CN109679933B

CN109679933B - Organophosphorus degrading enzyme OPHC2 mutant and application thereof

Info

Publication number: CN109679933B
Application number: CN201811625972.4A
Authority: CN
Inventors: 李光雷; 李立功
Original assignee: Beijing Schengenbiya Bioengineering Technology Co ltd
Current assignee: Beijing Schengenbiya Bioengineering Technology Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-09-15
Anticipated expiration: 2038-12-28
Also published as: CN109679933A

Abstract

The invention discloses an organophosphorus degrading enzyme OPHC2 mutant and application thereof. The invention discloses an organophosphorus degrading enzyme mutant, wherein the 226 th amino acid of wild organophosphorus degrading enzyme is mutated into histidine from leucine or the 239 th amino acid is mutated into isoleucine to obtain the mutant; or mutant obtained by simultaneously mutating leucine to histidine at amino acid 226 and phenylalanine to isoleucine at amino acid 239 of wild organophosphorus degrading enzyme. The invention further discloses application of the organophosphorus degrading enzyme mutant in degrading pyrethroid pesticides. According to the invention, through carrying out site-specific mutagenesis on the organophosphorus degrading enzyme, the obtained mutant has obvious improvement on thermal stability and pH stability, the catalytic efficiency of the pyrethroid pesticide is greatly improved, and the degradation effect of the organophosphorus degrading enzyme on the pyrethroid pesticide is obviously improved.

Description

Organophosphorus degrading enzyme OPHC2 mutant and application thereof

Technical Field

The invention relates to an organophosphorus degrading enzyme OPHC2 mutant, further relates to application of the organophosphorus degrading enzyme OPHC2 mutant in degrading pyrethroid pesticides, and belongs to the field of genetic engineering modification of organophosphorus degrading enzyme OPHC 2.

Background

The pesticide is a special chemical, on one hand, the pesticide plays an important role in the aspects of insect damage prevention, labor intensity reduction, new species culture and the like, and brings great economic benefit to human beings; on the other hand, the utilization rate of the pesticide is low, according to the report of the literature, the amount of the pesticide really acting on poisoning and killing the plant diseases and insect pests only accounts for 10% -20% of the application amount, most of the rest pesticide is remained on the surface of the plant or permeates into soil and water to cause serious pollution to the soil and water, harm is brought to the environment where people live, and meanwhile, the pesticide remained on agricultural products directly influences the health of human beings.

The organophosphorus pesticide has the characteristics of high efficiency, low residue, low use cost and the like, is a widely used pesticide type in agriculture, and is widely popularized all over the world. Organophosphorus pesticides once occupied more than half of the insecticide market. The organophosphorus pesticide is mainly used for preventing and treating plant diseases, insects and pests, is various in varieties, high in pesticide effect, wide in application, easy to decompose, generally does not accumulate in human bodies and livestock bodies, and is an extremely important compound in the pesticide; however, there are many varieties which have strong acute toxicity to human and livestock. From 2005, China has taken measures to eliminate and limit the production, sale and use of toxic and highly toxic organophosphorus pesticides, which causes the yield and use of organophosphorus pesticides to decline year by year.

In recent years, with the development of science and technology, some biomimetic pesticides such as pyrethroid pesticides, carbamate pesticides, neonicotinoid pesticides and the like have been rapidly increasing. The annual sales of pyrethroid insecticides is in the range of $ 13 to $ 14 billion, accounting for about 20% of the world's sales, and the use of pyrethroid insecticides is increasing further and has been developed as the second largest class of agricultural insecticide in the world. However, with the increasing call for environmental protection and human health concerns, the problems associated with the use of pyrethroid pesticides are gaining increasing attention. It is known that human beings exposed to pyrethroid insecticides have acute symptoms of dyspnea, cough, bronchospasm, nausea and vomiting, headache, etc., and also have skin allergy. Although the long-term effects of exposure to pyrethroid insecticides are not certain, studies have shown that pyrethroid insecticides are neurotoxins to which exposure of neonates and adults may produce developmental neurotoxicity, reproductive toxicity and immune system toxicity.

In order to not only give full play to the efficient protection effect of pesticides, but also solve the problem of pesticide residue, various researches are carried out at home and abroad, and various solving approaches such as chemical methods, physical methods, biological methods and the like are provided. In this respect, biological methods have the advantage of being non-toxic, residue-free, and free of secondary pollution. In particular, the microbial enzyme method plays a unique role in solving the pesticide residue. The method of using biological enzyme to eliminate the residue of pyrethroid pesticide has been successful. By studying the chemical structure and degradation pathway of pyrethroid pesticides, it is recognized that hydrolysis of ester bonds is a key step in degrading pyrethroid pesticides. Enzymes such as carboxylesterase, cytochrome C oxidase (P450s) and alcohol oxidase existing in the body of mammals can rapidly degrade pyrethroid pesticides containing ester bonds. Leersic reported pyrethroid insecticide hydrolase gene, the degradation effect of hydrolase expressed by the gene on 100pmm of various pyrethrins is more than 93% (Chinese patent CN 101429515B); liuyuhuan reports the application of esterase gene est816 and recombinant esterase thereof in the aspect of degrading pyrethroid pesticides, wherein the degradation rates of the recombinant esterase to cyhalothrin, cypermethrin, deltamethrin and deltamethrin are respectively 92.7%, 94.62%, 91.70% and 90.60% (Chinese patent CN 107058362A).

The organophosphorus degrading enzyme OPHC2 is an enzyme produced in a high-efficiency strain C2-1 for degrading organophosphorus pesticide, which is separated from polluted soil of a pesticide factory; the strain is gram-negative bacteria, the thallus is in a short rod shape, the size of the thallus is 0.8 mu m multiplied by 1.5 to 3.0 mu m, 1 to 3 polar flagella generally exist, no spores exist, and an oxidase experiment is positive and strictly aerobic. The bacillus can grow on an inorganic salt culture medium added with organophosphorus pesticide, and counteracts the pesticide to provide a carbon source for the growth of the bacillus. Identified by the national strain preservation detection center of the institute of microbiology of the Chinese academy of sciences, C2-1 is pseudoalcaligenes, and the enzyme which can degrade organophosphorus pesticides and is generated by the strain is organophosphorus degrading enzyme OPHC 2.

The wild organophosphorus degrading enzyme OPHC2 is not ideal in catalytic efficiency of degrading pyrethroid pesticides, and in addition, the wild organophosphorus degrading enzyme OPHC2 also has the problems of poor thermal stability, poor pH stability and the like, and the popularization and application of the organophosphorus degrading enzyme OPHC2 in practice are limited by the problems.

Disclosure of Invention

The invention aims to solve the first technical problem of providing an organophosphorus degrading enzyme OPHC2 mutant, wherein the catalytic performance, thermal stability and pH stability of the organophosphorus degrading enzyme OPHC2 on pyrethroid pesticides are obviously improved by carrying out site-directed mutagenesis on the organophosphorus degrading enzyme OPHC2, so that the degradation effect of the organophosphorus degrading enzyme OPHC2 on the pyrethroid pesticides is improved;

the invention aims to solve the second technical problem of providing the application of the organophosphorus degrading enzyme OPHC2 mutant in degrading pyrethroid pesticides.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention discloses an organophosphorus degrading enzyme OPHC2 mutant, wherein the 226 th amino acid of wild organophosphorus degrading enzyme OPHC2 (the nucleotide sequence of the coding gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2) is mutated from leucine (Leu) to histidine (His) to obtain the mutant (L226H).

The invention also discloses an organophosphorus degrading enzyme OPHC2 mutant, which is obtained by mutating the 239 th amino acid of wild organophosphorus degrading enzyme OPHC2 (the nucleotide sequence of the coding gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2) from phenylalanine (Phe) to isoleucine (Ile) (F239I); the invention further optimizes the codon of the coding gene of the mutant amino acid sequence, and the optimized nucleotide sequence is shown in SEQ ID NO. 4.

The invention further discloses an organophosphorus degrading enzyme OPHC2 mutant, which is a double-site mutant (L226HF239I) obtained by mutating the 226 th amino acid of wild organophosphorus degrading enzyme OPHC2 from leucine (Leu) to histidine (His) and mutating the 239 th amino acid from phenylalanine (Phe) to isoleucine (Ile); the codon of the coding gene of the mutant amino acid sequence is optimized, and the optimized nucleotide sequence is shown as SEQ ID NO. 5.

The invention further discloses a recombinant expression vector containing the coding gene of the organophosphorus degrading enzyme OPHC2 mutant and a recombinant host cell containing the recombinant expression vector.

The invention also discloses application of the organophosphorus degrading enzyme OPHC2 mutant in degrading pyrethroid pesticides. Wherein the pyrethroid pesticide includes but is not limited to: cypermethrin, beta-cypermethrin, lambda-cyhalothrin or beta-cyfluthrin.

The invention respectively constructs high-expression single-point and double-point mutated OPHC2 pichia pastoris recombinant expression strains. The determination result of the enzymatic kinetic constants shows that the catalytic efficiency of each modified protein of OPHC2 is higher than that of the wild-type protein, the catalytic efficiency of the single-point modified enzymes L226H, F239I and the double-mutation modified enzyme L226HF239I is 64 times, 110 times and 70 times of that of the wild-type OPHC2 respectively, and the catalytic efficiency of the single-point modified enzymes to carboxylic ester compounds is improved to a great extent.

The thermal stability detection result shows that more than 75% of enzyme activity remains after the double mutant enzyme L226HF239I is stored for 5 hours at 40 ℃, more than 60% of enzyme activity remains after 5 hours of heat preservation at 60 ℃, and more than 30% of enzyme activity remains after 3 hours of heat preservation at 70 ℃, which shows that the OPHC2 mutant enzyme after double mutation modification has better thermal stability. The detection result of the pH stability shows that the double-process modified enzyme L226HF239I has better pH stability, is preserved for 2 hours within the range of 5.5-10.5, and has the enzyme activity maintenance rate of more than 70%.

The detection result of the mutant enzyme on the degradation effect of the pyrethroid pesticide shows that the mutant enzyme L226HF239I has high degradation rate on the beta-cypermethrin and the beta-cyhalothrin, and the degradation rate is 100 percent; the degradation rate of the beta-cyfluthrin is 45.6 percent.

The invention also discloses a method for preparing the organophosphorus degrading enzyme OPHC2 mutant, which comprises the following steps: (1) cloning the gene for encoding the organophosphorus degrading enzyme OPHC2 mutant to a yeast expression plasmid to construct a yeast expression recombinant plasmid; (2) linearizing the yeast expression recombinant plasmid, and transforming yeast competent cells to obtain a recombinant yeast strain; (3) and (3) carrying out high-density fermentation culture on the recombinant yeast strain, carrying out induced expression on corresponding protein, and separating and purifying to obtain the recombinant yeast strain. Wherein, the yeast competent cell in the step (2) is a pichia pastoris KM71H competent cell; the inoculation amount of the recombinant yeast strain in the step (3) is 8-10%; the temperature of the whole fermentation process is 30 ℃; in the high-density fermentation process, the pH value of the thallus growth stage is 5.0, and the pH value after the induction period is 5.2-5.5.

The invention carries out an OPHC2 mutant enzyme 30L pichia pastoris high-density fermentation experiment, searches fermentation process parameters, determines the selected inoculum size to be 8-10%, sets the temperature to be 30 ℃ in the whole fermentation process, sets the pH value to be 5.0 in the thallus growth stage, sets the pH value to be 5.2-5.5 after the induction period, and adjusts the rotating speed and the ventilation volume according to the DO value and the wet weight condition of the thallus. The invention carries out 30L fermentation experiment of KM71H/pPICZaA-ophc2L226HF239I, the induction starts from about 24 hours of culture on a tank, the change range of the wet weight of thalli is smaller along with mixed feeding and continuous liquid supplement of the induction, the speed of Muts type methanol metabolism is slower, but the enzyme activity is continuously increased along with the prolonging of the induction time, and the enzyme activity reaches 143.75U/mL during 70 hours of fermentation.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention carries out site-directed mutagenesis on the organophosphorus degrading enzyme OPHC2 based on a genetic engineering means, improves the catalytic performance of the organophosphorus degrading enzyme OPHC2 on the pyrethroid pesticide, and further improves the degradation effect of the organophosphorus degrading enzyme OPHC2 on the pyrethroid pesticide. The modified enzyme of the invention obviously improves the catalytic performance of OPHC2 on aromatic ester compounds, can be used for degrading pyrethroid pesticides, widens the application field and action efficiency of OPHC2, and has important application prospects in the fields of pesticide residue elimination in food, water treatment, soil remediation and the like.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic A-cidRes.19:5081 (1991); Ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Cassol et al (1992); Rossolini et al, mol cell. Probes8:91-98 (1994)).

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell.

Drawings

FIG. 1 shows an alignment of OPHC2 with the key amino acids (214-244) of its homologous protein;

FIG. 2 is a SDS-PAGE electrophoresis of the shaking table level protein expression; wherein, 1: protein Marker, 2-7: the L226HF239I recombinant was induced for 48h in the supernatant; 8-13: the ophc2 recombinant was induced for 48h in the supernatant;

FIG. 3 is a SDS-PAGE electrophoresis of the horizontal protein expression by shaking table; wherein, 1-3: protein expression conditions of L226H 24h, 48h and 72h respectively; 4-6: the protein expression conditions of F239I 24h, 48h and 72h are respectively;

FIG. 4 shows the results of temperature stability determination of OPHC2 double mutant enzyme (L226HF 239I);

FIG. 5 shows the results of pH stability assay of OPHC2 double mutant enzyme (L226HF 239I);

FIG. 6 shows the measurement of the degradation effect of OPHC2 double mutant enzyme (L226HF239I) on cypermethrin and lambda-cyhalothrin;

FIG. 7 is a graph showing the relationship between the enzyme activity of OPHC2 double mutant enzyme (L226HF239I) and the fermentation time.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 detection of site-directed mutagenesis and mutants of organophosphorus degrading enzyme OPHC2

1. Experimental methods

1.1 determination of the site of mutation in OPHC2

The protein with homology to OPHC2 was found from NCBI's protein database using BLAST method, and the larger side chain ester substrate of OPHC2 was aligned with pocket amino acids using on-line protein alignment program COBALT on the web to find out the amino acids to be pre-mutated.

1.2 codon optimization and Gene Synthesis of mutant genes

And (3) removing an OPHC 272 bp signal peptide sequence by referring to a pichia pastoris codon optimization table, and carrying out codon optimization, wherein the optimized gene sequence is shown in a sequence table.

EcoRI and NotI restriction sites were added to the 5 'and 3' optimized OPHC2 mutant genes, respectively, to carry out gene synthesis.

1.3 construction of Yeast expression recombinant plasmid

The synthesized recombinant plasmids, pUC57-ophc2, pUC57-ophc2L226H (ophc2F239I/ophc2L226HF239I) and the vector pPICZaA were subjected to double digestion with EcoRI/NotI, recovered by electrophoresis, and ligated with T4DNA ligase, respectively. Thus, the desired gene was inserted between EcoRI and NotI sites on pPICZaA by using the site of enzymatic cleavage to form the recombinants pPICZaa-ophc2, pPICZaA-ophc2L226H, pPICZaA-ophc2F239I and pPICZaA-ophc2L226HF 239I.

1.4 transformation of Pichia pastoris by electric shock

(1) Linearization of recombinant plasmids

The recombinant expression plasmids pPICZaa-ophc2, pPICZaA-ophc2L226H, pPICZaA-ophc2F239I and pPICZaA-ophc2L226HF239I are extracted in a large amount. Respectively carrying out linearization treatment by PmeI, and detecting whether the enzyme digestion is complete by electrophoresis. And recovering the enzyme digestion fragments by using the PCR recovery kit.

(2) Preparation of Yeast competence

Firstly, selecting a single colony of Pichia pastoris KM71H, inoculating the single colony into a 100mL triangular flask containing 20mL YPD liquid culture medium, and culturing by shaking overnight at 30 ℃.

② overnight cultures were inoculated 1/20 into 300mL Erlenmeyer flasks containing 100mL YPD liquid Medium and grown to OD₆₀₀＝1.2。

③ centrifuging at 4 ℃ and 5000rpm for 5min, and collecting the thalli.

Fourthly, the precipitate is gently resuspended by using the ice precooled deionized water with the same volume, the centrifugation is carried out for 5min at the temperature of 4 ℃ and the speed of 5000rpm, and the thalli are collected.

And fifthly, gently resuspending the precipitate by using half volume of ice-precooled deionized water, centrifuging the precipitate at 4 ℃ and 5000rpm for 5min, and collecting thalli.

Sixthly, gently suspending and precipitating by using 1mol/L sorbitol precooled by ice with the volume of 1/10, centrifuging for 5min at the temperature of 4 ℃ and the rpm of 5000, and collecting thalli.

Seventhly, using 200 mu L of precooled 1mol/L sorbitol to gently resuspend the sediment, and packaging 80 mu L of each tube into ready-to-use pichia pastoris competent cells which are used as they are.

(3) Transformation of Pichia pastoris

After mixing 10. mu.g of linear DNA with 80. mu.L of yeast competent cells, the mixture was poured into a pre-cooled sterile cuvette (0.1cm, BioRad), and the cuvette was tapped to allow the mixture to fall to the bottom of the cuvette for electrotransformation.

After electrotransformation, L mL of precooled 1mol/L sorbitol is immediately added into an electric shocking cup, the transformation solution is taken out and placed into a 1.5mL centrifuge tube, incubated for 2h at 30 ℃, then coated on a 100 mu g/mL zeocin YPDS plate, and cultured at 30 ℃ until transformants appear, and the positive recon is obtained.

1.5 screening and identification of recombinants

(1) Positive recombinants induced expression

Picking single colony from the transformation plate, and respectively placing the single colony in BMGY culture medium for culturing for 48 h.

② centrifuging for 4min at 5000rpm, abandoning BMGY, changing into an induction culture medium BMMY, and carrying out induction culture for 48 h.

③ the culture is centrifuged at 12000rpm for 5min, and the supernatant is taken.

(2) Enzyme activity assay

Chrysanthemum esterase enzyme activity definition: the p-nitrophenol acetate generates yellow p-nitrophenol under the hydrolysis action of esterase. Measuring the generated amount of p-nitrophenol at 405nm by using a spectrophotometer, namely measuring the esterase activity. 1 unit of esterase has an activity (U) of pH8.0 and the amount of enzyme required to catalyze the formation of 1. mu. mol of p-nitrophenol per minute at 40 ℃.

Adding 100 μ L of diluted enzyme solution into 985 μ L of 50mmol/L Tris-Cl (pH8.0) buffer solution, adding 15 μ L of 50mg/mL p-nitrophenol acetate substrate solution, reacting at 40 deg.C for 3min, adding ice purified water to suspend the reaction, measuring OD405, and calculating enzyme activity according to standard curve formula.

(3) SDS-PAGE detection of Chrysanthemum esterase expression

Taking 20 μ L of crude enzyme solution, adding 5xSDS, PAGE sample buffer solution, boiling water bathing for 10min, taking out the prepared SDS-PAGE gel plate, adding 1 xprotein electrophoresis buffer solution, performing 80V electrophoresis for 30min, increasing voltage to 120V, performing electrophoresis for 1h, stopping, staining and decolorizing. And (5) observing an electrophoretogram, taking a picture and recording.

1.6 determination of the kinetic constants of the enzymology

Preparing 6 reaction systems by using p-nitrophenol acetate as a substrate, sequentially adding diluted enzyme liquid by using the same tube, measuring a light absorption value at 405nm, calculating enzyme activity, and then calculating a ratio of the enzyme activity to reaction time to determine Km and Vmax.

The enzyme activity was measured at 40 ℃ using p-nitrophenol acetate of various concentrations as a substrate in a 50mmol/L Tris-HCl buffer system (pH8.0), and the reaction rate of the enzyme was calculated. Km and Vmax are obtained according to a double reciprocal mapping method (Lineweaver-Burk method).

1.7 testing of thermal and pH stability

Taking an equal amount of OPHC2 mutant enzyme liquid, placing in a pH8.0Tris-HCl buffer solution system, placing in constant-temperature water bath at 40 ℃, 60 ℃ and 70 ℃ for 5h, sampling every 1h, rapidly cooling to room temperature, and determining residual enzyme activity at different temperatures according to an enzyme activity determination method, wherein the enzyme activity of the initial enzyme liquid is 100%.

And (3) taking an equal amount of OPHC2 mutant enzyme liquid, respectively placing the same amount of OPHC2 mutant enzyme liquid in buffer solution systems with pH5.6, 6.5, 7.5, 8.0, 8.5, 9.5 and 10.5, standing at room temperature for 2h, measuring residual enzyme activity according to an enzyme activity measuring method, and calculating relative enzyme activity. The pH stability of the OPHC2 mutant enzyme chrysanthemum esterase was determined with the enzyme activity of the initial enzyme solution as 100%.

1.8 detection of degradation effect of OPHC2 mutant enzyme on pyrethroid pesticides

Preparing a cypermethrin solution: weighing 10mg of cypermethrin or lambda-cyhalothrin standard sample, dissolving in 1mL of methanol, and uniformly mixing until the cypermethrin or lambda-cyhalothrin standard sample is completely dissolved. Preparing a high-efficiency cyhalothrin solution: weighing 10mg of lambda-cyhalothrin standard sample, dissolving in 1mL of methanol, and mixing until the sample is completely dissolved. OPHC2 double mutant enzyme (L226HF239I) solution preparation: 0.1g of the enzyme powder was dissolved in 50mL of 50mM Tris-HCl buffer (pH8.0), mixed until all was dissolved, and then placed at 4 ℃ for further use.

(1) OPHC2 double mutant enzyme (L226HF239I) for degrading cypermethrin

Control group 1: dissolving 25 mu L of cypermethrin solution in 50mL of 50mM Tris-HCl buffer solution with pH8.0, carrying out water bath reaction at 30 ℃, stirring for 0.5h, and immediately detecting the content of cypermethrin.

Experimental group 1: 12mL of OPHC2 double mutant enzyme (L226HF239I) solution was dissolved in 38mL of 50mM Tris-HCl buffer solution (pH8.0), 25. mu.L of cypermethrin solution was added, the mixture was reacted in a water bath at 30 ℃ and stirred for 0.5h, and the content of cypermethrin was immediately detected by gas chromatography.

(2) OPHC2 double mutant enzyme (L226HF239I) for degrading high-efficiency cyhalothrin

Control group 2: dissolving 25 mu L of efficient cyhalothrin solution in 50mL of 50mM Tris-HCl buffer solution with pH8.0, carrying out water bath reaction at 30 ℃, stirring for 0.5h, and immediately detecting the content of the efficient cyhalothrin.

Experimental group 2: 12mL of OPHC2 double mutant enzyme (L226HF239I) solution was dissolved in 38mL of 50mM Tris-HCl buffer solution (pH8.0), 25. mu.L of lambda-cyhalothrin solution was added, the mixture was reacted in a water bath at 30 ℃ and stirred for 0.5h, and then the content of lambda-cyhalothrin was immediately detected by gas chromatography.

1.930L mutant enzyme fermentation

(1) Stage of seed culture

The strain was inoculated into 40mL of YPD medium, cultured on a shaker at 30 ℃ for about 48 hours, transferred to 200mL of YPD medium, and cultured on a shaker at 30 ℃ for about 24 hours. The inoculum was inoculated by flame inoculation into a fermenter containing 12L of spent fermentation medium (about 10% inoculum size).

(2) Growth stage of thallus

Ventilating and stirring at 30 deg.C for about 11-14h, adding ammonia water to adjust pH to about 5.0, measuring residual sugar content and wet weight of thallus along with growth of strain, and determining specific carbon feeding time according to residual sugar content and dissolved oxygen.

(3) Carbon source feeding stage

When the reducing sugar test result is about 1%, carbon source feeding is started, 50% glucose (containing 12ml/LPTMl) is fed, and meanwhile, an inducer and PTM1 salt in the mixed feeding medium are also added into the carbon feeding medium. The reducing sugar, the wet weight of the thalli and microscopic examination are detected by sampling, and the glucose feeding speed is continuously adjusted. The pH value of the fermentation liquor is controlled to be maintained between 5.0 and 5.5 by continuously adding ammonia water, the air input is increased and the stirring speed is increased by increasing the pressure of the fermentation tank, and the dissolved oxygen in the fermentation tank is maintained between 20 and 50 percent.

(4) Carbon source methanol mixed feeding stage

When the wet weight of the thalli reaches a certain degree, stopping feeding the carbon source, entering a carbon source methanol mixed feeding stage, and mixing the carbon source glucose and an inducer methanol according to the weight ratio of 25% glucose: methanol-8: l, mixing, adding at a low flow rate, gradually increasing the adding speed, keeping for about 5-6h, simultaneously adding ammonia water to adjust pH to 5.2-5.5, and controlling the dissolved oxygen amount to be 20% -50%.

(5) Induced expression phase

Adding 80% methanol, adding at low flow rate, and gradually increasing the flow rate to maintain the methanol concentration within a reasonable range and maintain the dissolved oxygen content higher than 20%. Sampling once every 3-6 hours in the induction process to determine the activity and the wet weight of the pyrethrin enzyme, observing the shape of the thalli, increasing the sampling frequency when the activity of the enzyme is reduced, stopping fermentation when the activity of the enzyme is reduced for three times, and carrying out tank-placing treatment.

2. Results of the experiment

2.1 determination of the site of mutation in OPHC2

The active action site of OPHC2 (the nucleotide sequence of wild-type organophosphorus degrading enzyme OPHC2 is shown in SEQ ID NO.1, and the coded amino acid sequence is shown in SEQ ID NO. 2) is connected with metal auxiliary ions by histidine residues, hydrophobic residues form an ester group binding pocket, and the side chain of the hydrophobic residues determines the space structure of the ester group binding pocket and the recognition effect on a substrate, so that the analysis of the residues at the binding site provides a theoretical basis for the improvement of a substrate spectrum.

OPHC2 belongs to the beta-lactamase superfamily, and comes from a common ancestor with esterase, so OPHC2 has phosphotriesterase activity and also has esterase activity, but the esterase activity is lower, but other enzymes of the same family show higher esterase activity. In order to find out the key amino acids affecting the activity of OPHC2 esterase, proteins having homology to OPHC2 were found from NCBI using BLAST method. The larger side chain ester pocket amino acid was analyzed, and the results of the alignment revealed that OPHC2 at position 226 was Leu, the other homologous proteins were His, OPHC2 at position 239 was Phe, and the other homologous proteins were Ile (FIG. 1). Based on the analysis, 226 th Leu of OPHC2 is mutated into His (L226H) (the nucleotide sequence after codon optimization is shown in SEQ ID NO. 3); or the Phe at the position 239 is mutated into Ile (F239I) (the nucleotide sequence after codon optimization is shown in SEQ ID NO. 4); and at the same time, 226-bit Leu of OPHC2 is mutated into His, and 239-bit Phe is mutated into Ile (L226HF239I) (the nucleotide sequence after codon optimization is shown in SEQ ID NO. 5). Combining the coding sequence of the codon, mutating T at 677 site to A, mutating T at 716 site to A, so as to obtain the mutation of Leu at 226 site to His, and mutating Phe at 239 site to Ile.

2.2 construction of Yeast expression recombinant plasmids

The correctly detected recombinant plasmids pUC57-ophc2, pUC57-ophc2L226H (ophc2F239I/ophc2L226HF239I) and the vector pPICZaA are respectively subjected to double digestion treatment by EcoRI/NotI, and after electrophoretic recovery, the vector is subjected to T-type restriction enzyme digestion treatment₄The DNA ligases are ligated separately. Thus, the desired gene was inserted between EcoRI and NotI sites on pPICZaA by using the site of enzymatic cleavage to form the recombinants pPICZaa-ophc2, pPICZaA-ophc2L226H, pPICZaA-ophc2F239I and pPICZaA-ophc2L226HF 239I.

2.3 screening and identification of recombinant Yeast

The linearized recombinant plasmid is electrically transferred into a yeast strain, the yeast strain is screened by a bleomycin plate, the obtained clone is further induced and expressed on the level of a shaking table, and the recombinant with higher expression quantity is selected by measuring the activity of the pyrethrin enzyme. 12 recombinant strains of the pyrethrin enzyme pichia pastoris are obtained, and SDS-PAGE protein electrophoresis analysis results show that the chrysanthemum esterase (with the molecular weight of 36kD) is secreted into the fermentation liquor and accounts for more than 90 percent of the total protein content of the fermentation liquor. The expression level of ophc2-4 and L226HF239I-5 proteins was the highest (FIG. 2). The expression of L226H and F239I 24h, 48h and 72h proteins is shown in FIG. 3.

2.4 determination of the kinetic constants of the enzyme

The reciprocal of the reaction rate (1/v) was plotted against the reciprocal of the substrate p-nitrophenol acetate concentration (1/[ S ]), and the kinetic parameters of each mutant enzyme of OPHC2 and the original enzyme, pyrethrin enzyme, p-nitrophenol acetate, were calculated from the enzyme activity and enzyme concentration (Table 1).

TABLE 1 kinetic parameters of OPHC2 Proenzyme and P-nitrophenol acetate of each mutant enzyme

The results in table 1 show that the catalytic efficiency of each modified protein of OPHC2 is higher than that of the wild-type protein, and that the catalytic efficiency of the single-point modified enzymes L226H, F239I and the double-point modified enzyme L226HF239I is 64 times, 110 times and 70 times of that of the wild-type OPHC2, respectively, which means that the 226 th and 239 th, single-point mutation and double-point modification are benign, and the catalytic efficiency of the enzyme is greatly improved.

2.5 temperature and pH stability of the enzyme pyrethrin of the mutant enzyme

Taking an equal amount of L226HF239I-5 enzyme liquid, carrying out constant-temperature water bath for 5 hours at different temperatures, sampling every 1 hour, rapidly cooling to room temperature, measuring residual enzyme activity at different temperatures according to a method for measuring the enzyme activity of the pyrethrin enzyme, and taking the enzyme activity of the initial enzyme liquid as 100%, wherein the result is shown in figure 4, after the esterase is stored for 5 hours at 40 ℃, more than 75% of the enzyme activity remains, more than 60% of the enzyme activity remains after 5 hours of heat preservation at 60 ℃, and more than 30% of the enzyme activity remains after 3 hours of heat preservation at 70 ℃, which indicates that the esterase has better thermal stability.

Placing the same amount of L226HF239I-5 enzyme solution in different buffer solution systems for 2h, determining residual enzyme activity according to an enzyme activity determination method, and calculating relative enzyme activity. The pH stability of OPHC2-M esterase was determined with the enzyme activity of the initial enzyme solution as 100%, as shown in FIG. 5, the enzyme had better pH stability, and the enzyme activity maintenance rate was all above 70% when the enzyme was stored for 2h in the range of 5.5-10.5.

2.6 detection of degradation Effect of pyrethroid insecticides

The degradation rate of OPHC2 double mutant L226HF239I on different pyrethroid pesticides was measured by gas chromatography, and the results are shown in Table 2 and FIG. 6.

TABLE 2 degradation test results of pesticides

As can be seen from Table 2 and FIG. 6, the mutant enzyme has high degradation rate of the beta-cypermethrin and the beta-cyhalothrin, which are both 100%; the degradation rate of the high-efficiency cyfluthrin is the lowest and is only 45.6 percent.

2.730L mutant enzyme fermentation

Different methanol utilization phenotypes of pichia pastoris have different characteristics, so that different regulation and control modes are provided in the fermentation process, and the control of fermentation regulation plays an important role. The strain utilized by the invention is a Muts type KM71H/pPICZaA-ophc2L226HF239I strain, the methanol velocity is slow, methanol residue is strictly controlled to regulate and control, the DO value is regulated and controlled to be 30-50%, the flow rate of methanol is 1mL/h/L initial fermentation volume, after methanol is supplemented, the flow rate is increased by 10% per hour until the initial fermentation volume reaches 3mL/h/L, and then the flow rate can be slowly increased along with the increase of the fermentation volume so as to ensure the growth requirement of the bacteria.

According to the experience of fermentation in a plurality of fermentation tanks, the inoculation amount is 8-10%, the temperature is set to be 30 ℃ in the whole fermentation process, the pH value in the thallus growth stage is set to be 5.0, the pH value is set to be 5.2-5.5 after the induction period, and the rotating speed and the ventilation volume are adjusted according to the DO value and the wet weight condition of the thallus. In the invention, six batches of KM71H/pPICZaA-ophc2L226HF239I fermentation experiments of 30L are carried out totally, the induction starts from the culture on a tank for about 24 hours, the change range of the wet weight of the thalli is small along with the mixed feeding and the continuous liquid supplementation of the induction, the speed of methanol metabolism of Muts type is slow, the enzyme activity is continuously increased along with the prolonging of the induction time, and the enzyme activity reaches 143.75U/mL (figure 7) during the fermentation for 70 hours.

SEQUENCE LISTING

<110> Beijing Sendzia bioengineering technology, Inc

<120> organophosphorus degrading enzyme OPHC2 mutant and application thereof

<130>BJ-3010-180701A

<160>5

<170>PatentIn version 3.5

<210>1

<211>903

<212>DNA

<213>Pseudomonas pseudoalcaligenes

<400>1

gccgcaccgg cacaacagaa gacccaggta ccgggctact accgtatggc actcggtgac 60

ttcgaagtca ccgctctgta tgacggctac gtcgacctgc ctgccagcct gctcaagggc 120

atcgatgaca aggacctgca atcgctgctg gctcgcatgt tcgtggcgtc ggagaaaggc 180

gtgcagactg cggtcaacgc ctacctgatc aacactggcg acaacctggt gctgatcgat 240

accggcgccg cccagtgctt cggcccgact ctcggcgtgg tgcagaccaa cctcaaggca 300

tccggctacc agccggagca ggttgatacc gtgctgctca cccacctgca cccagaccat 360

gcctgcggcc tggtcaacgc cgacggcagc ccggcctacc ccaacgcgac cgtggaggtg 420

ccgcaggcgg aggctgaatt ctggctcgac gaggcgacca tggccaaggc ccccgaaggc 480

atgcaaggca tgttcaagat ggcgcaacag gcagtcgcac cctatgccaa gatgaacaag 540

ctcaagccct acaagacaga aggcgaattg ttgcctggcg tcagcctggt agcgagcccg 600

ggacacacgc ccggacatac ctcttacctg ttcaaatcgg gtggacagag cctgctggta 660

tggggcgaca ttctgcttaa ccacgccgtg cagttcgcca agcctgaagt ggtcttcgag 720

ttcgatgtcg acagcgacca ggccaggcaa tcccgccaac gcatcctggc cgaagcggcc 780

acagacaagc tgtgggtcgc tggtgcgcac ctgcccttcc ccggcctggg ccacgtacgc 840

aaggaagccc aaggctacgc ctgggtaccc gtcgagttca gcccgatccg tagcgaccgc 900

tga 903

<210>2

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<212>PRT

<213>Pseudomonas pseudoalcaligenes

<400>2

Ala Ala Pro Ala Gln Gln Lys Thr Gln Val Pro Gly Tyr Tyr Arg Met

1 5 10 15

Ala Leu Gly Asp Phe Glu Val Thr Ala Leu Tyr Asp Gly Tyr Val Asp

20 25 30

Leu Pro Ala Ser Leu Leu Lys Gly Ile Asp Asp Lys Asp Leu Gln Ser

35 40 45

Leu Leu Ala Arg Met Phe Val Ala Ser Glu Lys Gly Val Gln Thr Ala

50 55 60

Val Asn Ala Tyr Leu Ile Asn Thr Gly Asp Asn Leu Val Leu Ile Asp

65 70 75 80

Thr Gly Ala Ala Gln Cys Phe Gly Pro Thr Leu Gly Val Val Gln Thr

85 90 95

Asn Leu Lys Ala Ser Gly TyrGln Pro Glu Gln Val Asp Thr Val Leu

100 105 110

Leu Thr His Leu His Pro Asp His Ala Cys Gly Leu Val Asn Ala Asp

115 120 125

Gly Ser Pro Ala Tyr Pro Asn Ala Thr Val Glu Val Pro Gln Ala Glu

130 135 140

Ala Glu Phe Trp Leu Asp Glu Ala Thr Met Ala Lys Ala Pro Glu Gly

145 150 155 160

Met Gln Gly Met Phe Lys Met Ala Gln Gln Ala Val Ala Pro Tyr Ala

165 170 175

Lys Met Asn Lys Leu Lys Pro Tyr Lys Thr Glu Gly Glu Leu Leu Pro

180 185 190

Gly Val Ser Leu Val Ala Ser Pro Gly His Thr Pro Gly His Thr Ser

195 200 205

Tyr Leu Phe Lys Ser Gly Gly Gln Ser Leu Leu Val Trp Gly Asp Ile

210 215 220

Leu Leu Asn His Ala Val Gln Phe Ala Lys Pro Glu Val Val Phe Glu

225 230 235 240

Phe Asp Val Asp Ser Asp Gln Ala Arg Gln Ser Arg Gln Arg Ile Leu

245 250 255

Ala Glu Ala Ala Thr Asp Lys Leu TrpVal Ala Gly Ala His Leu Pro

260 265 270

Phe Pro Gly Leu Gly His Val Arg Lys Glu Ala Gln Gly Tyr Ala Trp

275 280 285

Val Pro Val Glu Phe Ser Pro Ile Arg Ser Asp Arg

290 295 300

<210>3

<211>903

<212>DNA

<213>Artifical sequence

<400>3

gccgcaccag cacaacagaa gacccaagtt ccaggttact acagaatggc tttgggtgac 60

ttcgaggtca ctgctttgta cgacggttac gtagacttgc ctgcttcttt gcttaagggt 120

atcgatgaca aggacctgca atctctgttg gctagaatgt tcgttgcttc tgagaaaggt 180

gttcaaactg ctgtcaacgc ttacttgatc aacactggtg acaacttggt tttgattgat 240

accggcgccg cccagtgttt tggtccaact ctcggtgttg tgcagaccaa ccttaaagct 300

tccggttacc aaccagagca ggttgatact gttttgctta cccacttgca cccagaccat 360

gcttgtggtt tggtcaacgc cgacggttcc ccagcctacc caaatgctac cgttgaggtt 420

ccacaagctg aggctgagtt ctggcttgac gaggctacca tggctaaggc ccctgaaggt 480

atgcaaggta tgttcaagat ggctcaacaa gctgtcgcac catacgctaa gatgaacaag 540

ttaaagccat acaagactga aggagagttg ttgcctggtg tctccttggt tgcttcccca 600

ggacacacgc caggacacac tagttacttg tttaaatctg gtggacaatc tttgctggtt 660

tggggtgaca ttctgcacaa ccacgccgtt caattcgcta agcctgaagt tgtcttcgag 720

ttcgatgtcg actccgacca agccagacaa tccagacaaa gaattttggc cgaagctgcc 780

acagacaagt tgtgggtcgc tggtgctcac ttgcctttcc caggtttggg acacgttaga 840

aaggaagctc aaggttacgc ctgggtacct gtcgagttct ctccaatccg ttccgacaga 900

taa 903

<210>4

<211>903

<212>DNA

<213>Artifical sequence

<400>4

gccgcaccag cacaacagaa gacccaagtt ccaggttact acagaatggc tttgggtgac 60

ttcgaggtca ctgctttgta cgacggttac gtagacttgc ctgcttcttt gcttaagggt 120

atcgatgaca aggacctgca atctctgttg gctagaatgt tcgttgcttc tgagaaaggt 180

gttcaaactg ctgtcaacgc ttacttgatc aacactggtg acaacttggt tttgattgat 240

accggcgccg cccagtgttt tggtccaact ctcggtgttg tgcagaccaa ccttaaagct 300

tccggttacc aaccagagca ggttgatact gttttgctta cccacttgca cccagaccat 360

gcttgtggtt tggtcaacgc cgacggttcc ccagcctacc caaatgctac cgttgaggtt 420

ccacaagctg aggctgagtt ctggcttgac gaggctacca tggctaaggc ccctgaaggt 480

atgcaaggta tgttcaagat ggctcaacaa gctgtcgcac catacgctaa gatgaacaag 540

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ggacacacgc caggacacac tagttacttg tttaaatctg gtggacaatc tttgctggtt 660

tggggtgaca ttctgcttaa ccacgccgtt caattcgcta agcctgaagt tgtcatcgag 720

ttcgatgtcg actccgacca agccagacaa tccagacaaa gaattttggc cgaagctgcc 780

acagacaagt tgtgggtcgc tggtgctcac ttgcctttcc caggtttggg acacgttaga 840

aaggaagctc aaggttacgc ctgggtacct gtcgagttct ctccaatccg ttccgacaga 900

taa 903

<210>5

<211>903

<212>DNA

<213>Artifical sequence

<400>5

gccgcaccag cacaacagaa gacccaagtt ccaggttact acagaatggc tttgggtgac 60

ttcgaggtca ctgctttgta cgacggttac gtagacttgc ctgcttcttt gcttaagggt 120

atcgatgaca aggacctgca atctctgttg gctagaatgt tcgttgcttc tgagaaaggt 180

gttcaaactg ctgtcaacgc ttacttgatc aacactggtg acaacttggt tttgattgat 240

accggcgccg cccagtgttt tggtccaact ctcggtgttg tgcagaccaa ccttaaagct 300

tccggttacc aaccagagca ggttgatact gttttgctta cccacttgca cccagaccat 360

gcttgtggtt tggtcaacgc cgacggttcc ccagcctacc caaatgctac cgttgaggtt 420

ccacaagctg aggctgagtt ctggcttgac gaggctacca tggctaaggc ccctgaaggt 480

atgcaaggta tgttcaagat ggctcaacaa gctgtcgcac catacgctaa gatgaacaag 540

ttaaagccat acaagactga aggagagttg ttgcctggtg tctccttggt tgcttcccca 600

ggacacacgc caggacacac tagttacttg tttaaatctg gtggacaatc tttgctggtt 660

tggggtgaca ttctgcacaa ccacgccgtt caattcgcta agcctgaagt tgtcatcgag 720

ttcgatgtcg actccgacca agccagacaa tccagacaaa gaattttggc cgaagctgcc 780

acagacaagt tgtgggtcgc tggtgctcac ttgcctttcc caggtttggg acacgttaga 840

aaggaagctc aaggttacgc ctgggtacct gtcgagttct ctccaatccg ttccgacaga 900

taa 903

Claims

1. An organophosphorus degrading enzyme OPHC2 mutant, which is characterized in that: the mutant is obtained by mutating the 226 th amino acid of wild type organophosphorus degrading enzyme OPHC2 shown in SEQ ID NO.2 from leucine to histidine.

2. A gene encoding the mutant organophosphorus degrading enzyme OPHC2 according to claim 1.

3. The encoding gene according to claim 2, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID No. 3.

4. An organophosphorus degrading enzyme OPHC2 mutant, which is characterized in that: the mutant is obtained by mutating the 239 th amino acid of wild type organophosphorus degrading enzyme OPHC2 shown in SEQ ID NO.2 from phenylalanine to isoleucine.

5. A gene encoding the mutant organophosphorus degrading enzyme OPHC2 according to claim 4.

6. The encoding gene as claimed in claim 5, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID No. 4.

7. An organophosphorus degrading enzyme OPHC2 mutant, which is characterized in that: the mutant is obtained by mutating the 226 th amino acid of wild type organophosphorus degrading enzyme OPHC2 shown in SEQ ID NO.2 from leucine to histidine and the 239 th amino acid from phenylalanine to isoleucine.

8. A gene encoding the mutant organophosphorus degrading enzyme OPHC2 according to claim 7.

9. The encoding gene according to claim 8, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID No. 5.

10. A recombinant expression vector comprising the coding gene of claim 2, 3, 5, 6, 8 or 9.

11. Use of the mutant of organophosphorus degrading enzyme OPHC2 according to claim 1, 4 or 7 for degrading pyrethroid insecticides.

12. A method for preparing the mutant of the organophosphorus degrading enzyme OPHC2 according to claim 1, 4 or 7, comprising the steps of:

(1) cloning a gene encoding the mutant of the organophosphorus degrading enzyme OPHC2 according to claim 1, 4 or 7 onto a yeast expression plasmid to construct a yeast expression recombinant plasmid; (2) linearizing the yeast expression recombinant plasmid, and transforming yeast competent cells to obtain a recombinant yeast strain; (3) and (3) carrying out high-density fermentation culture on the recombinant yeast strain, carrying out induced expression on corresponding protein, and separating and purifying to obtain the recombinant yeast strain.

13. The method according to claim 12, wherein the recombinant yeast strain of step (3) is inoculated in an amount of 8% to 10%; the temperature of the whole fermentation process is 30 ℃, the pH value of the thallus growth stage is 5.0, and the pH value after the induction period is 5.2-5.5.