CN103409387B

CN103409387B - Organophosphorus pesticide degrading enzyme mutant and preparation method thereof

Info

Publication number: CN103409387B
Application number: CN201310384286.3A
Authority: CN
Inventors: 徐建华; 胡德峰; 徐卡尔·翰
Original assignee: SHAOXING HUATAI BIOTECHNOLOGY Co Ltd
Current assignee: Feng Pengbo
Priority date: 2012-11-13
Filing date: 2012-11-13
Publication date: 2014-12-10
Anticipated expiration: 2032-11-13
Also published as: CN103409387A

Abstract

The invention provides an organophosphorus pesticide degrading enzyme mutant with excellent enzymatic activity and a preparation method of the mutant. The active centers of degrading enzymes are analyzed to obtain multiple enzyme mutants including a mutant M164F and a mutant L116M, and especially, the mutant M164F has higher enzymatic activity. The invention also provides a gene sequence of the degrading enzyme mutant subjected to pichia pastoris codon optimization, and a method for expressing genes by pichia pastoris. The preparation method is simple. By virtue of pichia pastoris optimized codon, the yield of the mutant is high. The organophosphorus pesticide degrading enzyme prepared by the method can be applied to the field of pesticide residue treatment of agricultural products such as fruits, vegetables and tea leaves, and has wide application prospects and great economic and social significance.

Description

A kind of opd mutant and preparation method thereof

The application is the divisional application of the Chinese patent application that on November 13rd, 2012 submits to, application number is 201210458400.8, denomination of invention is " a kind of opd mutant and preparation method thereof ".

Technical field

The present invention relates to genetically engineered field, specifically, relate to a kind of opd mutant and preparation method thereof.

Background technology

Organophosphorus pesticide (organophosphorus pesticides, OPs) is the most widely used one large class agricultural chemicals in agriculture production (Singh BK.2009.Organophosphorus-degrading bacteria:ecology and industrial applications.Nature Reviews Microbiology.7:156-164).Since nineteen forty-four, first Bayer A.G found that organophosphorus has insecticidal properties and produces the first sterilant " l605-thiophos ", organophosphorus is efficient as a class, the sterilant of wide spectrum is widely used, and has improved to a great extent the output of farm crop.According to statistics, the ultimate production of China's agricultural chemicals in 2010 exceedes 2,000,000 tons of (National Bureau of Statistics of the People's Republic of China China Statistical Yearbook-2010.Beijing: China Statistics Press, 2011:14-221), wherein organophosphorus pesticide product accounts for 80% of its total amount.The kind of organophosphorus pesticide commodity reaches more than hundreds of in the market, and institute of China is widely used, and (the huge white silk of repairing, Lee often puts down 2010 to comprise the sterilants such as Chlorpyrifos 94, Profenofos, acephate, diazinon and SD-1750.Organic phosphorous insecticide selectivity and security.Pesticide science and management.The kind of the sterilant such as 31:19-211), Kitazine, gram pest be clean just reaches kind more than 30.In addition other organophosphorus products that often use in China, also have (Zhang Chan, the Liao Xiao orchids 2010 such as organophosphorus rodenticide, organophosphorus herbicide, growth regulator.The microbial technology progress of organophosphorus pesticide.Guangxi Agricultural science.41:1079-10821)。Most of organophosphorus pesticide water insoluble (except Rogor, Trichlorphon), and be soluble in organic solvent is stablized under neutral and acidic conditions, not facile hydrolysis, under alkaline condition because hydrolysis was lost efficacy.

Organophosphorus pesticide belongs to the substituent of organochlorine pesticide, there is drug effect high, wide in variety, prevention and treatment range is wide, cost is low, than the easy degraded of organochlorine pesticide, the pollution to environment and the harm to the ecosystem and residual all advantages such as organochlorine pesticide is not general and outstanding, for increasing grain-production, disease preventing and treating is propagated and made a great contribution.But along with agriculture development, the usage quantity of organophosphorus pesticide is increasing, a large amount of organophosphorus pesticides remain in water and soil, not only earth's surface pesticide residue are exceeded standard, and cause food contamination, and pollute with groundwater resource by all means.

The symptoms such as organophosphorus pesticide is a kind of nervous poison, accumulates for a long time delay and can cause chronic poisoning in human body, causes human nerve dysfunction, perspires, blunt, off one's dot and speech disorder.The agricultural chemicals that contains organophosphorus enters food chain by modes such as food are residual, and it is rich long-pending in organism, to carry out biology, thereby causes the harm such as teratogenesis, carcinogenic, mutagenesis, finally threatens the mankind's existence and social Sustainable development.Therefore, under the trend of pay attention to day by day environment protection ability, strengthening the research of organic phosphorus pesticide degradation method, solve the pollution problem of organophosphorus pesticide to environment and food, is one of current problem in the urgent need to address of the mankind.

Organophosphorus pesticide pollution degradation technique can be divided into thermal destruction, photodegradation, chemical degradation and biological degradation.Biological degradation is, by biological effect, agricultural chemicals is decomposed into nontoxic or low toxicity micromolecular compound, and is finally degraded to water, CO ₂process with mineral substance.With respect to physics, chemical degradation technology, biological degradation has efficiently, thoroughly, the advantage of non-secondary pollution.Crop itself, microorganism are can both degrading organic phosphor pesticides residual, but the degraded of plant is very slow, and the cycle is very long, and microorganism, due to its powerful metabolism diversity, has larger advantage in organophosphorus pesticide degraded.Now, more and more come into one's own with microorganism or microbial source zymin deteriorating pesticide residue, the research of Microbial Degradation of Organophosphates has been become to a large important topic of environmental protection.

Opd microorganism can be divided into enzymatic and non-enzymatic reaction for the degraded of agricultural chemicals.So-called enzymatic reaction refers to that microorganism directly acts on agricultural chemicals with the extracellular enzyme of intracellular enzyme or secretion, and through a series of biochemical reactions, agricultural chemicals is degradable or resolve into the process of the nontoxic of molecular weight or the compound that toxicity is less the most at last.Change physics, the chemical property such as environment ion concentration, pH of agricultural chemicals by metabolism but not enzymatic form refers to microorganism, thereby indirectly impel the process of degrading pesticide.Enzymatic reaction is the principal mode of Degradation of Pesticides By Microorganisms.When organophosphorus pesticide enters after soil, microorganism can produce the degrading enzyme of degrading organic phosphor pesticides at once; Another kind is the enzyme system that microorganism itself there is no this organophosphorus pesticide of degradable, and when after agricultural chemicals entered environment, due to the needs of microorganism existence, restructuring or sudden change occur microorganism gene in the process conforming, and produce new degradation enzyme system.

Opd has been acknowledged as the most potential novel method of eliminating pesticide residue at present.Common are machine phosphorus insecticide degrading enzyme is mainly hydrolase, comprises Phosphoric acid esterase, parathion hydrolase, esterase, sulfenyl Ntn hydrolase, lyase etc., and they are mainly by cracking P-O, P-S, the keys such as P-N, are degraded organophosphorus pesticide, generate simple mineral compound.Because various organophosphorus pesticides have similar structure, just substituting group difference, so a kind of opd is degradable Determination of Organophosphorus Pesticide (Theriot CM often, Grunden AM.2011.Hydrolysis of organophosphorus compounds by microbial enzymes.Appl Microbiol Biotechnol.89,35-43).

Find that the earliest opd is before and after 1973.It is a kind of enzymes that are hydrolyzed phosphoric acid ester bond of these bacterium secretions that Sethunahan and Yoshida isolate can the degrade Flavobacterium of diazinon and thiophos the reason of finding its degraded of a strain from soil, organic phosphorus degrading enzyme (Sethunahan N, Yoshida is Flavobacterium that degrades diazinon and parathion.Can.J.Microbiol.19,873-875 T.1973.A).Munnecke in 1974 etc. detect the activity of phosphoesterase from false pseudomonas bacillus, find that it has Degradation to thiophos, the organophosphorus pesticides such as parathion-methyl, diazinon, Chlorpyrifos 94 all can effectively be degraded simultaneously, and this enzyme is not by solvent in agricultural chemicals and pesticide preparation is suppressed (MunneckeD.M., Hsieh P.D.1974.Microbial decontamination of parathion and p-nitrophenol in aqueous media.Applied Microbiol.28:212-217).

The eighties in 20th century; the discovery opds such as Munnecke more can be stood abnormal environment condition than the microbial cells that produces this fermentoid; as the degrading enzyme that derives from pseudomonas can keep high reactivity at 10% inorganic salt, 1% organic solvent, 50 DEG C; and the generation bacterium of this enzyme can not grow under same condition; and; the degradation effect of enzyme is better than microorganism itself far away, particularly more effective to the agricultural chemicals of lower concentration.Therefore, people's thinking turns to and utilizes opd from using microbe thalline purification pesticidal contamination.

1980, Brown etc. just carried out partial purification and the character of enzyme have been studied deriving from the opd of Flavobacterium, found that the optimal pH scope of enzyme reaction is 8-10; The activity of enzyme is not subject to the impact of metal ion, is suppressed (Brown KA.1980.Phosphotriesterases of Flavobacterium sp.Soil Biology and Biochemistry.12:105 – 112) by nonionic detergent.1989, the purifying such as Dumas obtained deriving from the opd of pseudomonas, found that this enzyme is monomer sphaeroprotein, and molecular weight is 39kDa, has a zine ion, and by sulfhydryl reagent competitive inhibition, metal chelator, EDTA etc. can make its inactivation.Except paraoxon, this enzyme also the multiple organic phosphorous insecticide of hydrolyzable as Chlorpyrifos 94, thiophos, Coumaphos, diazinon, (Dumas, the DP such as parathion-methyl, Caldwell, SR, Wild JR, Raushel FM.1989.Purification and properties of the phosphotriesterase from Pseudomonas diminuta.J Biol Chem, 264,19659-19665).

1996, Vanhooke etc. carry out the analysis of X-ray crystalline diffraction to deriving from the organic phosphorus degrading enzyme of Flavobacterium, find that this enzyme is dimer under field conditions (factors), on each monomer, contain 2 zine ions, form the needed bimetallic center (Vanhooke of enzymic catalytic reaction, J.L., Benning, M.M., Raushel, F.M.and Holden, H.M.1996.Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate.Biochemistry, 35, 6020-6025).These researchs are structurally or in the mechanism of action, have all had further understanding opd.

China recent years is also being done a lot of work and is having made great progress aspect organic phosphorus degrading enzyme bacterial screening.Chu Xiaona etc. have obtained Methyl Parathion Hydrolase in Pseudomonas sp.WBC-3 from pseudomonas.Dynamic analysis shows that this enzyme is non-specific organic phosphorus degrading enzyme, but the suitableeest substrate is parathion-methyl, in simple minimal medium, can reach 800mg/L to the tolerance concentration of parathion-methyl, in the substratum that contains glucose, can reach 2000mg/L(Chu Xiaona, Zhang Xianen, Chen Yali, Liu Hong, Song Donglin.2003。The preliminary study of pseudomonas WBC-23 Methyl Parathion Hydrolase character.Microorganism journal, 43:453-456).The separation and purification from aspergillus ornatus bacterium such as Liu Yuhuan goes out opd.This enzyme has good Degradation to organophosphorus pesticide Rogor, 45 DEG C of the optimal reactive temperatures of this enzyme, and optimal reaction pH7.2, more stable to heat, and can keep active in pH6-10 scope.Heavy metal Cu ²⁺this enzyme is had to obvious promoter action, and SDS is to it, inhibited (Liu Yuhuan and clock English are long.2000。Purifying and the character of aspergillus ornatus Z58 opd.Microorganism journal.40：430-434）。

Liu Yang etc. analyze the character of the organophosphorus pesticide lytic enzyme that derives from aspergillus oryzae LY-128, and the specificity of substrate is tested and shown, this enzyme not only can act on the organophosphorus pesticide containing P-O key; And can be hydrolyzed the organophosphorus pesticide containing P-S key.The optimal reactive temperature of this enzyme is 45 DEG C, and optimal pH 6.8, below 50 DEG C and activity stabilized within the scope of pH6.0-9.5.Hg ²⁺, Fe ³⁺, iodoacetic acid and NEM have strong restraining effect to this enzyme, and Cu ²⁺, sulfhydryl reagent and stain remover have activation (Liu Yang, Liu Yuhuan, Chen Zhishi, Lian Jie, Luo Weijia, Huang Xiao, Zhong Yingchang in various degree to enzyme.2003。The Purification and Characterization of aspergillus oryzae LY-128 broad spectrum organic phosphorus agricultural chemicals lytic enzyme.Fungus system.22,557-564）。Can find out thus, there is notable difference at aspects such as substrate specificity, susceptibility to chemical substance in several organophosphorus pesticide lytic enzymes of Purification and Characterization.

Wu Ningfeng etc. are separated to the high efficient strain of a degrading organophosphorus pesticide from insecticide factory's contaminated soil, pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) C2-1, and its zymologic property is studied.Optimum temperuture of its degraded parathion-methyl is 65 DEG C, and optimal pH is 9, and this enzyme has good thermostability and pH stability, insensitive to most metal ions and chemical reagent.The nucleotide sequence analysis of this enzyme amino acid sequence and encoding gene thereof shows, this enzyme does not have homology (Wu Ningfeng, Deng Minjie, Shi Xiuyun, Liang Guoyi, Yao Bin, Fan Yunliu with the organic phosphorus degrading enzyme of delivering at present.2003。A kind of separation and purification of new organic phosphorus degrading enzyme and zymologic property research.Science Bulletin 48,2446-2450).A large amount of genetic expression work has also further been done thereafter in same laboratory.

For opd is applied better in agriculture production, its mechanism of degradation also receives the concern that people are very large.This mode of action is mainly a series of enzymically hydrolyse reaction in fact.The organophosphorus pesticide that makes to enter in soil is broken down into the simple avirulent compounds such as carbonic acid gas completely, its whole process is as follows: first agricultural chemicals is adsorbed in microorganism surface, then permeates cell membranes enters cytolemma inside, the last degrading enzyme producing with Institute of Micro-biology in film is combined enzymatic reaction is occurred, to reach the object of degrading pesticide.Microorganism directly acts on the common mode of organophosphorus pesticide and mainly contains oxidation, dehydrogenation, reduction, hydrolysis, the reaction type such as synthetic.

At present, microorganism to the study metabolic pathways of organophosphorus pesticide more clearly be parathion-methyl.Microbial metabolism parathion-methyl, initial reaction is generally hydrolysis reaction, product is dimethyl thiophosphoric acid (dimethylthiophosphoricacid, and p-NP (p-nitrophenol DMTP), PNP), DMTP is non-toxic compound, and the toxicity of PNP has declined 100 times compared with its parent parathion-methyl, PNP is by producing 4-NC (4-nitrocatechol, 4-NC) and oxyhydroquinone (1,2, approach degradable (Bai Wenqin, Li Mei, Qiu Xinghui 4-benzenetriol), He Fengqin, cold glad husband..2004.。Pale bacillus B2 is to the research of parathion-methyl degradation pathway. Pesticide Science journal .6:48-54).

Opd content in original natural bacterial strain is too low, be difficult to a large amount of production, and production cost is high.Application opd preparation just must solve a critical problem-how industrialization cheapness is produced opd preparation.In recent years along with molecular biology and engineered development, for extensive cheap production of genetically engineered of organic phosphorus degrading zymin laid a good foundation, people attempt to build high-efficient biological reactor and improve the expression amount of opd.

At present from different microorganisms, be separated to Determination of Organophosphorus Pesticide degrading genes and carried out genetic expression.Serdar in 1985 etc. attempt first at expression in escherichia coli opd, though expression product has biologic activity, but expression amount does not also arrive 1/10(Serdar CM and Gibson DT.1985.Enzymatic hydrolysis of organo-phosphates:cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta, the Bio/Technol.3:567-571 of original natural bacterial strain).

Liu Zhi etc. are cloned into a methyl parathion hydrolase gene from degradation bacteria Peudomonas putida, and are converted in intestinal bacteria.He has also built each 1 strain of degradation bacteria of can degrade organochlorine and organophosphorus and the more than 3 kinds organophosphorus pesticide of simultaneously degrading simultaneously, engineering bacteria 1 strain of efficient degradation parathion-methyl and Furadan agricultural chemicals simultaneously, and under laboratory condition, degradation property is remarkable, enzymic activity improves 6 times, obtaining existing degradation of pesticide ability has again engineering strain (Liu Zhi, Hong Qing, the Xu Jianhong of Biological control function, Wu Jun, Zhang Xiaozhou, Zhang little Hua, Ma Aizhi, Zhu Jun, Li Shunpeng.2003。The clone of methyl parathion hydrolase gene and amalgamation and expression.Acta Genetica Sinica 9:1020-1026).

Fu state equality obtains Methyl Parathion Hydrolase encoding gene by PCR method, has built recombinant expression plasmid pET29a-mpd, is converted in Escherichia coli BL21 (DE3) and expresses.This enzyme optimal pH 8.6-8.8,15 DEG C of optimal reaction temperatures; Mn ²⁺, Zn ²⁺, Cu ²⁺can make enzymic activity increase by 15%～20%(Fu state flat, Cui Zhongli, Xu Wei, Wu Xuping, Li Shunpeng.2004。Recombinant expressed and purifying and the property research of Methyl Parathion Hydrolase.Microorganism journal.44:357-360）。

Yang etc. utilize pET expression system, and by the mpd gene heterogenous expression of Methyl Parathion Hydrolase (MPH), in intestinal bacteria, the specific enzyme activity of its expression is 1.4x10 ⁴u/mg albumen.Thick enzyme in Citrate trianion-phosphate buffered saline buffer (pH8.0) of 50mM can reduce approximately 98% the organophosphorus insecticide on wild cabbage that is sprayed at, removing toxic substances efficiency is better than water, washing composition, with commercially available enzyme product (Yang J, Yang C, Jiang H, Qiao is of methyl parathion hydrolase and its application in detoxification of organophosphates.Biodegradation.19:831-9 C.2008.Overexpression).

Model cloud six academicians of biotechnology research institute of the Chinese Academy of Agricultural Sciences, Wu Ningfeng researcher are from being filtered out the bacterium of the Determination of Organophosphorus Pesticide of can degrading the soil of organophosphorus pesticide pollution, therefrom clone the encoding gene (Deng Minjie of organic phosphorus degrading enzyme, Wu Ningfeng, Liang Guoyi, just know space, Yao Bin, Fan Yunliu.2004。The cloning and expression of a kind of new organic phosphorus degrading enzyme gene ophc2.Science Bulletin 49:1068-1072), and the organic phosphorus degrading enzyme that successfully utilized " pichia spp " high efficient expression, its expression amount is being 5.5g/L(Chu XY in 3 rising density fermentor tanks, Wu NF, Deng MJ, Tian J, Yao B, Fan YL.2006.Expression of organophosphorus hydrolase OPHC2in Pichia pastoris:purification and characterization.Protein Expr Purif.49:9-14).This achievement has been established the stable zymotechnique of gene engineering microzyme production organic phosphorus degrading enzyme in the pilot scale level of 3t fermentor tank, and its commodity " than sub-vegetable melon and fruit pesticide degradable enzyme " biotechnological formulation also comes into the market

Opd in natural bacterial strain, the degradation rate of large multipair organophosphorus pesticide is lower or effect is slower, can not reach the effect of expectation, can be by the way of genetically engineered or protein engineering, the catalytic activity of transformation degrading enzyme.254 and 257 histidine residues of opd OPH are positioned near of each monomer bimetal avtive spot, and these residues have determined the interaction of avtive spot and substrate.DiSioudi etc. utilize genetic engineering technique fixed point transformation oph, mutant H254R, the H257L producing, each avtive spot of H254R/H257L only contain 1 metal ion, enzyme after change has improved 2～30 times to the hydrolytic action of Systox (P-S is strong), and diisopropylfluorophosphate (P-F key) hydrolytic action is declined, H257L and the analogue of H254R/H257L mutant to NPPMP(nerve poison soman) hydrolysis ability improved respectively 11 times and 18 times.These results show, by changing the specificity of OPH to substrate to the pointed decoration in H254 and/or H257 site, this means the handiness that the change of metal content requirement has been increased to enzyme molecular structure, make large substrate easily enter into avtive spot, reduce the catalytic efficiency to little substrate simultaneously, thereby change catalysis characteristics (the diSioudi B of enzyme, Grimsley JK, Lai K, Wild JR.1999.Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.Biochemistry.38:2866-2872).

Two zine ions in Rochu etc. 2004 report opd active centre can be replaced by other transition-metal cations, and loss of activity not, but show different catalytic performances.The prerequisite of efficient enzyme mutant is the high thermostability of requirement.By to metallic cation zinc ²⁺, cobalt ²⁺and Cd ²⁺series of experiments, they find zinc ^{2+ can}obtain the highest enzyme stability, and cobalt ²⁺can obtain enzymatic activity high (Rochu D, Vigui é N, Renault F, Crouzier D, Froment MT, P.2004.Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase:temperature-and pH-dependence.Biochem is J.380:627-363 for Masson).

Especially Dong equals to have determined for 2005 that the gene alteration that the Methyl Parathion Hydrolase of Pseudomonas sp.WBC-3 is enzyme lays a good foundation.This enzyme is a zinc (II) dimer, the double-core zinc center that each subunit comprises mixing.These two kinds of ions are surrounded with octahedral structure by ligand.Between ion, distance is 3.5 dusts by amino-acid residue His147, His149, Asp151, His152, His234 and His302 and water molecules around.Asp255 and a water molecules are as the bridge between zine ion.Exist die aromatischen Aminosaeuren Trp179 in the ingress of catalytic center, Phe196 and Phe119.These three amino acid cause the K (m) of enzyme showing by L-Ala displacement to be increased, but catalytic activity disappears, show that these die aromatischen Aminosaeurens have important effect (Dong YJ at enzyme aspect the affinity of parathion-methyl, Bartlam M, Sun L, Zhou YF, Zhang ZP, Zhang CG, Rao Z, Zhang XE.2005.Crystal structure of methyl parathion hydrolase from Pseudomonas sp.WBC-3.J Mol Biol.353:655-663).

The report in 2008 such as Reeves is by site-directed mutagenesis and biological and physical analysis, can make organophosphor hydrolytic enzyme (OPH) mutant in keeping enzyme stability, improve catalytic capability (the Reeves TE of enzyme, Wales ME, Grimsley JK, Li P, Cerasoli DM, Wild JR.2008.Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.Protein Eng Des Sel.21:405-12).

The report in 2011 such as Su increases the lip-deep ionic linkage of enzyme by raising, can improve the thermostability of enzyme.By fork-like farm tool used in ancient China relatively from the organophosphor hydrolytic enzyme OPHC2(of pseudomonas C2-1 tool high thermal stability) and fork-like farm tool used in ancient China from Ochrobactrum Methyl Parathion Hydrolase (MPH_OCH), two proline(Pro) (Pro76 and Pro78) that are positioned on protein surface are suddenlyd change.Dibit point mutation (P76D/P78K), at inactivation 50%(T ₅₀) temperature be about 68 DEG C, higher than wild-type enzyme (64 DEG C), P76D(67 DEG C), P78K(59 DEG C).Structural analysis shows that the substituted residue of P76D/P78K (Asp76 and Lys78) may produce ionic linkage and increase electrostatic energy, then increased stability (the Su Y of enzyme, Tian J, Wang P, Chu X, Liu G, Wu N, Fan is the thermostability of a methyl parathion hydrolase by adding the ionic bond on protein surface.Appl Biochem Biotechnol.165:989-97 Y.2011.Improving).

The sudden change of the report glycine proline(Pro) in 2010 such as Tian also can increase the thermostability of enzyme.In the Methyl Parathion Hydrolase (MPH) of Ochrobactrum M231, use homology modeling and molecular dynamics simulation, meticulously select the mutational site of suitable glycine, they find that G194P has higher thermostability (Tian J, Wang P, Gao S, Chu X, Wu N, Y.2010.Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp.M231by rational engineering of a glycine to proline mutation.FEBS is J.277:4901-4908 for Fan).Same study group also finds that the reason that the OPHC2 in pseudomonas C2-1 has a high thermal stability may be C110-C146 disulfide linkage conformation (Chu XY, Tian J, Wu NF, Fan YL.2010.An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2.Appl Microbiol Biotechnol.88:125-31).

In known organic phosphorus degrading enzyme, although its aminoacid sequence, three-dimensional structure and enzymatic mechanism difference, they have some denominator.All these enzymes are lytic enzymes of metal dependent form, contain hydrophobic active centre, and its active centre has 3 pocket-like spaces, to be combined with 3 phosphide key groups of machine phosphorus insecticide.Along with increasing organic phosphorus degrading enzyme crystalline structure is elaborated, active centre and the catalytic mechanism of organic phosphorus degrading enzyme are further understood.Wherein most important and to study at most be the phosphoric triesterase (phosphotriesterase, PTE) in esterases.The crystal of this enzyme has the α spiral and the β dough sheet that alternately occur 8 times, namely TIM-bucket (TIM-Barrel) structure, due to the report of the enzyme crystal in multiple different fork-like farm tools used in ancient China source, zymophore and catalyst mechanism be comparatively clear (Bigley AN, Raushel FM.2012.Catalytic mechanisms for phosphotriesterases.Biochim.Biophy.Acta.doi:10:1016/j.bb apap.2012.04.004) all.

With respect to phosphoric triesterase PTH, the information known to Methyl Parathion Hydrolase (Methyl parathion hydrolase, MPH) is limited.The various bacteria that contains this enzyme can parathion-methyl as unique Carbon and nitrogen sources.By crystal structure analysis, this enzyme is 2 aggressiveness, and each monomer is α β/β α structure, and central authorities are β dough sheet, the α spiral that periphery is sandwich style, and metal ion is positioned at central active zone, is a kind of typical beta-lactam enzymatic structure.Molecular simulation research prediction organophosphorus pesticide dissociate group with by amino-acid residue F119, W179, the pocket of F196 composition interacts; Other 2 pocket sites are respectively by R72, L258, L273; And L65, L167 composition.α site and H152 in two metal sites, H302, D151 connects, β site and H147, H149, H234 connects (Dong YJ, Bartlam M, Sun L, Zhou YF, Zhang ZP, Zhang CG, Rao Z, Zhang XE.2005.Crystal structure of methylparathion hydrolase from Pseudomonas sp.WBC-3.J Mol Biol.353:655-663).The reports such as nearest Huang use molecular dynamics simulation, and by optionally changing basic aminoacids acidity, the acid stable of Methyl Parathion Hydrolase (OCHR-MPH) improves.Three mutant (K208E, K277D, and K208E/K277D) are more stable than wild-type.Their half life of enzyme is respectively 64 in the time of pH value 5.0,68,65 minutes, and wild-type is 39 minutes (Huang L, Wang P, Tian J, Jiang H, Wu N, Yang P, Yao B, Fan is the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues.Biotechnol Lett.34:1115-1121 Y.2012.Improving).

But take a broad view of PMH enzyme present Research, not only there is no the report of this enzymatic mechanism aspect, and to enzyme kinetics also know little (Bigley AN, Raushel FM.2012.Catalytic mechanisms for phosphotriesterases.Biochim.Biophy.Acta.doi:10:1016/j.bb apap.2012.04.004).In sum, the zymologic property of various degrading enzymes, the aspects such as substrate scope there are differences, but by molecular evolution and orthomutation, can obtain and there is property (for example enzymatic activity high more completely, better enzyme tolerance etc.) enzyme (Khare SD, Kipnis Y, Greisen P Jr, Takeuchi R, Ashani Y, Goldsmith M, Song Y, Gallaher JL, Silman I, Leader H, Sussman JL, Stoddard BL, Tawfik DS, Baker is redesign of amononuclear zinc metalloenzyme for organophosphate hydrolysis.Nat Chem Biol.8:294-300 D.2012.Computational).

Summary of the invention

For the problems referred to above, the invention provides a kind of opd mutant with better enzymic activity and preparation method thereof.By analyzing the active centre of degrading enzyme, obtain the mutant of multiple enzymes, found that mutant M164F and L116M, especially M164F has better enzymic activity.The present invention also provides a kind of degrading enzyme mutants gene order after pichia spp is codon optimized and has utilized the method for this gene of yeast expression, and its preparation method is simple, and due to the codon using after pichia spp is optimized, output is higher.Obtain opd and can be applied in fruit, vegetables, the fields such as Practice for Pesticide Residue in Agricultural Products processing such as tealeaves, are with a wide range of applications, economic implications and social effect.

Opd albumen has 300 amino-acid residues, and its gene order and aminoacid sequence are respectively as shown in SEQ ID No.2 and SEQ ID No.3.Contriver is by relatively and analyze other opd and comprise Methyl Parathion Hydrolase, choose and may be positioned at enzyme active center or near amino-acid residue suddenlys change, unexpected discovery determined on the basis in active centre, in the time that following two amino-acid residues are carried out to point mutation (L116 or M164), the opd mutant obtaining at least will strengthen more than 10% than the biologic activity of the zymoprotein not suddenling change.

Therefore, an object of the present invention is to provide a kind of opd mutant, its methionine(Met) (M) to 164 of opds or the leucine (L) of the 116th suddenly change, and described mutant has the aminoacid sequence as described in Seq ID No.8 or Seq ID No.13.The 164th amino acid X in wherein said Seq ID No.8 ₁for phenylalanine (F), tryptophane (W) or Isoleucine (I), be preferably phenylalanine.Work as X ₁during for phenylalanine, described opd mutant has the aminoacid sequence as described in Seq ID No.6.The 116th amino acid X in wherein said Seq ID No.13 ₁for methionine(Met) (M),, Isoleucine (I), phenylalanine (F) or tyrosine (Y), be preferably methionine(Met).Work as X ₁during for methionine(Met), described opd mutant has the aminoacid sequence as described in Seq ID No.11.

The present invention also provides the gene order of the above-mentioned opd mutant protein of encoding, and described gene order has the nucleotide sequence as described in Seq ID No.12 or Seq ID No.7.The Nucleotide N of 346-348 position in described sequence Seq ID No.12 ₁n ₂n ₃for the methionine(Met) of encoding respectively (M),, the codon of Isoleucine (I), phenylalanine (F) or tyrosine (Y).In the time of coding methionine(Met), described mutant gene has the nucleotide sequence as described in Seq ID No.10.The Nucleotide N of 490-492 position in described sequence Seq ID No.7 ₁n ₂n ₃for the codon of the phenylalanine of encoding respectively (F), tryptophane (W) or Isoleucine (I).In the time of coding phenylalanine, described mutant gene has the nucleotide sequence as described in Seq ID No.5.

The present invention also provides a kind of method that is prepared with machine phosphorus insecticide degrading enzyme mutants albumen, and described method comprises the steps:

(1) introduce restriction enzyme site at the two ends of opd mutant gene, cut and be operationally connected on Yeast expression carrier by enzyme,

(2) expression vector is transformed and imported in yeast, obtain recombination engineering bacteria through screening;

(3) high density fermentation recombination microzyme, centrifugal collection fermented supernatant fluid after abduction delivering, obtains restructuring opd mutant protein through separation and purification.

In aforesaid method step (1), the nucleotides sequence of the opd that can be AJ605330.1 according to global common sequence database GeneBank record sequence number is classified template as, introduce respectively restriction enzyme site at ripe opd two ends, preferably add the restriction enzyme site of restriction enzyme XhoI at front end, rear end adds the restriction enzyme site of restriction enzyme XbaI, and adds continuously two terminator codons at the restriction enzyme site front end of restriction enzyme XbaI.Two amino acid (LE) that XhoI causes and two basic aminoacidss (KR) codon of introducing belong to pichia spp signal α peptide, are also the inherent restriction enzyme sites of pichia spp.Codon preference according to pichia spp sexually revises nucleotide sequence, and the opd cDNA sequence designing (SEQ ID No.1) is carried out to chemosynthesis.This DNA sequence dna 4 amino acid of foremost coding in the time of Pichia anomala expression belong to signal peptide and digested removing finally obtains opd, and its aminoacid sequence is as shown in SEQ ID No.3.As previously mentioned, the operation of opd mutant gene is substantially similar therewith.

The opd gene of above-mentioned chemosynthesis can be cloned in various expression vectors and go by method well known in the art.The molecular cloning process of standard used is shown in the narration of (J. Pehanorm Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995.) such as J. Pehanorm Brookers.Many expression vectors and its corresponding host can buy from company, as Yeast expression carrier pPICZ-α-A, pHIL-D ₂, pPIC9, pHIL-S ₁(Invitrogen Corp.San Diego.California.USA), animal cell expression carrier pSVK3, pMSG(Amersham Pharmacia Biotech Inc.USA) etc.Preferred method is to Yeast expression carrier pPICZ-α-A by the nucleic acid clone of the target protein in code book invention or polypeptide, this plasmid is yeast integrative plasmid, with alcohol oxidase operon (AOX1) 5 ' sequence and 3 ' sequence, be used for facilitating encoding gene to be integrated into yeast chromosomal, and the expression of control coding gene.The Host Strains of these plasmids and correspondence etc. can obtain from Invitrogen Corp.San Diego.California.USA structure, and preferred promotor is AOX1.

In a preferred embodiment, opd gene and Yeast expression carrier pPICZ-α-A are carried out to double digestion, preferably respectively opd gene and Yeast expression carrier pPICZ-α-A are carried out to double digestion by restriction enzyme Xho I and restriction enzyme Xba I, opd gene after enzyme is cut is connected with Yeast expression carrier pPICZ-α-A, and the product after connecting is proceeded in bacillus coli DH 5 alpha.Choose restructuring bacterium colony pPICZ α A-OP, extract plasmid and confirm through double digestion and carry out sequencing, through confirm errorless after, continue sudden change work below.

By relatively and analyze other opd and comprise Methyl Parathion Hydrolase, choose and may be positioned at enzyme active center or near amino-acid residue suddenlys change, especially following two amino-acid residues carry out point mutation: L116 or M164, more preferably carry out following point mutation L116M or M164F.Point mutation can be undertaken by the conventional site-directed point mutation technology in this area, preferably according to green skies biotechnology research " site-directed point mutation test kit ", to these mutational site synthetic primers.The principle of this test kit is only need mutant plasmid by PCR-based synthetic, and the digestion of template plasmid based on Dpn I, transforms and cultivates and the qualification of cutting or check order of follow-up enzyme, can obtain the mutant plasmid of expecting.

While using this test kit, need first design length to be generally 30 two complementary primers more than base, in primer, contain the mutational site of expection.Then taking plasmid to be suddenlyd change as template, carry out pcr amplification reaction with these two primers.Can produce so the double-stranded plasmid in the mutational site of containing expection, but in this double-stranded plasmid, have two breaches (nick) site.The common fork-like farm tool used in ancient China of plasmid to be suddenlyd change comes from the bacteriums such as intestinal bacteria, the modification that can be methylated in bacterium, and the plasmid obtaining by pcr amplification in vitro can not be methylated.Process with methylase Dpn I like this, can digest plasmid template to be suddenlyd change, and make optionally to be remained by the pcr amplification plasmid that contains mutational site out.After the product transform bacteria that Dpn I was processed, have two breaches (nick) site to be repaired by intestinal bacteria in plasmid, the clone who obtains will contain the mutant plasmid of expection.

According to test kit requirement, synthetic following required primer

I、L116M：

1.pL116M_F：5’cacggtcttgctgactcatATGcaccctgatcatgcatgc3’；

2.pL116M_R：5’gcatgcatgatcagggtgCATatgagtcagcaagaccgtg3’；

II.、M164F

3.pM164F_F：5’ccagaaggaatgcaaggaTTCttcaaaatggctcaacaggc3’；

4.pM164F_R：5’gcctgttgagccattttgaaGAAtccttgcattccttctgg3’；

Taking the aforementioned plasmid that contains opd gene preparing as masterplate, carry out conventional site-directed point mutation PCR reaction.After PCR reaction, directly in PCR reaction system, add Dpn I to hatch, after DpnI digestion, can be directly used in conversion, or-20 DEG C save backup.

According to used competence bacterium, add as far as possible many postdigestive sudden change products of process Dpn I for transforming.Conventionally in every 100 microlitre competence bacteriums, can add 5-10 microlitre through the postdigestive sudden change product of Dpn I.Working method according to used competence bacterium operates, and before coated plate, by the way of centrifugal concentrating, the bacterium after all being converted, is all applied to and contains on suitable antibiotic flat board, overnight incubation.For the clone who obtains, can first extract the qualification of a small amount of plasmid enzyme restriction, whether conform to expected results with the size of inserting segment in plasmid with the size of the plasmid confirming to obtain.Get 3-5 enzyme cut qualification correct clone go order-checking, be the mutant clon that meets expected sequence with the clone who finally confirms to obtain.

Carrier can through transform or transfection to prokaryotic organism or eukaryote host.Transform required nucleic acid and remove available usual method to host cell, as: electroporation, prepare competent spheroplast etc.The cell that success transforms, contains the cell of DNA construct of the present invention, and the technology that can know by people is identified, if cell is through collecting and cracking, extracts DNA, then PCR method qualification.

The host who expresses target protein can be yeast, mammalian cell, bacterium, animal, plant etc., be preferably yeast, as yeast saccharomyces cerevisiae, pichia spp, candida yeasts, debaryomyces hansenii, Crewe are tieed up sub-yeast, female yeast or fission yeast etc., the more preferably pichia spp of belonging to of spore circle.Target protein or polypeptide may reside in host cell, can be also that secretion is out, preferred from host, are to secrete out from host.Secrete signal peptide used, preferably yeast MF signal α peptide.The nucleic acid of coding target protein, can be inserted into host chromosome, or exist with free plasmid form.

The host that can contain DNA construct of the present invention by cultivation, as recombination yeast, recombinant mammalian cells, recombinant bacteria, genetically modified animals and plants etc., produces opd mutant of the present invention.Concrete cultural method, can be with shaking flask or bio-reactor etc., bio-reactor preferably when production.Substratum should be able to provide thalline (or cell) growth and the required material of Product Expression, should comprise nitrogenous source, carbon source, pH buffer composition etc., and culture medium prescription generally should, according to different Objects of Development, obtain by test.Cultivation can divide two stages, and the first stage is mainly used in the growth of thalline (or cell), and subordinate phase is mainly used in abduction delivering target product.

The genetic engineering bacterium that structure is completed carries out liquid culture and fermentation, passes through methanol induction.By the medium centrifugal after methanol induction, collect supernatant liquor.Can be with the method separation and purification albumen of various albumen sepn, as saltout, the combination of the technology such as precipitation, ultrafiltration, liquid chromatography (LC) and these technology.Wherein liquid chromatography (LC) can be used gel exclusion, affine, ion-exchange, the chromatographic technique such as hydrophobic, anti-phase.

In a preferred embodiment, by adding the ammonium sulfate of 70% saturation ratio, 4 DEG C, centrifugation after 1 hour, is used the dialysis of 1mM Tris-HCl (pH8.0) damping fluid.Recombinase after purifying is homogeneous band through SDS-PAGE electrophoretic analysis, and purity exceedes 95%.Taking bovine serum albumin as standard, calculate opd, two mutant content all exceed 6.5 grams per liters, higher than 5.5g/L output (the Chu XY of the reports such as Chu, Wu NF, Deng MJ, Tian J, Yao B, Fan YL.2006.Expression of organophosphorus hydrolase OPHC2in Pichia pastoris:purification and characterization.Protein Expr Purif.49:9-14).

Expression product opd OP and mutant thereof are carried out to polyacrylamide gel electrophoresis detection, in gel imaging system, confirm to contain in sample solution expression product pesticide degradable enzyme OP and mutant, its detection molecules amount is consistent with theoretical value, is about 32.4kDa.

By using parathion-methyl to make substrate test restructuring degrading enzymatic activity, result shows: compared with wild-type organic phosphorus degrading enzyme OP, the enzyme specific activity that the enzyme specific activity of mutant M164F improves 23.6%, L116M has improved 13.2%.In order to understand the degrading activity of mutant enzyme to other organophosphorus pesticide, use liquid phase identification of spectrogram method, to triazophos, paraoxon, poison barnyard grass with poison and test, result proves that the enzyme specific activity of mutant M164F improves >20%, the enzyme specific activity >10% of L116M.Other mutant of M164 and L116 are compared with wild-type organic phosphorus degrading enzyme OP, and its specific enzyme activity all has increase to a certain degree.For example M164W, the specific enzyme activity of M164I exceedes 8% compared with wild-type organic phosphorus degrading enzyme OP; Such as L116I of other mutant of L116, L116F, L116Y exceedes 5% compared with wild-type organic phosphorus degrading enzyme OP.

Brief description of the drawings

The building process of Fig. 1 opd and mutant expression plasmid.

The recombinate SDS-PAGE gel electrophoresis figure of opd mutant M164F of Fig. 2.Number in the figure: sample cell 1 is molecular weight of albumen standard substance (kDa), is followed successively by from top to bottom: 71,55,35,26; Sample cell 2, the 2ul nutrient solution (0th day) of yeast after methanol induction; Sample cell 3,4 and 5: the 2ul nutrient solution (3rd, 4 and 5 day) of yeast after methanol induction.

Embodiment

The preparation method of embodiment 1, opd mutant M164F

1, the design of the opd gene OP after codon optimized and synthetic

Record the nucleotides sequence of the opd that sequence number is AJ605330.1 according to global common sequence database GeneBank and classify template as, add respectively the restriction enzyme site of restriction enzyme XhoI at ripe opd front end, rear end adds the restriction enzyme site of restriction enzyme XbaI, and adds continuously two terminator codons at the restriction enzyme site front end of restriction enzyme XbaI.Two amino acid (LE) that XhoI causes and two basic aminoacidss (KR) codon of introducing belong to pichia spp signal α peptide, are also the inherent restriction enzyme sites of pichia spp.Codon preference according to pichia spp sexually revises nucleotide sequence, and the opd cDNA sequence designing (SEQ ID No.1) is carried out to chemosynthesis.This DNA sequence dna 4 amino acid of foremost coding in the time of Pichia anomala expression belong to signal peptide and digested removing finally obtains opd, and its aminoacid sequence is as shown in SEQ ID No.3.

2, construction of recombinant plasmid

The construction of recombinant plasmid of opd and mutant gene as shown in Figure 1.

(1) the opd gene OP of chemosynthesis is carried out to double digestion by the plasmid pUC57-OP(Shanghai biotechnology company limited of containing OP gene by Xho I and Xba I) carry out double digestion: 20ug plasmid DNA, 20ul10X enzyme cutting buffering liquid, 5ul XhoI and 5ul XbaI, add water to cumulative volume 200ul, 37 DEG C, 6 hours.

(2) by vector plasmid pPICZ α A XhoI, XbaI carries out double digestion

PPICZ α A is carried out to double digestion: 20ug plasmid pPICZ α A(Invitrogen company), 20ul 10X enzyme cutting buffering liquid, 5ul XhoI and 5ul XbaI, add water to cumulative volume 200ul, and 37 DEG C, 6 hours.

3. opd gene and the vector plasmid after enzyme being cut with DNA gel recovery test kit reclaims, purifying

Taking 0.5 gram of agarose powder is added in the triangular flask that contains 50 milliliters of agarose gel electrophoresis damping fluids, agarose gel electrophoresis damping fluid in triangular flask is heated to seethe with excitement and agarose powder is fully dissolved, add the ethidium bromide solution of 5 μ 1, mix, after pour in agarose glue plate, insertion glue comb, solid after solidifying is called gel, take out glue comb, on gel, there is row's point sample hole, gel is put into the running gel box that contains agarose gel electrophoresis damping fluid, in the point sample hole of gel, add respectively the enzyme of 10 μ l to cut mixture and 2 μ 1DNA molecular weight standard product, open electric arteries and veins instrument switch, in the time that being 100 volts, electrophoresis apparatus voltage starts electrophoresis.

Get 1.5ml centrifuge tube and claim its weight, in full-automatic ultraviolet with visible analyser by required target DNA band, cut from gel, take 1.5ml centrifuge tube total amount and calculate the gel weight scaling off from gel.According to " DNA gel recovery test kit " method (Shanghai biotechnology company limited), reclaim opd gene OP and vector plasmid DNA.

4. the external connection of opd gene and vector plasmid DNA fragment

Opd gene and vector plasmid DNA fragment are pressed to the mixing of 4:1 mole ratio, add 1 μ l T4DNA ligase enzyme, in 1X T4DNA ligase enzyme damping fluid, 16 DEG C of child care are spent the night, and wait to transform.

5. the preparation of bacillus coli DH 5 alpha competent cell:

In 7ml test tube, add 3ml LB liquid nutrient medium and a bacillus coli DH 5 alpha list bacterium colony, mix, in shaking table (rotating speed is 180 revs/min, 37 DEG C) overnight incubation.Second day, in 250ml triangular flask, the bacterium liquid of 0.5ml overnight incubation is joined to 50ml LB liquid nutrient medium, under similarity condition, cultivate about 3 hours.Detect the 0D600 value of bacteria liquid, in the time of the 0D600=0.4 of bacterium liquid, the triangular flask that contains bacteria liquid is placed in to 0 DEG C of mixture of ice and water, place 20 minutes, for subsequent use.25ml bacterium liquid is joined in 50ml centrifuge tube, centrifugal 5 minutes (4000 revs/min, 4 DEG C), abandoning supernatant, adds the CaCl that 10ml is ice-cold ₂solution (50mM), mixes, 4 DEG C of child care 30 minutes.After half an hour, centrifugal (4000 revs/min, 4 DEG C), remove supernatant liquor, add the CaCl that fresh 10ml is ice-cold ₂solution (50mM).After centrifugal through the 3rd time, competence bacterium liquid is finally dissolved in the CaCl that 2ml is ice-cold ₂solution (50mM).Placement is stand-by after spending the night.

6. the conversion of bacillus coli DH 5 alpha competent cell

In 1.5ml centrifuge tube, add 100 μ 1 bacillus coli DH 5 alpha competent cells to be connected mixture with 8 μ 1 plasmids, mix, 1.5ml centrifuge tube is placed in to mixture of ice and water, place 20 minutes, rear taking-up is placed in 1.5ml centrifuge tube in the water-bath of 42 DEG C, places 90 seconds, takes out, again 1.5ml centrifuge tube is placed in to mixture of ice and water, places after 5 minutes.Add 1ml LB liquid nutrient medium, mix, 37 DEG C of shaking table incubations 1 hour, 3000 revs/min after centrifugal 5 minutes, abandoning supernatant, coats the thalline at the pipe end containing on the LB solid medium of 25 μ g/ml bleomycin, place 20 minutes, be placed under 37 DEG C of conditions, cultivate 16 hours, form bacterium colony.

7. the screening and identification of recombinant plasmid:

Choose the restructuring bacterium colony that contains plasmid pPICZ α A-OP, the UNIQ-10 pillar plasmid extraction test kit of using marine life Engineering Co., Ltd extracts plasmid.Recombinant plasmid, after double digestion confirms, then is delivered to Shanghai Sheng Gong Bioisystech Co., Ltd sequencing, after confirming, continues sudden change work below.

8. design and the preparation of opd mutant M164F gene

Analyze relatively Determination of Organophosphorus Pesticide lytic enzyme, choose relatively more conservative and may belong to enzyme active center or near amino-acid residue, the 164th methionine(Met) is changed into phenylalanine, i.e. M164F.According to the requirement of green skies biotechnology research institute " site-directed point mutation test kit ", to the synthetic following required primer in these mutational sites.5' holds primer: 5 ' ccagaaggaatgcaaggaTTCttcaaaatggctcaacaggc3 '; 3 ' end primer: 5 ' gcctgttgagccattttgaaGAAtccttgcattccttctgg3 '.The gene order producing after sudden change is as shown in SEQ ID No.5, and the recombinase aminoacid sequence producing is as SEQ ID No.6.

(1) site-directed point mutation reaction:

Remove nuclease water 37ul

Reaction buffer (10X) 5ul

Primer (10 μ M each) 2ul

DNTP mixture (2.5mM each) 4ul

Template plasmid to be suddenlyd change (0.5ug) 1ul

Cumulative volume 49ul

(2) add successively all ingredients according to order above, making cumulative volume is 49 microlitres.After mixing, add 1ul Pfu archaeal dna polymerase to mix.

According to following parameter, the initial sex change of PCR instrument: step 1(is set): circulation is 1,95 DEG C of temperature, 40 seconds time; Step 2(amplification): circulation is 18,95 DEG C of temperature, 40 seconds, sex change; 60 DEG C of annealing in 1 minute; 68 DEG C of 1.5 minutes/kb extend; Step 3(extends and completion): circulation is 1,95 DEG C of temperature, 40 seconds time; 72 DEG C 10 minutes; Step 4(temporarily deposits): circulation is 1, and 4 DEG C of temperature keep for a long time.

(3) Dpn I digestion: after PCR reaction, directly add 1 microlitre Dpn I in PCR reaction system, mix latter 37 DEG C and hatch 1 hour.After Dpn I digestion, can be directly used in conversion, or-20 DEG C save backup.

(4) transform, choose clone identification: according to used competence bacterium, add as far as possible many postdigestive sudden change products of process Dpn I for transforming.Conventionally in every 100 microlitre competence bacteriums, can add 5-10 microlitre through the postdigestive sudden change product of Dpn I.Working method according to used competence bacterium operates, and before coated plate, by the way of centrifugal concentrating, the bacterium after all being converted, is all applied to and contains on suitable antibiotic flat board, overnight incubation.For the clone who obtains, can first extract the qualification of a small amount of plasmid enzyme restriction, whether conform to expected results with the size of inserting segment in plasmid with the size of the plasmid confirming to obtain.Get 3 enzymes cut qualification correct clone go order-checking, finally confirm whole 3 clones be all expection mutant clon, choose pPICZ α A-M164F and make further work.

9, pichia yeast genetic engineering bacteria builds

(1) linearizing of recombinant plasmid

The 10X restriction enzyme digestion enzyme SacI damping fluid and 170 μ 1 deionized waters that in 1.5ml centrifuge tube, add 50 μ g recombinant plasmid pPICZ alpha A-M164F, 10 μ 1 restriction enzyme digestion enzyme SacI, 20 μ 1, mix, and is placed in 37 DEG C of water-baths, places 4 hours.Add 12 μ l5M NaCl, then add 636 μ l95% ethanol, place 15min, centrifugal 5min(12000rpm for-20 DEG C).By 1200 μ l75% washing with alcohol 3 times, after room temperature is dried, be dissolved in the sterilized water-20 DEG C preservation of 20 μ l, treat that electricity transforms.

(2) preparation of yeast X-33 competent cell

Upper at YPDS solid medium (1% yeast leaches+2% Tryptones+2% D-glucose+1M sorbyl alcohol+2% agar), inoculation pichia pastoris X-33 (Invitrogen company), is placed under 30 DEG C of conditions and cultivates 72 hours, observes white single bacterium colony.In 10ml centrifuge tube, add the YPDS liquid nutrient medium of 3ml and single bacterium colony of a pichia pastoris phaff X-33 bacterium, mix, under 30 DEG C of conditions, cultivate 12 hours.In 250ml triangular flask, add 0.5ml bacterium liquid and 50ml YPDS liquid nutrient medium, under 30 DEG C of conditions, cultivate 3 hours, detect the OD600 value of bacterium.In the time of OD600=1.5, in 50ml centrifuge tube, add 45ml bacterium liquid, 4 DEG C centrifugal (2500rpm, 5 minutes), abandon supernatant liquor, add 45ml deionized water, and centrifugal (1500rpm, 5 minutes) use washed with de-ionized water (totally three times) again.Thalline is dissolved in to the D-glucitol (1M) of 10ml0 DEG C, 4 DEG C centrifugal (1500rpm, 5 minutes).The competent cell finally making is dissolved in the D-glucitol (OD600=1) of 1M, for subsequent use.

(3) the preparing and screening of yeast positive recombinant

The pichia pastoris X-33 bacterium competence cell (0D600=1) that adds the linearizing vector plasmid of 10 μ g and 100 μ 1 in 4ml centrifuge tube, mixes.Vector plasmid is containing organophosphorus pesticide degrading enzyme gene mutant M164F.Under 0 DEG C of condition, it is in 70% ethanol that electric shock cup is put in to concentration, soaks after 30 minutes taking-up, in electric shock cup, put into 105 μ 1 mixed solutions, the cup that will shock by electricity is placed 5 minutes, after electric shock glass is put in electric conversion instrument, shock by electricity, electricimpulse is 25 microfarads, voltage is 1.4 kilovolts, and resistance is 200 ohm, and the electric shock time is 0.05 second.After electric shock finishes, the electric shock cup that taking-up contains mixed solution, after in electric shock cup, add 1ml D-glucitol (1M), mixture is added in 1.5ml centrifuge tube, in 30 DEG C of shaking tables, rotating speed is 225rpm, cultivates after 1 hour, gets the mixture after 100 μ 1 cultivate, and coated and contained on the YPDS solid medium that concentration is 25 μ g/mlZeocin, YPDS solid medium after coating is placed in 30 DEG C of incubators, cultivates 4 days, obtain white single bacterium colony.The single colony inoculation of whole whites obtaining above, to containing on the YPDS solid medium of 500 μ g/ml bleomycin, is placed in 30 DEG C of incubators, cultivates 3 days, observe single bacterium colony, the positive recombinant obtaining.

10, the abduction delivering of positive yeast recon

(1) shaking flask inducing culture

By the yeast of the opd mutant M164F expressing, be inoculated into 20ml BMGY liquid nutrient medium (1% yeast extract, 2% peptone, 100mM potassiumphosphate PH6.0,1.34%YNB4 × 10 in 100ml Erlenmeyer flask ^-5% vitamin H, 1% glycerine), bacterium liquid is placed under 30 DEG C of conditions, when shaking table is cultivated (200 revs/min) to OD600=5, get 8ml bacterium liquid centrifugal (4000rpm, 3 minutes), thalline is transferred to 20ml BMMY substratum (1% yeast extract in 100ml Erlenmeyer flask, 2% peptone, 100mM potassiumphosphate pH6.0,1.34%YNB4 × 10 ^-5% vitamin H, 0.5% methyl alcohol), 30 DEG C are continued to cultivate 120 hours, add the anhydrous methanol that 0.1ml concentration is 100% in culturing process every 12 hours.After cultivation finishes, get 20ml bacterium liquid, centrifugal 10 minutes (12000rpm), obtains supernatant liquor, is the opd mutant M164F of expression.

(2) fermentor tank inducing culture

Bacterial classification is seeded to 10ml YPD nutrient solution (1% yeast leaches+2% Tryptones+2% D-glucose), 30 DEG C, 240rpm, overnight incubation, just it proceeds to and contains fresh YPD nutrient solution (200ml), similarity condition overnight incubation.Then nutrient solution goes in 3 liters of fermentor tanks that contain 2 liters of basic minimal mediums and continues to cultivate.Basis minimal medium: 85% phosphoric acid 26.7ml/L, calcium sulfate 0.93g/L, potassium sulfate 18.2g/L, magnesium sulfate heptahydrate 14.9g/L, potassium hydroxide 4.13g/L, the rare metal substratum PTM1 of glucose 50g/L and 4.4ml/L.PTM1 consists of the following composition: cupric sulfate pentahydrate 6.0g/L, sodium iodide 0.08g/L, manganese sulfate monohydrate 3.0g/L, Sodium Molybdate Dihydrate 0.2g/L, boric acid 0.02g/L, cobalt chloride 0.5g/L, zinc chloride 20g/L, iron vitriol 65.0g/L, vitamin H 0.2g/L, sulfuric acid 5.0ml/L.

Fermentor tank pH is set to 5.0, dissolved oxygen amount >20%, and temperature is 30 DEG C, 750rpm.Treat that glucose consumption is complete, add 25% glucose solution that contains 1.2%PTM1 with every liter of 36ml per hour, continue 6 hours, carry out glucose cultivation to increase biomass.Treat that glucose consumption is complete, enter methanol induction and cultivate.First carry out the methyl alcohol laundering period, add the glucose that contains 11.1% methyl alcohol and 12ml/L PTM1, lasting 4 hours with every liter per hour.Add the methyl alcohol that contains 12ml/L PTM1 to carry out inducing culture until fermentation ends with every liter of 3ml per hour afterwards.

11, separating and purifying of restructuring opd and mutant.

By the nutrient solution after methanol induction, centrifugal (10000rpm, 4 DEG C, 10 minutes), collect supernatant liquor, add the ammonium sulfate of 70% saturation ratio, and 4 DEG C, centrifugation after 1 hour, is used the dialysis of 1mM Tris-HCl (pH8.0) damping fluid.Recombinase after purifying is homogeneous band through SDS-PAGE electrophoretic analysis, and purity exceedes 95%.Taking bovine serum albumin as standard, the content that calculates opd mutant M164F reaches 6.8 grams per liters, higher than 5.5g/L output (the Chu XY of the reports such as Chu, Wu NF, Deng MJ, Tian J, Yao B, Fan YL.2006.Expression of organophosphorus hydrolase OPHC2in Pichia pastoris:purification and characterization.Protein Expr Purif.49:9-14).

12, expression product opd mutant M164L is carried out to polyacrylamide gel electrophoresis detection

(1) preparation of gel

Get a clean 50ml centrifuge tube, add by following composition, fully mix

Resolving gel concentration 12%:6.5ml deionized water, 4.5ml40% acrylamide mixed solution, 3.9ml1.5MTris-HCl pH8.8,150 μ l10% sodium laurylsulfonates, 9 μ l TEMED, finally add 150 μ l10% ammonium persulphates

Spacer gel concentration 5%:2.96ml deionized water, 0.5ml40% acrylamide mixed solution, 0.5ml1.5M Tris-HCl pH6.8,40 μ l10% sodium laurylsulfonates, 4 μ l TEMED, finally add 40 μ l10% ammonium persulphates.

(2) polyacrylamide gel electrophoresis detects:

1) Coomassie brilliant blue staining fluid preparation

Get 100mg Coomassie brilliant blue, add 95% ethanol 50ml and 100ml acetic acid, mix, be diluted in 1000ml with deionized water, filter, obtain Coomassie brilliant blue dye liquor.

2) get 4% sodium laurylsulfonate, 12% glycerine, 50mM Tris-HCl, 2% thin base ethanol and 0.05% tetrabromophenol sulfonphthalein, mix, its pH value is adjusted to 6.8 with hydrochloric acid, form sample buffer, for subsequent use;

3) get the sample buffer of 6ul and the expression product opd OP of 2ul or its mutant, mix

4) in the point sample hole of gel, add respectively sample solution and the 5 μ 1 molecular weight of albumen standard substance of 5 μ 1, open electric arteries and veins instrument switch, in the time that being 100 volts, electrophoresis apparatus voltage starts electrophoresis, electrophoresis was closed electrophoresis apparatus after 3 hours, afterwards by Coomassie brilliant blue dye liquor dyeing 30 minutes, destainer decolouring, in gel imaging system, confirm to contain expression product degradation of pesticide enzyme mutant in sample solution, its detection molecules amount is 34kDa, consistent with theoretical value.

13, opd mutant M164F is active detects

(1) use parathion-methyl to make substrate test restructuring degrading enzymatic activity.

1) with methyl alcohol preparation 2ml parathion-methyl (10mg/ml)

2) preparation 10ml parathion-methyl DeR liquid: 7.5ml deionized water+0.5ml1MTris-HCl (pH8.0) damping fluid+1ml methyl alcohol+0.1M ZnCl ₂(10ul)+10mg/ml parathion-methyl (50ul).

3) experimental procedure

Get each restructuring organic phosphorus degrading enzyme and mutant 10ul, join 990ul deionized water, be mixed with 10 ^-2enzyme liquid; Get again 100ul 10 ^-2enzyme liquid joins 900ul deionized water, is mixed with 10 ^-3enzyme liquid.Add 900ul parathion-methyl DeR liquid and 10 at 1.5ml centrifuge tube ^-3enzyme liquid (100ul), adds after mixing and transfers in spectrophotometer under 412nm wavelength, measures optical density(OD).Add 900ul parathion-methyl DeR liquid and 100ul deionized water in contrast with 1.5ml centrifuge tube.Result shows: compared with wild-type organic phosphorus degrading enzyme OP, the enzyme specific activity of mutant M164F improves 23.6%((p<0.05).

(2) use liquid phase identification of spectrogram method, to triazophos, paraoxon, poison barnyard grass with poison and test, enzyme specific activity and the wild-type opd of result surface discontinuity body M164F more all exceed >20% (p<0.05).

The preparation method of embodiment 2 opd mutant L116M

1, the design of the opd gene OP after codon optimized and synthetic

2, construction of recombinant plasmid

(1) the opd gene OP of chemosynthesis is carried out to double digestion by Xho I and Xba I

By the plasmid pUC57-OP(Shanghai biotechnology company limited of containing OP gene) carry out double digestion: 20ug plasmid DNA, 20ul10X enzyme cutting buffering liquid, 5ul Xho I and 5ul Xba I, add water to cumulative volume 200ul, and 37 DEG C, 6 hours.

(2) by vector plasmid pPICZ α A Xho I, Xba I is carried out double digestion

PPICZ α A is carried out to double digestion: 20ug plasmid pPICZ α A, 20ul10X enzyme cutting buffering liquid, 5ul Xho I and 5ul Xba I, add water to cumulative volume 200ul, and 37 DEG C, 6 hours.

4. the external connection of opd gene and vector plasmid DNA fragment

Opd gene and vector plasmid DNA fragment are pressed to the mixing of 4:1 mole ratio, add 1 μ l T4DNA ligase enzyme, in 1X T4DNA ligase enzyme damping fluid, 16 DEG C of child care are spent the night, and wait to transform.5. the preparation of bacillus coli DH 5 alpha competent cell:

6. the conversion of bacillus coli DH 5 alpha competent cell

7. the screening and identification of recombinant plasmid:

Choose restructuring bacterium colony pPICZ α A-OP, the UNIQ-10 pillar plasmid extraction test kit of using marine life Engineering Co., Ltd extracts plasmid.Recombinant plasmid, after double digestion confirms, then is delivered to Shanghai Sheng Gong Bioisystech Co., Ltd sequencing, after confirming, continues sudden change work below.

8. design and the preparation of opd mutant L116M gene

Analyze relatively Determination of Organophosphorus Pesticide lytic enzyme, choose relatively more conservative and may belong to enzyme active center or near amino-acid residue, the 116th leucine is changed into methionine(Met), i.e. L116M.According to the requirement of green skies biotechnology research institute " site-directed point mutation test kit ", following required primer is synthesized in these mutational sites:

5' holds primer: 5 ' cacggtcttgctgactcatATGcaccctgatcatgcatgc3 '; 3 ' end primer: 5 ' gcatgcatgatcagggtgCATatgagtcagcaagaccgtg3 '

The gene order producing after sudden change is as shown in SEQ ID No.10, and the recombinase aminoacid sequence producing is as SEQ ID No.11.

(1) site-directed point mutation reaction:

Remove nuclease water 37ul

Reaction buffer (10X) 5ul

Primer (10uM each) 2ul

DNTP mixture (2.5mM each) 4ul

Template plasmid to be suddenlyd change (0.5ug) 1ul

Cumulative volume 49ul

(2) add successively all ingredients according to order above, making cumulative volume is 49 microlitres.After mixing, add 1ulPfu archaeal dna polymerase to mix.

(4) transform, choose clone identification: according to used competence bacterium, add as far as possible many postdigestive sudden change products of process Dpn I for transforming.Conventionally in every 100 microlitre competence bacteriums, can add 5-10 microlitre through the postdigestive sudden change product of Dpn I.Working method according to used competence bacterium operates, and before coated plate, by the way of centrifugal concentrating, the bacterium after all being converted, is all applied to and contains on suitable antibiotic flat board, overnight incubation.For the clone who obtains, can first extract the qualification of a small amount of plasmid enzyme restriction, whether conform to expected results with the size of inserting segment in plasmid with the size of the plasmid confirming to obtain.Get 3 enzymes cut qualification correct clone go order-checking, finally confirm 2 clones be all expection mutant clon, choose pPICZ α A-L116M and make further work.

9. pichia yeast genetic engineering bacteria builds

(1) linearizing of recombinant plasmid

The 10X restriction enzyme digestion enzyme SacI damping fluid and 170 μ 1 deionized waters that in 1.5ml centrifuge tube, add 50 μ g recombinant plasmid pPICZ alpha A-L116M, 10 μ 1 restriction enzyme digestion enzyme SacI, 20 μ 1, mix, and is placed in 37 DEG C of water-baths, places 4 hours.Add 12 μ l5M NaCl, then add 636 μ l95% ethanol, place 15min, centrifugal 5min(12000rpm for-20 DEG C).By 1200 μ l75% washing with alcohol 3 times, after room temperature is dried, be dissolved in the sterilized water-20 DEG C preservation of 20 μ l, treat that electricity transforms.

(2) preparation of yeast X-33 competent cell

Upper at YPDS solid medium (1% yeast leaches+2% Tryptones+2% D-glucose+1M sorbyl alcohol+2% agar), inoculation pichia pastoris X-33, is placed under 30 DEG C of conditions and cultivates 72 hours, observes white single bacterium colony.In 10ml centrifuge tube, add the YPDS liquid nutrient medium of 3ml and single bacterium colony of a pichia pastoris phaff X-33 bacterium, mix, under 30 DEG C of conditions, cultivate 12 hours.In 250ml triangular flask, add 0.5ml bacterium liquid and 50mlYPDS liquid nutrient medium, under 30 DEG C of conditions, cultivate 3 hours, detect the OD600 value of bacterium.In the time of OD600=1.5, in 50ml centrifuge tube, add 45ml bacterium liquid, 4 DEG C centrifugal (2500rpm, 5 minutes), abandon supernatant liquor, add 45ml deionized water, and centrifugal (1500rpm, 5 minutes) use washed with de-ionized water (totally three times) again.Thalline is dissolved in to the D-glucitol (1M) of 10ml0 DEG C, 4 DEG C centrifugal (1500rpm, 5 minutes).The competent cell finally making is dissolved in the D-glucitol (OD600=1) of 1M, for subsequent use.

(3) the preparing and screening of yeast positive recombinant

The pichia pastoris X-33 bacterium competence cell (0D600=1) that adds the linearizing vector plasmid of 10 μ g and 100 μ 1 in 4ml centrifuge tube, mixes.Vector plasmid is containing organophosphorus pesticide degrading enzyme gene mutant L116M.Under 0 DEG C of condition, it is in 70% ethanol that electric shock cup is put in to concentration, soaks after 30 minutes taking-up, in electric shock cup, put into 105 μ 1 mixed solutions, the cup that will shock by electricity is placed 5 minutes, after electric shock glass is put in electric conversion instrument, shock by electricity, electricimpulse is 25 microfarads, voltage is 1.4 kilovolts, and resistance is 200 ohm, and the electric shock time is 0.05 second.After electric shock finishes, the electric shock cup that taking-up contains mixed solution, after in electric shock cup, add 1ml D-glucitol (1M), mixture is added in 1.5ml centrifuge tube, in 30 DEG C of shaking tables, rotating speed is 225rpm, cultivates after 1 hour, gets the mixture after 100 μ 1 cultivate, and coated and contained on the YPDS solid medium that concentration is 25 μ g/mlZeocin, YPDS solid medium after coating is placed in 30 DEG C of incubators, cultivates 4 days, obtain white single bacterium colony.The single colony inoculation of whole whites obtaining above, to containing on the YPDS solid medium of 500 μ g/ml bleomycin, is placed in 30 DEG C of incubators, cultivates 3 days, observe single bacterium colony, the positive recombinant obtaining.

10. the abduction delivering of positive yeast recon

(1) shaking flask inducing culture

By the yeast of the opd mutant L116M expressing, be inoculated into 20ml BMGY liquid nutrient medium (1% yeast extract, 2% peptone, 100mM potassiumphosphate PH6.0,1.34%YNB4 × 10 in 100ml Erlenmeyer flask ^-5% vitamin H, 1% glycerine), bacterium liquid is placed under 30 DEG C of conditions, when shaking table is cultivated (200 revs/min) to OD600=5, get 8ml bacterium liquid centrifugal (4000rpm, 3 minutes), thalline is transferred to 20ml BMMY substratum (1% yeast extract in 100ml Erlenmeyer flask, 2% peptone, 100mM potassiumphosphate pH6.0,1.34%YNB4 × 10 ^-5% vitamin H, 0.5% methyl alcohol), 30 DEG C are continued to cultivate 120 hours, add the anhydrous methanol that 0.1ml concentration is 100% in culturing process every 12 hours.After cultivation finishes, get 20ml bacterium liquid, centrifugal 10 minutes (12000rpm), obtains supernatant liquor, is the opd mutant L116M of expression.

(2) fermentor tank inducing culture

Separation and the purifying of 11. restructuring opd mutant.

By the nutrient solution after methanol induction, centrifugal (10000rpm, 4 DEG C, 10 minutes), collect supernatant liquor, add the ammonium sulfate of 70% saturation ratio, and 4 DEG C, centrifugation after 1 hour, is used the dialysis of 1mM Tris-HCl (pH8.0) damping fluid.Recombinase after purifying is homogeneous band through SDS-PAGE electrophoretic analysis, and purity exceedes 95%.Taking bovine serum albumin as standard, the content that calculates opd mutant L116M reaches 7.2 grams per liters, higher than 5.5g/L output (the Chu XY of the reports such as Chu, Wu NF, Deng MJ, Tian J, Yao B, Fan YL.2006.Expression of organophosphorus hydrolase OPHC2in Pichia pastoris:purification and characterization.Protein Expr Purif.49:9-14).

12. couples of expression product opd mutant L116M carry out polyacrylamide gel electrophoresis detection

(1) preparation of gel

Get a clean 50ml centrifuge tube, add by following composition, fully mix.

Resolving gel concentration 12%:6.5ml deionized water, 4.5ml40% acrylamide mixed solution, 3.9ml1.5M Tris-HCl pH8.8,150 μ l10% sodium laurylsulfonates, 9 μ l TEMED, finally add 150 μ l10% ammonium persulphates.

(2) polyacrylamide gel electrophoresis detects:

1) Coomassie brilliant blue staining fluid preparation

4) in the point sample hole of gel, add respectively sample solution and the 5 μ 1 molecular weight of albumen standard substance of 5 μ 1, open electric arteries and veins instrument switch, in the time that being 100 volts, electrophoresis apparatus voltage starts electrophoresis, electrophoresis was closed electrophoresis apparatus after 3 hours, afterwards by Coomassie brilliant blue dye liquor dyeing 30 minutes, destainer decolouring, in gel imaging system, confirm to contain expression product degradation of pesticide enzyme mutant in sample solution, its detection molecules amount is consistent with theoretical value, is 32kDa.

13. opd mutant L116M are active to be detected

1) with methyl alcohol preparation 2ml parathion-methyl (10mg/ml)

3) experimental procedure

Get each restructuring organic phosphorus degrading enzyme and mutant 10ul, join 990ul deionized water, be mixed with 10 ^-2enzyme liquid; Get again 100ul 10 ^-2enzyme liquid joins 900ul deionized water, is mixed with 10 ^-3enzyme liquid.Add 900ul parathion-methyl DeR liquid and 10 at 1.5ml centrifuge tube ^-3enzyme liquid (100ul), adds after mixing and transfers in spectrophotometer under 412nm wavelength, measures optical density(OD).Add 900ul parathion-methyl DeR liquid and 100ul deionized water in contrast with 1.5ml centrifuge tube.Result shows: compared with wild-type organic phosphorus degrading enzyme OP, the enzyme specific activity of mutant L116M improves 13.2% (p<0.05).

(2) use liquid phase identification of spectrogram method, to triazophos, paraoxon, poison barnyard grass with poison and test, enzyme specific activity and the wild-type opd of result surface discontinuity body L116M more all exceed >10% (p<0.05).

Claims

1. an opd mutant, the aminoacid sequence of described mutant is as described in Seq ID No.11.

2. the gene of coding opd mutant as claimed in claim 1, the nucleotide sequence of described gene is as described in Seq ID No.10.

3. carry the carrier of gene as claimed in claim 2.

4. carrier according to claim 3, described carrier is selected from Yeast expression carrier pPICZ-α-A, pHIL-D ₂, pPIC9 or pHIL-S ₁.

5. carrier according to claim 4, described carrier is Yeast expression carrier pPICZ-α-A.

6. a host system, it is obtained through conversion or transfection yeast host by carrier claimed in claim 3.

7. host system according to claim 6, is characterized in that: described yeast is selected from yeast saccharomyces cerevisiae, pichia spp, candida yeasts, debaryomyces hansenii, Crewe and ties up that sub-yeast, spore circle are female belongs to yeast or fission yeast.

8. host system according to claim 7, is characterized in that: described yeast is pichia spp.

9. prepare a method for opd mutant as claimed in claim 1, described method comprises the steps:

10. method according to claim 9, is characterized in that: in described method steps (1), introduce XhoI/XbaI restriction enzyme site at opd mutant gene two ends.

11. methods according to claim 10, it is characterized in that: in described method steps (1), add the restriction enzyme site of restriction enzyme XhoI at opd mutant gene front end, rear end adds the restriction enzyme site of restriction enzyme XbaI, and adds continuously two terminator codons at the restriction enzyme site front end of restriction enzyme XbaI.

12. methods according to claim 9, is characterized in that: in described method steps (1), described carrier is selected from Yeast expression carrier pPICZ-α-A, pHIL-D ₂, pPIC9 or pHIL-S ₁.

13. methods according to claim 12, is characterized in that: in described method steps (1), described carrier is Yeast expression carrier pPICZ-α-A.

14. methods according to claim 9, it is characterized in that: in described method steps (2), described yeast is selected from yeast saccharomyces cerevisiae, pichia spp, candida yeasts, debaryomyces hansenii, Crewe and ties up that sub-yeast, spore circle are female belongs to yeast or fission yeast.

15. methods according to claim 14, is characterized in that: in described method steps (2), described yeast is pichia spp.

16. methods according to claim 9, it is characterized in that: in described method steps (3), high density fermentation recombination microzyme is divided into two stages, and the first stage is mainly used in the growth of thalline, and subordinate phase is mainly used in abduction delivering target product.

17. methods according to claim 9, is characterized in that: in described method steps (3), separation purification method is selected from and saltouts, the combination of precipitation, ultrafiltration, liquid chromatography (LC) technology and these technology.

18. methods according to claim 17, is characterized in that: in described method steps (3), liquid chromatography (LC) adopts gel exclusion, affine, ion-exchange, hydrophobic or reversed phase chromatography technology.