Recombinant plasmid vector, preparation method of enzyme for hydrolyzing organophosphate, carbamate and pyrethroid insecticides and preparation using enzyme as functional component

Abstract

The invention relates to a process for producing three recombinases capable of hydrolyzing organophosphorus, carbamate and pyrethroid insecticides, and preparations containing these enzymes. The present invention provides recombinant plasmid DNA vectors in which the protein secretion system has been modified to improve the ability of recombinant proteins to secrete into the space around the plasmid or culture medium. A nucleotide sequence for coding a PelB (butyric acid lyase B) signal sequence is deleted and substituted by a nucleotide sequence for coding a LamB (maltodextrin or phage lambda receptor) signal sequence, so that the secretion efficiency of the recombinant protein is higher. The invention discloses three recombinant plasmid DNA (deoxyribonucleic acid) vectors which are constructed by utilizing the improved plasmid DNA vectors and are used for expressing recombinant proteins. The recombinant protein comprises (a) a recombinant organophosphate hydrolase having at least 80% sequence identity with the amino acid sequence of methyl parathion hydrolase (MPH) (Protein Database (PDB) ID: 1P9E) as shown in SEQ ID NO: 1, wherein the recombinant organophosphate hydrolase has hydrolytic activity on organophosphate insecticides; (b) a recombinant carbamate hydrolase having at least 80% sequence identity with the amino acid sequence of L-aminopeptidase-D-Ala-esterase / amidase (aryl amidase) (MAH) (PDB ID: 1B65), as shown in SEQ ID NO: 2, wherein the recombinant carbamate hydrolase has hydrolytic activity on carbamate insecticides; and (c) a recombinant pyrethroid hydrolase having at least 80% sequence identity with the amino acid sequence of the pyrethroid hydrolytic carboxylesterase (EstA) (PDB ID: 4CCY) as shown in SEQ ID NO: 3, wherein the recombinant pyrethroid hydrolase has hydrolytic activity on a pyrethroid insecticide. The embodiment of the invention further provides three recombinant bacterial strains, an escherichia coli strain BL21 (DE3) is used as a host cell, and each recombinant plasmid DNA vector is used for transformation. The invention also discloses culture conditions of the recombinant strain, wherein the culture conditions comprise culture medium composition, induction conditions and enzyme harvesting and enzyme activity detection methods. In addition, the embodiment of the invention also provides a preparation which contains the three recombinases as functional components.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of enzyme biotechnology. Background Art

[0002] Insecticides, including pesticides, are synthetic chemicals widely used in modern agriculture to eliminate pests during crop production and post-harvest in order to maintain crop yield and quality. In addition, they are used to prevent insect infestation in some types of dried foods (e.g., dried fish, beef jerky, etc.). The intensive use of these harmful chemicals in agriculture leads to their contamination and accumulation in agricultural soil and water, crop products, and food, thereby posing a threat to the health of farmers and consumers. Long-term pesticides are harmful to humans through consumption and accumulation, even at low concentrations, because these pesticides can cause acute and / or chronic toxicity. Acute toxicity symptoms include stimulation of the respiratory system, digestive system, nervous system, and muscles. Chronic toxicity symptoms include adverse effects on the liver, kidneys, immune system, and endocrine glands (hormone system); genetic changes and mutations that may cause cancer; entomopathogenic effects; and stopped brain development in infants and young children. In addition, the rejection of agricultural products with excessive pesticide residues by trading partner countries has a negative impact on the economics of exporting companies and related exporting countries.

[0003] There are several standards for classifying pesticides. Pesticides can be divided into eight groups based on their form or compound state [1], such as water dispersible powders (WDP), emulsifiable concentrates (EC), and suspension concentrates (SC) [Lin et al. (2023) Microbiology, 11, 364]; [2] at least 29 groups based on their mechanism of action, such as acetylcholinesterase inhibitors, which affect the nervous system, cause sodium balance disorders, and act as chloride channel blockers [Insecticide Resistance Action Committee, IRAC (2023): irac-online.org]; and [3] four categories based on their chemical type or chemical structure, namely (i) organophosphorus, i.e. compounds with phosphorus as the main substituent; (ii) carbamates, i.e. compound derivatives of carbamic acid; (iii) pyrethroids, i.e. synthetic derivatives of natural pyrethroids; and (iv) organochlorines, i.e. compounds containing at least one carbon-chlorine bond [Zacharia, JT (2011) ISBN 978-953-51-5154-8]. Due to their high toxicity to humans and their high persistence in the environment, organochlorines are banned in most countries.

[0004] Organophosphate, carbamate, and pyrethroid insecticides are widely used in agriculture and residential settings. Organophosphate and carbamate residues have been reported in random samples of vegetables and fruits from fresh markets and supermarkets in amounts exceeding legal limits, including kale, morning glory [Wanwimolruk et al. (2015) Environ Health Prev Med 20(3), 204; Wanwimolruk et al. (2016) Peer J 4, 2432], mangosteen [Phopin et al. (2017) J Sci Food Agric 97, 832], and mushrooms [Phutphat et al. (2016)]. Organophosphate and carbamate insecticides are potent inhibitors of acetylcholinesterase, an enzyme that hydrolyzes acetylcholine and plays an important role in the normal functioning of the central nervous system in humans and mammals. Exposure to these pesticides at low concentrations can cause nausea, dizziness, vomiting, cramps, convulsions, respiratory failure, sympathetic hyperexcitation, skeletal muscle paralysis or coma. If subjects are exposed to high concentrations of organophosphates and carbamates without treatment, they may die within 24 hours. Long-term exposure can cause brain cancer and leukemia.

[0005] The presence, persistence and bioaccumulation of pesticide residues in the environment have serious adverse effects not only on humans and organisms in the food chain, but also on the ecosystem. This leads to the contamination of soil, water and sediments. Soil contamination in agricultural areas can cause soil infertility and have adverse effects on plant growth, development and yield. In addition, the contamination of crop products with these residues, especially when the concentration exceeds the standard limit, often leads to the rejection of these products at border checkpoints, resulting in economic losses.

[0006] Current methods of washing fruits and vegetables vary in their effectiveness in removing pesticides. The Pesticide Division of the Department of Agriculture, Ministry of Agriculture and Cooperatives of Thailand reported that washing with running water for two minutes reduced pesticide residues in fruits and vegetables by 25-39%, heating reduced them by 48-50%, and soaking in a chemical detergent for 10 minutes followed by washing with running water reduced them by 22-36%. https: / / shorturl.at / dtJV7]. However, these washing techniques also have some disadvantages. Washing fruits and vegetables with running water must last for a long time and requires a large amount of water, which cannot be reused. Conversely, the residues of chemicals used to wash fruits and vegetables may cause harm to consumers or have a negative impact on the quality of the above-mentioned agricultural products. For example, especially in factories, exposure to high concentrations of sodium bicarbonate residues from chemical washing may cause severe headaches, diarrhea, nausea or vomiting, irritability and respiratory restrictions. Inhalation of potassium permanganate and chlorine vapors may even irritate the nose, throat and lungs of factory operators, causing coughing and / or shortness of breath, while contact with the residues of these chemicals can harm the digestive system and may cause blindness if they come into contact with the eyes. Certain washing techniques can also reduce the quality of crops. For example, washing fruits and vegetables with vinegar can cause fruits and vegetables to wilt, while also leaving the agricultural products with an unpleasant taste and pungent odor. In addition, heating, steaming or boiling fresh fruits and vegetables in water only reduces their nutritional value. Therefore, this method is not suitable for exporting fresh crops.

[0007] In addition to physical and chemical treatments, enzyme-catalyzed biological reactions can also effectively degrade pesticides. Enzymes are natural protein biomacromolecules that are harmless to humans and the environment. Therefore, pesticide-degrading enzymes can be used as functional ingredients in fruit and vegetable cleaning agents to minimize pesticide residues on fruits and vegetables before and after harvest. In addition, they can be used as bioremediation agents to treat groundwater and surface water contaminated by pesticides to improve the quality of drinking water.

[0008] According to scientific research, various microbial enzymes are able to catalyze the hydrolysis of organophosphate, carbamate, and pyrethroid insecticides [Bhatt et al. (2021) J Haz Mat 411(5), 125026]. These enzyme catalytic activities occur naturally in microbial metabolism (such as metabolism in bacteria) to transform and detoxify insecticide molecules and metabolize them into carbon and energy sources. Nevertheless, the catalytic properties and efficiency of these insecticide-degrading enzymes vary, and most of them are intracellular enzymes, which limits their application.

[0009] Heterologous gene expression and transformation methods in bacterial host cells (such as Escherichia coli and Bacillus subtilis) have been disclosed in the prior art [Kataoka et al. (2011) AMB Express 1(1), 1; Kataoka et al. (2013) J Biosci Bioeng 115(5), 475; Mahipant et al. (2019) J Gen Appl Microbiol 65, 96]. In general, recombinant host cells must meet important criteria, such as producing the protein or enzyme of interest at a high or acceptable yield, being able to efficiently secrete the protein or enzyme, and being easy to operate and maintain.

[0010] The present embodiment relates to a process for producing recombinant enzymes, each of which is capable of hydrolyzing organophosphate, carbamate and pyrethroid insecticides, and formulations thereof. It also relates to the construction of plasmid DNA vectors for the extracellular expression of insecticide hydrolases in recombinant host cells (i.e., Escherichia coli strain BL21 (DE3)) so that these enzyme cocktails can be used as functional ingredients in the formulation of final products in the desired proportions. Summary of the invention

[0011] Embodiments of the present invention relate to a method for producing three recombinant enzymes capable of hydrolyzing each class of organophosphate, carbamate and pyrethroid insecticides and formulations containing these enzymes.

[0012] The present invention provides a recombinant plasmid DNA vector in which the protein secretion system has been modified to enhance the secretion of the recombinant protein into the space surrounding the plasmid or into the culture medium. The nucleotide sequence encoding the PelB (tyric acid lyase B) signal sequence is deleted for peri-plastid localization and replaced with a nucleotide sequence encoding the LamB (maltose porin or bacteriophage lambda receptor) signal sequence to make the secretion of the recombinant protein more efficient.

[0013] The present invention discloses three recombinant plasmid DNA vectors for expressing recombinant proteins constructed using the above improved plasmid DNA vector. The recombinant proteins include the following: (a) a recombinant organophosphate hydrolase having at least 80% sequence identity with the amino acid sequence of methyl parathion hydrolase (MPH) (Protein Data Bank (PDB) ID: 1P9E) shown in SEQ ID NO: 1, wherein the recombinant organophosphate hydrolase has hydrolysis activity on organophosphate pesticides. (b) a recombinant carbamate hydrolase having at least 80% sequence identity with the amino acid sequence of L-aminopeptidase-D-Ala-esterase / amidase (arylamidase) (MAH) (PDB ID: 1B65) shown in SEQ ID NO:2, wherein the recombinant carbamate hydrolase has hydrolysis activity against carbamate pesticides. (c) a recombinant pyrethroid hydrolase having at least 80% sequence identity with the amino acid sequence of pyrethroid hydrolyzing carboxyl esterase (EstA) (PDB ID: 4CCY) shown in SEQ ID NO: 3, wherein the recombinant pyrethroid hydrolase has hydrolysis activity against pyrethroid insecticides.

[0014] This embodiment further provides three recombinant bacterial strains, using the Escherichia coli strain BL21 (DE3) as a host cell and transformed with each of the above-mentioned recombinant plasmid DNA vectors.

[0015] The embodiments of the present invention also disclose the culture conditions of the above-mentioned recombinant strain, including culture medium composition, induction conditions, and enzyme harvesting and enzyme activity detection methods.

[0016] In addition, an embodiment of the present invention provides a preparation comprising the above three recombinant enzymes as functional components for hydrolyzing organophosphate, carbamate and pyrethroid insecticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A map of the 6646-base pair expression vector pEL-OP is shown, which consists of a modified pET22b(+) with a LamB signal sequence and the gene encoding MPH for production of the recombinant enzyme methylparathion hydrolase in bacterial host cells.

[0018] Figure 2 Shown is a map of the 7144-base pair expression vector pEL-CM, which consists of modified pET22b(+) with a LamB signal sequence and the gene encoding MAH for production of the recombinant enzyme L-aminopeptidase-D-Ala-esterase / amidase (arylamidase) in bacterial host cells.

[0019] Figure 3 Shown is a map of the 6418-base pair expression vector pEL-PY, which consists of a modified pET22b(+) with a LamB signal sequence and a gene encoding EstA, for production of recombinant pyrethroid hydrolyzing carboxylesterase in bacterial host cells.

[0020] Figure 4 The protein expression of crude enzymes extracted from recombinant host cells during fermentation in a 5-liter fermentor for 0 to 16 hours is shown, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of each recombinant enzyme is indicated, namely 34.8 kDa methylparathion hydrolase or MPH, 56.5 kDa L-aminopeptidase-D-Ala-esterase / amidase or arylamidase, and 30 kDa pyrethroid hydrolyzing carboxylesterase. DETAILED DESCRIPTION

[0021] As used herein, the singular forms "a", "an", "and", and "the" include plural nouns unless the context clearly dictates otherwise. Thus, "a cell" includes a plurality of cells, and "a protein" includes one or more proteins and their equivalents. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary ability and skill in the art to which the present disclosure belongs.

[0022] The present invention relates to the recombinant expression of three enzymes consisting of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in Escherichia coli BL21 (DE3) host cells. As a result of the above recombinant expression, the cells have the ability to produce and secrete the three recombinant enzymes, and ultimately produce a preparation containing these enzymes. Each of the three recombinant enzymes can hydrolyze a specific class of pesticides, namely organophosphates, carbamates and pyrethroids. Therefore, the above recombinant enzymes and preparations can be used to biodegrade and detoxify organophosphate, carbamate and pyrethroid pesticide residues on the surface of fruits and vegetables and in pesticide-contaminated environments.

[0023] The term "methylparathion hydrolase" refers to an enzyme whose systematic name is aryl dialkyl triphosphate phosphohydrolase and whose Enzyme Commission number is EC3.1.8.1. This enzyme catalyzes the hydrolysis of organophosphorus compounds (including phosphonic acids and phosphonic esters). The "recombinant methylparathion hydrolase" in the embodiments of the present invention is a methylparathion hydrolase that is naturally expressed in bacteria other than the host cell of the present invention, i.e., a heterologous methylparathion hydrolase whose amino acids are not modified or are preferably modified by one or more deletions, insertions or substitutions. In a preferred embodiment, the recombinant methylparathion hydrolase is a naturally occurring methylparathion hydrolase derived from bacteria other than the host cell of the present invention, preferably Ochrobactrum sp.

[0024] The recombinant methyl parathion hydrolase disclosed herein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. The amino acid sequence of the recombinant methyl parathion hydrolase can be obtained from the amino acid sequence of methyl parathion hydrolase of Gram-negative bacteria, such as Ochrobactrum sp., Stutzerimonas stutzeri, Pseudomonas putida, Pseudomonas sp., Caballeronia zhejiangensis, Stenotrophomonas sp., Achromobacter sp., Stenotrophomonas maltophilia, Burkholderia sp. and Cupriavidus sp.

[0025] The term "L-aminopeptidase-D-Ala-esterase / amidase or aryl amide enzyme" refers to an enzyme whose systematic name is D-stereospecific aminopeptidase and whose Enzyme Commission number is EC3.4.11.19. This enzyme catalyzes the hydrolytic cleavage of amide bonds. Substrates with amide groups include D-amino acid amides, methyl esters and amide pesticides, such as carbamate pesticides and herbicides, organophosphorus pesticides with amide groups and acetanilide herbicides. The "recombinant L-aminopeptidase-D-Ala-esterase / amidase or aryl amide enzyme" in the embodiments of the present invention is an L-aminopeptidase-D-Ala-esterase / amidase or aryl amide enzyme that is naturally expressed in bacteria other than the host cell of the embodiments of the present invention, i.e., a heterologous L-aminopeptidase-D-Ala-esterase / amidase or aryl amide enzyme, whose amino acids are not modified or have been modified, preferably by one or more deletions, insertions or substitutions. In a preferred embodiment, the recombinant L-aminopeptidase-D-Ala-esterase / amidase or arylamidase is a naturally occurring enzyme derived from bacteria other than the host cell of the embodiments of the present invention, preferably from Ochrobactrum sp.

[0026] The recombinant L-aminopeptidase-D-Ala-esterase / amidase or aryl amidase of the present disclosure comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2. The amino acid sequence of the recombinant L-aminopeptidase-D-Ala-esterase / amidase or aryl amidase can be selected from Gram-negative bacteria, such as Ochrobactrum sp, Gemmobacternanjingensis and Starkeya sp.

[0027] The term "pyrethroid hydrolyzing carboxyl esterase" refers to an enzyme whose systematic name is pyrethroid hydrolase and whose Enzyme Commission number is EC3.1.1.88. This enzyme catalyzes the hydrolysis of pyrethroid insecticides. The "recombinant pyrethroid hydrolyzing carboxyl esterase" in the embodiments of the present invention is a pyrethroid hydrolyzing carboxyl esterase that is naturally expressed in bacteria other than the host cells of the embodiments of the present invention, i.e., a heterologous pyrethroid hydrolyzing carboxyl esterase, whose amino acids are not modified or are preferably modified by one or more deletions, insertions or substitutions. In a preferred embodiment, the recombinant pyrethroid-hydrolyzing carboxyl esterase is a naturally occurring pyrethroid-hydrolyzing carboxyl esterase derived from bacteria other than the host cells of the embodiments of the present invention, preferably from Bacillus.

[0028] The recombinant pyrethroid-hydrolyzing carboxyl esterase disclosed in the present invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, and the amino acid sequence of the recombinant pyrethroid-hydrolyzing carboxyl esterase can be obtained from the amino acid sequence of pyrethroid-hydrolyzing carboxyl esterase of Gram-positive bacteria, such as Bacillus, Bacillus subtilis, Bacillus thuringiensis, Bacillus Widmannii and Bacillus trophotrophicus.

[0029] The term "signal sequence" refers to a short N-terminal amino acid sequence that mediates the transfer of any linked or fused polypeptide or protein or recombinant protein to the secretory pathway of a cell or host cell. The signal sequence of the present invention is a 75-base nucleotide sequence encoding the amino acid sequence of LamB (maltose porin or bacteriophage lambda receptor), which is identical to the maltose porin of Gram-negative bacteria and can be obtained from Escherichia coli, Salmonella enterica, Klebsiella pneumoniae or Shigella sonnei.

[0030] The terms "nucleic acid construct" and "gene construct" are used interchangeably herein to refer to nucleic acid molecules isolated from naturally occurring genes, nucleic acid molecules synthesized according to natural gene sequences, or nucleic acid molecules modified in a specific manner to contain nucleic acid fragments. When the nucleic acid construct contains the control sequences required for expressing the recombinase coding sequence, "nucleic acid construct" and "gene construct" are synonymous with "expression cassette".

[0031] The terms "expression vector" and "expression construct" are used interchangeably herein. An expression vector is a plasmid DNA vector designed for gene expression, which produces a protein encoded by a gene by housing a protein synthesis machinery, and is therefore used to introduce a specific gene into a host cell. The expression vector described in the present disclosure is constructed using an expression vector pET22b(+) with a modified signal sequence, in which the nucleotide sequence encoding PelB is deleted and replaced with a nucleotide sequence encoding LamB to produce a more efficient secreted recombinant protein. The expression construct is operably linked to other nucleotides for expression and is introduced into a host cell so that the expression vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0032] The expression vector is used to produce recombinant methyl parathion hydrolase (MPH) by any method currently recognized in the art. The method described in the present disclosure includes codon optimization, gene synthesis, cloning the gene encoding methyl parathion hydrolase (MPH) into a modified expression vector creating pEL-OP according to SEQ ID NO: 1, and transformation into a bacterial host cell. The expression vector is transformed into the host cell Escherichia coli DH5-α in order to increase the number of expression vectors and evaluate the accuracy of the expression construct. Subsequently, the expression vector is transformed into the host cell Escherichia coli BL21 (DE3), and the host cell is then cultured under the conditions described in the present disclosure to produce secreted methyl parathion hydrolase (MPH).

[0033] The expression vector is used to produce recombinant L-aminopeptidase-D-Ala-esterase / amidase or aryl amidase (MAH) by any method currently accepted in the art. The method described in the present disclosure includes codon optimization, gene synthesis, cloning the gene encoding L-aminopeptidase-D-Ala-esterase / amidase or aryl amidase (MAH) according to SEQ ID NO:2 into a modified expression vector for creating pEL-CM, and transforming into a bacterial host cell. The expression vector is transformed into a host cell (Escherichia coli DH5-α) for multiplying the number of expression vectors and evaluating the accuracy of the expression construct. Subsequently, the expression vector is transformed into a host cell Escherichia coli BL21 (DE3), and the host cell is then cultured under the conditions described in the present disclosure to produce secretory L-aminopeptidase-D-Ala-esterase / amidase or aryl amidase (MAH).

[0034] The expression vector is used to produce recombinant pyrethroid hydrolyzing carboxyl esterase (EstA) by any method currently accepted in the art. The method described in the present disclosure includes codon optimization, gene synthesis, cloning the gene encoding pyrethroid-hydrolyzing carboxyl esterase (EstA) into a modified expression vector creating pEL-PY according to SEQ ID NO:3, and transforming into a bacterial host cell. The expression vector is transformed into the host cell Escherichia coli DH5-α for breeding the number of expression vectors and evaluating the accuracy of the expression construct. Subsequently, the expression vector is transformed into the host cell Escherichia coli BL21 (DE3), and the host cell is then cultured under the conditions described in the present disclosure to produce secreted pyrethroid hydrolyzing carboxyl esterase (EstA).

[0035] The host cells described in the present disclosure express functional recombinant enzymes, including methyl parathion hydrolase (MPH), L-aminopeptidase-D-Ala-esterase / amidase or arylamidase (MAH) and pyrethroid hydrolyzing carboxylesterase (EstA). Such host cells are capable of secreting the recombinant enzymes into the extracellular medium. The term "functional" refers to the ability of the expressed enzyme to hydrolyze its insecticide substrate, i.e., to hydrolyze organophosphates, carbamates and pyrethroids, respectively. The activity of each enzyme can be measured by any appropriate method known in the art to assess the hydrolysis activity to each substrate group, preferably using the method of the embodiments of the present invention described below.

[0036] The first aspect of the embodiment of the present invention refers to three expression vectors, namely pEL-OP, pEL-CM and pEL-PY, which preferably constitute a group. In addition, these three recombinases are expressed by the host cell of the present invention. Preferably, the above three recombinases consist of enzymes having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, as shown in Table 1.

[0037] The culture medium and conditions of the host cells used to produce the recombinase are in accordance with methods known in the art. For example, in a laboratory or industrial bioreactor, the host cells are cultured in a suitable culture medium and under conditions capable of expressing the recombinase by shaking flask culture and small-scale or large-scale fermentation (including continuous fermentation, batch fermentation or feeding batch fermentation). The culture is carried out in a suitable nutrient medium consisting of a carbon source, a nitrogen source, an inorganic salt and an antibiotic. The seed culture culture conditions described in the present disclosure include: at 35 to 37 degrees Celsius, a shaking speed of 200 rpm, in a LuriaBertani (LB) medium containing 100-micrograms / ml ampicillin, 1% to 5% (volume ratio) of the inoculum is used to culture the host cells producing each recombinase for 16 hours. The seed culture of each recombinant host strain is cultured separately or together in a volume ratio of 1:1:1.

[0038] The second aspect of the embodiments of the present invention relates to the cultivation of recombinant host cells and the induction of enzyme production. The cultivation of the enzyme production stage described in the present disclosure requires the recombinant host cells to grow in SPY medium so as to produce each recombinant enzyme under specified conditions. The pH value of the SPY medium is 7 to 8, and contains a composition of one or more ingredients, including 1 to 10 grams per liter of soy peptone, soy flour or soy protein; 1 to 10 grams per liter of yeast extract; and / or 5 to 10 grams per liter of sodium chloride. The above ingredients also include one or more coenzymes, including 1 to 10 millimoles of trace elements magnesium chloride, cobalt chloride, dicobalt chloride, copper chloride, manganese chloride and / or ferric chloride. At least one component that stimulates enzyme expression is also added to the composition, wherein the component includes 1 to 10 millimoles of galactose or 5 to 20 millimoles of lactose. Adding 100 micrograms / ml of ampicillin to the composition is optional, but more desirable. At this stage, the seed culture can be inoculated in one of two ways: either 5% (volume ratio) of the three recombinant host strains are inoculated separately, or 15% (volume ratio) of the three recombinant host strains are inoculated at a volume ratio of 1:1:1 under the above conditions. The recombinant cells are grown at 35 to 37 degrees Celsius and a shaking speed of 200 rpm for 3 to 4 hours until the cell optical density measured by a spectrophotometer at a wavelength of 600 nanometers reaches 0.6 to 0.8.

[0039] The second aspect further includes the induction conditions for enzyme expression and production. The induction conditions for the recombinant host cells used to produce enzymes described in the present disclosure include the addition of an enzyme expression induction composition containing one or more components, i.e., 1 to 10 millimoles of galactose or 5 to 20 millimoles of lactose, in the above-mentioned recombinant cells grown, in addition to the enzyme secretion enhancement solution, 5% to 20% (weight to volume) of sucrose, 1% to 2% (weight to volume) of glycine and 1% to 2% (weight to volume) of Tween-20. The recombinant cells are further grown at 30 to 37 degrees Celsius and a shaking speed of 200 rpm for at least 16 to 20 hours. Enzyme activity is analyzed at intervals using the method described in the following example. Methods well known in the art, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be selected to determine the expression of the protein. At a specified time, the recombinant cells are removed by centrifugation or 0.22-micron membrane filtration under conditions of 4 to 10 degrees Celsius. The recovery of the crude enzyme product adopts a membrane filtration method.

[0040] The third aspect of the present invention relates to formulating three recombinant enzymes as functional ingredients into a liquid form for manufacturing a final product. The formulation is prepared in a 10 mM glycine buffer at a pH of 7.5-8.5 and consists of 30-50% by volume of methyl parathion hydrolase, 5-15% by volume of L-aminopeptidase-D-Ala-esterase / amidase (Arylamidase) and 5-15% by volume of pyrethroid hydrolyzing carboxylesterase and 30-40% by volume of glycerol as a stabilizer. Example Example 1. Detection of the enzymatic activity of methylparathion hydrolase by hydrolyzing an organophosphorus substrate.

[0041] The amino acid sequence of methylparathion hydrolase (EC3.1.8.1) is shown in SEQ ID NO: 1, and is a hydrolase that catalyzes the hydrolysis of organophosphorus compounds (including phosphonic acids and phosphonic esters). The activity of the enzyme is measured using the same procedure as glucoamylase activity (amyloglucosidase activity) using a specific substrate for methylparathion hydrolase in accordance with the guidelines in the Food Additives Specification and Food Chemical Codex.

[0042] Methylparathion hydrolase activity is determined using methylparathion (O,O-dimethyl O-(4-nitrophenyl) phosphate; CAS No. 950-35-6) as a substrate comparable to organophosphorus compounds. In this assay, the enzyme reaction mixture (final volume of 750 μl) containing 50 to 100 mM Tris-HCl buffer (pH 8.0), 0.1 mM cobalt chloride, 0.8 mM methylparathion and the appropriate amount of the corresponding enzyme (protein concentration of approximately 0.8 ± 0.2 mg per ml) is incubated at 37 ± 2 degrees Celsius for 5 minutes. The amount of p-nitrophenol released as a hydrolysis product (yellow product) is measured using a spectrophotometer at an absorbance wavelength of 410 nm. Enzyme activity is calculated using a p-nitrophenol standard curve or the formula shown in the figure below, with a molar extinction coefficient (ε) of 11,933 M -1 cm -1 One unit of methylparaben hydrolysis activity was defined as the amount of enzyme required to release one trace of p-nitrophenol per minute. The protein concentration of the enzyme was determined using the Lowry protein assay (Thermo Scientific TM The specific activity of the enzyme is determined by the Pierce23240) or BCA method (AppliChem, A7787 0500), and bovine serum albumin is used as the protein standard curve. The specific activity of the enzyme is defined as the unit of enzyme per milligram of protein. The recombinant methyl parathion hydrolase produced under shake flask conditions can produce 10 to 15 units per milliliter, while the recombinant methyl parathion hydrolase produced in 5 liter to 300 liter fermenters can produce 45 to 50 units per milliliter. Example 2. Detection of the enzyme activity of L-aminopeptidase-D-Ala-esterase / amidase or arylamidase by hydrolysis of carbamate substrates.

[0043] L-aminopeptidase-D-Ala-esterase / amidase or arylamidase (EC3.4.11.19), whose amino acid sequence is shown in SEQ ID NO:2, is a hydrolase that catalyzes the hydrolytic cleavage of the amide bond of substrates having an amide group, including D-amino acid amides, methyl esters and amide pesticides, such as carbamate pesticides and herbicides, organophosphorus pesticides having an amide group and acetanilide herbicides. Enzyme activity assays were performed according to the reference methods described in Ozturk et al. (2016) Environmental Microbiology 18(12), 4878-4887 (doi.org / 10.1111 / 1462-2920.13409) and Zhang et al. (2019) Ecotoxicology and Environmental Safety 15(167), 122-129 (doi: 10.1016 / j.ecoenv.2018.09.127.).

[0044] For the L-aminopeptidase-D-Ala-esterase / amidase or arylamidase activity assay, the enzyme reaction mixture (final volume of 1 ml), containing 10 to 50 mM glycine buffer (pH 8.0), 0.8 to 1.0 mM carbaryl as a representative of carbamate insecticides and an appropriate amount of the corresponding enzyme (protein concentration of approximately 0.8 ± 0.2 mg per ml), was incubated for 15 min at 37 ± 2 degrees C. The amount of carbaryl hydrolyzed or the amount of 1-naphthol released as a hydrolysis product was measured using a reversed-phase high performance liquid chromatography (HPLC) equipped with a photodiode array detector, equipped with a C18 column (4.6 mm inner diameter, 250 mm length) and an acetonitrile-water mobile phase (70:30 by volume) at a flow rate of 1 ml per minute. The monitoring wavelengths were 212 nm and 280 nm, at which the retention times of carbaryl and 1-naphthol were 10.30 ± 0.09 min and 11.66 ± 0.06 min, respectively. The reduction in substrate concentration (carbaryl; Merck 32055; CAS No. 63-25-2) or the formation of product (1-naphthol; Merck N100; CAS No. 90-15-3) was determined by the HPLC peak height ratio relative to its standard calibration curve. The protein concentration of the enzyme was determined by Lowry protein assay (Thermo Scientific TMThe specific activity of the enzyme is defined as the units of enzyme per milligram of protein. Under shake flask conditions, 3 to 5 units of enzyme can be produced per milliliter of recombinant arylamidase (MAH), while 10 to 15 units of enzyme can be produced per milliliter in a 5- to 300-liter fermenter. Example 3. Detection of the enzymatic activity of pyrethroid hydrolyzing carboxyl esterase by hydrolyzing pyrethroid substrate.

[0045] The pyrethroid hydrolyzing carboxyl esterase (EC3.1.1.88) having the amino acid sequence shown in SEQ ID NO: 3 is an enzyme capable of catalyzing the hydrolysis of pyrethroid pesticides. The enzyme activity was determined using a colorimetric method according to the reference method described in Pengpumkiat et al. (2020) Sensors 2020, 20, 4107 (doi: 10.3390 / s20154107).

[0046] Cypermethrin (Merck 239900; CAS No. 52315-07-8) was used as a representative of pyrethroid insecticides to determine the pyrethroid hydrolyzing carboxylesterase activity. The enzyme reaction mixture (final volume of 1 ml) contained 10 to 50 mM glycine buffer (pH 8.6), 0.8 to 1.0 mM cypermethrin and an appropriate amount of the corresponding enzyme (protein concentration of approximately 0.8 ± 0.2 mg per ml) and was incubated at 37 ± 2 degrees Celsius for 5 minutes. Then, ammonium acetate (10% by volume) and ninhydrin solution in ethanol (4% by volume) were added to the mixture to react with the free cyanide released during the hydrolysis process to form a purple product. The purple product was quantified using a spectrophotometer with an absorption wavelength of 570 nm. The enzyme activity was calculated based on the amount of pyrethroid hydrolysis product using a cyanide standard calibration curve. The protein concentration of the enzyme was determined using the Lowry protein assay (Thermo Scientific TM The specific activity of the enzyme is defined as the units of enzyme per milligram of protein. The recombinant pyrethrin hydrolyzing carboxylesterase produced under shake flask conditions can produce 6 to 10 units per milliliter, while the recombinant pyrethrin hydrolyzing carboxylesterase produced in 5 to 300 liter fermenters can produce 15 to 20 units per milliliter.

Claims

1. A recombinant plasmid vector set for producing a recombinant enzyme capable of hydrolyzing organophosphates, carbamates and pyrethroid insecticides, the recombinant plasmid vector set comprising: a first recombinant vector having a gene encoding a first recombinase, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1; a second recombinant vector having a gene encoding a second recombinase, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2; and a third recombinant vector having a gene encoding a third recombinase, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3; and wherein each of the first recombinant plasmid vector, the second recombinant plasmid vector and the third recombinant plasmid vector is transformed into a single bacterial host cell, respectively; and wherein the single bacterial host cell is modified to express a recombinase. 2 . The recombinant plasmid vector set according to claim 1 , wherein the first recombinant enzyme having an amino acid sequence at least 80% identical to that of SEQ ID NO: 1 is a recombinant methyl parathion hydrolase.

3. The recombinant plasmid vector set according to claim 1, wherein the second recombinant enzyme having an amino acid sequence at least 80% identical to that of SEQ ID NO: 2 is a recombinant L-aminopeptidase-D-Ala-esterase / amidase or arylamidase. 4 . The recombinant plasmid vector set according to claim 1 , wherein the third recombinase having an amino acid sequence at least 80% identical to that of SEQ ID NO: 3 is a recombinant pyrethroid hydrolyzing carboxylesterase. The recombinant plasmid vector set according to claim 1 , wherein each of the single bacterial host cells is an Escherichia coli strain BL21 (DE3).

6. A method for producing a recombinant enzyme capable of hydrolyzing organophosphate, carbamate and pyrethroid insecticides, wherein the method comprises: The single bacterial host cell according to claim 5 is cultured in a culture medium, and the recombinant enzyme is collected from the culture medium.

7. A formulation for manufacturing a final product, said formulation comprising a recombinant enzyme capable of hydrolyzing organophosphate, carbamate and pyrethroid insecticides, wherein said recombinant enzyme is used as a functional ingredient of said formulation and is produced by a single bacterial host cell according to claim 5.

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