CN102876642B - Preparation method for pyrethroids pesticide degrading enzyme - Google Patents

Preparation method for pyrethroids pesticide degrading enzyme Download PDF

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CN102876642B
CN102876642B CN201210412214.0A CN201210412214A CN102876642B CN 102876642 B CN102876642 B CN 102876642B CN 201210412214 A CN201210412214 A CN 201210412214A CN 102876642 B CN102876642 B CN 102876642B
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enzyme
fermentation
temperature
degrading enzyme
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CN102876642A (en
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黄皓
罗华建
刘玉焕
梁卫驱
胡珊
刘远星
罗诗
莫忠兴
李艳芳
陈仕丽
徐匆
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DONGGUAN RESEARCH CENTER OF AGRICULTURAL SCIENCE
Sun Yat Sen University
National Sun Yat Sen University
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DONGGUAN RESEARCH CENTER OF AGRICULTURAL SCIENCE
National Sun Yat Sen University
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Abstract

The invention relates to the technical field of a preparation method for pesticide degrading enzyme, in particular to a preparation method for pyrethroids pesticide degrading enzyme. The preparation method for the pyrethroids pesticide degrading enzyme comprises the following steps of: 1, taking recombinant escherichia coli (E.coliBL21(DE3)/pET-28a-est825) as a production strain, and storing the production strain at the temperature of 86DEG C below zero by a glycerol tube; 2, carrying out seed culture; 3, carrying out fermentation culture; 4, carrying out feed supplement and fermentation culture; 5, carrying out lactose induction; and 6, preparing enzyme powder. According to the preparation method for the pyrethroids pesticide degrading enzyme, the recombinant escherichia coli is adopted to efficiently express the pyrethroids pesticide degrading enzyme, the yield of the finally obtained degrading enzyme powder is 25-40g/, the enzyme activity of the enzyme powder is 48000-60000U/g, the yield of the pyrethroids pesticide degrading enzyme is greatly improved, the use cost of the pyrethroids pesticide degrading enzyme is lowered, and an effective, economic and feasible path is provided for the mass production of the pyrethroids pesticide degrading enzyme.

Description

A kind of preparation method of pyrethrin degrading enzyme
Technical field
The present invention relates to preparation method's technical field of pesticide degradable enzyme, relate in particular to a kind of preparation method of pyrethrin degrading enzyme.
Background technology
Pyrethrin is broad spectrum insecticide, has the feature of quick-acting, efficient, low toxicity, low residue, and the control of 140 various pests is had to special efficacy.But, while farm crop being carried out to pest control with agricultural chemicals, will make more or less more residual agricultural chemicals of surface of farm crop, especially pyrethrin degradation speed is slow, not only HUMAN HEALTH is worked the mischief, and the ecosystem is also affected.For this reason, people adopt microorganism to degrade to agricultural chemicals, and microorganism is various to the mode of action of agricultural chemicals, and majority belongs to by enzymatic reaction degrading pesticide.
The Liu Yuhuan of Zhongshan University etc. have cloned by the method for the high-throughput functional screening in (a kind of novel esterases and application thereof, ZL201010547206.8) klebsiellathe new gene est825 in sp.ZD112 with pyrethrin degradation capability, has built recombinant expressed intestinal bacteria e.colibL21 (DE3)/pET-28a-est825, zymologic property research is found, the recombinase of producing by this recombination bacillus coli reaches respectively 80.82% to the degradation rate of cyhalothrin, Cypermethrin, fenvalerate and Deltamethrin at normal temperatures, 82.98%, 69.23% and 69.65%, pyrethrin degrading enzyme degradation efficiency is high, safety non-toxic, environmental adaptation are strong, has a good application prospect.
At present, there is the new gene by building a pyrethrin degrading enzyme of genomic library clone both at home and abroad, and build can high this gene of efficient expression the correlative study such as engineering bacteria, as the Yan Yanchun of Shandong Agricultural University etc., (a kind of high enzyme of engineering bacteria is lived and the degraded of immobilized cell to agricultural chemicals, China Environmental Science, 1999, 19 (5)) by resistance northern house five the anti-organophosphorus pesticide gene clone with subspecies in plasmid pRL-439, obtain high enzyme engineering bacteria alive, the enzyme of expressing is to two class difficult degradation agricultural chemicals (organochlorines, chrysanthemum ester class) degradable is more than 90%, but not mentioned yield of enzyme, (detoxication enzyme of a kind of super engineering bacteria and expression thereof and its construction process and the application such as the Qiao Chuanling of Institute of Zoology, Academia Sinica, ZL200410042712.6) clone respectively removing toxic substances esterase and hydrolase gene, this gene is cloned into respectively on expression vector pETDuet-1, vector construction hexose transport protein pETDuet-b1-opd, finally transform and obtain recombinant bacterial strain and carry out inducing culture, this super engineering bacteria and the detoxication enzyme Malathion of can simultaneously degrading, thiophos, effective cypermethrin, the organic acid acetic such as acetofenate and organophosphorus insecticide, but not mentioned degradation rate and yield of enzyme, (the Hydrolysis of pyrethroids by carboxylesterases from Lucilia cuprina and Drosophila melanogaster with active sites modified by vitro mutagenesis such as Heidari Rama insect Biochemistry and Molecular Biology2005,35 (6): the avtive spot that 597 ~ 609) changes the Procaine esterase that derives from lucilia and housefly by mutation technique, make this enzyme to the degradation capability of suitable, trans pyrethrin than wild increasing many times, and mutational site difference, also can affect it to degradation capability suitable, trans pyrethrin.Stok Jeanette E etc. (Identification, expression, and purification of a pyrethroid-hydrolyzing carboxylesterase from mouse liver microsomes, journal of Biological Chemistry279 (28): the carboxylesterase gene of 29863 ~ 29869) having cloned 1 energy degradation pyrethrin pesticide from Mouse Liver particulate, and in baculovirus express, purifying, also study this enzyme to catalytic constant suitable, trans pyrethrin, again the carboxylesterase gene of the 2nd hydrolysis pyrethrin of screening and separating from the liver particle cDNA library of mouse.
But vacant about adopting the technology of genetic engineering bacterium high density fermentation technique to high-efficiency production pyrethrin degrading enzyme both at home and abroad at present, therefore this area is in the urgent need to the method for exploitation high yield pyrethrin degrading enzyme.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, a kind of preparation method of pyrethrin degrading enzyme is provided, this preparation method utilizes recombination bacillus coli High-efficient Production pyrethrin degrading enzyme.
The present invention is achieved through technology.
A preparation method for pyrethrin degrading enzyme, comprises the following steps:
1) with recombination bacillus coli e.colibL21 (DE3)/pET-28a-est825} as produce bacterial classification, with glycerine pipe by produce bacterial classification at the temperature of-86 ℃, preserve; Wherein, the nucleotide sequence of gene est825 is as shown in SEQ ID NO.1;
2) seed culture: by the production bacterial classification in glycerine pipe by 2 ~ 4% inoculum size access shake-flask seed substratum, take rotating speed as 200 ~ 220r/min, temperature cultivates in constant-temperature table as 37 ℃, incubation time as the culture condition of 8 ~ 10h; In the fermenting process of recombination bacillus coli, plasmid stability is extremely important to Product Expression, higher temperature as 37 ℃ at, the plasmid stability of recombination bacillus coli is higher.
3) fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75 ~ 4.5L fermention medium is housed and carries out fermentation culture with 2 ~ 5% inoculum size, the time of fermentation culture is 24 ~ 28h, during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 20% ~ 25%; Fermentation scale of the present invention is not limited to 7.5L, can suitably expand according to actual needs fermentation scale.
4) fed-batch fermentation is cultivated: after fermentation culture 4 ~ 6h, and the mixing feed supplement liquid of stream glycerol adding and yeast powder, the specific growth rate of controlling thalline is 0.1 ~ 0.2h -1, the lasting time of fed-batch fermentation cultivation stage is 10 ~ 14h; The mode that mixing feed supplement liquid adds by stream is supplemented, feed rate with can maintain thalli growth and to express pyrethrin degrading enzyme required and to maintain dissolved oxygen more stable in fermentation system and pH value as according to setting, the Nutrious fermented source of assurance recombination bacillus coli.
5) lactose-induced: fed-batch fermentation is cultured to the OD of thalline 600nmvalue reaches at 100 ~ 140 o'clock, disposable interpolation lactose solution, the concentration that makes fermented liquid lactose is 5 ~ 8g/L, cool the temperature to 30 ~ 35 ℃ and start induction, the afterflow of Induction Process relaying adds the mixing feed supplement liquid in step 4), the concentration that maintains glycerine in fermented liquid is 1 ~ 2g/L, lactose-induced time remaining 8 ~ 10h; Adopt and lactose-inducedly have advantages of that cost is low, environmental protection, harmless.
6) enzyme powder preparation: after fermentation ends, centrifugal collection thalline at the temperature of 4 ~ 8 ℃, the phosphate buffered saline buffer of pH6.5 ~ 7.0 for thalline is mixed with to the bacteria suspension of mass concentration 15% ~ 20%, by bacteria suspension at the temperature of 4 ℃, high pressure fragmentation obtains broken liquid under the pressure of 150Mpa, broken liquid is carried out at the temperature of 4 ℃ to high speed centrifugation and make supernatant liquor, or filter and make filtered liquid with low temperature mocromembrane, more supernatant liquor or filtered liquid vacuum lyophilization are made to enzyme powder.
Wherein, described step 2) in, the pH of described shake-flask seed substratum is 7.0, described shake-flask seed substratum comprises following component: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, kantlex 50 ~ 100mg/L.
Wherein, in described step 3), the pH of described fermention medium is 6.8, and described fermention medium comprises following component: glycerine 5 ~ 8g/L, peptone 3 ~ 10g/L, yeast powder 3 ~ 8g/L, (NH 4) 2sO 45 ~ 10g/L, MgSO 47H 2o 1 ~ 3g/L, KH 2pO 43.4 ~ 6.85g/L, K 2hPO 412H 2o 5.68 ~ 11.35g/L, liquid microelement 1 ~ 2mL/L, kantlex 50 ~ 100mg/L.The present invention adopts the raw materials such as glycerine, peptone and yeast powder to produce, and is suitable for scale operation.
Wherein, described liquid microelement comprises following component: CaCl 22.0g/L, ZnSO 47H 2o 5.0g/L, MnSO 4h 2o 0.5g/L, Na 2-EDTA 18.0g/L, FeSO 47H 2o 10.0g/L, CuSO 45H 2o 2.5g/L, CoCl 26H 2o 0.2g/L, (NH 4) 6mo 7o 244H 2o 0.1g/L.
Wherein, described liquid microelement is in being dissolved in sterilized water, then uses the liquid microelement after the membrane filtration degerming of 0.22 μ m.
Wherein, described step 3) is specially: fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75 ~ 4.5L fermention medium is housed and carries out fermentation culture with 2 ~ 5% inoculum size, the time of fermentation culture is 24 ~ 28h, and during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 20% ~ 25%, air flow is 0.5 ~ 4vvm, mixing speed 200 ~ 1000r/min, 37 ℃ of culture temperature, the pH that stream adds the hydrochloric acid soln controlled fermentation liquid of 20% ~ 25% ammoniacal liquor and 3mol/L is 6.8 ~ 7.0.
Wherein, in described step 4), the mixing feed supplement liquid of described glycerine and yeast powder comprises following component: glycerine 300 ~ 500g/L, yeast powder 60 ~ 100g/L, kantlex 50 ~ 100mg/L.
Wherein, the measuring method of glycerol content:
Adopt HPLC(high performance liquid chromatography): Zorbax Extend-C18(4.6mm × 250mm, m) chromatographic column of 5 μ, 37 ℃ of column temperatures, sample size 10 μ l, moving phase: pure water, flow velocity: 1.0mL/min.At fed-batch fermentation stage detection one time fermentation per hour liquid glycerol concentration, monitoring glycerine residual volume; At same detection glycerol concentration per hour of lactose-induced stage, by controlling feed rate control glycerine residual at 1 ~ 2g/L.
Wherein, the measuring method of cell concentration:
Adopt spectrophotometry under 600nm wavelength, to detect the absorbance value of fermented liquid, determine induction starting point according to cell concentration.
Wherein, the measuring method of the enzyme activity of pyrethrin degrading enzyme:
Take p-NP acetic ester (pNPA) as substrate, adopt spectrophotometry, enzyme work is defined as: in the time of 40 ℃ of pH6.5, temperature, to generate the required enzyme amount of 1 μ mol p-NP be a Ge Meihuo unit (U) to per minute catalytic hydrolysis pNPA.Concrete measuring method is as follows:
(1) substrate mother liquor preparation: take 18.1mg pNPA and be dissolved in 1mL methyl alcohol, be made into the substrate mother liquor of 0.1M, and substrate mother liquor is stored for future use at 4 ℃;
(2) measure liquid preparation: get substrate mother liquor 0.4mL, slowly join in Sodium phosphate dibasic-citrate buffer solution of 9.6mL pH6.5, and fully mix, prepare mensuration liquid;
(3) enzyme activity determination: get 5 μ l enzyme liquid and add in the mensuration liquid of 200 μ l, make enzyme liquid and mensuration liquid react 4min at the temperature of 40 ℃ and make reaction solution, do blank with inactivator liquid simultaneously, reaction solution is diluted to and gets 300 μ l after certain multiple and add enzyme plate, measure light absorption value at 405nm wavelength place, the typical curve doing according to the light absorption value under the concentration of p-NP and 405nm, draws the amount of p-NP that enzymatic reaction generates, and then calculates enzyme activity.
Beneficial effect of the present invention is: the present invention utilizes recombination bacillus coli high density fermentation technology, employing intestinal bacteria e.colithe high efficient expression pyrethrin of BL21 (DE3)/pET-28a-est825} degrading enzyme, adopts lactose to induce fermented liquid in fermenting process, have advantages of that cost is low, environmental protection, harmless, after fermentation ends, and fermented liquid OD 600nmvalue reaches 125 ~ 165; fermented liquid degradation enzyme activity is 2000 ~ 3000U/mL; the degradation enzyme powder yield 25 ~ 40g/L finally making; the work of enzyme powder enzyme is 48000 ~ 60000U/g; greatly improve the output of pyrethrin degrading enzyme; reduce the use cost of pyrethrin degrading enzyme, for large-scale production pyrethrin degrading enzyme provides effective, economic, a feasible approach.
Accompanying drawing explanation
Fig. 1 is the fermenting process graphic representation of embodiments of the invention one.
Fig. 2 is the fermenting process graphic representation of embodiments of the invention two.
Fig. 3 is the fermenting process graphic representation of embodiments of the invention three.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment mono-.
The preparation method of a kind of pyrethrin degrading enzyme of the present embodiment, comprises the following steps:
1) with recombination bacillus coli e.colibL21 (DE3)/pET-28a-est825} as produce bacterial classification, with glycerine pipe by produce bacterial classification at the temperature of-86 ℃, preserve.
2) seed culture: by the production bacterial classification in glycerine pipe by 2% inoculum size access shake-flask seed substratum, take rotating speed as 220r/min, temperature cultivates in constant-temperature table as 37 ℃, incubation time as the culture condition of 9h; The pH of described shake-flask seed substratum is 7.0, and described shake-flask seed substratum comprises following component: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, kantlex 50mg/L.
3) fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 4L fermention medium is housed and carries out fermentation culture with 4% inoculum size, the time of fermentation culture is 26h, during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 20%, air flow is 0.5 ~ 4vvm, mixing speed 200 ~ 1000r/min, 37 ℃ of culture temperature, the pH that stream adds the hydrochloric acid soln controlled fermentation liquid of 25% ammoniacal liquor and 3mol/L is 6.8;
In the present embodiment, the pH of described fermention medium is 6.8, and described fermention medium comprises following component: glycerine 5g/L, peptone 5g/L, yeast powder 3g/L, (NH 4) 2sO 45g/L, MgSO 47H 2o 2.5g/L, KH 2pO 46.85g/L, K 2hPO 412H 2o 11.35g/L, liquid microelement 1.5mL/L, kantlex 50mg/L;
In the present embodiment, described liquid microelement comprises following component: CaCl 22.0g/L, ZnSO 47H 2o 5.0g/L, MnSO 4h 2o 0.5g/L, Na 2-EDTA 18.0g/L, FeSO 47H 2o 10.0g/L, CuSO 45H 2o 2.5g/L, CoCl 26H 2o 0.2g/L, (NH 4) 6mo 7o 244H 2o 0.1g/L;
In the present embodiment, described liquid microelement is in being dissolved in sterilized water, then uses the liquid microelement after the membrane filtration degerming of 0.22 μ m.
4) fed-batch fermentation is cultivated: after fermentation culture 4h, and the mixing feed supplement liquid of stream glycerol adding and yeast powder, the specific growth rate of controlling thalline is 0.1 ~ 0.2h -1, the lasting time of fed-batch fermentation cultivation stage is 12h; The mixing feed supplement liquid of described glycerine and yeast powder comprises following component: glycerine 500g/L, yeast powder 100g/L, kantlex 50mg/L;
5) lactose-induced: fed-batch fermentation is cultured to the OD of thalline 600nmvalue reaches at 120 o'clock, disposable interpolation lactose solution, and the concentration that makes fermented liquid lactose is 5g/L, cool the temperature to 33 ℃ and start induction, the afterflow of Induction Process relaying adds the mixing feed supplement liquid in step 4), and the concentration that maintains glycerine in fermented liquid is 1 ~ 2g/L, lactose-induced time remaining 10h.
6) enzyme powder preparation: after fermentation ends, centrifugal collection thalline at the temperature of 4 ℃, thalline is mixed with to the bacteria suspension of mass concentration 15% with the phosphate buffered saline buffer of pH6.8, by bacteria suspension at the temperature of 4 ℃, high pressure fragmentation obtains broken liquid under the pressure of 150Mpa, broken liquid is carried out at the temperature of 4 ℃ to high speed centrifugation and make supernatant liquor, or filter and make filtered liquid with low temperature mocromembrane, more supernatant liquor or filtered liquid vacuum lyophilization are made to enzyme powder.
In the present embodiment, after fermentation ends, fermented liquid OD 600nmvalue reaches 165, and fermented liquid degradation enzyme activity is 3000U/mL, the degradation enzyme powder yield 40g/L finally making, and enzyme powder enzyme is lived as 60000U/g.Fermenting process curve is shown in Fig. 1.
embodiment bis-.
The preparation method of a kind of pyrethrin degrading enzyme of the present embodiment, comprises the following steps:
1) with recombination bacillus coli e.colibL21 (DE3)/pET-28a-est825} as produce bacterial classification, with glycerine pipe by produce bacterial classification at the temperature of-86 ℃, preserve.
2) seed culture: by the production bacterial classification in glycerine pipe by 2% inoculum size access shake-flask seed substratum, take rotating speed as 200r/min, temperature cultivates in constant-temperature table as 37 ℃, incubation time as the culture condition of 10h; The pH of described shake-flask seed substratum is 7.0, and described shake-flask seed substratum comprises following component: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, kantlex 50mg/L.
3) fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75L fermention medium is housed and carries out fermentation culture with 2% inoculum size, the time of fermentation culture is 28h, and during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 20%, air flow is 0.5 ~ 4vvm, mixing speed 200 ~ 1000r/min, 37 ℃ of culture temperature, the pH that stream adds the hydrochloric acid soln controlled fermentation liquid of 25% ammoniacal liquor and 3mol/L is 6.9;
In the present embodiment, the pH of described fermention medium is 6.8, and described fermention medium comprises following component: glycerine 8g/L, peptone 5g/L, yeast powder 5g/L, (NH 4) 2sO 48g/L, MgSO 47H 2o 1g/L, KH 2pO 44.57g/L, K 2hPO 412H 2o 7.57g/L, liquid microelement 1mL/L, kantlex 50mg/L;
In the present embodiment, described liquid microelement comprises following component: CaCl 22.0g/L, ZnSO 47H 2o 5.0g/L, MnSO 4h 2o 0.5g/L, Na 2-EDTA 18.0g/L, FeSO 47H 2o 10.0g/L, CuSO 45H 2o 2.5g/L, CoCl 26H 2o 0.2g/L, (NH 4) 6mo 7o 244H 2o 0.1g/L;
In the present embodiment, described liquid microelement is in being dissolved in sterilized water, then uses the liquid microelement after the membrane filtration degerming of 0.22 μ m.
4) fed-batch fermentation is cultivated: after fermentation culture 6h, and the mixing feed supplement liquid of stream glycerol adding and yeast powder, the specific growth rate of controlling thalline is 0.1 ~ 0.2h -1, the lasting time of fed-batch fermentation cultivation stage is 12h; The mixing feed supplement liquid of described glycerine and yeast powder comprises following component: glycerine 400g/L, yeast powder 80g/L, kantlex 50mg/L;
5) lactose-induced: fed-batch fermentation is cultured to the OD of thalline 600nmvalue reaches at 140 o'clock, disposable interpolation lactose solution, and the concentration that makes fermented liquid lactose is 6g/L, cool the temperature to 30 ℃ and start induction, the afterflow of Induction Process relaying adds the mixing feed supplement liquid in step 4), and the concentration that maintains glycerine in fermented liquid is 1 ~ 2g/L, lactose-induced time remaining 10h.
6) enzyme powder preparation: after fermentation ends, centrifugal collection thalline at the temperature of 4 ℃, thalline is mixed with to the bacteria suspension of mass concentration 18% with the phosphate buffered saline buffer of pH6.5, by bacteria suspension at the temperature of 4 ℃, high pressure fragmentation obtains broken liquid under the pressure of 150Mpa, broken liquid is carried out at the temperature of 4 ℃ to high speed centrifugation and make supernatant liquor, or filter and make filtered liquid with low temperature mocromembrane, more supernatant liquor or filtered liquid vacuum lyophilization are made to enzyme powder.
In the present embodiment, after fermentation ends, fermented liquid OD 600nmvalue reaches 150, and fermented liquid degradation enzyme activity is 2600U/mL, the degradation enzyme powder yield 33g/L finally making, and enzyme powder enzyme is lived as 52000U/g.Fermenting process curve is shown in Fig. 2.
embodiment tri-.
The preparation method of a kind of pyrethrin degrading enzyme of the present embodiment, comprises the following steps:
1) with recombination bacillus coli e.colibL21 (DE3)/pET-28a-est825} as produce bacterial classification, with glycerine pipe by produce bacterial classification at the temperature of-86 ℃, preserve.
2) seed culture: by the production bacterial classification in glycerine pipe by 4% inoculum size access shake-flask seed substratum, take rotating speed as 220r/min, temperature cultivates in constant-temperature table as 37 ℃, incubation time as the culture condition of 8h; The pH of described shake-flask seed substratum is 7.0, and described shake-flask seed substratum comprises following component: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, kantlex 100mg/L.
3) fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75L fermention medium is housed and carries out fermentation culture with 2% inoculum size, the time of fermentation culture is 25h, during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 20%, air flow is 0.5 ~ 4vvm, mixing speed 200 ~ 1000r/min, 37 ℃ of culture temperature, the pH that stream adds the hydrochloric acid soln controlled fermentation liquid of 25% ammoniacal liquor and 3mol/L is 7.0;
In the present embodiment, the pH of described fermention medium is 6.8, and described fermention medium comprises following component: glycerine 6g/L, and peptone 10g/L, yeast powder 8g/L, (NH4) 2sO 410g/L, MgSO 47H 2o 3g/L, KH 2pO 43.4g/L, K 2hPO 412H 2o 5.68g/L, liquid microelement 2mL/L, kantlex 100mg/L;
In the present embodiment, described liquid microelement comprises following component: CaCl 22.0g/L, ZnSO 47H 2o 5.0g/L, MnSO 4h 2o 0.5g/L, Na 2-EDTA 18.0g/L, FeSO 47H 2o 10.0g/L, CuSO 45H 2o 2.5g/L, CoCl 26H 2o 0.2g/L, (NH 4) 6mo 7o 244H 2o 0.1g/L;
In the present embodiment, described liquid microelement is in being dissolved in sterilized water, then uses the liquid microelement after the membrane filtration degerming of 0.22 μ m.
4) fed-batch fermentation is cultivated: after fermentation culture 5h, and the mixing feed supplement liquid of stream glycerol adding and yeast powder, the specific growth rate of controlling thalline is 0.1 ~ 0.2h -1, the lasting time of fed-batch fermentation cultivation stage is 10h; The mixing feed supplement liquid of described glycerine and yeast powder comprises following component: glycerine 300g/L, yeast powder 60g/L, kantlex 100mg/L;
5) lactose-induced: fed-batch fermentation is cultured to the OD of thalline 600nmvalue reaches at 100 o'clock, disposable interpolation lactose solution, and the concentration that makes fermented liquid lactose is 8g/L, cool the temperature to 35 ℃ and start induction, the afterflow of Induction Process relaying adds the mixing feed supplement liquid in step 4), and the concentration that maintains glycerine in fermented liquid is 1 ~ 2g/L, lactose-induced time remaining 10h.
6) enzyme powder preparation: after fermentation ends, centrifugal collection thalline at the temperature of 4 ℃, thalline is mixed with to the bacteria suspension of mass concentration 20% with the phosphate buffered saline buffer of pH6.5, by bacteria suspension at the temperature of 4 ℃, high pressure fragmentation obtains broken liquid under the pressure of 150Mpa, broken liquid is carried out at the temperature of 4 ℃ to high speed centrifugation and make supernatant liquor, or filter and make filtered liquid with low temperature mocromembrane, more supernatant liquor or filtered liquid vacuum lyophilization are made to enzyme powder.
In the present embodiment, after fermentation ends, fermented liquid OD 600nmvalue reaches 125, and fermented liquid degradation enzyme activity is 2000U/mL, the degradation enzyme powder yield 25g/L finally making, and enzyme powder enzyme is lived as 48000U/g.Fermenting process curve is shown in Fig. 3.
In embodiments of the invention one to embodiment tri-, the measuring method of glycerol content:
Adopt HPLC(high performance liquid chromatography): Zorbax Extend-C18(4.6mm × 250mm, m) chromatographic column of 5 μ, 37 ℃ of column temperatures, sample size 10 μ l, moving phase: pure water, flow velocity: 1.0mL/min.At fed-batch fermentation stage detection one time fermentation per hour liquid glycerol concentration, monitoring glycerine residual volume; At same detection glycerol concentration per hour of lactose-induced stage, by controlling feed rate control glycerine residual at 1 ~ 2g/L.
The measuring method of cell concentration:
Adopt spectrophotometry under 600nm wavelength, to detect the absorbance value of fermented liquid, determine induction starting point according to cell concentration.
The measuring method of the enzyme activity of pyrethrin degrading enzyme:
Take p-NP acetic ester (pNPA) as substrate, adopt spectrophotometry, enzyme work is defined as: in the time of 40 ℃ of pH6.5, temperature, to generate the required enzyme amount of 1 μ mol p-NP be a Ge Meihuo unit (U) to per minute catalytic hydrolysis pNPA.Concrete measuring method is as follows:
(1) substrate mother liquor preparation: take 18.1mg pNPA and be dissolved in 1mL methyl alcohol, be made into the substrate mother liquor of 0.1M, and substrate mother liquor is stored for future use at 4 ℃;
(2) measure liquid preparation: get substrate mother liquor 0.4mL, slowly join in Sodium phosphate dibasic-citrate buffer solution of 9.6mL pH6.5, and fully mix, prepare mensuration liquid;
(3) enzyme activity determination: get 5 μ l enzyme liquid and add in the mensuration liquid of 200 μ l, make enzyme liquid and mensuration liquid react 4min at the temperature of 40 ℃ and make reaction solution, do blank with inactivator liquid simultaneously, reaction solution is diluted to and gets 300 μ l after certain multiple and add enzyme plate, measure light absorption value at 405nm wavelength place, the typical curve doing according to the light absorption value under the concentration of p-NP and 405nm, draws the amount of p-NP that enzymatic reaction generates, and then calculates enzyme activity.
The above embodiment, it is preferred embodiments of the present invention, be not to limit the scope of the present invention, the equivalence of doing according to structure, feature and principle described in the present patent application the scope of the claims therefore all changes or modifies, and all should comprise in patent claim of the present invention.
< 110 > Zhongshan University
< 120 > novel esterases and application thereof
＜160＞?3
＜210＞?1
＜211＞?825
＜212＞?DNA
The full length DNA sequence of < 221 > novel esterases
＜400＞?1
Figure 824865DEST_PATH_IMAGE001

Claims (3)

1. a preparation method for pyrethrin degrading enzyme, is characterized in that: comprise the following steps:
1) with recombination bacillus coli e.colibL21 (DE3)/pET-28a-est825} as produce bacterial classification, with glycerine pipe by produce bacterial classification at the temperature of-86 ℃, preserve; Wherein, the nucleotide sequence of gene est825 is as shown in SEQ ID NO.1;
2) seed culture: by the production bacterial classification in glycerine pipe by 2 ~ 4% inoculum size access shake-flask seed substratum, take rotating speed as 200 ~ 220r/min, temperature cultivates in constant-temperature table as 37 ℃, incubation time as the culture condition of 8 ~ 10h;
The pH of described shake-flask seed substratum is 7.0, and described shake-flask seed substratum comprises following component: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, kantlex 50 ~ 100mg/L;
3) fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75 ~ 4.5L fermention medium is housed and carries out fermentation culture with 2 ~ 5% inoculum size, the time of fermentation culture is 24 ~ 28h, during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 25%;
The pH of described fermention medium is 6.8, and described fermention medium comprises following component: glycerine 5 ~ 8g/L, peptone 3 ~ 10g/L, yeast powder 3 ~ 8g/L, (NH 4) 2sO 45 ~ 10g/L, MgSO 47H 2o 1 ~ 3g/L, KH 2pO 43.4 ~ 6.85g/L, K 2hPO 412H 2o 5.68 ~ 11.35g/L, liquid microelement 1 ~ 2mL/L, kantlex 50 ~ 100mg/L;
Described liquid microelement comprises following component: CaCl 22.0g/L, ZnSO 47H 2o 5.0g/L, MnSO 4h 2o 0.5g/L, Na 2-EDTA 18.0g/L, FeSO 47H 2o 10.0g/L, CuSO 45H 2o 2.5g/L, CoCl 26H 2o 0.2g/L, (NH 4) 6mo 7o 244H 2o 0.1g/L;
4) fed-batch fermentation is cultivated: after fermentation culture 4 ~ 6h, and the mixing feed supplement liquid of stream glycerol adding and yeast powder, the specific growth rate of controlling thalline is 0.1 ~ 0.2 h -1, the lasting time of fed-batch fermentation cultivation stage is 10 ~ 14h;
The mixing feed supplement liquid of described glycerine and yeast powder comprises following component: glycerine 300 ~ 400g/L, yeast powder 60 ~ 100g/L, kantlex 50 ~ 100mg/L;
5) lactose-induced: fed-batch fermentation is cultured to the OD of thalline 600nmvalue reaches at 100 ~ 140 o'clock, disposable interpolation lactose solution, the concentration that makes fermented liquid lactose is 5 ~ 8g/L, cool the temperature to 30 ~ 35 ℃ and start induction, the afterflow of Induction Process relaying adds the mixing feed supplement liquid in step 4), the concentration that maintains glycerine in fermented liquid is 1 ~ 2g/L, lactose-induced time remaining 8 ~ 10h;
6) enzyme powder preparation: after fermentation ends, centrifugal collection thalline at the temperature of 4 ~ 8 ℃, the phosphate buffered saline buffer of pH6.5 ~ 7.0 for thalline is mixed with to the bacteria suspension of mass concentration 15% ~ 20%, by bacteria suspension at the temperature of 4 ℃, high pressure fragmentation obtains broken liquid under the pressure of 150Mpa, broken liquid is carried out at the temperature of 4 ℃ to high speed centrifugation and make supernatant liquor, or filter and make filtered liquid with low temperature mocromembrane, more supernatant liquor or filtered liquid vacuum lyophilization are made to enzyme powder;
The measuring method of the enzyme activity of the pyrethrin degrading enzyme making is:
Take p-NP acetic ester (pNPA) as substrate, adopt spectrophotometry, enzyme work is defined as: in the time of 40 ℃ of pH6.5, temperature, to generate the required enzyme amount of 1 μ mol p-NP be a Ge Meihuo unit (U) to per minute catalytic hydrolysis pNPA, and concrete measuring method is as follows:
(1) substrate mother liquor preparation: take 18.1mg pNPA and be dissolved in 1mL methyl alcohol, be made into the substrate mother liquor of 0.1M, and substrate mother liquor is stored for future use at 4 ℃;
(2) measure liquid preparation: get substrate mother liquor 0.4mL, slowly join in Sodium phosphate dibasic-citrate buffer solution of 9.6mL pH6.5, and fully mix, prepare mensuration liquid;
(3) enzyme activity determination: get 5 μ l enzyme liquid and add in the mensuration liquid of 200 μ l, make enzyme liquid and mensuration liquid react 4min at the temperature of 40 ℃ and make reaction solution, do blank with inactivator liquid simultaneously, reaction solution is diluted to and gets 300 μ l after certain multiple and add enzyme plate, measure light absorption value at 405nm wavelength place, the typical curve doing according to the light absorption value under the concentration of p-NP and 405nm, draws the amount of p-NP that enzymatic reaction generates, and then calculates enzyme activity.
2. the preparation method of a kind of pyrethrin degrading enzyme according to claim 1, is characterized in that: described liquid microelement is in being dissolved in sterilized water, then uses the liquid microelement after the membrane filtration degerming of 0.22 μ m.
3. the preparation method of a kind of pyrethrin degrading enzyme according to claim 1, it is characterized in that: described step 3) is specially: fermentation culture: cultured seed liquor is accessed in the 7.5L fermentor tank that 3.75 ~ 4.5L fermention medium is housed and carries out fermentation culture with 2 ~ 5% inoculum size, the time of fermentation culture is 24 ~ 28h, and during the fermentation by passing into sterile air and change mixing speed to carry out controlled fermentation liquid oxygen dissolving value and maintain 25%, air flow is 0.5 ~ 4vvm, mixing speed 200 ~ 1000r/min, 37 ℃ of culture temperature, the pH that stream adds the hydrochloric acid soln controlled fermentation liquid of 20% ~ 25% ammoniacal liquor and 3mol/L is 6.8 ~ 7.0.
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