CN1330760C - Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process - Google Patents
Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process Download PDFInfo
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- CN1330760C CN1330760C CNB2005101125804A CN200510112580A CN1330760C CN 1330760 C CN1330760 C CN 1330760C CN B2005101125804 A CNB2005101125804 A CN B2005101125804A CN 200510112580 A CN200510112580 A CN 200510112580A CN 1330760 C CN1330760 C CN 1330760C
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- degrading enzyme
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- phosphorus degrading
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000003805 vibration mixing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The present invention discloses a novel artificially synthesized organophosphorous catabolic enzyme gene which is suitable for being expressed in eukaryotic cells. The artificial gene has a nucleotide sequence represented by SEQ ID No. 1. The gene of the present invention is obtained by a method that molecular modification and molecular embellishment are carried out to the original organophosphorous catabolic enzyme gene and then the organophosphorous catabolic enzyme gene is prepared through artificial synthesis. Compared with the original gene, the recombination gene after modification has totally 154 basic groups altered and implicates 135 amino acids, and the G +C content is changed from 62.89 % to 50.27%. The present invention is suitable for efficient expression in eukaryotic cells. The present invention also provides a recombination expression carrier comprising the gene, a host cell comprising the recombination expression carrier, and a method for preparing recombination organophosphorous catabolic enzyme.
Description
Technical field
The present invention relates to a kind of artificial gene, relate in particular to a kind of gene of coding organic phosphorus degrading enzyme of synthetic, the recombinant yeast expression vector that comprises this gene, the preparation method of this recombinant yeast expression vector institute's transformed host cells and reorganization organic phosphorus degrading enzyme belongs to the genetically engineered field.
Background technology
Show that according to China Statistical Yearbook about 1,000,000 tons of China's chemical pesticide annual production occupies the second place of the world.Wherein the sterilant output of high poison ranks first in China's agricultural chemicals, accounts for 70% of agricultural chemicals ultimate production.China is again the use big country of agricultural chemicals, and about 4,400,000,000 mu times of annual chemical prevention area only just can be retrieved 4,800 ten thousand tons of the losses that disease and pest causes for vegetables, comprises grain, cotton, fruit, and the total value of retrieving is about 50,000,000,000 yuan.Yet, the residue problem ubiquity of agricultural chemicals.Simultaneously, majority is the organophosphorus pesticide of high poison in sterilant, but the utilization ratio of agricultural chemicals has only 10-20%, its residual soil and water body of entering, havoc farmland ecosystem, and endangered people ' s health, also directly influenced marketing of agricultural products and foreign exchange earning.
With tealeaves is example, and about 200,000 tons of China's annual outlet tealeaves occupy the second in the world, about 400,000,000 dollars of the trade amount of money, and export volume accounts for 1/3 of China's Tea Production total amount.European Union is a staple market of China's tea export, and the volume of trade reaches 7,000 ten thousand dollars.But over the past two years, European Union was on the increase the gainer of tealeaves Pesticide Residue, and limit value significantly reduces, and had seriously hindered the export to Europe of China's tealeaves.
China is large agricultural country, and the development green agricultural product is the direction of 21st century agricultural development.The world market has not existed to the trade barrier of agricultural products in China, and technology barriers, especially the pesticide residue problem then becomes main limiting factor.Solve the pesticide residual contamination of agricultural-food, not only can improve the quality of agricultural products in China, satisfy the demand of people, help foreign exchange earning simultaneously, increase peasant's income the safety agricultural-food.Therefore, how to remove pesticide residue, become the problem that presses for solution.
Organophosphorus pesticide is the primary categories in the agricultural chemicals, is that agriculture production is requisite.But organophosphorus pesticide has the function that suppresses human acetylcholinesterase, and the people is existed the different toxicity of degree.Along with the raising of people's quality of life and the reinforcement of environmental consciousness, the residual toxicity problem of organophosphorus pesticide more and more is subjected to people's attention.
From Sethunarhan in 1973 and Yoshida (Sethunathan, N., and T.Yoshida.AFlavobacterium that degrades diazinon and parathion.Can.J.Microbiol.1973.19:873-875) from the soil of contacted organophosphorus pesticide, isolated since the Flavobacterium that first strain has diazinon and thiophos degrading activity, people constantly find out some soil microorganisms (bacteriums, fungi) can degrading organic phosphor pesticides, this mainly is because it can secrete a kind of enzyme liberating organophosphorus, product after the degraded can be used as the carbon of microorganism growth simultaneously, nitrogen, the phosphorus source, for its growth provides nutrition [(Chaudhry, G.R., A.N.Ali, and W.B.Wheeler.Isolation of a methyl parathion-degradingPseudomonas sp.that possesses DNA homologous to the opd gene from aFlavobacterium sp.Appl.Environ.Microbiol.1988.54:288-293; Zboinska E., et al:Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.Appl Environ Microbiol.1992Sep; 58 (9): 2993-9.; Li Shubin, all benevolence is superfine: the research of aspergillus M-2 degrading organic phosphor pesticides (acephatemet).The microbiology circular, 1999,26 (1): 27-30)].
Organophosphorus contains three phosphide keys usually, so often be called as phosphotriester.General organophosphorus is divided into two types, and the one, phosphorus passes through two keys and combines (P=O) with oxygen, as acephatemet, omethoate, SD-1750 etc.; Another kind is that phosphorus passes through two keys and combines (P=S) with sulphur, as thiophos, parathion-methyl, Volaton, isocarbophos, Chlorpyrifos 94 (Le Siben) etc.There are some researches show, if being hydrolyzed, a phosphoric acid ester bond in the organophosphorus will reduce its toxicity greatly, with the thiophos is example, to make its toxicity reduce by 100 times of (Serdar, C.M., and D.T.Gibson.Enzymatic hydrolysis of organophosphates:cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta.Bio/Technology.1985.3:567-571; Serdar, C.M.Hydrolysis of cholinesteraseinhibitors using parathion hydrolase.U.S.patent.December 1996.5,589,386).Therefore, the phosphide key of destruction organophosphorus is to reduce organophosphorus pesticide toxicity efficient ways.
(Organophosphorus acid hydrolase, EC3.1.8.1) degradable organophosphorus pesticide molecule destroy the phosphide key of organophosphorus and make its detoxification opd.Because various organophosphorus pesticides all have similar structure, so substituting group difference just is a kind of opd multiple organophosphorus pesticide of degradable often.Opd has been acknowledged as the most potential novel method (Yu Yunlong etc., 1996, the present Research of microbe degradation of pesticides and development strategy, environmental science progress, 1996, Vol.4, No.3,28～36) of eliminating pesticide residue at present.For the molecular biology research of opd along with people to the understanding of pesticide residue and deepen continuously.People such as DiSioudi (DiSioudi, B., Miller, C., Lai, K., Grimsley, J.and Wild, J.Rational design of organophosphorushydrolase for altered substrate specificities.Chem.Biol.Interact.1999,14:211-223; DiSioudi, B., Grimsley, J.Lai, K., and Wild, J.Modification of nearactive site residues organophosphorus hydrolase reduces metal stoichometry andalters substrate specificity.Biochemistry, 1999,38 (10): 2866-2872) studied the structure of its encoding gene, and carried out molecular modification, make it change substrate specificity, thereby can have degradation efficiency efficiently multiple organophosphorus pesticide.Simultaneously, people also with the gene clone of degradation of pesticide enzyme in intestinal bacteria, and make it at cell surface expression, increase its degradation efficiency [Wu CF, Valdes JJ, Rao G, Bentley WE.Enhancement of organophosphorus hydrolase yield in Escherichiacoli using multiple gene fusion.Biotechnol Bioeng.2001,75 (1): 100-103; Cho CM, Mulchandani A, Chen W.Bacterial cell surface display of organophosphorushydrolase for selective screening of improved hydrolysis of organophosphate nerveagents.Appl.Environ.Microbiol.2002,68 (4): 2026-2030].
Wu Ningfeng etc. separate the bacterium that obtains a high-efficiency degradation organophosphorus pesticide from the soil of pesticidal contamination, (this bacterium is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center through being accredited as the false Alcaligenes of false unit cell (Pseudomonas pseudoalcaligenes), preserving number is CGMCC1150), simultaneously, this bacteriogenic organic phosphorus degrading enzyme OPHC2 has been carried out research [the Wu Ningfeng of separation and purification and zymologic property, Deng Minjie, et al:Isolation, purification andcharacterization of a new organphosphorus hydrolase OPHC2.Chinese ScienceBulletin, 2004,49 (3): 268-272], and therefrom cloned the encoding gene [WuNingfeng of organic phosphorus degrading enzyme, Deng Minjie, et al:Cloning and expression of ophc2, a neworganphosphorus hydrolase gene. Chinese Science Bulletin, 2004,49 (12): 1245-1249] (nucleotide sequence of encoding gene is logined in GenBank, and accession number is AJ605330).
But the expression amount of organic phosphorus degrading enzyme in former bacterium and reorganization prokaryotic expression system intestinal bacteria is not high, and the expression amount in former bacterium is about the 0.2U/mL fermented liquid, and abduction delivering is 2U/mL only also in intestinal bacteria, does not reach the purpose of cheap scale operation.Utilize yeast expression system can allow foreign gene is inserted on the carrier that contains signal peptide, the foreign protein of expression can be secreted in the nutrient solution supernatant, do not need broken wall just can obtain zymoprotein, simplified the after-processing technology of zymoprotein, be particularly conducive to the scale operation of zymoprotein.Therefore, necessary former organophosphorus degrading enzyme gene is carried out molecular modification and modification, make improved organic phosphorus degrading enzyme gene can in yeast cell, efficiently express the reorganization organic phosphorus degrading enzyme, produce the reorganization organic phosphorus degrading enzyme for large-scale industrialization is cheap and open up a new approach.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome former organophosphorus degrading enzyme gene (accession number of this gene in GenBank the is AJ605330) defective that expression efficiency is not high, former organophosphorus degrading enzyme gene is carried out molecular modification and modification, obtain a kind of artificial gene that can in yeast cell, efficiently express organic phosphorus degrading enzyme.
The present invention's technical problem at first to be solved realizes by following technological approaches:
A kind of artificial sequence oligodeoxynucleotide of the organic phosphorus degrading enzyme of encoding, it has the nucleotide sequence shown in the SEQ ID NO:1, and this artificial sequence oligodeoxynucleotide can efficiently express the reorganization organic phosphorus degrading enzyme in yeast cell.
Exogenous gene cloning is expressed to host cell, can be subjected to the influence of many factors, comprise the stability of promotor efficient, 5 ' end non-coding area sequence, effective termination of transcribing, foreign protein and foreign structural gene itself etc.The molecular biology latest Progress of combined influence gene efficient expression of the present invention, the organic phosphorus degrading enzyme gene that derives from prokaryotic micro-organisms has been carried out molecular modification, it can especially can be efficiently expressed in yeast expression system in eukaryotic expression system.The present invention is based on the following overall principle former organophosphorus degrading enzyme gene is carried out molecular modification: do not change the coded aminoacid sequence of original lactase gene 1.; According to the selection of pichia spp codon deflection to codon be optimized (Zhao Xiang, Huo Keke, Li Yuyang. the codon usage analysis of pichia spp, biotechnology journal .2000,16 (3): 308～311); 3. avoid some may reduce the sequence of expression.Nucleotide sequence after the molecular modification is shown in SEQ ID NO:1, improved organic phosphorus degrading enzyme gene is compared with original gene, has changed 154 bases altogether, relates to 135 amino acid, G+C content becomes 50.27% by original 62.89%, is more suitable for expressing in eukaryotic cell.
Another technical problem to be solved by this invention is to make up to contain the recombinant yeast expression vector of nucleotide sequence shown in the SEQ ID NO.1 and obtain the recombinant yeast cell that contains this recombinant yeast expression vector.
Another technical problem to be solved by this invention realizes by following technological approaches:
A kind of recombinant yeast expression vector contains the nucleotide sequence shown in the SEQ ID NO.1.
Recombinant yeast expression vector of the present invention can make up by the ordinary method of this area and form, promptly just the nucleotide sequence shown in the SEQID NO.1 is inserted between the suitable restriction enzyme site of Yeast expression carrier, makes that the nucleotide sequence shown in the SEQ ID NO:1 is exercisable to be connected with the yeast cell to express regulating and controlling sequence.As the most preferred embodiment of the present invention, be preferably the nucleotide sequence shown in the SEQ ID NO:1 is inserted between the SnaBI and NotI restriction enzyme site on the plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it, obtain recombinant yeast expression vector pPIC9-ophc2-m.
The constructed recombinant yeast expression vector of the present invention can be by conventional method transformed host cell, and described host cell can be pichia spp cell (Pichic pastoris), cerevisiae (Saccharomyces cerevisiae) or saccharomyces lactis cell (Hansenula polymorpha).As the most preferred embodiment of the present invention, be preferably recombinant yeast expression vector pPIC9-ophc2-m is transformed pichia spp cell (Pichic pastoris) GS115, obtain recombinant yeast cell: pichia spp cell (Pichic pastoris) GS115/pPIC9-ophc2-m.
Another technical problem to be solved by this invention provides a kind of method for preparing organic phosphorus degrading enzyme.
Another technical problem to be solved by this invention realizes by following technological approaches:
A kind of method for preparing organic phosphorus degrading enzyme may further comprise the steps:
Cultivate with the recombinant yeast cell that recombinant yeast expression vector of the present invention transformed, induce the expression of reorganization organic phosphorus degrading enzyme, reclaim and the expressed organic phosphorus degrading enzyme of purifying.
In the above-mentioned method for preparing organic phosphorus degrading enzyme, preferred, described recombinant yeast expression vector is Yeast expression carrier pPIC9-ophc2-m; Described recombinant yeast cell is pichia spp cell (Pichicpastoris) GS115/pPIC9-ophc2-m.
It is proteic more than 90% that reorganization organic phosphorus degrading enzyme content in fermented liquid of Pichia anomala expression of the present invention accounts for total secretion, only see the existence of a small amount of foreign protein by SDS-PAGE, therefore only need fermented liquid is handled through desalting and purifying, need not the reorganization organic phosphorus degrading enzyme that further processing can obtain purifying.
Another technical problem to be solved by this invention provides a kind of method of utilizing recombinant yeast cell of the present invention [pichia spp cell (Pichic pastoris) GS115/pPIC9-ophc2-m] large scale fermentation production reorganization organic phosphorus degrading enzyme.
Another technical problem to be solved by this invention realizes by following technological approaches:
A kind of method of utilizing recombinant yeast cell of the present invention [pichia spp cell (Pichic pastoris) GS115/pPIC9-ophc2-m] large scale fermentation production reorganization organic phosphorus degrading enzyme, by seed culture, the thalli growth stage, carbon source is fed the stage, carbon source-methanol is mixed the step of feed stage and five conventional fermentative production enzymes of methanol induction expression phase and is formed, wherein when the methanol induction expression phase, the following condition that adopts is carried out abduction delivering reorganization organic phosphorus degrading enzyme: add methyl alcohol that 3.0ml contains 12ml/l PTM1 with the abduction delivering organic phosphorus degrading enzyme of recombinating to per 1 hour stream of per 1 liter of fermented liquid, the inductive time is 120-144 hour; The pH value that stream adds ammoniacal liquor adjusting fermented liquid is 5.4-5.6.
In sum, in yeast cell, to efficiently express having synthesized of success of the present invention the artificial organic phosphorus degrading enzyme gene of reorganization organic phosphorus degrading enzyme, and the recombinant expression vector and the screening that have successfully made up this gene obtain the engineering strain (this project bacterial strain called after GS-OPHC2-55) of high expression level, set up the recombinate best approach of organic phosphorus degrading enzyme of the method that prepare organic phosphorus degrading enzyme of recombinating and large-scale industrialization fermentative production.The present invention recombinate organic phosphorus degrading enzyme at the expression amount of 3 liters of fermentor tanks up to 6 grams per liters, the zymologic property analysis revealed behind the recombinase purifying, its zymologic property is good; Degradation experiment to different organophosphorus pesticides shows that it has higher enzymic activity and degraded broad spectrum, produces the reorganization organic phosphorus degrading enzyme for large-scale industrialization and opens up a new way.
Description of drawings
Fig. 1 yeast recombinant plasmid pPIC9-ophc2-m enzyme is cut qualification result;
The PCR detected result of the organic phosphorus degrading enzyme gene on Fig. 2 yeast chromosomal;
The proteic SDS-PAGE electrophoretogram of reorganization organic phosphorus degrading enzyme in Fig. 3 fermented liquid;
The recombinate SDS-PAGE electrophoretogram of organic phosphorus degrading enzyme protein purification of Fig. 4;
Fig. 5 temperature is to the influence of reorganization organic phosphorus degrading enzyme reaction;
The recombinate temperature stability of organic phosphorus degrading enzyme of Fig. 6 the present invention;
Fig. 7 pH is to the recombinate influence of organic phosphorus degrading enzyme reaction of the present invention;
The recombinate pH stability of organic phosphorus degrading enzyme of Fig. 8 the present invention.
Further describe preparation method of the present invention and beneficial effect by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
One, test materials and reagent
1, biochemical reagents, enzyme, test kit:
Restriction enzyme is that TaKaRa and BioLabs company product, ligase enzyme are that Promega company product, Taq enzyme are that worker bio-engineering corporation product, T are given birth in Shanghai
4Polynueleotide kinase is a TaKaRa company product; DNA recovery test kit is purchased in Bao Bio-Engineering Company, T carrier connection test kit and is purchased the company in Promega, acrylamide, N, and N '-methylene fork acrylamide, no amino acid yeast nitrogen (YNB), vitamin H (Biotin), agarose are available from Sigma company.Other biochemical reagents are all purchased in Sigma, Promega and Beijing chemical reagents corporation.
2, plasmid and bacterial strain:
Pichia spp (Pichic pastoris) expression system (comprising yeast F-strain Pichic pastorisGS115, plasmid pPIC9) is available from American I nvitrogen Corporation company; It is Time Technology company limited that the pBS-T carrier is purchased in sky, Beijing.
3, substratum:
1) perfect medium YPD: yeast extract 10g/L, peptone 20g/L, glucose 20g/L (solid medium contains 1.5% agar).
2) transform substratum RDB: yeast nitrogen (Yeast Nitrogen Base W/O amino acids, YNB) 13.4g/L, glucose 20g/L, vitamin H 4 * 10
-4G/L, sorbyl alcohol 186g/L, 0.05g/L L-glutamic acid, 0.05g/L methionine(Met), 0.05g/L Methionin, 0.05g/L leucine, 0.05g/L Isoleucine, agar powder 20g/L.
3) select substratum MD:YNB13.4g/L, glucose 20g/L, vitamin H 4 * 10
-4G/L, agar powder 20g/L.
4) select substratum MM:YNB13.4g/L, methyl alcohol 5mL/L, vitamin H 4 * 10
-4G/L, agar powder 20g/L.
5) abduction delivering substratum BMGY: yeast extract 10g/L, peptone 20g/L, yeast nitrogen (YNB) 13.4g/L, vitamin H 4 * 10
-4G/L, glycerine 10mL, pH6.0.
6) abduction delivering substratum BMMY: yeast extract 10g/L, peptone 20g/L, yeast nitrogen (YNB) 13.4g/L, vitamin H 4 * 10
-4G/L, methyl alcohol 5mL/L, pH6.0.
7) recombination yeast fermention medium: phosphoric acid 26.7mL/L, CaSO
40.93g/L, K
2SO418.2g/L, MgSO
47H
2O14.9g/L, KOH4.13g/L, glucose 50g/L.
8) used trace salt solution (PTM1) in the fermentation: copper sulfate 6.0g/L, sodium iodide 0.08g/L, manganous sulfate 3.0g/L, Sodium orthomolybdate 0.2g/L, boric acid 0.02g/L, cobalt chloride 0.5g/L, zinc chloride 20g/L, ferrous sulfate 65g/L, vitamin H 0.25g/L, sulfuric acid 5mL/L.
9) LB perfect medium: NaCl, 10g/L; Peptone; 10g/L; Yeast powder, 5g/L.Sterilized 20 minutes for 121 ℃, solid medium adds 1.5% agar.
4, dna sequencing, primer synthesize:
Entrust Jikang Biotechnology Co Ltd, Shanghai to carry out dna sequencing; Entrust the primer of synthetic all the experiment usefulness of Beijing AudioCodes biotech company.
Synthetic and the clone of [embodiment 1] reorganization organic phosphorus degrading enzyme gene
1, is partial to according to the selection of pichia spp codon, [this gene order sees for details: Wu Ningfeng not changing original organic phosphorus degrading enzyme gene, Deng Minjie, et al:Cloning and expression ofophc2, a new organophosphorus hydrolase gene.Chinese Science Bulletin, 2004,49 (12): 1245-1249] under the prerequisite of coded aminoacid sequence, the codon of organic phosphorus degrading enzyme gene mature protein coding sequence has been carried out optimization design.
Improved organic phosphorus degrading enzyme gene is compared with original gene, has changed 154 bases altogether, relates to 135 amino acid, and G+C content becomes 50.27% by original 62.89%, is adapted at expressing in the pichia spp.
Improved complete genome sequence is divided into A, B, C three big sections, and then further big fragment is divided into the small segment of size about 50bp, A:C1-1-C1-6, complementary strand CR1-1-CR1-6; B:C2-1-C2-7, complementary strand CR2-1-CR2-7; C:C3-1-C3-6, complementary strand CR3-1-CR3-6; Totally 38 sections oligo DNA fragments are synthesized respectively.The segmental synthetic work of oligo DNA is finished by AudioCodes biotech company.
2, synthetic DNA small segment is used deionized water dissolving respectively, and making its final concentration is 20pmol/L, again they is carried out phosphorylation respectively, in 10 μ l reaction systems, adds 3UT
4Polynueleotide kinase and Triphosaden (ATP, final concentration are 0.2mmol/L), 37 ℃ of water bath heat preservation 30min.Then, each DNA small segment is merged into three pipes respectively by A, B, C group, 95 ℃ of water bath heat preservation 5min naturally cool to room temperature.
3, for ease of carrying out clone operations, and consider the correct direction of insertion of target gene fragment on carrier, in the time of synthetic three sections big Segment A, B, C, designed XhoI, SnaBI restriction enzyme site successively, added the HindIII restriction enzyme site at 3 ' end at the segmental 5 ' end of A; Add HindIII and SpeI restriction enzyme site respectively at B section 5 ' end and 3 ' end, add SpeI and NotI restriction enzyme site respectively at C section 5 ' end and 3 ' end.Agarose detects the qualified big fragment of each bar and directly is connected with the pBS-T carrier of handling well with the respective limits restriction endonuclease (is Time Inc. available from the sky); electric shock is transformed into e. coli jm109; send to order-checking, examining order is finished by Jikang Biotechnology Co Ltd, Shanghai.
4, the correct big fragment of order-checking is linked to be complete ophc2-m gene again: earlier A and B are spliced on pBS-T, then segment C is downcut to link and have on the segmental pBS-T of AB, obtain having the recon pBS-ophc2-m of complete modifying gene, the recombinant plasmid transformed e. coli jm109, coating LB (containing ammonia benzyl 100 μ g/mL) flat board, selecting single bacterium colony cultivates, extract the plasmid of culture, carry out that enzyme is cut, electrophoresis detection, show that enzyme slitting band is consistent with the synthetic clip size, preliminary identification has obtained positive recombinant clone.For further conclusive evidence, this recon has been carried out sequencing, examining order is finished by last sea base Kanggong department, and the sequencing result further confirms in the successful tram that is inserted into the pBS-T carrier of the nucleotide sequence shown in the SEQ ID NO:1.
[embodiment 2] yeast construction of recombinant plasmid
Carry out the double digestion processing with SnaBI/NotI respectively with detecting correct recombinant plasmid pBS-ophc2-m and plasmid pPIC9, electrophoresis is used T after reclaiming
4Dna ligase (Promega company) connects.Like this, utilize restriction enzyme site that the goal gene orientation is inserted between the SnaBI and NotI site on the pPIC9, form recon pPIC9-ophc2-m, the enzyme of recon is cut and is identified and see Fig. 1.Thereby, goal gene is cloned into AOX1 promotor downstream, and has formed correct reading frame with signal coding sequence.
The screening of [embodiment 3] conversion, detection and high expression level organic phosphorus degrading enzyme engineering bacteria
1, the acceptor zymic transforms: extract the prepared yeast recombinant expression plasmid pPIC9-ophc2-m of embodiment 2 in a large number, get 10 μ g and make linearization process with excessive slightly BglII, whether the electrophoresis detection enzyme is cut complete, the plasmid DNA that linearizing is good phenol chloroform, each extracting of chloroform once, ethanol sedimentation, centrifugal, abandon supernatant liquor, 70% ethanol washes twice, the sterilized water dissolving.Then, after getting linear DNA 1～5 μ g and 80 μ L yeast GS115 competent cells mixing, inject the aseptic electric shock cup (0.2cm of precooling, BioRad) in, rap the electric shock cup, make mixture fall into electric shock cup bottom, going up setting voltage at electric shock instrument (BioRad) is 2.5kV, electric capacity is 25 μ F, and resistance is 400 Ω, carries out electric conversion operation.After electricity transforms, add the 1mol/L Sorbitol Powder of 1mL precooling immediately in the electric shock cup, be coated with the RDB flat board behind the mixing immediately, every plate is coated with 200 μ L.Be inverted flat board in 28～30 ℃ of incubators, be cultured to transformant and (about 60 hours) occur.
, be connected to successively on MM, the MD solid medium from transforming the dull and stereotyped picking list bacterium colony of going up with aseptic toothpick, be inverted flat board, be cultured to transformant and (about 48 hours) occur in 28～30 ℃ of incubators.Screening growth on the MD flat board normal but on the MM flat board poor growth or clone's (his of not growing fully
+, mut
s).In order to screen the restructuring yeast strains that obtains high expression level, directly detect the expression of organic phosphorus degrading enzyme in the inducing culture.With his+mut
sTransformant is at first cultivated in the BMGY substratum, treat that it grows to state of saturation after, the centrifugal 4min of 5000rpm, abandon BMGY, change to inducing culture BMMY, behind inducing culture 48h, with the centrifugal 3min of culture 10000rpm, get supernatant liquor and carry out the organic phosphorus degrading enzyme assay as follows:
Get 100 μ L enzyme liquid adding to be measured and contain in the system of 5 μ L10mg/mL parathion-methyls and 900 μ L50mmol/L Tris-Cl (pH9.0) damping fluids, 37 ℃ are incubated 10 minutes, add 1mL10% trichoroacetic acid(TCA) termination reaction, add 1mL10%Na again
2CO
3The solution colour developing, 410nm measures the OD value, calculates the content of hydrolysate p-nitrophenol and the activity of enzyme.The activity unit of an enzyme (U) is defined as at 37 ℃, and per minute discharges the required enzyme amount of 1 μ mol p-nitrophenol.
Organic phosphorus degrading enzyme enzyme unit alive calculation formula:
Wherein: 3 * 10
-3: reaction cumulative volume (L); N: the extension rate of enzyme liquid; 10
1.: the enzymic activity conversion in the 100 μ L dilution enzyme liquid is the enzymic activity of 1mL; 10
2.: the reaction times.
Table 1 has been listed the enzyme assay result.According to measurement result, the 4 strain recons that the expression of enzymes amount is high are 7
#, 9
#, 55
#, 67
#, wherein 55
#Recon expression organic phosphorus degrading enzyme is the highest, with its called after GS-OPHC2-55, is used for the engineering strain of embodiment 4 and embodiment 5 fermentative Production reorganization organic phosphorus degrading enzyme.
The enzyme assay result of the different transformants of table 1
| Bacterial strain | Enzyme U/mL alive |
| 7 | 2.221 |
| 9 | 2.707 |
| 15 | 1.267 |
| 20 | 1.134 |
| 27 | 1.709 |
| 48 | 1.947 |
| 55 | 2.486 |
| 59 | 1.568 |
| 67 | 2.071 |
| 69 | 1.788 |
| 77 | 1.886 |
| 83 | 1.674 |
2, the PCR of the organic phosphorus degrading enzyme gene on the yeast chromosomal detects: cultivate 10mL recombination yeast 48h to be detected for 28 ℃, centrifugal collection thalline, grind into powder in liquid nitrogen, add pre-ice-cold extracting solution 0.4mL (50mmol/L Tris-HCl pH8.0,150mmol/L NaCl, 100mmol/L EDTA pH8.0), vibration mixing; Add 50 μ L10%SDS, 37 ℃ of insulation 1h; Add 75 μ L5mol/LNaCl again, mixing gently, (65 ℃ are incubated 10-20min down for 10%CTAB, 0.7mol/LNaCl) mixed solution to add 65 μ L CTAB/NaCl then, use isopyknic phenol successively: chloroform (1: 1), chloroform extracting, supernatant liquor precipitates with after 70% washing with alcohol 2 times vacuum-drying with the isopropanol precipitating of final concentration 75%, water-soluble, obtain the genome of recombination yeast.With it is template, 5 ' end primer ophc2-5-2:tacgtagccgcaccggcacaacagaag (SEQ ID NO:2), 3 ' end primer ophc2-3:tcagcggtcg ctacggatcgg (SEQ ID NO:3) carries out pcr amplification, and the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min.Amplified production is carried out agarose gel electrophoresis, and the result is presented at about 900bp place and a band occurred, conforms to insertion gene size, has confirmed that the organic phosphorus degrading enzyme gene has been incorporated into (see figure 2) on the yeast chromosomal.
[embodiment 4] 3 liters of ferment tank production reorganization organic phosphorus degrading enzyme
Choose the highest transformant GS-OPHC2-55 of organic phosphorus degrading enzyme expression amount on the shaking table level
#Come fermentative production reorganization organic phosphorus degrading enzyme as engineering strain, 3 liters of fermentor tanks of employing.
1, seed culture: picking transformant GS-OPHC2-55 at first
#Single colony inoculation in the 20mLYPD liquid nutrient medium, 28 ℃ of shaking table overnight incubation are transferred in the 200mLYPD substratum with 10% inoculum size again, 28 ℃ of shaking tables are cultivated 24h, are inoculated in 10% inoculum size then to begin fermentation in the 2L fermention medium.
2, the thalli growth stage: insert seed liquor with 10% inoculum size, 18-24h is cultivated in 30 ℃ of aeration-agitations, stream adds ammoniacal liquor to regulate pH is about 4.5-5.0 simultaneously, in culturing process along with the growth of bacterial strain, dissolved oxygen amount in the substratum will reduce gradually by 100%, dissolved oxygen amount will be increased to more than 80% once again after carbon source runs out of, and the thalline weight in wet base will reach 85g/L this moment.
3, carbon source is fed the stage: stream adds 25% glucose (comprising 12mL/L PTM1 trace salt solution), and the stream dosage is 36/h/L, and stream adds ammoniacal liquor to regulate pH is about 4.5-5.0 simultaneously, cultivates 5-6h.Adjust air flow and make dissolved oxygen amount all the time greater than 20%, the thalline weight in wet base will reach 150g/L this moment.
4, carbon source-methanol is mixed and to be fed the stage: stream adds 25% glucose: methyl alcohol (8: 1) is cultivated 4h, and stream dosage is 9mL/h/L, and stream adds ammoniacal liquor to regulate pH is 5.2-5.5 simultaneously, controls dissolved oxygen amount all the time greater than 20%.
5, the abduction delivering stage: stream adds methyl alcohol (containing 12mL/L PTM1), and the stream dosage is that per 1 hour stream of per 1 liter of fermented liquid adds 3.0mL methyl alcohol with abduction delivering, and stream adds ammoniacal liquor to regulate pH is 5.4-5.6 simultaneously, and dissolved oxygen amount is all the time greater than 20%.In inducing process, once measure the activity of the organic phosphorus degrading enzyme of expressing, and carry out the accumulation of SDS-PAGE monitoring expression amount simultaneously every the 12h sampling.
Carried out three 3L ferment tank experiments altogether, the result shows, before methyl alcohol is not induced, in strain culturing and carbon source are fed two stages, thalline increases fast, and its weight in wet base can reach 150～200g/L, and detect less than the organic phosphorus degrading enzymic activity this moment in fermented liquid supernatant.Along with the prolongation of the induction time of methyl alcohol, the organophosphorus enzymic activity in the fermented liquid increases gradually, induces that the organic phosphorus degrading enzymic activity is about 10.1U/mL (table 2) in the 120h secondary fermentation liquid.SDS-PAGE shows in the fermented liquid organic phosphorus degrading enzyme expressing quantity simultaneously also in continuous accumulation (see figure 3), induces that the expression amount of organic phosphorus degrading enzyme reaches 6 grams per liters behind the 120h.
Organic phosphorus degrading enzyme is through the methanol induction different time in 3 liters of fermentor tanks of table 2
The weight in wet base of enzymic activity and thalline
| Fermentation time (hour) | Enzyme (U/mL) alive | Thalline weight in wet base (g/L) |
| 12 | 3.452 | 152 |
| 24 | 4.278 | 176 |
| 36 | 5.966 | 196 |
| 48 | 6.365 | 210 |
| 60 | 6.945 | 223 |
| 72 | 8.369 | 225 |
| 84 | 9.014 | 231 |
| 96 | 9.249 | 260 |
| 108 | 9.46 | 269 |
| 120 | 10.146 | 273 |
[embodiment 5] 3 tons of ferment tank production reorganization organic phosphorus degrading enzyme
Choose the highest transformant GS-OPHC2-55 of organic phosphorus degrading enzyme expression amount on the shaking table level
#Come fermentative production reorganization organic phosphorus degrading enzyme as engineering strain, 3 tons of fermentor tanks of employing.The volume of substratum is 1.5 tons, and its main fermentation step is identical with embodiment 4, specifically is divided into following several stages:
1, seed culture: scrape bacterium from the inclined-plane, be seeded in the 500mL triangular flask that the 200mLYPD substratum is housed, after cultivating 20-22 hour on 28 ℃ of shaking tables, be forwarded in the 1000mL triangular flask that the 400mLYPD substratum is housed, after continuing to cultivate 20-22 hour on 28 ℃ of shaking tables, change in 0.5 ton the seeding tank that the 200L substratum is housed, inoculum size is 4%, cultivate the content that begins to measure thalline weight in wet base and glucose after 16 hours, when the content of glucose reduces to 0 (about 20 hours), the weight in wet base of thalline reaches 70-80g/L, seed can be transferred in 3 tons the fermentor tank.
2, the thalli growth stage: 29 ℃ of-30 ℃ of aeration-agitations are cultivated, and it is about 4.5-5.0 that stream adds ammoniacal liquor adjusting pH, the content of sampling and measuring thalline weight in wet base and glucose after 12 hours, at this moment the thalline weight in wet base reaches 90g/L, sugar content reduces to 0.2%, and sugar runs out of substantially, enters carbon source and feeds the stage.
3, carbon source is fed the stage: begin to add glucose (technical grade 25%), additional amount is 60L/h, adds 7 hours altogether, and stream adds ammoniacal liquor to regulate pH is about 4.5-5.0 simultaneously, and the thalline weight in wet base of this moment reaches about 140g/L, enters to mix to feed the stage.
4, the carbon source-methanol mixing is fed the stage: main purpose of this stage is to allow yeast cell adapt to inductor methyl alcohol, be generally 5-6 hour, stream adds 25% glucose 16L/h, methyl alcohol 2L/h, stream adds ammoniacal liquor to regulate pH is 5.2-5.5 simultaneously, mixing the stage of feeding and finish back thalline weight in wet base and do not have variation substantially, still is about 140g/L.
5, the abduction delivering stage: main purpose of this stage is the cell high density fermentation, simultaneously under the inducing of inductor methyl alcohol, target protein is efficiently expressed, generally to induce 120-144 hour, the stream dosage of methyl alcohol is that per 1 hour stream of per 1 ton of fermented liquid adds 3 liters of methyl alcohol with abduction delivering for the stream dosage, and stream adds ammoniacal liquor to regulate pH is 5.4-5.6 simultaneously.Along with the prolongation of the induction time of methyl alcohol, the weight in wet base of thalline constantly increases, and the organophosphorus enzymic activity in the fermented liquid also increases gradually, and the weight in wet base of following jar of preceding thalline reaches about 240g/L, and the organic phosphorus degrading enzymic activity is about 20.0U/mL in the fermented liquid.
[embodiment 6] the present invention the recombinate purifying and the zymologic property analysis of organic phosphorus degrading enzyme
It is proteic more than 90% that reorganization organic phosphorus degrading enzyme content in fermented liquid of Pichia anomala expression accounts for total secretion in the above embodiment of the present invention, only see the existence of a small amount of foreign protein by SDS-PAGE, therefore only need fermented liquid is handled through desalting and purifying, need not the reorganization organic phosphorus degrading enzyme that further processing can obtain purifying.
Concrete desalting and purifying treatment process is as follows: Tris-Cl (pH8.0) the damping fluid balance desalting column Hiprep Desalting 26/10 that at first uses 0.02mol/L, flow velocity is 5mL/min, get sample on the 2mL fermented liquid behind 2 column volumes of ready to balance, still use 2 column volumes of Tris-Cl (pH8.0) buffer solution elution of 0.02mol/L, flow velocity is 2mL/min, fraction collection, every pipe 1mL.Measure the organic phosphorus degrading enzymic activity of solution in each collection tube respectively, choose an enzyme the highest pipe alive, and verify that through SDS-PAGE the reorganization organic phosphorus degrading enzyme behind this pipe purifying is single band, the results are shown in Figure 4.Carry out the research of following zymologic property with the enzyme behind this purifying:
1, recombinate organic phosphorus degrading enzyme reaction optimum temperuture and THERMAL STABILITY of the present invention: the reorganization organic phosphorus degrading enzyme with purifying is a material, after under differing temps, carrying out enzymatic reaction, (organic phosphorus degrading activity determination method among the embodiment 2) measures its enzymic activity as mentioned above, obtains its optimal reactive temperature; Difference treat enzyme liquid under 65 ℃ and 75 ℃, the treatment time is 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, handles back survey enzymic activity under 37 ℃, with definite its thermostability.The result shows that its optimal reactive temperature is 65 ℃.Temperature is when being lower than 65 ℃, and along with temperature rising enzymic activity progressively rises, but relative enzyme work still has 23.9% in the time of 20 ℃.Be higher than 65 ℃ of enzyme activities and descend rapidly, 29.2% (see figure 5) of maximum vigor is arranged during to 80 ℃.Fig. 6 is the thermostability curve of reorganization organic phosphorus degrading enzyme, and 65 ℃ of these enzymes are very stable, are incubated after 30 minutes, and its relative enzyme work has 78.6%; 75 ℃ of down insulations 30 minutes, its enzymic activity descends to some extent, but enzyme work still has 66.8% relatively, illustrates that the present invention's organic phosphorus degrading enzyme of recombinating has good heat endurance.
2, the present invention's optimal pH and pH stability of organic phosphorus degrading enzyme reaction of recombinating: the reorganization organic phosphorus degrading enzyme with purifying is a material, carries out enzymatic reaction under different pH as mentioned above, has measured its optimal pH; Enzyme liquid after 30 minutes, is surveyed enzymic activity to determine its pH stability in 37 ℃ of insulations in the damping fluid of different pH; Measurement result shows that its optimal reaction pH is 9, but in the scope of pH5.8-10, the relative vigor of enzyme illustrates that all more than 60% it has very wide pH enzymolysis scope, sees Fig. 7.After being incubated 30 minutes under the different pH, the relative effect of vigor of enzyme is little, also reaches 51.3% even its relative enzyme of pH5 is lived, and stablize (see figure 8) more under alkaline condition.
[embodiment 7] the present invention degradation experiment of organic phosphorus degrading enzyme of recombinating to different organophosphorus pesticides
The embodiment of the invention 6 purified reorganization organic phosphorus degrading enzymes are used to the different organophosphorus pesticide of degrading, and experimental result sees Table 3.
Table 3 recombinase is to the degradation experiment of different organophosphorus pesticides
| Pesticide name | Handle the agricultural chemicals detected level (mg/kg) of sample without organic phosphorus degrading enzyme | Agricultural chemicals detected level (mg/kg) after the present invention recombinates organic phosphorus degrading enzyme processing sample | Degradation rate (%) |
| Parathion-methyl | 19.35 | Do not detect | 100 |
| Omethoate | 30.75 | 1.26 | 95.9 |
| Sumithion | 2.15 | 0.29 | 86.5 |
| Thiophos | 13.8 | 4.3 | 68.8 |
| SD-1750 | 36.3 | 11.45 | 68.5 |
| Volaton | 60.8 | 22.65 | 62.7 |
| The Malathion | 11.4 | 4.9 | 57.0 |
| Tiguvon | 10.2 | 5.6 | 45.1 |
| Chlorpyrifos 94 | 24.0 | 14.0 | 41.7 |
Annotate: the enzyme reaction system is: and the 1.9mL damping fluid (50mM Tris-HCl, pH8.0)+0.1mL organic phosphorus degrading enzyme liquid (0.5U)+5 μ L agricultural chemicals.Temperature of reaction: 37 ℃; Reaction times: 20 minutes.
Experimental result shows that the present invention recombinates organic phosphorus degrading enzyme to the tangible degradation effect of multiple organophosphorus pesticide tool.
Sequence table .txt
SEQUENCE LISTING
＜110〉Scientia Agricultura Sinica research institute biotechnology research institute
＜120〉preparation method of reorganization organic phosphorus degrading enzyme gene, its expression vector and reorganization organic phosphorus degrading enzyme
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Claims (8)
1. the gene of the coding organic phosphorus degrading enzyme of a transformation is characterized in that having the nucleotide sequence shown in the SEQ ID NO:1.
2. the recombinant yeast expression vector that contains the gene of claim 1.
3. according to the recombinant yeast expression vector of claim 2, it is characterized in that described recombinant yeast expression vector is pPIC9-ophc2-m.
4. recombinant yeast expression vector transformed yeast cells with claim 2 or 3.
5. according to the yeast cell of claim 4, it is characterized in that described yeast cell is pichia spp cell (Pichic pastoris) GS115.
6. method for preparing the organic phosphorus degrading enzyme of recombinating may further comprise the steps:
Cultivate the recombinant yeast cell that recombinant yeast expression vector transformed, induce the expression of reorganization organic phosphorus degrading enzyme, reclaim and the expressed reorganization organic phosphorus degrading enzyme of purifying with claim 2 or 3.
7. according to the method for claim 6, it is characterized in that described recombinant yeast expression vector is pPIC9-ophc2-m, and the host cell of described recombinant yeast cell is pichia spp cell (Pichicpastoris) GS115.
8. the method for a yeast cell large scale fermentation production reorganization organic phosphorus degrading enzyme that utilizes claim 4 or 5, form by seed culture, thalli growth stage, carbon source cultivation stage, carbon source-methanol mixed culture stage and five steps of methanol induction expression phase, it is characterized in that: when the methanol induction expression phase, per 1 hour stream adds methyl alcohol that 3.0ml contains 12ml/lPTM1 to induce the expression of reorganization organic phosphorus degrading enzyme in per 1 liter of fermented liquid, and the inductive time is 120-144 hour; The pH value that stream adds ammoniacal liquor adjusting fermented liquid is 5.4-5.6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053037A2 (en) * | 1998-04-15 | 1999-10-21 | Prodigene, Inc. | Optimized nucleotide sequence encoding organophosphorous hydrolase and methods of use for same |
| RU2232807C1 (en) * | 2002-11-26 | 2004-07-20 | Химический факультет МГУ им. М.В. Ломоносова | Recombinant plasmid dna ptrcte-oph and producer of enzyme organophosphate hydrolase |
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2005
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053037A2 (en) * | 1998-04-15 | 1999-10-21 | Prodigene, Inc. | Optimized nucleotide sequence encoding organophosphorous hydrolase and methods of use for same |
| RU2232807C1 (en) * | 2002-11-26 | 2004-07-20 | Химический факультет МГУ им. М.В. Ломоносова | Recombinant plasmid dna ptrcte-oph and producer of enzyme organophosphate hydrolase |
Non-Patent Citations (3)
| Title |
|---|
| 一种新的有机磷降解酶基因ophc2的克隆与表达 邓敏捷等，科学通报，第49卷第11期 2004 * |
| 一种新的有机磷降解酶基因ophc2的克隆与表达 邓敏捷等，科学通报，第49卷第11期 2004;有机磷农药降解酶及其基因工程研究进展 伍宁丰等，生物技术通报，第5期 2003 * |
| 有机磷农药降解酶及其基因工程研究进展 伍宁丰等，生物技术通报，第5期 2003 * |
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