CN1330760C - Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process - Google Patents

Abstract

The invention discloses an artificially synthesized organophosphate degrading enzyme gene suitable for expression in eukaryotic cells. The artificial gene has the nucleotide sequence shown in SEQ ID NO:1. The gene of the present invention is prepared by molecular transformation and modification of the original organophosphate degrading enzyme gene, and then artificially synthesized. Compared with the original gene, the modified recombinant gene has changed 154 bases in total, involving 135 amino acids, G The +C content changed from 62.89% to 50.27%, suitable for high-efficiency expression in eukaryotic cells. The invention also provides a recombinant expression vector containing the above gene, a host cell containing the recombinant expression vector and a method for preparing the recombinant organophosphate degrading enzyme.

Description

Recombinant organophosphate degrading enzyme gene, its expression vector and preparation method of recombinant organophosphate degrading enzyme

technical field

The present invention relates to an artificial gene, in particular to an artificially synthesized gene encoding an organophosphate degrading enzyme, a recombinant yeast expression vector containing the gene, a host cell transformed by the recombinant yeast expression vector, and preparation of the recombinant organophosphate degrading enzyme The method belongs to the field of genetic engineering.

Background technique

According to the China Statistical Yearbook, the annual output of chemical pesticides in my country is about 1 million tons, ranking second in the world. Among them, the output of highly toxic pesticides ranks first among pesticides in my country, accounting for 70% of the total output of pesticides. my country is also a big country in the use of pesticides. The area of chemical control is about 4.4 billion mu per year. For vegetables alone, 48 million tons of losses caused by pests and diseases can be recovered. Including grain, cotton, and fruits, the total value recovered is about 50 billion yuan. . However, the problem of pesticide residues is ubiquitous. At the same time, most of the pesticides are highly toxic organophosphorus pesticides, but the utilization rate of pesticides is only 10-20%, and their residues enter the soil and water bodies, seriously destroying the farmland ecosystem, endangering people's health, and directly affecting The sale of agricultural products and export earnings.

Taking tea as an example, my country exports about 200,000 tons of tea every year, ranking second in the world, with a trade value of about US$400 million, accounting for 1/3 of my country's total tea production. The EU is an important market for my country's tea exports, with a trade volume of US$70 million. However, in the past two years, the number of items stipulated by the European Union on the limits of pesticide residues in tea has been increasing, and the limits have been greatly reduced, which has seriously hindered the export of Chinese tea to Europe.

my country is a big agricultural country, and the development of green agricultural products is the direction of agricultural development in the 21st century. The trade barriers to my country's agricultural products in the international market no longer exist, but technical barriers, especially pesticide residues, have become the main limiting factors. Solving the pesticide residue pollution of agricultural products can not only improve the quality of agricultural products in our country, meet people's demand for safe agricultural products, but also help earn foreign exchange through export and increase farmers' income. Therefore, how to remove pesticide residues has become an urgent problem to be solved.

Organophosphorus pesticides are the main category of pesticides and are essential for agricultural production. However, organophosphorus pesticides have the function of inhibiting human acetylcholinesterase, and have different degrees of toxicity to humans. With the improvement of people's quality of life and the strengthening of environmental protection awareness, the residual toxicity of organophosphorus pesticides has attracted more and more attention.

Since 1973, Sethunarhan and Yoshida (Sethunathan, N., and T. Yoshida. AFlavobacterium that degrades diazinon and parathion. Can. J. Microbiol. 1973.19: 873-875) isolated the first strain from soil exposed to organophosphorus pesticides Since Flavobacterium with diazinon and parathion degrading activity, people have continuously found that some soil microorganisms (bacteria, fungi) can degrade organophosphorus pesticides, mainly because it can secrete an enzyme to degrade organophosphate, and at the same time degrade The final product can be used as carbon, nitrogen, and phosphorus sources for microbial growth, providing nutrients for its growth [(Chaudhry, G.R., A.N.Ali, and W.B.Wheeler. Isolation of a methyl parathion-degrading Pseudomonas 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, Zhou Renchao et al.: Study on the degradation of organophosphorus pesticide (methamidophos) by Aspergillus M-2. Microbiology Bulletin, 1999, 26(1): 27-30)].

Organophosphorus usually contains three phosphoester bonds, so it is often called a phosphate triester. Generally, organic phosphorus is divided into two types, one is that phosphorus is combined with oxygen through a double bond (P=O), such as methamidophos, omethoate, dichlorvos, etc.; the other is that phosphorus is combined with sulfur through a double bond (P =S), such as parathion, methyl parathion, phoxim, isocarbophos, chlorpyrifos (Lesben), etc. Studies have shown that if a phosphate bond in organic phosphorus is hydrolyzed, its toxicity will be greatly reduced. Taking parathion as an example, it will reduce its toxicity by 100 times (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 cholinesterase inhibitors using parathion hydrolase. U.S. patent. December 1996.5, 589, 386). Therefore, breaking the phosphoester bonds of organophosphorus is an effective method to reduce the toxicity of organophosphorus pesticides.

Organophosphate pesticide degrading enzyme (Organophosphorus acid hydrolase, EC3.1.8.1) can degrade organophosphorus pesticide molecules, destroy the phosphate bond of organophosphorus and detoxify it. Since various organophosphorus pesticides have similar structures but different substituents, one organophosphorus pesticide degrading enzyme can often degrade many organophosphorus pesticides. Organophosphorus pesticide degrading enzymes have been recognized as the most potential new method for eliminating pesticide residues (Yu Yunlong et al., 1996, Research Status and Development Strategies of Microbial Degradation of Pesticides, Progress in Environmental Science, 1996, Vol.4, No. 3, 28-36). The research on molecular biology of organophosphorus pesticide degrading enzymes continues to deepen along with people's understanding of pesticide residues. DiSioudi et al. (DiSioudi, B., Miller, C., Lai, K., Grimsley, J. and Wild, J. Rational design of organophosphorushhydrolase 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 its The structure of the coding gene has been modified, and the substrate specificity has been changed, so that it can have high degradation efficiency for various organophosphorus pesticides. At the same time, people also cloned the pesticide degrading enzyme gene into Escherichia coli and expressed it on the cell surface to 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 organophosphorushhydrolase for selective screening of improved hydrolysis of organophosphate nerve agents.Appl.En80.Microbi2 (4): 2026-2030].

Wu Ningfeng et al. isolated a bacterium that efficiently degrades organophosphorus pesticides from pesticide-contaminated soil, and was identified as Pseudomonas pseudoalcaligenes (this bacterium is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee. , the preservation number is CGMCC1150), and at the same time, the organic phosphorus degrading enzyme OPHC2 produced by this bacterium was isolated and purified and its enzymatic properties were studied [Wu Ningfeng, Deng Minjie, et al: Isolation, purification and characterization of a new organphosphorus hydrolase OPHC2. Chinese Science Bulletin, 2004, 49 (3): 268-272], and cloned the coding gene of organophosphate degrading enzyme [WuNingfeng, Deng Minjie, et al: Cloning and expression of ophc2, a neworganphosphorus hydrolase gene. Chinese Science Bulletin, 2004, 49(12): 1245-1249] (the nucleotide sequence of the coding gene has been registered in GenBank, the accession number is AJ605330).

However, the expression level of organophosphate degrading enzymes in the original bacteria and the recombinant prokaryotic expression system E. coli is not high. The expression level in the original bacteria is about 0.2 U/mL fermentation broth, and the induced expression in E. coli is only 2 U/mL. The purpose of cheap mass production cannot be achieved. The use of yeast expression system allows the insertion of foreign genes into the carrier containing signal peptide, so that the expressed foreign protein can be secreted into the supernatant of the culture medium, and the enzyme protein can be obtained without breaking the wall, which reduces the loss of the enzyme protein The post-processing technology is especially beneficial to the large-scale production of enzyme proteins. Therefore, it is very necessary to carry out molecular transformation and modification of the original organophosphate degrading enzyme gene, so that the modified organophosphate degrading enzyme gene can efficiently express recombinant organophosphate degrading enzyme in yeast cells, and provide industrialized large-scale and low-cost production of recombinant organophosphate degrading enzymes. Enzymes carve out a new pathway.

Contents of the invention

The first technical problem to be solved by the present invention is to overcome the defect of low expression efficiency of the original organophosphate degrading enzyme gene (the gene's accession number in GenBank is AJ605330), carry out molecular transformation and modification of the original organophosphate degrading enzyme gene, and obtain a An artificial gene capable of highly expressing organophosphate degrading enzymes in yeast cells.

The technical problem to be solved in the present invention is achieved by the following technical approaches:

An artificial nucleotide sequence encoding an organophosphate degrading enzyme has the nucleotide sequence shown in SEQ ID NO: 1, and the artificial nucleotide sequence can efficiently express the recombinant organophosphate degrading enzyme in yeast cells.

Cloning of foreign genes into host cells for expression will be affected by many factors, including promoter efficiency, 5' non-coding region sequence, effective termination of transcription, stability of foreign proteins, and foreign structural genes themselves. The present invention comprehensively affects the latest research progress in molecular biology of high-efficiency expression of genes, and molecularly transforms the organophosphate degrading enzyme gene derived from prokaryotic microorganisms, so that it can be used in eukaryotic expression systems, especially in yeast expression systems. Express. The present invention carries out molecular modification to the original organophosphate degrading enzyme gene based on the following general principles: 1. The amino acid sequence encoded by the original lactase gene is not changed; 2. The codon is optimized according to the selection bias of the Pichia pastoris codon ( Zhao Xiang, Hawke Ke, Li Yuyang. Analysis of codon usage in Pichia pastoris, Acta Biological Engineering. 2000, 16(3): 308-311); 3. Avoid some sequences that may reduce expression. The modified nucleotide sequence is shown in SEQ ID NO: 1. Compared with the original gene, the modified organophosphate degrading enzyme gene has changed 154 bases, involving 135 amino acids, and the G+C content is determined by The original 62.89% becomes 50.27%, which is more suitable for expression in eukaryotic cells.

Another technical problem to be solved by the present invention is to construct a recombinant yeast expression vector containing the nucleotide sequence shown in SEQ ID NO.1 and to obtain recombinant yeast cells containing the recombinant yeast expression vector.

Another technical problem to be solved by the present invention is achieved through the following technical approaches:

A recombinant yeast expression vector contains the nucleotide sequence shown in SEQ ID NO.1.

The recombinant yeast expression vector of the present invention can be constructed by conventional methods in the art, that is, the nucleotide sequence shown in SEQ ID NO.1 is inserted between the suitable restriction enzyme sites of the yeast expression vector, so that the SEQ ID The nucleotide sequence represented by NO: 1 is operably linked to the yeast cell expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the nucleotide sequence shown in SEQ ID NO: 1 is inserted between SnaBI and the NotI restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence Located downstream of and regulated by the AOX1 promoter, the recombinant yeast expression vector pPIC9-ophc2-m is obtained.

The recombinant yeast expression vector constructed in the present invention can be transformed into host cells by conventional methods, and the host cells can be Pichia pastoris cells (Pichic pastoris), beer yeast cells (Saccharomyces cerevisiae) or lactic acid yeast cells (Hansenula polymorpha). As a most preferred embodiment of the present invention, it is preferred to transform the recombinant yeast expression vector pPIC9-ophc2-m into Pichia pastoris GS115 to obtain recombinant yeast cells: Pichia pastoris GS115/pPIC9 -ophc2 -m.

Another technical problem to be solved by the present invention is to provide a method for preparing organophosphate degrading enzyme.

Another technical problem to be solved by the present invention is achieved through the following technical approaches:

A method for preparing organophosphate-degrading enzymes, comprising the steps of:

The recombinant yeast cell transformed with the recombinant yeast expression vector of the present invention is cultivated, the expression of the recombinant organophosphate degrading enzyme is induced, and the expressed organophosphate degrading enzyme is recovered and purified.

In the above method for preparing organophosphate degrading enzymes, preferably, the recombinant yeast expression vector is the yeast expression vector pPIC9-ophc2-m; the recombinant yeast cell is Pichicpastoris GS115/pPIC9-ophc2- m.

The content of the recombinant organophosphate-degrading enzyme expressed by Pichia pastoris in the present invention accounts for more than 90% of the total secreted protein in the fermentation broth, and only a small amount of miscellaneous protein can be seen through SDS-PAGE, so the fermentation broth only needs to be desalted and purified. Purified recombinant organophosphate degrading enzymes were obtained without further treatment.

Another technical problem to be solved by the present invention is to provide a method for large-scale fermentation and production of recombinant organophosphate degrading enzymes using the recombinant yeast cells [Pichic pastoris (Pichic pastoris) GS115/pPIC9-ophc2-m] of the present invention.

Another technical problem to be solved by the present invention is achieved through the following technical approaches:

A method for producing recombinant organophosphate degrading enzymes by large-scale fermentation of recombinant yeast cells [Pichic pastoris (Pichic pastoris) GS115/pPIC9-ophc2-m] of the present invention, including seed cultivation, thalline growth stage, and carbon source feeding stage, carbon source-methanol mixed feeding stage, and methanol-induced expression stage. Add 3.0ml of methanol containing 12ml/l PTM1 to 1 liter of fermentation broth every 1 hour to induce the expression of recombinant organophosphate degrading enzyme, and the induction time is 120-144 hours; add ammonia water to adjust the pH of the fermentation broth to 5.4-5.6.

In summary, the present invention has successfully synthesized the artificial organophosphate degrading enzyme gene capable of efficiently expressing the recombinant organophosphate degrading enzyme in yeast cells, and successfully constructed the recombinant expression vector of the gene and screened to obtain highly expressed engineering strains ( The engineering strain is named GS-OPHC2-55), and a method for preparing a recombinant organophosphate degrading enzyme and an optimal method for producing the recombinant organophosphate degrading enzyme through industrialized large-scale fermentation have been established. The expression level of the recombinant organophosphorus degrading enzyme of the present invention is as high as 6 grams per liter in a 3-liter fermenter, and the analysis of the enzymatic properties of the recombinant enzyme after purification shows that its enzymatic properties are excellent; the degradation experiments of different organophosphorus pesticides show that its It has high enzyme activity and broad-spectrum degradation, and opens up a new way for industrialized large-scale production of recombinant organophosphate degrading enzymes.

Description of drawings

Fig. 1 Restriction identification result of yeast recombinant plasmid pPIC9-ophc2-m;

The PCR detection result of the organophosphate degrading enzyme gene on Fig. 2 yeast chromosome;

The SDS-PAGE electrophoresis pattern of recombinant organophosphate degrading enzyme protein in Fig. 3 fermented liquid;

The SDS-PAGE electrophoresis pattern of Fig. 4 recombinant organophosphorus degrading enzyme protein purification;

The influence of Fig. 5 temperature on the reaction of recombinant organophosphate degrading enzyme;

The temperature stability of Fig. 6 recombinant organophosphate degrading enzyme of the present invention;

The influence of Fig. 7 pH on the reaction of recombinant organophosphate degrading enzyme of the present invention;

Fig. 8 pH stability of the recombinant organophosphate degrading enzyme of the present invention.

The following examples will further describe the preparation method and beneficial effects of the present invention. It should be understood that these examples are for illustrative purposes only, and in no way limit the protection scope of the present invention.

Detailed ways

1. Test materials and reagents

1. Biochemical reagents, enzymes, kits:

Restriction enzymes are products of TaKaRa and BioLabs, ligases are products of Promega Company, Taq enzymes are products of Shanghai Sangon Bioengineering Company, _T4 polynucleotide kinase is products of TaKaRa Company; Bioengineering Company and T vector ligation kit were purchased from Promega Company, and acrylamide, N,N′-methylene acrylamide, amino acid-free yeast nitrogen source (YNB), biotin (Biotin), and agarose were purchased from Sigma Company. Other biochemical reagents were purchased from Sigma, Promega and Beijing Chemical Reagent Company.

2. Plasmids and strains:

Pichic pastoris expression system (including yeast recipient strain Pichic pastorisGS115, plasmid pPIC9) was purchased from Invitrogen Corporation of the United States; pBS-T vector was purchased from Beijing Tianwei Times Technology Co., Ltd.

3. Medium:

1) Complete medium YPD: yeast extract 10g/L, peptone 20g/L, glucose 20g/L (solid medium containing 1.5% agar).

2) Transformation medium RDB: Yeast Nitrogen Base W/O amino acids (YNB) 13.4g/L, glucose 20g/L, biotin 4×10 ^-4 g/L, sorbitol 186g/L, 0.05 g/L glutamic acid, 0.05g/L methionine, 0.05g/L lysine, 0.05g/L leucine, 0.05g/L isoleucine, agar powder 20g/L.

3) Selection medium MD: YNB 13.4g/L, glucose 20g/L, biotin 4×10 ^-4 g/L, agar powder 20g/L.

4) Selection medium MM: YNB 13.4g/L, methanol 5mL/L, biotin 4×10 ^-4 g/L, agar powder 20g/L.

5) Induced expression medium BMGY: yeast extract 10g/L, peptone 20g/L, yeast nitrogen source (YNB) 13.4g/L, biotin 4×10 ^-4 g/L, glycerol 10mL, pH 6.0.

6) Induced expression medium BMMY: yeast extract 10g/L, peptone 20g/L, yeast nitrogen source (YNB) 13.4g/L, biotin 4×10 ^-4 g/L, methanol 5mL/L, pH 6.0 .

7) Recombinant yeast fermentation medium: phosphoric acid 26.7mL/L, CaSO ₄ 0.93g/L, K ₂ SO4 18.2g/L, MgSO ₄ 7H ₂ O 14.9g/L, KOH 4.13g/L, glucose 50g/L .

8) Trace salt solution (PTM1) used in fermentation: copper sulfate 6.0g/L, sodium iodide 0.08g/L, manganese sulfate 3.0g/L, sodium molybdate 0.2g/L, boric acid 0.02g/L, chlorine Cobalt chloride 0.5g/L, zinc chloride 20g/L, ferrous sulfate 65g/L, biotin 0.25g/L, sulfuric acid 5mL/L.

9) LB complete medium: NaCl, 10g/L; peptone; 10g/L; yeast powder, 5g/L. Sterilize at 121°C for 20 minutes, and add 1.5% agar to the solid medium.

4. DNA sequencing and primer synthesis:

Entrust Shanghai Jikang Biotechnology Co., Ltd. for DNA sequencing; entrust Beijing Aoke Biotechnology Company to synthesize all primers for experiments.

[Example 1] Synthesis and cloning of recombinant organophosphate degrading enzyme gene

1. According to the codon selection bias of Pichia pastoris, without changing the original organophosphate degrading enzyme gene [see the gene sequence: Wu Ningfeng, Deng Minjie, et al: Cloning and expression ofophc2, a new organophosphorus hydrolase gene.Chinese Science Bulletin, 2004, 49 (12): 1245-1249] under the premise of the encoded amino acid sequence, the codon of the mature protein coding sequence of the organophosphate degrading enzyme gene was optimized.

Compared with the original gene, the modified organophosphate degrading enzyme gene has changed 154 bases, involving 135 amino acids, and the G+C content has changed from 62.89% to 50.27%, which is suitable for expression in Pichia pastoris.

Divide the modified whole gene sequence into three segments A, B, and C, and then further divide the large segment into small segments of about 50bp in size, A: C1-1-C1-6, complementary chain CR1-1-CR1- 6; B: C2-1-C2-7, complementary chain CR2-1-CR2-7; C: C3-1-C3-6, complementary chain CR3-1-CR3-6; a total of 38 oligomeric DNA fragments respectively to synthesize. The synthesis of oligomeric DNA fragments was completed by Aoke Biotechnology Company.

2. Dissolve the synthesized small DNA fragments with deionized water to make the final concentration 20pmol/L, and then phosphorylate them respectively. In a 10μl reaction system, add _3UT4 polynucleotide kinase and adenosine triphosphate (ATP , the final concentration is 0.2mmol/L), and incubated in a 37°C water bath for 30min. Then, the small DNA fragments were combined into three tubes according to groups A, B, and C respectively, kept in a water bath at 95°C for 5 minutes, and cooled naturally to room temperature.

3. In order to facilitate the cloning operation and consider the correct insertion direction of the target gene fragment on the vector, when synthesizing the three large fragments A, B, and C, the 5' end of the A fragment was sequentially designed to be digested with XhoI and SnaBI site, a HindIII restriction site was added at the 3' end; HindIII and SpeI restriction sites were added at the 5' and 3' ends of the B segment, respectively, and SpeI and NotI were added at the 5' and 3' ends of the C segment Restriction sites. Each large fragment that passed the agarose test was directly connected to the pBS-T carrier (purchased from Tianwei Times Company) treated with the corresponding restriction endonuclease, transformed into Escherichia coli JM109 by electric shock, and sent for sequencing. The sequencing work was carried out by Shanghai Jikang Biotechnology Co., Ltd. was completed.

4. Sequencing the correct large fragments and connecting them into a complete ophc2-m gene: first splicing A and B on pBS-T, then cutting out fragment C and connecting it to pBS-T with AB fragments to obtain The recombinant pBS-ophc2-m with complete genetic modification, the recombinant plasmid transformed into Escherichia coli JM109, spread on LB (containing 100 μg/mL of ampicillin) plate, selected a single colony for cultivation, extracted the cultured plasmid, carried out enzyme digestion and electrophoresis detection, It was shown that the digested band was consistent with the size of the synthesized fragment, and it was preliminarily determined that a positive recombinant clone was obtained. In order to further confirm, the sequence of the recombinant was determined, and the sequencing work was completed by Shanghai Jikang Company. The sequence determination results further confirmed that the nucleotide sequence shown in SEQ ID NO: 1 had been successfully inserted into the correct position of the pBS-T vector. in position.

[Example 2] Construction of yeast recombinant plasmid

The correctly detected recombinant plasmid pBS-ophc2-m and plasmid pPIC9 were subjected to double enzyme digestion treatment with SnaBI/NotI respectively, recovered by electrophoresis, and ligated with T ₄ DNA ligase (Promega Company). In this way, the target gene was directionally inserted between the SnaBI and NotI sites on pPIC9 by using the restriction site to form the recombinant pPIC9-ophc2-m. See Figure 1 for the restriction restriction identification of the recombinant. Thus, the target gene was cloned downstream of the AOX1 promoter, and formed a correct reading frame with the signal peptide coding sequence.

[Example 3] Transformation, detection and screening of highly expressed organophosphate degrading enzyme engineering bacteria

1. Transformation of recipient yeast: Extract a large amount of the yeast recombinant expression plasmid pPIC9-ophc2-m prepared in Example 2, take 10 μg and use a slightly excessive amount of BglII for linearization treatment, electrophoresis to detect whether the enzyme digestion is complete, and the linearized plasmid The DNA was extracted once with phenol-chloroform and chloroform respectively, precipitated with ethanol, centrifuged, discarded the supernatant, washed twice with 70% ethanol, and dissolved in sterile water. Then, mix 1-5 μg of linear DNA with 80 μL of yeast GS115 competent cells, pour it into a pre-cooled sterile electric shock cup (0.2 cm, BioRad), tap the electric shock cup to make the mixture fall into the bottom of the electric shock cup, (BioRad) set the voltage at 2.5 kV, the capacitance at 25 μF, and the resistance at 400Ω, and performed the electroconversion operation. Immediately after electroporation, add 1 mL of pre-cooled 1 mol/L sorbitol to the electroshock cup, and coat the RDB plate immediately after mixing, 200 μL per plate. Place the plates upside down in an incubator at 28-30°C, and culture until transformants appear (about 60 hours).

Pick a single colony from the transformation plate with a sterile toothpick, inoculate it on MM and MD solid medium in turn, invert the plate and place it in an incubator at 28-30°C, and cultivate until transformants appear (about 48 hours). The clones (his ⁺ , mut ^s ) that grow normally on MD plates but grow slowly or not at all on MM plates were screened. In order to screen highly expressed recombinant yeast strains, the expression of organophosphate degrading enzymes in the induction medium was directly detected. The his+mut ^s transformant was first cultured in BMGY medium, after it grew to saturation, centrifuged at 5000rpm for 4min, discarded BMGY, replaced with induction medium BMMY, after induction culture for 48h, centrifuged the culture at 10000rpm for 3min, Get supernatant and carry out organophosphate degrading enzyme activity determination according to the following method:

Take 100 μL of the enzyme solution to be tested and add it to a system containing 5 μL of 10 mg/mL methyl parathion and 900 μL of 50 mmol/L Tris-Cl (pH9.0) buffer solution, incubate at 37 ° C for 10 minutes, add 1 mL of 10% trichloroacetic acid to terminate the reaction, and then Add 1 mL of 10% Na ₂ CO ₃ solution for color development, measure the OD value at 410 nm, and calculate the content of p-nitrophenol in the hydrolyzed product and the activity of the enzyme. An enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute at 37°C.

Organophosphate degrading enzyme enzyme activity unit calculation formula:

Among them: 3×10 ^-3 : total reaction volume (L); N: dilution factor of enzyme solution; 10 ^① : convert the enzyme activity in 100 μL of diluted enzyme solution to 1 mL of enzyme activity; 10 ^② : reaction time.

Table 1 lists the enzyme activity assay results. According to the measurement results, the four recombinants with high enzyme expression were 7 ^# , 9 ^# , 55 ^# , and 67 ^# , among which the 55 ^# recombinant expressed the highest organophosphate degrading enzyme, which was named GS-OPHC2-55 and used for Engineering strains for producing recombinant organophosphate-degrading enzymes by fermentation in Examples 4 and 5.

Table 1 Enzyme activity assay results of different transformants

Strains  Enzyme activity U/mL 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. PCR detection of organophosphate-degrading enzyme genes on yeast chromosomes: Cultivate 10 mL of the recombinant yeast to be tested at 28 ° C for 48 hours, collect the bacteria by centrifugation, grind them into powder in liquid nitrogen, add 0.4 mL of pre-ice-cooled extract (50 mmol/L Tris-HCl (pH8.0, 150mmol/L NaCl, 100mmol/L EDTA (pH8.0), shake and mix; add 50μL of 10% SDS, incubate at 37℃ for 1h; then add 75μL of 5mol/L NaCl, mix gently, and then add 65μL CTAB/NaCl (10% CTAB, 0.7mol/LNaCl) mixture, keep warm at 65°C for 10-20min, extract with equal volumes of phenol:chloroform (1:1), and chloroform successively, and use the final concentration of 75% for the supernatant Precipitate with isopropanol, wash the precipitate twice with 70% ethanol, dry in vacuum, dissolve in water, and obtain the genome of recombinant yeast. Using it as a template, 5' end primer ophc2-5-2: tacgtagccgcaccggcacaacagaag (SEQ ID NO: 2), 3' end primer ophc2-3: tcagcggtcg ctacggatcgg (SEQ ID NO: 3), PCR amplification, PCR reaction conditions It is: 94°C 5min; 94°C 45sec, 55°C 45sec, 72°C 1min, 30 cycles; 72°C 10min. The amplified product was subjected to agarose gel electrophoresis, and the results showed that a band appeared at about 900 bp, which was consistent with the size of the inserted gene, confirming that the organophosphate degrading enzyme gene had been integrated on the yeast chromosome (see Figure 2).

[Example 4] 3 liters of fermentation tank fermentation production recombinant organophosphate degrading enzyme

The transformant GS-OPHC2-55 ^# with the highest expression of organophosphate degrading enzymes at the shaker level was selected as an engineering strain, and a 3-liter fermenter was used to ferment and produce recombinant organophosphate degrading enzymes.

1. Seed culture: first pick a single colony of the transformant GS-OPHC2-55 ^# and inoculate it in 20mLYPD liquid medium, culture it overnight on a shaker at 28°C, and then transfer it to 200mLYPD medium with a 10% inoculation amount, and inoculate it in 28mLYPD medium. Cultivate on a shaker at ℃ for 24 hours, then inoculate 2L fermentation medium with 10% inoculum size to start fermentation.

2. Bacterial growth stage: Inoculate the seed liquid with 10% inoculum amount, cultivate with aeration and stirring at 30°C for 18-24 hours, and at the same time add ammonia water to adjust the pH to about 4.5-5.0. During the cultivation process, as the strain grows, the culture medium The amount of dissolved oxygen in the plant will gradually decrease from 100%, and when the carbon source is exhausted, the dissolved oxygen amount will rise to more than 80% again, and the wet weight of the bacteria will reach 85g/L at this time.

3. Carbon source feeding stage: add 25% glucose (including 12mL/L PTM1 trace salt solution), the feeding amount is 36/h/L, and at the same time, add ammonia water to adjust the pH to about 4.5-5.0, and cultivate for 5-6h . Adjust the ventilation rate so that the dissolved oxygen is always greater than 20%, and the wet weight of the bacteria will reach 150g/L at this time.

4. Carbon source-methanol mixed feeding stage: add 25% glucose:methanol (8:1) to incubate for 4 hours, the feeding rate is 9mL/h/L, and at the same time, add ammonia water to adjust the pH to 5.2-5.5 to control dissolved oxygen The amount is always greater than 20%.

5. Induction expression stage: Feed methanol (containing 12mL/L PTM1), the feed amount is 3.0mL methanol per 1 liter of fermentation broth every 1 hour to induce expression, and feed ammonia water to adjust the pH to 5.4-5.6 at the same time. Oxygen is always greater than 20%. During the induction process, samples were taken every 12 hours to measure the activity of the expressed organophosphate degrading enzyme, and at the same time, SDS-PAGE was carried out to monitor the accumulation of the expression amount.

A total of three 3L fermenter fermentation experiments were carried out. The results showed that before methanol was not induced, the bacteria grew rapidly during the two stages of strain cultivation and carbon source feeding, and their wet weight could reach 150-200g/L. Organophosphate-degrading enzyme activity was not detected in the supernatant of the fermentation broth. With the prolongation of methanol induction time, the activity of organophosphorus enzymes in the fermentation broth gradually increased, and the activity of organophosphorus degrading enzymes in the fermentation broth was about 10.1 U/mL after 120 hours of induction (Table 2). SDS-PAGE also showed that the protein expression of organophosphate degrading enzymes in the fermentation broth was also continuously accumulating (see Figure 3), and the expression of organophosphate degrading enzymes reached 6 g/L after induction for 120 h.

Table 2 Organophosphorus degrading enzymes induced by methanol for different time in 3 liter fermentor

Enzyme activity and wet weight of bacteria

Fermentation time (hours) Enzyme activity (U/mL) Bacterial wet weight (g/L) 12 3.452 152 twenty four 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

[Example 5] 3 tons of fermentation tank fermentation production recombinant organophosphate degrading enzyme

The transformant GS-OPHC2-55 ^# with the highest expression of organophosphate degrading enzymes at the shaker level was selected as an engineering strain, and a 3-ton fermenter was used to ferment and produce recombinant organophosphate degrading enzymes. The volume of culture medium is 1.5 tons, and its main fermentation step is identical with embodiment 4, specifically is divided into following stages:

1. Seed culture: Scrape off the bacteria from the slope, inoculate into a 500mL Erlenmeyer flask containing 200mL of YPD medium, cultivate on a shaker at 28°C for 20-22 hours, then transfer to a 1000mL Erlenmeyer flask containing 400mL of YPD medium After continuing to cultivate on a shaker at 28°C for 20-22 hours, transfer to a 0.5-ton seed tank with 200L medium, the inoculum size is 4%, and start to measure the wet weight of the thalline and the content of glucose after 16 hours of cultivation. When the content of glucose drops to 0 (about 20 hours), the wet weight of the thalline reaches 70-80g/L, and the seeds can be transferred to a 3-ton fermenter.

2. Bacteria growth stage: 29°C-30°C aerated and stirred culture, add ammonia water to adjust the pH to about 4.5-5.0, take samples after 12 hours to measure the wet weight of the bacteria and the content of glucose, at this time the wet weight of the bacteria reaches 90g/ L, the sugar content is reduced to 0.2%, the sugar has been basically consumed, and it enters the stage of carbon source feeding.

3. Carbon source feeding stage: Add glucose (industrial grade 25%), and add 60L/h for a total of 7 hours. At the same time, add ammonia water to adjust the pH to about 4.5-5.0. When the wet weight reaches about 140g/L, it enters the stage of mixed feeding.

4. Carbon source-methanol mixed feeding stage: the main purpose of this stage is to adapt the yeast cells to the inducer methanol, usually for 5-6 hours, add 25% glucose 16L/h, methanol 2L/h, and add ammonia water at the same time Adjust the pH to 5.2-5.5, and the wet weight of the bacteria basically did not change after the mixed feeding stage was completed, which was still about 140g/L.

5. Induced expression stage: The main purpose of this stage is high-density fermentation of cells, and at the same time, the target protein is efficiently expressed under the induction of the inducer methanol. Generally, it takes 120-144 hours to induce, and the feed rate of methanol is 1 ton of fermentation broth was fed with 3 liters of methanol every 1 hour to induce expression, and ammonia water was added to adjust the pH to 5.4-5.6. With the prolongation of methanol induction time, the wet weight of the bacteria continues to increase, and the activity of organophosphorus enzymes in the fermentation broth also gradually increases. The wet weight of the bacteria before entering the tank reaches about 240g/L. The activity is about 20.0U/mL.

[Example 6] Purification and enzymatic property analysis of recombinant organophosphate degrading enzyme of the present invention

In the above-mentioned embodiments of the present invention, the recombinant organophosphate-degrading enzyme expressed by Pichia pastoris accounts for more than 90% of the total secreted protein in the fermentation broth, and only a small amount of miscellaneous protein can be seen by SDS-PAGE, so it is only necessary to pass the fermentation broth through Desalting and purification treatment, the purified recombinant organophosphate degrading enzyme can be obtained without further treatment.

The specific desalination and purification treatment method is as follows: First, equilibrate the desalting column Hiprep Desalting 26/10 with 0.02mol/L Tris-Cl (pH8.0) buffer solution, the flow rate is 5mL/min, and take 2mL of fermentation broth after equilibrating 2 column volumes Load the sample, still use 0.02mol/L Tris-Cl (pH8.0) buffer to elute for 2 column volumes, flow rate is 2mL/min, collect in portions, 1mL per tube. The organophosphate-degrading enzyme activity of the solution in each collection tube was measured respectively, and the tube with the highest enzyme activity was selected, and the purified recombinant organophosphate-degrading enzyme in this tube was verified by SDS-PAGE as a single band. The results are shown in Figure 4. With this purified enzyme, the following enzymatic properties were studied:

1. Research on the optimal temperature and thermal stability of the reaction of the recombinant organophosphate degrading enzyme of the present invention: using the purified recombinant organophosphate degrading enzyme as material, after carrying out the enzymatic reaction at different temperatures, as described above (organophosphorus in Example 2) Degrading enzyme activity assay method) to measure its enzyme activity to obtain its optimum reaction temperature; treat the enzyme solution at 65°C and 75°C respectively, and the treatment time is 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes. Enzyme activity was measured at 37°C to determine its thermal stability. The results showed that the optimum reaction temperature was 65℃. When the temperature is lower than 65°C, the enzyme activity gradually increases with the increase of temperature, but the relative enzyme activity is still 23.9% at 20°C. The enzyme activity decreased rapidly above 65°C, and reached 29.2% of the maximum activity at 80°C (see Figure 5). Figure 6 is the thermal stability curve of the recombinant organophosphate degrading enzyme. The enzyme is very stable at 65°C, and its relative enzyme activity is 78.6% after incubation for 30 minutes; The enzyme activity is still 66.8%, indicating that the recombinant organophosphate degrading enzyme of the present invention has good heat resistance.

2. The optimal pH and pH stability of the reaction of the recombinant organophosphate degrading enzyme of the present invention: using the purified recombinant organophosphate degrading enzyme as a material, the enzymatic reaction was carried out as described above at different pHs, and the optimal pH was determined; After incubating the enzyme solution in buffers with different pH at 37°C for 30 minutes, measure the enzyme activity to determine its pH stability; the measurement results show that the optimum reaction pH is 9, but within the range of pH 5.8-10 , the relative activities of the enzymes are all above 60%, indicating that they have a wide range of pH enzymolysis, as shown in Figure 7. After incubation at different pH for 30 minutes, the relative activity of the enzyme has little effect, even at pH 5, its relative activity reaches 51.3%, and it is more stable under alkaline conditions (see Figure 8).

[Example 7] Degradation experiment of different organophosphorus pesticides by recombinant organophosphorus degrading enzyme of the present invention

The recombinant organophosphorus degrading enzyme purified in Example 6 of the present invention was used to degrade different organophosphorus pesticides, and the experimental results are shown in Table 3.

Table 3 Degradation experiments of different organophosphorus pesticides by recombinant enzymes

Pesticide name Detected amount of pesticides in samples without organophosphate degrading enzyme treatment (mg/kg) Pesticide detection amount (mg/kg) after the sample is processed by recombinant organophosphate degrading enzyme of the present invention Degradation rate(%) Methyl parathion 19.35 not detected 100 Omethoate 30.75 1.26 95.9 Fenitrothion 2.15 0.29 86.5 parathion 13.8 4.3 68.8 Dichlorvos 36.3 11.45 68.5 Phoxim 60.8 22.65 62.7 Malathion 11.4 4.9 57.0 Fenthion 10.2 5.6 45.1 Chlorpyrifos 24.0 14.0 41.7

Note: The enzyme reaction system is: 1.9mL buffer solution (50mM Tris-HCl, pH8.0) + 0.1mL organophosphate degrading enzyme solution (0.5U) + 5μL pesticide. Reaction temperature: 37°C; reaction time: 20 minutes.

Experimental results show that the recombinant organophosphorus degrading enzyme of the present invention has obvious degradation effects on various organophosphorus pesticides.

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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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