Preparation method of cypermethrin degradation esterase

Abstract

The invention provides a preparation method of cypermethrin degradation esterase, and belongs to the technical field of bioengineering. The esterase expressed based on the gene Est13 has the advantages of being stable in protein structure, resistant to low temperature and good in pyrethroid degradation function, saccharomyces cerevisiae conforming to food-grade host bacteria is selected to construct a eukaryotic expression system, no toxic by-products are generated in the whole expression process, and the method is suitable for industrial production. The activity and the quality stability of the esterase produced by the saccharomyces cerevisiae genetically engineered bacterium can be effectively improved, the degradation rate of residual pyrethroid pesticides in agricultural products is remarkably improved, and the saccharomyces cerevisiae genetically engineered bacterium can be applied to the field of food more safely and greenly.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing cypermethrin-degrading esterase. Background Art

[0002] Pyrethroid pesticides are a class of highly effective and low-toxic biomimetic insecticides widely used in agriculture and household pest control. Due to their mobility and accumulation, these pesticides often have varying degrees of residues in the environment and food. A large number of studies have shown that pyrethroids have reproductive and neurotoxicity, and can also cause intestinal flora disorders and "three-hazard" risks, which have a significant negative impact on food safety and human health. Cypermethrin (CY) is a common type and representative of pyrethroid pesticides. The key link in its degradation is the cleavage of ester bonds to generate two intermediates, chrysanthemic acid and cyanohydrin. The generated α-cyano-3-phenoxybenzyl alcohol can be further oxidized to 3-phenoxybenzoic acid. In order to eliminate the hazards of pesticide residues, people have explored various technologies, such as leaching, nanofiltration, photodegradation, ozone oxidation, screening of microbial degradation bacteria, etc., but these physical, chemical, and microbial methods are limited in the removal of pesticides in food, feed, or the human body, and many microbial degradation bacteria have great safety hazards.

[0003] The essence of microbial degradation of pyrethroids is an enzymatic reaction, in which degradation enzymes play a key role. This process involves hydrolases and oxidoreductases. Hydrolases mainly include esterases, peptidases, etc., and oxidoreductases mainly include oxygenases, peroxidases, etc. The main mechanism of microbial degradation of pyrethroids is that esterases break the ester bonds of pesticides, produce carboxylic acids and alcohols, and then undergo oxidative decomposition. Esterase hydrolysis is the first key reaction for the degradation and detoxification of pyrethroid pesticides. The preparation of degradation esterases by genetically engineered bacteria can realize industrial production, especially the exploration of cloning and expressing degradation esterases by food-grade genetically engineered bacteria is of great significance.

[0004] Genetically engineered bacterial expression systems can be divided into prokaryotic expression systems and eukaryotic expression systems. Prokaryotic expression system hosts mainly include Escherichia coli and Bacillus subtilis, while eukaryotic expression system hosts mainly include Saccharomyces cerevisiae, Pichia pastoris, and filamentous fungi such as Aspergillus niger and Aspergillus oryzae. Although Escherichia coli is the most commonly used expression system, its safety issues have seriously hindered its application in the food, feed, medical and other industries.

[0005] Pyrethroid pesticide residues are often detected in vegetables and fruits in many countries and regions, so when eating vegetables and fruits, their surface contamination puts people at greater risk of exposure to residual pesticides. Traditional tap water washing is less effective for hydrophobic pesticides; fruit and vegetable detergents, whose main components are surfactants, can regulate cell permeability and promote the entry of pesticides and their metabolites into fruits and vegetables, which may pose health risks. Therefore, it is of great significance to develop food-grade genetically engineered bacteria to clone and express degradative esterases. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing cypermethrin-degrading esterase.

[0007] The present invention provides a method for preparing a cypermethrin-degrading esterase, the method comprising the following steps:

[0008] (a) inoculating genetically engineered yeast Saccharomyces cerevisiae into a culture medium, fermenting, and obtaining a fermentation liquid;

[0009] (b) transferring the fermentation broth to a culture medium, inducing expression under the action of an inducer and a carbon source, collecting the bacterial cells, breaking the bacterial cells, centrifuging, collecting the supernatant, and obtaining cypermethrin-degrading esterase;

[0010] The genetically engineered yeast of Saccharomyces cerevisiae contains a gene encoding a cypermethrin-degrading esterase.

[0011] Furthermore, in step (a), the culture medium is a SD-H liquid culture medium containing glucose, the added amount of glucose is 0.1% to 5%, preferably 0.5%; the liquid volume of the SD-H liquid culture medium is 5 to 30%, preferably 20%;

[0012] In step (b), the volume ratio of the fermentation broth to the culture medium is 1:0.5-1.5, preferably 1:1; the culture medium is SGR-H culture medium; the inducer is galactose, and the addition amount is 1-3%, preferably 2%; the carbon source is raffinose, and the addition amount is 0.5-1.5%, preferably 1%;

[0013] The NCBI accession number of the gene encoding cypermethrin-degrading esterase is OR400756.

[0014] Furthermore, in step (a), the inoculation amount of the genetically engineered yeast of Saccharomyces cerevisiae is 0.5-2.5%, preferably 1%; the fermentation broth is the fermentation broth washed with phosphate buffer; the fermentation conditions are: at 20-40°C, at 100-250rpm for 2-48h, preferably: at 30°C, at 220rpm for 4h.

[0015] Furthermore, in step (b), the induction expression conditions are: a rotation speed of 100 to 250 rpm, an induction temperature of 20 to 40°C, and an induction time of 6 to 48 hours, preferably: a rotation speed of 220 rpm, an induction temperature of 30°C, and an induction time of 12 hours. In step (a), the preparation method of the genetically engineered yeast of cerevisiae comprises the following steps:

[0016] (1) Construction of recombinant plasmid;

[0017] (2) The recombinant plasmid is transferred into Saccharomyces cerevisiae cells to obtain genetically engineered Saccharomyces cerevisiae.

[0018] Furthermore, in step (1), the recombinant plasmid comprises an expression vector and a gene encoding a cypermethrin-degrading esterase, and the expression vector is pESC-HIS;

[0019] In step (2), the Saccharomyces cerevisiae cell is a Saccharomyces cerevisiae INVSc1 cell.

[0020] Furthermore, in step (1), the preparation method of the recombinant plasmid comprises the following steps: the expression vector and the gene encoding the cypermethrin-degrading esterase are connected in a PCR instrument at 10 to 25° C. for 5 to 20 hours under the action of DNA ligase to obtain the recombinant plasmid.

[0021] Furthermore, in step (1), the preparation method of the recombinant plasmid comprises the following steps: the DNA ligase is T4 DNA ligase; and the recombinant plasmid is obtained by connecting at 16° C. for 7 to 17 hours in a PCR instrument.

[0022] The present invention also provides a method for testing the activity of cypermethrin-degrading esterase, which comprises the following steps: a buffer solution, a cypermethrin-acetone solution and a cypermethrin-degrading esterase constitute a reaction system, acetonitrile is added after the degradation is completed, ultrasonication, centrifugation, supernatant filtration, and detection;

[0023] The degradation temperature is 0-45°C and the degradation time is 10-150 minutes.

[0024] Furthermore, the degradation temperature is 25°C and the time is 120 minutes.

[0025] The present invention provides a food-grade cypermethrin-degrading esterase-producing yeast genetic engineering bacteria and its application. Based on the advantages that the esterase expressed by the gene Est13 has a stable protein structure, is resistant to low temperatures, and has a good pyrethroid degradation function, the present invention selects a yeast saccharomyces cerevisiae that meets the food-grade host bacteria to construct a eukaryotic expression system, and obtains the best experimental conditions for inducing expression by optimizing the amount of glucose added, the amount of liquid, the rotation speed, the inoculation amount, the expansion culture time, the induction temperature, the amount of galactose added, and the induction time. No toxic byproducts are generated during the whole expression process, which can effectively improve the activity and quality stability of the esterase produced by the yeast genetic engineering bacteria, significantly improve the degradation rate of residual pyrethroid pesticides in agricultural products, and be more safely and greenly applied in the field of food.

[0026] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0027] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the effect of reaction temperature on the expression of recombinant esterase.

[0029] Figure 2 is the effect of degradation time on the expression of recombinant esterase.

[0030] Figure 3 It is the effect of glucose addition on the expression of recombinant esterase.

[0031] Figure 4 The effect of liquid volume on the expression of recombinant esterase.

[0032] Figure 5 The effect of rotation speed on the expression of recombinant esterase.

[0033] Figure 6 is the effect of inoculum size on the expression of recombinant esterase.

[0034] Figure 7 The effect of expansion time on the expression of recombinant esterase.

[0035] Figure 8 The effect of induction temperature on the expression of recombinant esterase.

[0036] Fig. 9 The effect of galactose addition on the expression of recombinant esterase.

[0037] Fig.10 The effect of induction time on the expression of recombinant esterase. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0039] The "overnight" referred to in the present invention is 12±5 hours.

[0040] Example 1. Construction of recombinant plasmid

[0041] The genomic DNA of Bacillus subtilis J6 (NCBI accession number: OR400756, full length 741bp) was extracted, and specific primers were designed according to the target gene to amplify and recover the target fragment, connect it with the pMD18-T vector, transform it into E.coli DH5α, and screen positive clones by histidine-deficient plate SD-H. The plasmid containing the target fragment Est13 in E.coli DH-5α was extracted and used as a template in the PCR reaction. The primer F / R with restriction site was used for PCR amplification. The amplification program was 94℃30s; 94℃10s; 65℃30s; 72℃30s, 10cycles, -1℃ / cycle; 94℃10s, 55℃30s, 72℃30s, 10cycles: 72℃2min, 4℃end. The target gene fragment Est13 containing restriction site was obtained. The target gene and pESC-HIS expression vector were double-digested with EcoRI and NotI restriction endonucleases, and the enzyme digestion reaction system was mixed evenly and placed in a 37°C constant temperature water bath for 2 hours. The target fragment and expression vector were recovered and purified using a PCR purification kit. The target gene after enzyme digestion was connected to the pESC-HIS expression vector in a PCR instrument at 16°C overnight under the action of T4 DNA ligase to obtain the recombinant plasmid pESC-HIS-Est13 with the target fragment.

[0042] Example 2: Transformation of recombinant genetically engineered bacteria

[0043] The constructed recombinant plasmid pESC-HIS-Est13 was transformed into the competent cell Saccharomyces cerevisiae INVSc1. The specific steps are as follows: (1) Insert the carrier DNA into the float and place it in a 95℃ water bath for 3 minutes. After heating, quickly insert it into ice. (2) Take 100μL of INVSc1 competent cells melted on ice, add 5μg of pre-cooled target plasmid, 10μL of pre-treated carrier DNA, and 500μL of PEG / LiAc in sequence and pipette several times to mix, and place it in a 30℃ water bath for 30 minutes. (3) Place it in a 42℃ water bath for 15 minutes. (4) Centrifuge at 5000×g for 60 seconds and discard the supernatant. Resuspend in 400μL of ddH2O and centrifuge for 60 seconds and discard the supernatant. (5) Resuspend in 100μL of ddH2O, apply to SD-H plate, and culture at 30℃ for 48-96 hours. Positive clones from the SD-H plate were randomly selected for colony PCR verification and product sequencing verification (sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing), and the verified correct pESC-HIS-Est13 / INVSc1 recombinant bacteria were stored in glycerol.

[0044] Example 3: Inducible expression of recombinase Est13

[0045] In a 500mL fermentation container, the recombinant genetic engineering bacteria were inoculated at 1% (v / v) into 100mL (i.e., the liquid volume was 20%) SD-H liquid medium containing 2% glucose, and cultured at 30°C, 220rpm for 24h (OD value was about 0.4), and washed with phosphate buffer to obtain a fermentation broth. The fermentation broth was centrifuged at 5000×g, 4°C for 15min, the supernatant was removed, and after centrifugation and washing with PBS once, it was resuspended in an equal volume of SGR-H medium, and 1% (v / v) raffinose and 2% (v / v) galactose after membrane sterilization were added to the resuspended culture, and induced at 30°C, 220rpm for 24h. After induction, the cells were collected by centrifugation (5000×g, 4°C, 15 min), washed with an appropriate amount of PBS (pH 7.0) buffer, centrifuged at 5000×g, 4°C for 15 min, collected, resuspended with PBS (pH 7.0) buffer to 1 / 10 of the original volume, and crushed with an ultrasonic cell disruptor in an ice bath with an output power of 40 W, working for 2 s, stopping for 2 s, and a total of 30 min of ultrasonic crushing. After the crushing, the cells were centrifuged at 4°C and 5000×g for 15 min, and the supernatant was collected, which was the crude enzyme solution and stored at 4°C for later use.

[0046] Example 4: Verification of the degradation function of the recombinase Est13

[0047] Taking cypermethrin as substrate, the esterase activity was calculated by detecting the degradation amount of cypermethrin in the reaction system. The reaction system was 3 mL, that is, 0.1 mL of cypermethrin-acetone solution (containing 0.3 mg / mL of cypermethrin) was added to 2.4 mL of PBS (pH 7.0) buffer, and 0.5 mL of the crude enzyme solution obtained in Example 3 was added after mixing, and the reaction was carried out at 25° C. for 120 min. After the reaction was completed, 3 mL of acetonitrile was added immediately, ultrasonicated for 30 min, mixed, and 1.5 mL was centrifuged (12000 r / min, 10 min), the supernatant was passed through a 0.22 μm organic filter membrane, and the filtrate was taken for HPLC detection.

[0048] The results showed that the crude enzyme solution obtained in Example 3 had a degradation rate of 43.78% for 0.3 mg / mL cypermethrin in 120 min.

[0049] Example 5: Inducible expression of recombinase Est13

[0050] In a 500mL fermentation container, the recombinant genetic engineering bacteria were inoculated at 1% (v / v) into 100mL (i.e., the liquid volume was 20%) SD-H liquid medium containing 2% glucose, and cultured at 30°C, 220rpm for 4h (OD value was about 0.4), and washed with phosphate buffer to obtain a fermentation broth. The fermentation broth was centrifuged at 5000×g, 4°C for 15min, the supernatant was removed, and after centrifugation and washing once with PBS, it was resuspended in an equal volume of SGR-H medium, and 1% (v / v) raffinose and 2% (v / v) galactose after membrane sterilization were added to the resuspended culture, and induced at 30°C, 220rpm for 4h. After induction, the cells were collected by centrifugation (5000×g, 4°C, 15 min), washed with an appropriate amount of PBS (pH 7.0) buffer, centrifuged at 5000×g, 4°C for 15 min, collected, resuspended with PBS (pH 7.0) buffer to 1 / 10 of the original volume, and crushed with an ultrasonic cell disruptor in an ice bath with an output power of 40 W, working for 2 s, stopping for 2 s, and a total of 30 min of ultrasonic crushing. After the crushing, the cells were centrifuged at 4°C and 5000×g for 15 min, and the supernatant was collected, which was the crude enzyme solution and stored at 4°C for later use.

[0051] Example 6: Verification of the degradation function of the recombinase Est13

[0052] Taking cypermethrin as substrate, the esterase activity was calculated by detecting the degradation amount of cypermethrin in the reaction system. The reaction system was 3 mL, that is, 0.1 mL of cypermethrin-acetone solution (containing 0.3 mg / mL of cypermethrin) was added to 2.4 mL of PBS (pH 7.0) buffer, and 0.5 mL of the crude enzyme solution obtained in Example 5 was added after mixing, and the reaction was carried out at 25° C. for 120 min. After the reaction was completed, 3 mL of acetonitrile was added immediately, ultrasonicated for 30 min, mixed, and 1.5 mL was centrifuged (12000 r / min, 10 min), the supernatant was passed through a 0.22 μm organic filter membrane, and the filtrate was taken for HPLC detection.

[0053] The results showed that the crude enzyme solution obtained in Example 5 had a degradation rate of 47.74% for 0.3 mg / mL cypermethrin in 120 min.

[0054] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0055] Experimental Example 1: Screening of degradation conditions for recombinase Est13

[0056] 1. Optimum degradation temperature

[0057] The reaction system described in Example 4 was placed in a constant temperature shaking incubator at 0, 4, 10, 15, 20, 25, 30, 37, and 45° C. and shaken for 4 h. Figure 1 The results showed that when the degradation temperature was 25℃, the degradation rate was significantly higher than that at other temperatures (P<0.05).

[0058] 2. Best time for degradation

[0059] The reaction system described in Example 4 was placed in a constant temperature shaking incubator at 25° C. and shaken for 10, 30, 60, 90, 120, and 150 min, respectively. Figure 2 The results showed that the degradation rate increased significantly within 10 to 120 minutes (P < 0.05), but there was no significant change when it was greater than 120 minutes (P > 0.05), so the optimal degradation time was 120 minutes.

[0060] Experimental Example 2: Screening of induction conditions

[0061] According to the esterase activity test method of Example 4, the degradation function of the recombinant enzyme obtained under different induction conditions on cypermethrin was tested in batches.

[0062] 1. Effect of glucose addition on esterase expression

[0063] Under the conditions of inoculation amount of 1% (v / v), expansion time of 24h, galactose addition of 2% (v / v), induction temperature of 30℃, induction time of 24h, liquid volume of 20% and rotation speed of 220rpm, the effects of glucose addition of 0.5%, 1%, 1.5%, 2%, 3% and 5% on esterase expression were studied. Figure 3 It shows that when the addition amount of glucose is 0.5%, its degradation rate is greater than that of other addition amounts. It means that when the addition amount of glucose is 0.5%, the esterase activity in the obtained crude enzyme solution is the best, so 0.5% is the best addition amount of glucose.

[0064] 2. Effect of liquid volume on esterase expression.

[0065] Under the conditions of inoculation volume of 1% (v / v), expansion time of 24h, glucose addition of 2% (v / v), galactose addition of 2% (v / v), induction temperature of 30°C, induction time of 24h, and rotation speed of 220rpm, the effects of liquid filling volume of 5%, 10%, 15%, 20%, 25%, and 30% on esterase expression were studied. Figure 4 The results showed that the degradation rate increased significantly within the range of 5% to 20% (P < 0.05), and decreased significantly between 20% and 30% (P < 0.05), so the optimal liquid filling volume was 20%.

[0066] 3. Effect of rotation speed on esterase expression

[0067] Under the conditions of inoculation amount of 1% (v / v), expansion time of 24h, glucose addition amount of 2% (v / v), galactose addition amount of 2% (v / v), induction temperature of 30°C, liquid volume of 20% and induction time of 24h, the effects of culture rotation speed of 0rpm, 50rpm, 100rpm, 160rpm, 220rpm and 250rpm on esterase expression were studied. Figure 5 It shows that the degradation rate significantly increases within the range of 0-220 rpm (P<0.05), and significantly decreases between 220-250 rpm (P<0.05), so the optimal speed is 220 rpm.

[0068] 4. Effect of inoculum size on esterase expression

[0069] Under the conditions of expansion time of 24h, glucose addition of 2% (v / v), galactose addition of 2% (v / v), induction temperature of 30℃, induction time of 24h, liquid volume of 20%, and rotation speed of 220rpm, the effects of inoculation amount of 0.5%, 1%, 1.5%, 2%, and 2.5% on esterase expression were studied. Figure 6The results showed that the degradation rate was significantly different within the inoculum size of 1% to 2.5% (P < 0.05). Although there was no significant difference between 0.5% and 1% (P > 0.05), the degradation rate of 1% inoculum size was slightly higher than that of 0.5%, so the optimal inoculum size was 1%.

[0070] 5. Effect of expansion time (i.e. shaking culture time) on esterase expression

[0071] Under the conditions of inoculation amount of 1% (v / v), glucose addition amount of 2% (v / v), galactose addition amount of 2% (v / v), induction temperature of 30°C, induction time of 24h, liquid volume of 20% and rotation speed of 220rpm, the effects of expansion time of 4h, 10h, 16h, 24h, 36h and 48h on esterase expression were studied. Figure 7 The results showed that when the culture time was 4h, the degradation rate was significantly higher than that at other times (P<0.05), so 4h was the optimal culture time.

[0072] 6. Effect of induction temperature on esterase expression

[0073] Under the conditions of inoculation amount of 1% (v / v), expansion time of 4h, glucose addition of 2% (v / v), galactose addition of 2% (v / v), induction time of 24h, liquid volume of 20% and rotation speed of 220rpm, the effects of induction temperatures of 20℃, 25℃, 30℃, 35℃ and 40℃ on esterase expression were studied. Figure 8 The results showed that when the induction temperature was 30℃, the degradation rate was significantly higher than that at other temperatures (P<0.05), so 30℃ was the optimal induction temperature.

[0074] 7. Effect of galactose addition on esterase expression

[0075] Under the conditions of inoculation amount of 1% (v / v), expansion time of 4h, glucose addition of 2% (v / v), induction temperature of 30℃, induction time of 24h, liquid volume of 20% and rotation speed of 220rpm, the effects of galactose addition of 1%, 1.5%, 2%, 2.5% and 3% on esterase expression were studied. Fig. 9 The results showed that although there was no significant difference in the addition amount of galactose from 1% to 3% (P>0.05), the degradation rate of 2% addition was higher than that of other addition amounts, so the optimal galactose addition amount was 2%.

[0076] 8. Effect of induction time on esterase expression

[0077] Under the conditions of inoculation amount of 1% (v / v), expansion time of 4h, glucose addition of 2% (v / v), galactose addition of 2% (v / v), induction temperature of 30℃, liquid volume of 20% and rotation speed of 220rpm, the effects of induction time of 6h, 12h, 24h, 36h and 48h on esterase expression were studied. Fig.10The results showed that there was no significant difference in degradation rate between 12h and 48h (P>0.05), but the effect achieved was the same. In order to save time cost, the optimal induction time was 12h.

[0078] After optimization of the above experimental examples, the optimal glucose addition amount is 0.5%, the optimal liquid filling amount is 20%, the optimal inoculation amount is 1%, the optimal rotation speed is 220rpm, the optimal expansion time is 4h, the optimal induction temperature is 30℃, the optimal galactose addition amount is 2%, and the optimal induction time is 12h.

[0079] Experimental Example 2: Removal of Cypermethrin Residues in Fruits and Vegetables by Recombinant Enzymes

[0080] Contaminated samples were obtained by immersing lettuce and grapes in a 5 mg / L cypermethrin solution for 30 min. Except for the unwashed group, all samples were immersed in water or Est13 enzyme solution for 10 or 20 min, and finally washed once in water to complete the cleaning process.

[0081] Table 1 shows the removal rate of cypermethrin residues on fruits and vegetables in different cleaning methods. As can be seen from the table, the enzyme solution cleaning effect is significant after HPLC detection. The cypermethrin residue on lettuce decreased by 48.36% after 20 minutes of enzyme solution treatment, while it only decreased by 11.32% after the same time of water washing. For grapes, the cypermethrin removal rate was 35.42% after 20 minutes of enzyme solution treatment, which is about 10 times that of water.

[0082] Table 1 Removal rate of cypermethrin residues on fruits and vegetables in different cleaning methods

[0083]

[0084] Note: “-” means no subtraction.

[0085] In summary, the present invention provides a method for preparing a cypermethrin-degrading esterase. The present invention is based on the advantages that the esterase expressed by the gene Est13 has a stable protein structure, is resistant to low temperatures, and has good pyrethroid degradation function. The esterase is selected from a food-grade host bacteria, Saccharomyces cerevisiae, to construct a eukaryotic expression system. No toxic byproducts are generated during the entire expression process. The activity and quality stability of the esterase produced by the genetically engineered bacteria of Saccharomyces cerevisiae can be effectively improved, and the degradation rate of residual pyrethroid pesticides in agricultural products can be significantly improved, and it can be applied to the food field more safely and greenly.

Claims

1. A method for preparing a cypermethrin-degrading esterase, characterized in that: The method comprises the following steps: (a) inoculating genetically engineered yeast Saccharomyces cerevisiae into a culture medium, fermenting, and obtaining a fermentation liquid; (b) transferring the fermentation broth to a culture medium, inducing expression under the action of an inducer and a carbon source, collecting the bacterial cells, breaking the bacterial cells, centrifuging, collecting the supernatant, and obtaining cypermethrin-degrading esterase; The genetically engineered yeast of Saccharomyces cerevisiae contains a gene encoding a cypermethrin-degrading esterase.

2. The method according to claim 1, characterized in that In step (a), the culture medium is a SD-H liquid culture medium containing glucose, the added amount of glucose is 0.1% to 5%, preferably 0.5%; the liquid volume of the SD-H liquid culture medium is 5 to 30%, preferably 20%; In step (b), the volume ratio of the fermentation broth to the culture medium is 1:0.5-1.5, preferably 1:1; the culture medium is SGR-H culture medium; the inducer is galactose, and the addition amount is 1-3%, preferably 2%; the carbon source is raffinose, and the addition amount is 0.5-1.5%, preferably 1%; The NCBI accession number of the gene encoding cypermethrin-degrading esterase is OR400756.

3. The method according to claim 1, characterized in that In step (a), the inoculation amount of the genetically engineered yeast of Saccharomyces cerevisiae is 0.5-2.5%, preferably 1%; the fermentation broth is the fermentation broth washed with phosphate buffer; the fermentation conditions are: at 20-40°C, at 100-250rpm for 2-48h, preferably: at 30°C, at 220rpm for 4h.

4. The method according to claim 1, characterized in that: In step (b), the conditions for inducing expression are: a rotation speed of 100-250 rpm, an induction temperature of 20-40°C, and an induction time of 6-48 h, preferably: a rotation speed of 220 rpm, an induction temperature of 30°C, and an induction time of 12 h.

5. The method according to claim 1, characterized in that In step (a), the method for preparing the genetically engineered yeast Saccharomyces cerevisiae comprises the following steps: (1) Construction of recombinant plasmid; (2) The recombinant plasmid is transferred into Saccharomyces cerevisiae cells to obtain genetically engineered Saccharomyces cerevisiae.

6. The method according to claim 5, characterized in that In step (1), the recombinant plasmid comprises an expression vector and a gene encoding a cypermethrin-degrading esterase, and the expression vector is pESC-HIS; In step (2), the Saccharomyces cerevisiae cell is a Saccharomyces cerevisiae INVSc1 cell.

7. The method according to claim 6, characterized in that In step (1), the preparation method of the recombinant plasmid comprises the following steps: the expression vector and the gene encoding the cypermethrin-degrading esterase are connected in a PCR instrument at 10 to 25° C. for 5 to 20 hours under the action of DNA ligase to obtain the recombinant plasmid.

8. The method according to claim 7, characterized in that In step (1), the preparation method of the recombinant plasmid comprises the following steps: the DNA ligase is T4 DNA ligase; the ligation is performed at 16° C. in a PCR instrument for 7 to 17 hours to obtain a recombinant plasmid.

9. A method for testing the activity of cypermethrin-degrading esterase, characterized in that: The method comprises the following steps: a buffer solution, a cypermethrin-acetone solution and a cypermethrin-degrading esterase constitute a reaction system, after the degradation is completed, acetonitrile is added, ultrasound is performed, centrifugation is performed, supernatant is filtered, and detection is performed; The degradation temperature is 0-45°C and the degradation time is 10-150 minutes.

10. The method according to claim 9, characterized in that The degradation temperature is 25° C. and the degradation time is 120 min.

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