET-28a-est825 as producing strain, will with glycerol pipe
Produce strain to preserve at a temperature of-86 DEG C.
2. seed culture: the strain that produces in glycerol pipe is accessed shake-flask seed culture medium by the inoculum concentration of 1.5%, to turn
Speed for 220r/min, temperature be 37 DEG C, condition of culture in constant-temperature table, cultivate 10h；The pH of described shake-flask seed culture medium is
7.0, described shake-flask seed culture medium is the LB culture medium containing kanamycin 50mg/L.
3. fermentation culture: cultured seed liquor is accessed the fermentation equipped with 5.5L fermentation medium with the inoculum concentration of 4%
Through row fermentation culture in tank, the time of fermentation culture is 26h, maintains oxygen dissolving value 20%, and ventilation is 3vvm, speed of agitator
1000r/min, fermentation temperature 37 DEG C, the pH using the ammonia of 25% and the hydrochloric acid solution of 3mol/L to control fermentation liquid is 6.8；Send out
The pH of ferment culture medium is 6.8, and this experiment fermentation medium comprises: 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,
Kanamycin 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 is cultivated: after fermentation culture 4h, stream glycerol adding and the mixing feed supplement liquid of yeast powder, controls the ratio of thalline
Growth rate is 0.1-0.2h-1, fed-batch fermentation cultivation stage duration is 7h；The mixing feed supplement of described glycerol and yeast powder
Liquid includes following components: glycerol 500g/L, yeast powder 100g/L, kanamycin 50mg/L；
5. carry out fermentation culture: when fed-batch fermentation cultivates the OD to thalline600nmWhen value reaches about 130, disposably add
The mixing inducing solution (molar concentration rate is 7:3) of lactose+IPTG, making the concentration of above-mentioned mixing induction in fermentation liquid is 9/L, fall
Start induction, Induction Process relaying afterflow glycerol adding and the mixing feed supplement liquid of yeast powder to temperature to 30 DEG C, maintain in fermentation liquid
The concentration of glycerol is 1g/L, and the time of induction continues 7h.
6. post processing: after fermentation ends, centrifugal collection thalline at a temperature of 4-8 DEG C, by thalline with containing 0.005mol/
The phosphate buffered saline of the pH6.5 of L Phenylmethanesulfonyl fluoride becomes the bacteria suspension of mass concentration 20%, by bacteria suspension at 4 DEG C
At a temperature of, 20KHz ultrasonication, broken liquid is centrifugal prepares supernatant, and lyophilizing i.e. obtains enzyme powder, and enzyme powder yield is 267g/L.Enzyme is lived
For 70000U/g.
Embodiment 2
1. using recombination bacillus coli E.coli BLR (DE3)/pET-28a-est825 as producing strain, will with glycerol pipe
Produce strain to preserve at a temperature of-86 DEG C.
2. seed culture: the strain that produces in glycerol pipe is accessed shake-flask seed culture medium by the inoculum concentration of 1.5%, to turn
Speed for 220r/min, temperature be 37 DEG C, condition of culture in constant-temperature table, cultivate 10h；The pH of described shake-flask seed culture medium is
7.0, described shake-flask seed culture medium is the LB culture medium containing kanamycin 50mg/L.
3. fermentation culture: cultured seed liquor is accessed the fermentation equipped with 5.5L fermentation medium with the inoculum concentration of 4%
Through row fermentation culture in tank, the time of fermentation culture is 26h, maintains oxygen dissolving value 20%, and ventilation is 3vvm, speed of agitator
1000r/min, fermentation temperature 37 DEG C, the pH using the ammonia of 25% and the hydrochloric acid solution of 3mol/L to control fermentation liquid is 6.8；Send out
The pH of ferment culture medium is 6.8, and this experiment fermentation medium comprises: 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, kanamycin 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 is cultivated: after fermentation culture 4h, stream glycerol adding and the mixing feed supplement liquid of yeast powder, controls the ratio of thalline
Growth rate is 0.1-0.2h-1, fed-batch fermentation cultivation stage duration is 9h；The mixing feed supplement of described glycerol and yeast powder
Liquid includes following components: glycerol 500g/L, yeast powder 100g/L, kanamycin 50mg/L；
5. carry out fermentation culture: when fed-batch fermentation cultivates the OD to thalline600nmWhen value reaches about 130, disposably add
The mixing inducing solution (molar concentration rate is 7:3) of lactose+IPTG, making the concentration of above-mentioned mixing induction in fermentation liquid is 9/L, fall
Start induction, Induction Process relaying afterflow glycerol adding and the mixing feed supplement liquid of yeast powder to temperature to 30 DEG C, maintain in fermentation liquid
The concentration of glycerol is 1g/L, and the time of induction continues 7h.
6. post processing: after fermentation ends, centrifugal collection thalline at a temperature of 4-8 DEG C, by thalline with containing 0.005mol/
The phosphate buffered saline of the pH6.5 of L Phenylmethanesulfonyl fluoride becomes the bacteria suspension of mass concentration 20%, by bacteria suspension at 4 DEG C
At a temperature of, 20KHz ultrasonication, broken liquid is centrifugal prepares supernatant, and lyophilizing i.e. obtains enzyme powder, and enzyme powder yield is 315g/L.Enzyme is lived
For 72000U/g.
Described above is the detailed description for the preferable possible embodiments of the present invention, but embodiment is not limited to this
Bright patent claim, the equal change completed under the technical spirit suggested by all present invention or modification change, all should belong to
In the contained the scope of the claims of the present invention.
Claims (1)
1. the method improving chrysanthemum esters digestive enzyme expression, comprises the following steps:
1) preparation produces strain E. coli BLR (DE3)/pET-28a-est825；
2) seed culture is carried out；
3) fermentation culture is carried out: when fed-batch fermentation cultivates the OD to thalline600nmWhen value reaches 120-140, disposably add lactose+
The mixing inducing solution of isopropylthio galactose (IPTG), making the concentration of above-mentioned mixing induction liquid in fermentation liquid is 9-13g/L,
Described lactose is 7:3 with the molar concentration rate of isopropylthio galactose, and temperature is down to 30-35 DEG C and is started induction, Induction Process
Relaying afterflow glycerol adding and the mixing feed supplement liquid of yeast powder, maintaining the concentration of glycerol in fermentation liquid is 1-2g/L, the time of induction
Continue 6-7h；
4), after fermentation ends, centrifugal collection thalline at a temperature of 4-8 DEG C, by thalline with containing 0.005mol/L benzyl sulphonyl
The phosphate buffered saline of the pH6.5-7.0 of fluorine becomes the bacteria suspension of mass concentration 15%-20%, by bacteria suspension at 4-8 DEG C
At a temperature of 20KHz ultrasonication, broken liquid is centrifugal prepares supernatant, and lyophilizing i.e. obtains enzyme powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610531523.8A CN106191002B (en) | 2016-07-07 | 2016-07-07 | A method of improving pyrethroids class degrading enzyme expression quantity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610531523.8A CN106191002B (en) | 2016-07-07 | 2016-07-07 | A method of improving pyrethroids class degrading enzyme expression quantity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106191002A true CN106191002A (en) | 2016-12-07 |
| CN106191002B CN106191002B (en) | 2019-11-15 |
Family
ID=57472560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610531523.8A Active CN106191002B (en) | 2016-07-07 | 2016-07-07 | A method of improving pyrethroids class degrading enzyme expression quantity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106191002B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985627A (en) * | 2010-11-17 | 2011-03-16 | 中山大学 | Novel esterase and application thereof |
| CN102876642A (en) * | 2012-10-25 | 2013-01-16 | 东莞市农业科学研究中心 | Preparation method for pyrethroids pesticide degrading enzyme |
-
2016
- 2016-07-07 CN CN201610531523.8A patent/CN106191002B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985627A (en) * | 2010-11-17 | 2011-03-16 | 中山大学 | Novel esterase and application thereof |
| CN102876642A (en) * | 2012-10-25 | 2013-01-16 | 东莞市农业科学研究中心 | Preparation method for pyrethroids pesticide degrading enzyme |
Non-Patent Citations (1)
| Title |
|---|
| 黄皓等: "重组大肠杆菌发酵表达菊酯类农药降解酶培养基优化", 《广东农业科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106191002B (en) | 2019-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bashan et al. | Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013) | |
| JP2022000024A (en) | Nitrogen fixation using refactored nif clusters | |
| Saxena | Applied microbiology | |
| CN109486702A (en) | A kind of composite bacteria agent and preparation method thereof enhancing salt tolerance of wheat | |
| CN106062177B (en) | Corynebacteria microorganism with the improved ability for generating L-lysine and the method for using it to generate L-lysine | |
| CN105316306B (en) | A kind of fermentation process efficiently producing keratinase using recombination bacillus coli | |
| US11293027B2 (en) | Recombinant Bacillus subtilis with improved 2′-fucosyllactose production, and construction method thereof | |
| CN108753671A (en) | A kind of the recombination bacillus coli engineering bacteria and its zymotechnique of high yield cutinase | |
| CN109468259A (en) | A kind of culture medium for promoting gemma to generate | |
| JPH0697994B2 (en) | Method for producing agricultural chemical-producing bacteria | |
| CN100562565C (en) | The gene recombined engineering bacterium of Spinosad microbial inoculum | |
| DE102007021001A1 (en) | Expression system for the antibiotic-free production of polypeptides | |
| Phaff | Industrial microorganisms | |
| CN100510097C (en) | Production of zeaxanthin by Phaffia | |
| CN106191002A (en) | A kind of method improving chrysanthemum esters digestive enzyme expression | |
| Prisa et al. | Improving the quality of ornamental bulbous with plant growth-promoting rhizobacteria (PGPR) | |
| CN106497961A (en) | A kind of genetic engineering modified method for improving bacillus biomass | |
| CN109053867A (en) | A kind of cultural method of high density fermentation expression recombinant rotavirus Δ VP8* antigen | |
| CN101886046B (en) | Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof | |
| CN106754420A (en) | Using the method for blue-green algae mud culture Trichoderma | |
| CN110184291A (en) | A kind of building and its application of the non-methanol induction of Pichia pastoris expression vector of sequestered | |
| CN108018305B (en) | Method for preventing and treating radopholus similis by using radopholus similis accompanying bacteria mediation | |
| CN106995793A (en) | A kind of fermentation process in high density for recombinating bG-CSF engineering bacteria | |
| CN114729301A (en) | Fermentation method of recombinant bacillus spores | |
| CN105087725A (en) | Method for increasing expression level of soluble recombinant proteins in escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191012 Address after: 431800 33 Green House Road No. 12, No. 1, green road, Xinshi Town, Jingshan, Jingmen, Hubei. Applicant after: Li Fen Address before: Nanning City, the Guangxi Zhuang Autonomous Region Qingxiu District Shuangyong road 530021 No. 22 Guangxi Medical University 2008 undergraduate students dormitory Applicant before: Long Yan Ni |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191223 Address after: 314400 No.11, Weisan Road, Nongfa District, Chang'an Town, Haining City, Jiaxing City, Zhejiang Province Patentee after: Haining hi tech Zone Science and Innovation Center Co., Ltd Address before: 431800 33 Green House Road No. 12, No. 1, green road, Xinshi Town, Jingshan, Jingmen, Hubei. Patentee before: Li Fen |