A hydrolase and its application in biocatalytic synthesis of 4″-methylamino-5′-hydroxy-avermectin

Abstract

The invention provides a hydrolase and application thereof in biocatalytic synthesis of 4"-methylamino-5'-hydroxy-avermectin. The amino acid sequence of the hydrolase is selected from one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. The hydrolase screened by the invention can replace the deprotection reaction in the synthesis process of emamectin benzoate with a biocatalytic method. 5-O-allylformyl-4"-methylamino-avermectin is used as a substrate, and the product 4"-methylamino-5'-hydroxy-avermectin is obtained by the hydrolysis of esterase, thereby avoiding a complicated chemical synthesis process and providing a new way for the biosynthesis of the product 4"-methylamino-5'-hydroxy-avermectin.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a hydrolase and application thereof in biocatalytic synthesis of 4″-methylamino-5′-hydroxy-avermectin. Background Art

[0002] Avermectin (AVM) is a highly effective anthelmintic and insecticide produced by the actinomycete Streptomyces avermitilis MA-4680. Structurally, it is a 16-membered pentacyclic lactone derivative. By modifying the structure of its parent, new derivatives with higher activity, stronger selectivity and better safety can be developed. Avermectin insecticides have become one of the best bio-source insecticides since the 1980s due to their high efficacy, unique insecticidal mechanism, broad insecticidal spectrum and safety for humans and animals. They have also become a major new insecticide variety across the fields of medicine (veterinary drugs) and pesticides. Since its discovery, it has spread rapidly around the world as a commercial veterinary drug to control various types of diseases caused by nematodes and arthropod parasites.

[0003] However, the original drug of avermectin is highly toxic. At present, the dosage of avermectin preparations in some areas has been too large, and the residues exceed the standard. Moreover, avermectin products are mainly emulsifiable concentrates, which emit a lot of organic matter and are not conducive to environmental safety. Relatively speaking, emamectin benzoate is a derivative of avermectin (its 4″ position is replaced by methylamino), which is moderately toxic, and the preparation has low toxicity and is not affected by wind and rain. Compared with avermectin, its activity against Lepidoptera is 1-3 levels higher. Emamectin benzoate has higher activity, less residue, lower toxicity, and better safety. Moreover, the preparation is advanced and beneficial to environmental protection.

[0004] The synthesis of emamectin benzoate in the prior art mainly includes steps such as protection, oxidation, amination, reduction and deprotection. The reaction process uses a variety of protective agents, oxidants, reducing agents, deacidifying agents, dehydrating agents and various catalysts. Each step is extremely strict on reaction temperature and pressure, and has high requirements on equipment. In addition, each step of the reaction produces waste gas and waste water. Moreover, the intermediate product needs to be separated and desolventized before the next step of reaction can be carried out. In short, the chemical synthesis of emamectin benzoate has the main problems of high cost and high pollution.

[0005] Biocatalysis, also known as biotransformation (bioconversion), refers to adding exogenous natural or synthetic organic compounds as substrates to an active biological system or enzyme system, and cultivating them under suitable conditions, so that the substrate interacts with the enzyme in the system, thereby producing structural changes. Its essence is an enzyme-catalyzed reaction. Biocatalytic reactions are characterized by mild conditions, high efficiency and high selectivity (chemical, regional and stereoselectivity), and are considered to be a resource-saving and environmentally friendly technology.

[0006] Esterase refers to a class of hydrolases that can catalyze the hydrolysis of ester bonds. The esterases usually referred to are carboxylesterases, which can hydrolyze carboxylates into alcohols and carboxylic acids. Esterases are widely available, most of which are animal pancreatic esterases and microbial esterases. As biocatalysts, microbial esterases have high catalytic activity, substrate and reaction specificity, and have been widely used in food, medicine, chemical industry, energy and environmental protection. Esterases can catalyze many reactions, especially they can complete esterification, transesterification, transesterification and other reactions in organic phases, and most of them have high stereoselectivity, and can specifically prepare many chiral compounds (such as pesticides, optically active drugs) and their precursors that are difficult to synthesize by chemical methods. In addition, these catalytic reactions have the advantages of mild reaction conditions, not easily affected by the environment, easy separation and purification of products, and no need for coenzymes. Therefore, topics related to esterases have become one of the hot spots in enzymology research.

[0007] From the perspective of the global pesticide market, the sales of biological pesticides and transgenic pesticides only account for 10% of the total global pesticide sales, but this figure is less than 5% in my country, which also shows that avermectin has a good market expansion space in my country. As farmers' awareness of the characteristics of avermectin and its derivative pesticide varieties increases, it is the most popular and competitive product in the current biological pesticide market. With the large-scale use of avermectin and avermectin on rice, the last major market development has been completed. It is expected that the direction of development will mainly be the market space for avermectin to replace avermectin.

[0008] In summary, the above existing methods have complicated steps; therefore, how to develop a new strain of bacteria for biotransformation has become a technical problem that needs to be solved urgently. Summary of the invention

[0009] The present invention aims to provide a catalytic product and its application in the biocatalytic synthesis of 4″-methylamino-5′-hydroxy-avermectin, and screen out a hydrolase, which can replace the deprotection reaction in the synthesis process of emamectin benzoate with a biocatalytic method. 5-O-allylformyl-4″-methylamino-avermectin is used as a substrate, and the product 4″-methylamino-5′-hydroxy-avermectin is obtained by the hydrolysis of esterase, which can avoid the complicated chemical synthesis process and provide a new way for the biosynthesis of the product 4″-methylamino-5′-hydroxy-avermectin.

[0010] In a first aspect of the present invention, a hydrolase is provided, wherein the amino acid sequence of the hydrolase is selected from one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

[0011] In a second aspect of the present invention, a nucleic acid molecule encoding the hydrolase is provided, wherein the nucleotide sequence of the nucleic acid molecule is selected from one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

[0012] In the third aspect of the present invention, a hydrolase expression vector is provided, wherein the expression vector comprises a nucleic acid molecule encoding the hydrolase.

[0013] Furthermore, the expression vector comprises one of a prokaryotic expression vector and a viral vector.

[0014] In a fourth aspect of the present invention, a method for preparing a hydrolase expression vector is provided, the method comprising:

[0015] Obtaining a target gene fragment of the nucleic acid molecule of the hydrolase;

[0016] The target gene fragment and the pET28α expression vector were double-digested with BamHI and XhoI, and then ligated to obtain the expression vector pET28α-ycaC.

[0017] In the fifth aspect of the present invention, a recombinant bacterium or an engineered cell line comprising the expression vector is provided.

[0018] In the sixth aspect of the present invention, there is provided an application of the hydrolase or the nucleic acid molecule or the expression vector or the recombinant bacterium or the engineered cell line in catalyzing the synthesis of 4″-methylamino-5′-hydroxy-avermectin from 5-O-allylformyl-4″-methylamino-avermectin.

[0019] In the sixth aspect of the present invention, a catalytic product is provided, comprising at least one of the hydrolase, the nucleic acid molecule, the expression vector, the recombinant bacteria or the engineered cell line.

[0020] In the seventh aspect of the present invention, a method for catalyzing 5-O-allylformyl-4″-methylamino-avermectin to synthesize 4″-methylamino-5′-hydroxy-avermectin is provided, the method comprising: using 5-O-allylformyl-4″-methylamino-avermectin as a substrate, and using the CAR protein to catalyze the synthesis product 4″-methylamino-5′-hydroxy-avermectin.

[0021] Furthermore, the temperature of the catalytic reaction is 25-35°C.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] The present invention provides a catalytic product and its application in biocatalytic synthesis of 4″-methylamino-5′-hydroxy-avermectin. The present invention screens out a hydrolase, which can replace the deprotection reaction in the synthesis process of emamectin benzoate with a biocatalytic method. 5-O-allylformyl-4″-methylamino-avermectin is used as a substrate, and the product 4″-methylamino-5′-hydroxy-avermectin is obtained by the hydrolysis of esterase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a diagram showing the double enzyme digestion identification results of the expression vector pET28α-ycaC in the example of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0029] The following is a detailed description of a catalytic product of the present application and its application in the biocatalytic synthesis of 4″-methylamino-5′-hydroxy-avermectin in combination with embodiments and experimental data.

[0030] Example 1 Construction of hydrolase expression vector and engineering bacteria

[0031] 1. Materials and Methods

[0032] 1. Test materials

[0033] E.coil DH5α and E.coil BL21 (DE3) strains were purchased from Beijing Biolab Technology Co., Ltd.; plasmid pET28α was purchased from Fenghui Biotechnology. BCA protein concentration determination kit was purchased from Beijing Solebow Technology Co., Ltd.

[0034] Trifluoroacetic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; agarose, tris(hydroxymethylaminomethane) hydrochloride, ethylenediaminetetraacetic acid, nucleic acid dye 4S Green, glycine, sodium dodecyl sulfate, bromophenol blue, glycerol, dithiothreitol, Coomassie brilliant blue, anhydrous ethanol, glacial acetic acid, acrylamide, N,N-methylenebisacrylamide, ammonium persulfate, N,N,N,N-tetramethylethylenediamine, sucrose, glycerol, and isopropyl-beta-D-thiogalactopyranoside were all purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0035] Kanamycin was purchased from Saiguo Biotechnology Co., Ltd.

[0036] Yeast extract, peptone, chromatography-grade methanol, chromatography-grade acetonitrile, protein marker, BamHI and XhoI restriction endonucleases were purchased from Thermo Fisher Scientific; nucleic acid marker was purchased from Anolun (Beijing) Biotechnology Co., Ltd.; plasmid minipreparation kit and gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0037] The synthesis of 5'-O-allylformyl-4"-methylamino-avermectin was carried out according to the method in the literature (Fan Liqiyan, Zhang Jianlin, Liu Yougang. A method for synthesizing emamectin benzoate with improved processability, Guangdong Chemical Industry, 2010, 37(209): 49-50, 54).

[0038] The standard product of 4″-methylamino-5′-hydroxy-avermectin (emamectin benzoate) was purchased from Shanghai Harling Biotechnology Co., Ltd.

[0039] LB medium (liquid, solid): 5 g·L-1 yeast powder, 10 g·L-1 tryptone, 10 g·L-1 sodium chloride. If LB solid medium is prepared, add 2% agar. Sterilize at 121°C under a pressure of 0.105 MPa for 20 min. When culturing recombinant strains, add kanamycin to a final concentration of 50 μg·mL-1 in the LB medium.

[0040] 2. Test instruments

[0041] Table 1

[0042]

[0043]

[0044] 2. Test methods

[0045] 1. Screening, cloning and vector construction of hydrolase genes

[0046] (1) The SCI Finder database was used to search and screen microbial enzymes that catalyze aromatic carboxylates with similar functional groups, and the NCBI online database was used to mine enzyme gene sequences. The ycaC gene (NCBI ID: WP_042895670.1) was selected for amino acid sequence modification:

[0047] Fragment 1, the amino acid residues at positions 19 and 20 are changed to A and A, and the amino acid sequence of the hydrolase after modification is shown in SEQ ID NO: 1;

[0048] Fragment 2, the amino acid sequence of the hydrolase after modification is shown in SEQ ID NO: 3;

[0049] Fragment 3, the amino acid sequence of the hydrolase after modification is shown in SEQ ID NO: 5;

[0050] Fragment 4, the amino acid sequence of the hydrolase after modification is shown in SEQ ID NO: 7;

[0051] Fragment 5: the amino acid sequence of the hydrolase after modification is shown in SEQ ID NO: 9;

[0052] (2) The codons of the ycaC gene were optimized according to the preference of E. coli and sent to GenScript to synthesize the following four gene fragments:

[0053] Fragment 1: the nucleotide sequence is shown in SEQ ID NO: 2;

[0054] Fragment 2: the nucleotide sequence is shown in SEQ ID NO: 4;

[0055] Fragment 3: the nucleotide sequence is shown in SEQ ID NO: 6;

[0056] Fragment 4: the nucleotide sequence is shown in SEQ ID NO: 8;

[0057] Fragment 5: the nucleotide sequence is shown in SEQ ID NO: 10;

[0058] Fragments 1 to 5 all carry a Histag tag and a restriction enzyme cleavage site;

[0059] (3) After obtaining the target gene and pET28α expression vector, add double distilled water, endonuclease buffer, enzyme substrate, and restriction endonuclease into the PCR tube respectively to perform double enzyme digestion reaction, and the order of addition is from most to least.

[0060] The target gene and pET28α were placed at 37°C for double enzyme digestion. The reaction system was as follows:

[0061] ycaC fragment double enzyme digestion system:

[0062] Table 2

[0063]

[0064] pET28α double restriction enzyme digestion system:

[0065] Table 3

[0066]

[0067] After double digestion reaction was carried out at 37°C for 3 h using BamHI and Xho I restriction endonucleases, Loading Buffer was added to terminate the reaction, and the double digestion product was purified and recovered according to the instructions of the gel recovery kit.

[0068] (4) After being cut by the same restriction endonuclease, the double-digestion products have the same sticky ends and can be connected to a complete plasmid by DNA ligase. The ycaC gene and pET28α double-digestion products with the same sticky ends are placed in the same PCR tube. The ligation reaction uses a 10μL system, and the target gene and plasmid digestion products are added in a ratio of 3:1, mixed with 1μL T4 DNA ligase, and connected overnight at 16°C. The successfully connected plasmid is named pET28α-ycaC.

[0069] (5) The ligation product is transformed into E. coli DH5α competent cells. The transformation steps are as follows:

[0070] a. First adjust the temperature of the constant temperature water bath to 42°C.

[0071] b. Take out a tube (100 μl) of competent bacteria from the -80°C ultra-low temperature freezer, immediately melt it with your fingers, then insert it into ice and place it in an ice bath for 10 minutes.

[0072] c. Add 10 μl of the ligation product pET28α-ycaC, shake gently and place on ice for 20 minutes.

[0073] d. Shake it gently and place it in a 42℃ water bath for 90 seconds of heat shock, then quickly return it to ice and let it stand for 5 minutes.

[0074] e. Add 900 μL of LB medium without antibiotics into each of the above tubes and mix gently. Then fix them on the spring frame of the shaker and shake them at 37 degrees Celsius for 50 minutes.

[0075] f. Add 300 μl of the above transformation mixture taken out from the clean bench to each solid LB plate culture dish containing appropriate antibiotics, and evenly apply it using a glass coating rod that has been burned with an alcohol lamp and cooled.

[0076] g. Place the marked culture dish in a 37°C constant temperature incubator for 30-60 minutes. After the liquid on the surface has penetrated into the culture medium, turn it upside down and place it in a 37°C constant temperature incubator overnight.

[0077] (6) Identify the recombinant plasmid from the grown single colonies: Pick the single colonies grown on the above LB solid culture medium, inoculate them into 5 mL of LB culture medium containing kanamycin, and culture them overnight at 37°C and 200 r·min-1. Take part of the bacterial solution and store the strain in a glycerol tube. Take an appropriate amount of the bacterial solution and extract the recombinant plasmid according to the instructions of the plasmid extraction kit. The extracted recombinant plasmid is double-digested with BamHI and Xho I, using the same double-digestion system as pET28α. The products after double-digestion are analyzed by 1% agarose gel electrophoresis to identify the target gene fragment and the vector fragment. The double-digestion identification results are as follows: Figure 1 As shown, the recombinant plasmids that were positive after double enzyme digestion were sent to Shanghai Bioengineering for sequence determination. Four expression vectors pET28α-ycaC were successfully obtained.

[0078] Example 2 Protein expression and purification

[0079] 1. Expression and extraction of hydrolase enzymes

[0080] The recombinant plasmid verified by sequencing in Example 1 was transferred into the E. coli expression host cell BL21 (DE3) using the same transformation method as DH5α. The bacterial solution was spread on an LB plate containing 50 μg / mL kanamycin and cultured overnight at 37°C. After the colonies grew, a single clone was picked and inoculated into 50 mL LB liquid culture medium for culture. 37°C, 220 r·min -1 After shaking culture for 12 hours, transfer to 300 mL LB medium at a ratio of 2% to expand the culture. 37°C, 220 r·min -1 The mixture was shaken and cultured for 2 h until OD600 reached 0.5, and 300 μL of 1 mol·L -1 The inducer isopropyl-β-D-thiogalactoside (IPTG) was added, the culture temperature was lowered to 16°C, and the shaking culture was continued for 16 hours. The induced bacterial solution was centrifuged at 10000rpm for 5 minutes, the precipitate was collected, and it was resuspended in 50mmol·L -1 In PBS buffer, cells were disrupted using an ultrasonic disruptor (operating for 3 seconds, interval of 7 seconds, duration of 10 minutes, ultrasonic power of about 200 W). -1Centrifuge for 5 min and collect the supernatant.

[0081] The supernatant was slowly passed through a nickel column Ni-NTA equilibrated with pre-cooled lysate for 3 to 5 times, and the impurities were washed away by gradient method using pH 9.5 Washbuffer (imidazole concentrations were 20 and 40 mmol / L, respectively). The target protein was then washed off with an eluent (containing 250 mmol / L imidazole), and the eluted protein solution was added to a dialysis bag (Rida Henghui, molecular weight cutoff 12-14 KD) and dialyzed with PBS overnight to obtain a purified protein solution. The purified protein solution was spectrophotometrically determined using a BCA protein concentration determination kit. The protein concentration was diluted to 200 μg / mL with 50 mmol / L PBS buffer to obtain a catalytic protein sample solution.

[0082] Example 3 Synthesis of 4″-methylamino-5′-hydroxy-avermectin from 5-O-allylformyl-4″-methylamino-avermectin by biocatalysis

[0083] 1. Biocatalysis

[0084] Experimental group 1: Weigh 0.1 g of 4″-methylamino-5′-allylformyl-avermectin and dissolve it in 10 mL of methanol. Add 300 μL of catalytic protein sample solution and 200 μL of substrate into a 1.5 mL EP tube, dilute to 1 mL with buffer, and shake at 200 rpm and 30°C for 10 h using a constant temperature and speed rotary shaker. Dry the reaction solution with nitrogen in a fume hood, dilute to 1 mL with ethyl acetate, and filter through a 0.45 μm microporous nylon membrane to obtain test sample solution 1.

[0085] Experimental group 2: weigh 0.1 g of 4″-methylamino-5′-allylformyl-avermectin, perform the same operation as above, add the protein sample solution of fragment 2, and obtain test sample solution 2;

[0086] Experimental group 3: weigh 0.1 g of 4″-methylamino-5′-allylformyl-avermectin and perform the same operation as above, add the protein sample solution of fragment 3 to obtain test sample solution 3;

[0087] Experimental group 4: weigh 0.1 g of 4″-methylamino-5′-allylformyl-avermectin and perform the same operation as above, add the protein sample solution of fragment 4 to obtain test sample solution 4;

[0088] Experimental group 5: weigh 0.1 g of 4″-methylamino-5′-allylformyl-avermectin and perform the same operation as above, add the protein sample solution of fragment 5 to obtain test sample solution 5;

[0089] The above experimental groups 1-5 also set up control groups:

[0090] Control group: The control catalytic system is as follows: the catalytic protein sample solution is kept at 100℃ for 20min to obtain an inactivated enzyme solution. 0.1g 4″-methylamino-5′-allylformyl-avermectin is dissolved in 10mL methanol, 300μL of the inactivated enzyme solution and 200μL of the substrate are added to a 1.5mL EP tube, and the volume is adjusted to 1mL with 50mmol / L PBS buffer, and a constant temperature and speed rotary shaker is used to shake at 200rpm and 30℃ for 10h. The reaction solution is blown dry with nitrogen in a fume hood, and the volume is redivided to 1mL with ethyl acetate, and filtered with a 0.45μm microporous nylon membrane to obtain a control group sample solution.

[0091] 2. Enzyme activity detection

[0092] 4″-methylamino-5′-hydroxy-avermectin content detection method: HPLC detection conditions: Agilent HP1100 liquid chromatograph; C18 chromatographic column (4.6mm×250mm); column temperature: 30°C; mobile phase acetonitrile: water (plus 0.1% trifluoroacetic acid) volume 75:25. Flow rate: 1ml / min; detection wavelength: 245nm; injection volume 5μl. The difference between the peak area of ​​4″-methylamino-5′-hydroxy-avermectin of the test sample 1 and the peak area of ​​4″-methylamino-5′-hydroxy-avermectin of the control sample is the peak area of ​​4″-methylamino-5′-hydroxy-avermectin generated by enzyme catalysis. According to the content of the standard and the corresponding peak area, the content of 4″-methylamino-5′-hydroxy-avermectin produced by enzyme catalysis is calculated.

[0093] The enzyme activity unit is defined as: the amount of enzyme required to catalyze the substrate per hour to produce 1 μg of 4″-methylamino-5′-hydroxy-avermectin is 1U. When determining the enzyme activity, three test sample solutions 1 or control sample solutions are prepared according to the catalytic conditions in 2.3.

[0094] Specific enzyme activity was calculated as enzyme activity divided by protein content.

[0095] 3. Results

[0096] (1) When the test sample 1 and the control sample were analyzed by HPLC, a single peak was obtained at a retention time of 5 min. The retention time of the peak was the same as that of the 4″-methylamino-5′-hydroxy-avermectin standard sample.

[0097] The specific enzyme activity was determined to be 20.52 U / mg after three tests. 蛋白 , 21.82U / mg 蛋白 , 20.65U / mg 蛋白 The standard deviation of the enzyme activity was smaller, indicating that the detection method was feasible and effective. The results also showed that the enzyme could convert 5-O-allylformyl-4"-methylamino-avermectin into 4"-methylamino-5'-hydroxy-avermectin.

[0098] (2) Sample 2 was tested three times and the specific enzyme activity was 3.02 U / mg 蛋白 .

[0099] (3) Sample 3 was tested three times, and the specific enzyme activity was 9.02 U / mg 蛋白 .

[0100] (4) Sample 4 was tested three times and the specific enzyme activity was 7.82 U / mg 蛋白 .

[0101] (5) Sample 5 was tested three times and the specific enzyme activity was 2.86 U / mg 蛋白 .

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0103] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0104] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0105] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations. Sequence Listing <110> Hubei Zhongnong Huawei Bioengineering Co., Ltd. <120> A hydrolase and its application in biocatalytic synthesis of 4"-methylamino-5'-hydroxy-avermectin <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <400> 1 His His His His His His Met Ala Gln Asp Tyr Val Arg Leu Asp Lys 1 5 10 15 Asp Asn Ala Ala Val Leu Leu Val Ala Ala Gln Ala Gly Leu Leu Ser 20 25 30 Leu Val Arg Asp Ile Asp Pro Asp Lys Phe Lys Asn Asn Val Leu Ala 35 40 45 Leu Ala Asp Ala Ala Lys Tyr Phe Asn Leu Pro Thr Ile Leu Thr Thr 50 55 60 Ser Phe Glu Asn Gly Pro Asn Gly Pro Leu Val Pro Glu Leu Lys Glu 65 70 75 80 Leu Phe Pro Asp Ala Pro Phe Ile Pro Arg Pro Gly Gln Ile Asn Ala 85 90 95 Trp Asp Asn Glu Asn Phe Val Lys Ala Val Lys Ala Thr Gly Lys Lys 100 105 110 Gln Leu Ile Ile Ala Gly Val Val Thr Glu Val Cys Val Ala Phe Pro 115 120 125 Ala Leu Ala Ala Leu Ala Glu Gly Phe Glu Val Phe Val Ile Thr Asp 130 135 140 Ala Ser Gly Thr Phe Asn Glu Leu Thr Arg Asp Ala Ala Trp Asp Arg 145 150 155 160 Met Ser Lys Ala Gly Ala Gln Leu Met Thr Trp Phe Gly Met Ala Cys 165 170 175 Glu Leu His Arg Asp Trp Arg Asn Asp Val Glu Gly Leu Gly Ala Leu 180 185 190 Phe Ser Asn His Ile Pro Asp Tyr Arg Asn Leu Met Thr Ser Tyr Ser 195 200 205 Phe Asn Thr Ser Gln Lys 210 <210> 2 <211> 662 <212> DNA <213> Artificial Sequence <400> 2 cggatccatg catcatcatc accaccacat ggctcaggac tacgttcgtc tggataaaga 60 taacgctgca gttctgctgg tggccgctca ggctggtctg ctgtccctgg ttcgtgatat 120 tgacccggat aaatttaaaa ataacgttct ggcactggct gacgcagcta aatacttcaa 180 tctgcctact atcctgacca cctcttttga aaacggtccg aacggtccgc tggtgcctga 240 actgaaagaa ctgttcccgg atgcgccgtt cattccgcgt ccgggtcaga tcaacgcatg 300 ggacaatgag aactttgtta aagctgtaaa agctaccggt aaaaaacagc tgatcatcgc 360 gggcgttgtt accgaagtat gcgttgcttt cccggcgctg gcagcgctgg cggagggttt 420 cgaagtattc gtcatcaccg atgcttctgg caccttcaac gaactgaccc gtgacgcagc 480 gtgggaccgc atgtccaagg caggcgccca gctgatgacc tggtttggca tggcgtgcga 540 gctgcaccgt gattggcgca acgacgttga aggtctgggc gcgctgtttt ccaaccacat 600 cccggactat cgcaatctga tgacctccta ctctttcaac acttcccaga aataactcga 660 gg 662 <210> 3 <211> 214 <212> PRT <213> Artificial Sequence <400> 3 His His His His His His Met Ala Gln Asp Tyr Val Arg Leu Asp Lys 1 5 10 15 Asp Asn Ala Ala Val Leu Leu Val Asp His Gln Ala Gly Leu Leu Ser 20 25 30 Leu Val Arg Asp Ile Asp Pro Asp Lys Phe Lys Asn Asn Val Leu Ala 35 40 45 Leu Ala Asp Ala Ala Lys Tyr Phe Asn Leu Pro Thr Ile Leu Thr Thr 50 55 60 Ser Phe Glu Asn Gly Pro Asn Gly Pro Leu Val Pro Glu Leu Lys Glu 65 70 75 80 Leu Phe Pro Asp Ala Pro Phe Ile Pro Arg Pro Gly Gln Ile Asn Ala 85 90 95 Trp Asp Asn Glu Asn Phe Val Lys Ala Val Lys Ala Thr Gly Lys Lys 100 105 110 Gln Leu Ile Ile Ala Gly Val Val Thr Glu Val Cys Val Ala Phe Pro 115 120 125 Ala Leu Ala Ala Leu Ala Glu Gly Phe Glu Val Phe Val Ile Thr Asp 130 135 140 Ala Ser Gly Thr Phe Asn Glu Leu Thr Arg Asp Ala Ala Trp Asp Arg 145 150 155 160 Met Ser Lys Ala Gly Ala Gln Leu Met Thr Trp Phe Gly Met Ala Cys 165 170 175 Glu Leu His Arg Asp Trp Arg Asn Asp Val Glu Gly Leu Gly Ala Leu 180 185 190 Phe Ser Asn His Ile Pro Asp Tyr Arg Asn Leu Met Thr Ser Tyr Ser 195 200 205 Phe Asn Thr Ser Gln Lys 210 <210> 4 <211> 662 <212> DNA <213> Artificial Sequence <400> 4 cggatccatg catcatcacc accaccatat ggcgcaggac tatgttcgtc tggacaaaga 60 taatgccgca gtactgctgg ttgaccacca ggcaggcctg ctgagcctgg tacgtgacat 120 cgacccggac aagttcaaaa acaatgttct ggcactggca gatgctgcga aatacttcaa 180 cctgccgacc attctgacca cttcctttga gaacggccca aacggtccgc tggttccgga 240 actgaaagaa ctgttcccag atgcaccgtt tattccgcgt ccgggccaga ttaacgcatg 300 ggacaacgaa aacttcgtta aagcggtgaa agctactggc aagaaacagc tgatcattgc 360 tggcgtggtt actgaagttt gcgttgcatt cccggcgctg gcagcactgg cagaaggctt 420 cgaggtcttc gtgatcacgg acgcttccgg taccttcaac gaactgaccc gcgatgcagc 480 gtgggaccgt atgagcaaag ctggtgcgca gctgatgacc tggtttggta tggcatgtga 540 gctgcatcgt gactggcgta acgacgtgga aggcctgggc gcgctgttct ccaaccacat 600 cccggattac cgtaacctga tgacttccta ttccttcaac acctcccaga aataactcga 660 gg 662 <210> 5 <211> 214 <212> PRT <213> Artificial Sequence <400> 5 His His His His His His Met Ala Gln Asp Tyr Val Arg Leu Asp Lys 1 5 10 15 Asp Asn Ala Ala Val Leu Leu Val Ala Ala Gln Ala Gly Leu Leu Ser 20 25 30 Leu Val Arg Asp Ile Asp Pro Asp Lys Phe Lys Asn Asn Val Leu Ala 35 40 45 Leu Ala Asp Ala Ala Lys Tyr Phe Asn Leu Pro Thr Ile Leu Thr Thr 50 55 60 Ser Phe Glu Thr Gly Pro Asn Gly Pro Leu Val Pro Glu Leu Lys Glu 65 70 75 80 Leu Phe Pro Asp Ala Pro Phe Ile Pro Arg Pro Gly Gln Ile Asn Ala 85 90 95 Trp Asp Asn Glu Asn Phe Val Lys Ala Val Lys Ala Thr Gly Lys Lys 100 105 110 Gln Leu Ile Ile Ala Gly Val Val Thr Glu Val Cys Val Ala Phe Pro 115 120 125 Ala Leu Ala Ala Leu Ala Glu Gly Phe Glu Val Phe Val Ile Thr Asp 130 135 140 Ala Ser Gly Thr Phe Asn Glu Leu Thr Arg Asp Ala Ala Trp Asp Arg 145 150 155 160 Met Ser Lys Ala Gly Ala Gln Leu Met Thr Trp Phe Gly Met Ala Cys 165 170 175 Glu Leu His Arg Asp Trp Arg Asn Asp Val Glu Gly Leu Gly Ala Leu 180 185 190 Phe Ser Asn His Ile Pro Asp Tyr Arg Asn Leu Met Thr Ser Tyr Ser 195 200 205 Phe Asn Thr Ser Gln Lys 210 <210> 6 <211> 662 <212> DNA <213> Artificial Sequence <400> 6 cggatccatg catcaccacc accaccacat ggcacaggat tatgtacgtc tggacaaaga 60 taacgctgct gttctgctgg tggccgcgca agcaggcctg ctgtctctgg ttcgcgatat 120 cgacccagac aaattcaaaa acaatgttct ggcgctggca gacgctgcta aatactttaa 180 cctgccgacc atcctgacca cgtctttcga gaccggtccg aacggtccgc tggttccgga 240 gctgaaagaa ctgttcccgg atgcaccgtt catcccgcgt ccgggccaga tcaacgcttg 300 ggataacgaa aacttcgtaa aagctgttaa agcaaccggt aaaaaacagc tgattatcgc 360 aggtgttgtg acggaagtgt gtgtcgcgtt cccagcactg gcagccctgg cggagggctt 420 tgaggtattt gttattaccg atgcaagcgg cacctttaac gaactgaccc gtgatgcggc 480 ttgggatcgt atgtccaaag ccggtgcgca gctgatgacc tggttcggta tggcatgcga 540 gctgcatcgt gactggcgca acgacgtaga aggtctgggt gcgctgtttt ctaaccatat 600 cccggactac cgtaacctga tgactagcta ctctttcaac accagccaaa aataactcga 660 gg 662 <210> 7 <211> 214 <212> PRT <213> Artificial Sequence <400> 7 His His His His His His Met Ala Gln Asp Tyr Val Arg Leu Asp Lys 1 5 10 15 Asp Asn Ala Ala Val Leu Leu Val Asp Ala Gln Ala Gly Leu Leu Ser 20 25 30 Leu Val Arg Asp Ile Asp Pro Asp Lys Phe Lys Asn Asn Val Leu Ala 35 40 45 Leu Ala Asp Ala Ala Lys Tyr Phe Asn Leu Pro Thr Ile Leu Thr Thr 50 55 60 Ser Phe Glu Asn Gly Pro Asn Gly Pro Leu Val Pro Glu Leu Lys Glu 65 70 75 80 Leu Phe Pro Asp Ala Pro Phe Ile Pro Arg Pro Gly Gln Ile Asn Ala 85 90 95 Trp Asp Asn Glu Asn Phe Val Lys Ala Val Lys Ala Thr Gly Lys Lys 100 105 110 Gln Leu Ile Ile Ala Gly Val Val Thr Glu Val Cys Val Ala Phe Pro 115 120 125 Ala Leu Ala Ala Leu Ala Glu Gly Phe Glu Val Phe Val Ile Thr Asp 130 135 140 Ala Ser Gly Thr Phe Asn Glu Leu Thr Arg Asp Ala Ala Trp Asp Arg 145 150 155 160 Met Ser Lys Ala Gly Ala Gln Leu Met Thr Trp Phe Gly Met Ala Cys 165 170 175 Glu Leu His Arg Asp Trp Arg Asn Asp Val Glu Gly Leu Gly Ala Leu 180 185 190 Phe Ser Asn His Ile Pro Asp Tyr Arg Asn Leu Met Thr Ser Tyr Ser 195 200 205 Phe Asn Thr Ser Gln Lys 210 <210> 8 <211> 662 <212> DNA <213> Artificial Sequence <400> 8 cggatccatg caccatcatc atcaccatat ggcgcaagat tatgtgcgtc tggacaaaga 60 taacgcggcg gttctgctgg tcgacgcgca ggcgggtctg ctgtctctgg tacgtgatat 120 tgatccggac aaattcaaaa acaacgtgct ggccctggct gacgcagcga aatacttcaa 180 cctgccgacc atcctgacga ccagcttcga aaacggtccg aacggtccgc tggttcctga 240 actgaaagaa ctgttcccgg acgcgccgtt catcccacgt ccaggtcaga tcaacgcttg 300 ggacaacgaa aattttgtca aagcagtcaa agcaacgggc aaaaagcagc tgatcattgc 360 cggcgttgtt accgaggtgt gtgtagcgtt cccggcactg gcagccctgg cggaaggctt 420 cgaagttttt gtaattaccg acgccagcgg cactttcaat gaactgactc gtgatgcagc 480 ttgggatcgt atgtctaaag cgggtgcgca gctgatgact tggtttggca tggcttgcga 540 actgcaccgc gattggcgta acgacgtgga gggtctgggc gcgctgttct ctaaccacat 600 cccggactac cgcaatctga tgacctctta ctctttcaac accagccaga aataactcga 660 gg 662 <210> 9 <211> 214 <212> PRT <213> Artificial Sequence <400> 9 His His His His His His Met Ala Gln Asp Tyr Val Arg Leu Asp Lys 1 5 10 15 Asp Asn Ala Ala Val Leu Leu Val Asp His Gln Ala Gly Leu Leu Ser 20 25 30 Leu Val Arg Asp Ile Asp Pro Asp Lys Phe Lys Asn Asn Val Leu Ala 35 40 45 Leu Ala Asp Ala Ala Lys Tyr Phe Asn Leu Pro Thr Ile Leu Thr Thr 50 55 60 Ser Phe Glu Asn Gly Pro Asn Gly Pro Leu Val Pro Glu Leu Lys Glu 65 70 75 80 Leu Phe Pro Asp Ala Pro Phe Ile Pro Arg Pro Gly Gln Ile Asn Ala 85 90 95 Trp Asp Asn Glu Asn Phe Val Lys Ala Val Lys Ala Thr Gly Lys Lys 100 105 110 Gln Leu Ile Ile Ala Gly Val Val Thr Glu Val Cys Val Ala Phe Pro 115 120 125 Ala Leu Ala Ala Leu Ala Glu Gly Phe Glu Val Phe Val Ile Thr Asp 130 135 140 Ala Ser Gly Thr Phe Asn Glu Leu Thr Arg Asp Ala Ala Trp Asp Arg 145 150 155 160 Met Ser Lys Ala Gly Ala Gln Leu Met Thr Trp Phe Gly Met Ala Cys 165 170 175 Glu Leu His Arg Asp Trp Arg Asn Asp Val Glu Gly Leu Gly Ala Leu 180 185 190 Phe Ser Asn His Ile Pro Asp Tyr Arg Asn Leu Met Thr Ser Tyr Ser 195 200 205 Phe Asn Thr Ser Gln Lys 210 <210> 10 <211> 662 <212> DNA <213> Artificial Sequence <400> 10 cggatccatg caccatcatc accaccatat ggcgcaggat tatgttcgtc tggacaaaga 60 <h2 style=";text-align:left;direction:ltr">caacgcggca gttctgctgg ttgaccacca ggcgggtctg ctgtccctgg ttcgtgatat 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgatccggat aaattcaaga acaacgttct ggcgctggct gacgcagcca aatactttaa 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cctgccgacg atcctgacca ctagcttcga aaacggtccg aacggtccac tggtgccaga 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> actgaaagaa ctgtttccgg acgctccatt catccctcgt ccgggtcaga tcaacgcatg 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggataatgaa aacttcgtaa aagcagttaa agctaccggt aaaaaacagc tgattatcgc 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tggcgtagta accgaggtgt gcgttgcttt cccagctctg gcagcactgg cagaaggttt 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgaagttttc gtaatcactg acgcttccgg cacctttaat gaactgaccc gtgacgctgc 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttgggatcgc atgtctaaag ctggtgcgca gctgatgact tggttcggta tggcctgcga 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> actgcaccgt gactggcgca acgatgttga aggcctggggc gcgctgttct ctaaccacat 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cccggattac cgtaacctga tgacgtccta ctcttttaac accagccaga aataactcga 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gg 662

Claims

1. A hydrolase, characterized in that The amino acid sequence of the hydrolase is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. A hydrolase expression vector, characterized in that: The expression vector contains the nucleic acid molecule encoding claim 2.

4. A hydrolase expression vector according to claim 3, characterized in that: The expression vector includes one of a prokaryotic expression vector and a viral vector.

5. A method for preparing a hydrolase expression vector, characterized in that: The method comprises: Obtaining the target gene fragment of the nucleic acid molecule of the hydrolase according to claim 2; The target gene fragment and the pET28α expression vector were double-digested with BamHI and XhoI, and then ligated with enzymes to obtain the expression vector.

6. A recombinant bacterium comprising the expression vector according to any one of claims 3-4.

7. Use of the hydrolase according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to any one of claims 3-4, or the recombinant bacterium according to claim 6 in catalyzing the synthesis of 4"-methylamino-5'-hydroxy-avermectin from 5-O-allylformyl-4"-methylamino-avermectin.

8. A catalytic product, characterized in that It comprises at least one of the hydrolase according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to any one of claims 3-4, and the recombinant bacteria according to claim 6.

9. A method for catalyzing 5-O-allylformyl-4"-methylamino-avermectin to synthesize 4"-methylamino-5'-hydroxy-avermectin, characterized in that: The method comprises: using 5-O-allylformyl-4"-methylamino-avermectin as a substrate and using the hydrolase according to claim 1 to catalyze the synthesis of the product 4"-methylamino-5'-hydroxy-avermectin.

10. The method according to claim 9, characterized in that The temperature of the catalytic reaction is 25-35°C.

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