Deacetylase mutant and application thereof in synthesis of clethodim

Abstract

The invention belongs to the field of genetic engineering and enzymology, and particularly relates to a deacetylase mutant and application thereof in clethodim synthesis, the deacetylase mutant is obtained by performing single-point mutation or multi-point mutation on the 272th site, the 92th site, the 203th site, the 304th site, the 305th site, the 306th site and the 270th site of an amino acid sequence shown in SEQ ID NO.1, and the mutant with improved specificity on a substrate chloroacetamide is obtained. The mutant Y305E, the mutant Y305E / G304L and the mutant Y305E / H306F can catalyze a substrate chloroacetamide to synthesize chloramine and are used for subsequent clethodim production, compared with a wild type, the relative enzyme activities of the mutants Y305E, Y305E / G304L and Y305E / H306F are improved by 7.8 times, 9.6 times and 10.3 times respectively, the catalytic efficiency of the Y305E is improved by 3.2 times compared with that of the wild type, and the catalytic efficiency of the Y305E / H306F is improved by 4.2 times compared with that of the wild type. When the deacetylase mutant provided by the invention is used for synthesizing chloramine, the reaction condition is mild, the deacetylase mutant is harmless to the environment, the reaction time is short, and the deacetylase mutant has the application prospect of large-scale production of chloramine and subsequent products.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzymology, and particularly relates to a deacetylase mutant and an application thereof in synthesizing clethodim. Background Art

[0002] O-3-Chloro-2-propenylhydroxylamine (chloroamine for short) is an important pesticide intermediate, mainly used in the production of cyclohexenone herbicides, such as clethodim, cycloxydim and tralkoxydim, etc. This type of herbicide is a post-emergence selective herbicide, commonly used to control grass weeds and volunteer cereal crops in broad-leaved crop fields. Cyclohexenone herbicides can inhibit the activity of plant meristems and are widely used to control annual and perennial grasses in various broad-leaved crops such as soybeans, cotton, and flax, and are highly safe for broad-leaved crops. They are widely used because of their high efficiency, low toxicity, and good selectivity.

[0003] At present, the industrial production methods of chloroamines include the hydrolysis of acetone oxime ether, the hydrolysis of phthalimide, the oxime method, etc. However, the chemical production of chloroamines generally has the problem of high pollution or high impurities, which is generally not conducive to industrial promotion.

[0004] Biocatalysis is increasingly used in the synthesis of organic compounds. Its advantages of high efficiency, specificity and mildness can overcome the drawbacks of chemical production of chloroamines. Therefore, the development of enzymes that can catalyze the production of chloroamines and their in-depth research are of great practical significance. Summary of the invention

[0005] In view of the demand for enzymes that efficiently catalyze the production of chloroamines in the prior art, the present invention provides a deacetylase mutant and its application in the synthesis of clethodim. The specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides a deacetylase mutant, wherein the mutant is obtained by single-point mutation or multi-point mutation at positions 272, 92, 203, 304, 305, 306, and 270 of the amino acid sequence shown in SEQ ID NO.1.

[0007] Furthermore, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 according to one of the following mutation forms:

[0008] (1) Tyrosine at position 305 is mutated to alanine, phenylalanine, leucine, asparagine, glutamic acid or valine;

[0009] (2) Glutamic acid at position 272 is mutated to lysine, arginine, or histidine;

[0010] (3) tyrosine at position 305 mutated to glutamic acid, and alanine at position 92 mutated to methionine or leucine;

[0011] (4) tyrosine at position 305 mutated to glutamic acid, and glutamic acid at position 272 mutated to lysine, arginine, or histidine;

[0012] (5) tyrosine at position 305 mutated to glutamic acid, and tryptophan at position 203 mutated to cysteine ​​or glycine;

[0013] (6) Tyrosine at position 305 mutated to glutamic acid, and aspartic acid at position 270 mutated to methionine, serine, or tyrosine;

[0014] (7) tyrosine at position 305 mutated to glutamic acid, and histidine at position 306 mutated to phenylalanine;

[0015] (8) The tyrosine at position 305 was mutated to glutamic acid, and the glycine at position 304 was mutated to leucine.

[0016] The invention constructs a mutant with improved substrate specificity of chloroacetamide by rationally modifying the key site of histone deacetylase 10 (hHDAC10) from Homo sapiens that determines substrate specificity.

[0017] In a second aspect, the present invention provides a gene encoding the above-mentioned deacetylase mutant.

[0018] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned encoding gene.

[0019] In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned encoding gene.

[0020] In a fifth aspect, the present invention provides the use of the above-mentioned deacetylase mutant or the above-mentioned genetically engineered bacteria in catalyzing the synthesis of chloroamines from chloroacetamide.

[0021] In a sixth aspect, the present invention provides the use of the above-mentioned deacetylase mutant or the above-mentioned genetically engineered bacteria in the production of clethodim.

[0022] In a seventh aspect, the present invention provides a method for producing chloroamines, comprising: using chloroacetamide as a substrate, the crude enzyme solution, pure enzyme or immobilized enzyme of the above-mentioned deacetylase mutant, or the wet bacteria or crude enzyme solution of the above-mentioned genetically engineered bacteria as a catalyst to form a reaction system, and reacting to obtain chloroamines.

[0023] Furthermore, the reaction time is 16 hours.

[0024] By using the mutant provided by the present invention as a catalyst, chloroacetamide can be efficiently catalyzed into chloroamines for the subsequent production of clethodim, with a short reaction time, a high reaction conversion rate, and little environmental pollution, which is conducive to the large-scale production of chloroamines and clethodim.

[0025] Furthermore, the amount of catalyst added in the reaction system is greater than 20 g / L; and the substrate concentration in the reaction system is 20 mM to 40 mM.

[0026] Furthermore, the amount of catalyst added to the reaction system is 25 g / L.

[0027] Through experiments on the effect of catalyst dosage on substrate conversion rate in the present invention, it was found that the optimum concentration of the catalyst is 25 g / L. When the concentration exceeds 25 g / L, the rate of increase of conversion rate slows down or does not change much.

[0028] Furthermore, the reaction system includes 0-0.5 mM zinc ions.

[0029] Furthermore, the reaction system includes 0.25 mM zinc ions.

[0030] In the present invention, it is found that adding zinc ions during the catalytic reaction can effectively enhance the enzymatic activity of deacetylase. Specifically, 0.25 mM and 0.5 mM zinc ions have an enhancing effect on the enzymatic activity of deacetylase, and increasing the concentration of zinc ions to 0.75 mM will inhibit the deacetylase.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention rationally transforms the key sites that determine the substrate specificity of the deacetylase hHDAC10, constructs a mutant with improved specificity for the substrate chloroacetamide, which can catalyze the substrate chloroacetamide to synthesize chloroamines. Compared with the wild type, the relative enzyme activities of the mutants Y305E, Y305E / G304L, and Y305E / H306F are respectively 7.8 times, 9.6 times, and 10.3 times higher than those of the wild type, wherein the catalytic efficiency of Y305E is 3.2 times higher than that of the wild type, and the catalytic efficiency of Y305E / H306F is 4.2 times higher than that of the wild type. When the deacetylase mutant provided by the present invention is used to synthesize chloroamines, the reaction conditions are mild, harmless to the environment, and the reaction time is short, and the application prospect of large-scale production of chloroamines and subsequent products is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure is a schematic diagram of the reaction of synthesizing chloroamine from chloroacetamide catalyzed by deacetylase in the present invention, and deacetylase represents deacetylase.

[0034] Figure 2 Schematic diagram of the structural comparison between chloroacetamide and acetyl polyamine compounds; wherein acetyl polyamine compound is N 8 -Acetylspermidine, acetylputrescine.

[0035] Figure 3 Schematic diagram of the relative enzyme activity of different mutants catalyzing the synthesis of chloroamines.

[0036] Figure 4 Schematic diagram of the amino acids surrounding the -Cl in chloroacetamide when the deacetylase binds to the substrate molecule. DETAILED DESCRIPTION

[0037] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0038] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0040] The culture medium used in the embodiment of the present invention is composed of:

[0041] LB liquid medium (g / L): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is water, pH 7.0, high pressure steam sterilization at 115°C for 30 min.

[0042] LB solid medium is LB liquid medium with 1.5%-2.0% agar powder added.

[0043] TB liquid culture medium (g / L): peptone 12 g / L, yeast extract 24 g / L, K2HPO4·3H2O 12.54 g / L, KH2PO4 2.31 g / L, glycerol 5 g / L, solvent is water, pH 7.2±0.2, high pressure steam sterilization at 115℃ for 30 min.

[0044] 50 mg / mL kanamycin (Kan) stock solution: Weigh 0.5 g of kanamycin and place it on a clean bench for UV sterilization for 15-20 min, then dissolve it in 10 mL of sterile water, filter it with a 0.22 μm filter membrane for sterilization, then divide it into portions and store it in a -20°C refrigerator for later use.

[0045] 0.1 M isopropyl-β-D-thiogalactoside (IPTG) mother solution: Weigh 1.2 g IPTG and place it on a clean bench for UV sterilization for about 15 minutes, then dissolve it in 50 mL sterile water. Filter it with a 0.22 μm filter membrane for sterilization, then divide it into portions and store it in a -20°C refrigerator for later use.

[0046] The reaction diagram of the wild-type deacetylase and the deacetylase mutant catalyzing the synthesis of chloroamine from chloroacetamide is shown in the following figure: Figure 1 shown.

[0047] In the present invention, the amino acid sequence of the wild-type deacetylase is shown in SEQ ID NO.1, the nucleotide sequence of the TAT solubility tag used is shown in SEQ ID NO.2, and the nucleotide sequence of the thrombin site sequence is shown in SEQ ID NO.3.

[0048] SEQ ID NO.1 amino acid sequence:

[0049] MGTALVYHEDMTATRLLWDDPECEIERPERLTAALDRLRQRGLEQRCLRLSAREASEEELGLVHSPEYVSLVRETQVLGKEELQALSGQFDAIYFHPSTFHCARLAAGAGLQLVDAVLTGAVQNGLALVRPPGHHGQRAAANGFCVFNNVAIAAAHAKQKHGLHRIL VVDWDVHHGQGIQYLFEDDPSVLYFSWHRYEHGRFWPFLRESDADAVGRGQGLGFTVNLPWNQVGMGNADYVAAFLHLLLPLAFEFDPELVLVSAGFDSAIGDPEGQMQATPECFAHLTQLLQVLAGGRVCAVLEGGYHLESLAESVCMTVQTLLGDPAPPLSGPMA PCQSALESIQSARAAQAPHWKSLQQQDVTAVPMSPSSHSPEGRPPPLLPGGPVCKAAASAPSSLLDQPCLCPAPSVRTAVALTTPDITLVLPPDVIQQEASALREETEAWARPHESLAREEALTALGKLLYLLDGMLDGQVNSGIAATPASAAAATLDVAVRRGLSH GAQRLLCVALGQLDRPPDLAHDGRSLWLNIRGKEAAALSMFHVSTPLPVMTGGFLSCILGLVLPLAYGFQPDLVLVALGPGHGLQGPHAALLAAMLLRGLAGGRVLALLEENSTPQLAGILARVLNGEAPPSLGPSSVASPEDVQALMYLRGQLEPQWKMLQCHPHLVA

[0050] SEQ ID NO.2:

[0051] TATGGCCGCAAAAAACGCCGCCAGCGCCGCCGC

[0052] SEQ ID NO.3:

[0053] GGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCA.

[0054] Example 1 Screening of wild-type deacetylase, construction of engineered bacteria and detection of enzyme activity

[0055] 1. Screening of wild-type deacetylase

[0056] Through the screening of literature reports, databases, and bioinformatics methods, we found deacetylases and amidases that catalyze similar substrates on the brenda website. Then, we found the enzymatic properties and catalytic reaction formulas of the corresponding deacetylases in the UniProt database to find the best deacetylase hHDAC10. We optimized it according to the codon preference of Escherichia coli, introduced EcoRⅠ restriction site, XhoⅠ restriction site, His tag and terminator into the sequence published in GenBank, and handed it over to Nanjing GenScript Biotechnology Co., Ltd. for synthesis to obtain the wild-type deacetylase hHDAC10 gene.

[0057] 2. Construction of wild-type deacetylase recombinant engineering bacteria

[0058] The wild-type gene (hHDAC10 gene) obtained in step 1 was connected to the plasmid pET28a(+) to construct a recombinant plasmid, which was then transformed into E. coli BL21 (DE3) competent cells were used to construct wild-type recombinant engineered bacteria. The transformation steps were as follows: thawing Escherichia coli BL21 (DE3) competent cells on ice for 2 minutes, adding the recombinant plasmid, flicking the tube wall to mix, placing on ice for 30 minutes, heat shocking in a 42°C water bath for 90 seconds, incubating on ice for 2 minutes, adding LB liquid culture medium without antibiotics, resuscitating at 37°C for 45-60 minutes, discarding part of the supernatant after centrifugation, and then spreading on LB solid culture medium plates containing 50 mg / mL kanamycin resistance, culturing at 37°C for 12 hours, picking a single colony and inoculating it into LB liquid culture medium containing 50 mg / mL kanamycin, culturing at 180 rpm, 37°C for 7-8 hours, and entrusting Hangzhou Qingke Biotechnology Co., Ltd. to sequence, and the wild-type recombinant engineered bacteria with successful sequencing were preserved in glycerol tubes to construct wild-type recombinant engineered bacteria E. coli BL21 (DE3)-pET28a (+) -hHDAC10.

[0059] 3. Determination of the activity of wild-type deacetylase recombinant engineering bacteria in transforming substrates

[0060] (1) Wet cell preparation

[0061] The wild-type recombinant engineered bacteria screened in step 2 were streaked and inoculated in LB solid medium containing 50 mg / mL kanamycin and cultured at 37°C for 12 hours. A single colony was picked and inoculated into LB liquid medium containing 50 mg / mL kanamycin, and cultured at 180 rpm and 37°C for 7-8 hours. Then, the inoculum was transferred to TB liquid medium containing 50 mg / mL kanamycin at a volume concentration of 2%, and the flask was shaken at 37°C and 180 rpm until the OD value reached 0.6-0.8, and IPTG was added at a final concentration of 0.1 M, and the expression was induced at 16°C and 180 rpm for 18 hours. The bacterial solution was centrifuged at 8000 rpm for 10 minutes, and the wet bacterial precipitate was collected and stored at -20°C.

[0062] (2) Substrate conversion capacity test

[0063] In a 25mL reaction bottle, add 1x PBS Buffer (pH7.2-7.6), 25g / L wet bacteria and a final concentration of 20mM substrate chloroacetamide to form a 10mL reaction system, react at 35°C and 600rpm in a water bath equipped with a magnetic stirrer for 16h, take 200μL of the reaction solution and boil it at high temperature for 10min to terminate the reaction. Then centrifuge at 12000rpm for 1min, take 100μL of the supernatant of the reaction solution, dilute it to 1mL with methanol, add 250μL of ultrapure water and mix thoroughly, pass through a 0.22μm organic membrane, and use high performance liquid chromatography to detect the peak area of ​​substrate chloroacetamide and product chloroamine in the filtrate, and calculate the content according to their respective standard curves.

[0064] Substrate standard curve: Accurately weigh chloroacetamide powder and dissolve it in methanol to prepare a 50mM substrate solution. Dilute it with methanol to 10mM, 20mM, 30mM, and 40mM, and use high performance liquid chromatography to detect the peak area. Use substrate concentration as the horizontal axis and liquid phase peak area as the vertical axis to develop a standard curve, and obtain the substrate standard curve y(mAU*min)=4.97x(mM)+6.161.

[0065] Product standard curve: Accurately weigh the product chloroamine standard solution, dissolve it with methanol to prepare a 50mM product solution, dilute it with methanol to 10mM, 20mM, 30mM, and 40mM, use high performance liquid chromatography to detect the peak area, and formulate a standard curve with product concentration as the horizontal coordinate and liquid phase peak area as the vertical coordinate to obtain the product standard curve y(mAU*min)=1.74x(mM)+6.26.

[0066] HPLC determination conditions: Use Thermo Fisher HPLC, C18 chromatographic column, column temperature 40°C, UV absorbance 210nm, mobile phase (phosphoric acid adjusted to pH = 2.0) methanol: water = 3:7 (volume ratio), then add 5mL of NaOH, and perform HPLC analysis on the substrate and product simultaneously. The substrate peaks at around 7.9min, and the product peaks at around 4.1min.

[0067] Inclusion bodies are high-density, insoluble protein particles wrapped by membranes formed when foreign genes are expressed in prokaryotic cells, especially when they are efficiently expressed in Escherichia coli. In order to solve the problem of many enzyme-producing inclusion bodies, a polypeptide tag (TAT solubilizing tag, nucleotide sequence as shown in SEQ ID NO.2) was inserted into the front end of the thrombin site sequence (nucleotide sequence as shown in SEQ ID NO.3), fused with the N-terminus of the target gene, recorded as recombinant vector pET28a (+) -TAT-hHDAC10, transformed into E. coli BL21 (DE3) host cells, and recombinant engineering bacteria were constructed to construct E. coli BL21 (DE3) -pET28a (+) -TAT-hHDAC10 for subsequent experiments.

[0068] As shown in Table 5, the yield of the recombinant engineering bacteria E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 catalyzing 20 mM substrate in 16 h was 6.9%.

[0069] Example 2 Preparation of crude deacetylase enzyme solution and enzyme activity detection method

[0070] 1. Wet cell preparation

[0071] Wild-type recombinant engineered bacteria E. coli BL21 (DE3) -pET28a (+) -TAT-hHDAC10 were streaked and inoculated in LB solid medium containing 50 mg / mL kanamycin and cultured at 37 ° C for 12 hours. A single colony was picked and inoculated into LB liquid medium containing 50 mg / mL kanamycin, 180rpm, 37 ° C for 7-8 hours, and then transferred to TB liquid medium containing 50 mg / mL kanamycin at a volume concentration of 2%. The inoculum was cultured at 37 ° C and 180rpm until the OD value reached 0.6-0.8, and IPTG was added at a final concentration of 0.1M, and the expression was induced at 16 ° C and 180rpm for 18 hours. The bacterial solution was centrifuged at 8000rpm for 10 minutes, and the wet bacterial precipitate was collected and stored at -20 ° C.

[0072] 2. Preparation of crude enzyme solution

[0073] The wet bacteria obtained in step (1) were suspended in 1x PBS Buffer (pH 7.2-7.6) at a concentration of 25 g / L, and the bacterial suspension was subjected to ultrasonic disruption in an ice bath under the conditions of 250 W power, 2 s ultrasonication, 4 s pause, and disruption for 30 min. The bacterial solution was clear and transparent. After disruption, the suspension was centrifuged at 4000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution.

[0074] 3. Enzyme activity detection

[0075] Enzyme activity assay conditions: In a 25mL reaction bottle, add 1x PBS Buffer (pH7.2-7.6), the amount of crude enzyme solution added is 25mg / L in terms of protein content, and the final concentration of 20mM substrate chloroacetamide constitutes a 10mL reaction system. In a water bath equipped with a magnetic stirrer, react at 35°C and 600rpm for 16h, take 200μL of the reaction solution and boil at high temperature for 10min to terminate the reaction. Then centrifuge at 12000rpm for 1min, take 100μL of the supernatant of the reaction solution, add methanol to dilute to 1mL, add 250μL of ultrapure water and mix thoroughly, pass through a 0.22μm organic membrane, take the filtrate and use the steps and conditions of high performance liquid chromatography described in Example 1 to detect the substrate and product concentrations. The relative enzyme activity assay results of the wild-type deacetylase are shown in Table 2.

[0076] Enzyme activity is defined as the amount of enzyme that can convert 1 micromole of substrate (μmol) per minute under the above assay conditions.

[0077] Definition of relative enzyme activity: The original enzyme activity is defined as 100%, and the amount of enzyme that can convert 1 micromole of substrate (μmol) per minute compared with the original enzyme activity is the relative enzyme activity.

[0078] Example 3 Screening of deacetylase mutants and construction of engineered bacteria

[0079] According to literature reports, the substrate specificity of the deacetylase hHDAC10 (amino acid sequence as shown in SEQ ID NO.1) is determined by two aspects: first, the glutamate at position 272 can react with acetylpolyamine compounds, such as N 8 - The specific NH2 of acetyl spermidine and acetyl putrescine forms electrostatic interactions. Secondly, hHDAC10 has a unique 3 10 The helix constrains the active site to accommodate long and narrow substrates. It was observed that both chloroacetamide and acetylpolyamine compounds are long and narrow substrates, which is consistent with one of their specificities. However, Figure 2 As shown, the specific -NH2 in the acetyl polyamine compound that can form a specific electrostatic interaction with E272 corresponds to -Cl in chloroacetamide. Therefore, according to the charge interaction, the following mutations were performed:

[0080] First round of mutation: Glutamic acid at position 272 was mutated into positively charged lysine, arginine, and histidine

[0081] The plasmid pET28a(+)-TAT-hHDAC10 of the wild-type recombinant engineering bacteria E.coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 was extracted, and PCR amplification was performed using the primers E272K-F / R, E272R-F / R, and E272H-F / R in Table 1. The PCR product was added with 1 μL of DpnI and placed in a 37°C shaker for digestion for more than 2 hours. Take 10 μL of the digested PCR product, add it to 100 μL of E.coli BL21(DE3) competent cell suspension in an ice bath, let it stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, quickly put it on ice for 2 minutes, add 600 μL of LB liquid culture medium to the tube, and culture it at 37°C, 180 rpm for 45-60 minutes. Take 100 μL of bacterial solution and apply it to LB solid culture medium plate containing 50 mg / mL kanamycin. After the bacterial solution is completely absorbed by the culture medium, invert and culture at 37°C for 12 hours. Pick a single colony and inoculate it into LB liquid culture medium containing 50 mg / mL kanamycin. Culture at 180rpm and 37°C for 7-8 hours. Centrifuge at 12000rpm for 10 minutes, collect the bacteria, extract the plasmid, and entrust Hangzhou Qingke Biotechnology Co., Ltd. to sequence the plasmid. Screen the engineering bacteria corresponding to the correctly sequenced plasmid, and test the relative enzyme activity using the method of Example 2. The results are shown in Table 2. The relative enzyme activities of mutants E272K, E272R, and E272H are 2.88 times, 2.02 times, and 1.93 times higher than those of the wild type, respectively. PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25 μL, dNTPs 1 μL, forward primer 2 μL, reverse primer 2 μL, template DNA 1 μL, Phanta Max Super-Fidelity DNA Polymerase 0.5 μL, add ddH2O to 50 μL.

[0082] The PCR amplification conditions were: 95°C for 5 min; 32 cycles of (95°C for 15 s, 60°C for 15 s, 72°C for 4 min); 72°C for 5 min.

[0083] Table 1. Primers of related genes used for mutants

[0084]

[0085]

[0086] The 305NNK mutation is a degenerate codon design method in gene mutation library construction technology. NNK represents a specific base combination, where N represents the four bases A / T / G / C and K represents the two bases G / T.

[0087] Second round of mutation: Mutate other amino acids around the substrate chloroacetamide-Cl to alanine

[0088] Using the primers in Table 1, the same operation as the first round of mutation was performed, such as Figure 4 As shown in the deacetylase hHDAC10, the glutamic acid at position 22, glycine at position 143, tyrosine at position 305, aspartic acid at position 265, aspartic acid at position 172, histidine at position 174, tryptophan at position 203, histidine at position 134, histidine at position 135, phenylalanine at position 202, cysteine ​​at position 145, glycine at position 303, and isoleucine at position 25 around the substrate chloroacetamide-Cl were mutated to alanine to obtain E22A, G143A, Y305A, D265A, D172A, H174A, W203A, H134A, H135A, F202A, C145A, G303A, and I25A. The relative enzyme activity was tested using the method of Example 2. The results are shown in Table 2. Y305A is a positive mutation, and the enzyme activity of Y305A is more than 4 times that of the wild type. The others are basically negative mutations, and the enzyme activity is significantly reduced after alanine mutation.

[0089] Table 2 Relative enzyme activities of wild type and its mutants

[0090]

[0091] The third round of mutation: The enzyme activity of Y305A was significantly improved by alanine scanning, and the Y305 site was subjected to saturation mutation. The results are shown in Table 3, and the enzyme activity of Y305E was improved the most. Next, the other amino acids around the substrate chloroacetamide-Cl were mutated to alanine, and the enzyme activity was significantly improved or decreased. The mutant Y305E was used as a template for saturation double mutation, and the Y305 site was selected. Saturation double mutation was performed at the spatial range points D270, H306, and G304.

[0092] Table 3 Relative enzyme activities of wild type and its mutants

[0093]

[0094] Using the primers in Table 1 and mutant Y305E as a template, the same method as the first round of mutation was used to mutate the alanine at position 92 to methionine and leucine; the glutamic acid at position 272 to lysine, arginine, and histidine; the tryptophan at position 203 to cysteine ​​and glycine; the aspartic acid at position 270 to methionine, serine, and tyrosine; the histidine at position 306 to phenylalanine; and the glycine at position 304 to leucine.

[0095] The relative enzyme activity was tested by the method of Example 2. The results are shown in Table 4. The enzyme activities of mutants Y305E / A92M, Y305E / A92L, Y305E / E272R, Y305E / E272H, Y305E / E272K, Y305E / W203C, Y305E / W203G, Y305E / D270S, Y305E / D270M, Y305E / D270Y, Y305E / H306F, and Y305E / G304L were all improved, among which the enzyme activity of mutant Y305E / H306F was the most improved.

[0096] After three rounds of mutation, mutant Y305E and combined mutants Y305E / H306F and Y305E / G304L were selected as dominant mutants, and the corresponding recombinant engineering bacteria were E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E, E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / H306F, and E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / G304L, respectively.

[0097] Table 4 Relative enzyme activities of mutant Y305E and its combined mutants

[0098]

[0099]

[0100] Example 4 Determination of kinetic parameters of dominant mutants

[0101] The wild-type E. coli BL21 (DE3) -pET28a (+) -TAT -hHDAC10 constructed in Example 1, the recombinant engineered bacteria E. coli BL21 (DE3) -pET28a (+) -TAT -hHDAC10-Y305E and E. coli BL21 (DE3) -pET28a (+) -TAT -hHDAC10-Y305E / H306F corresponding to the superior mutants screened in Example 3 were respectively prepared into crude enzyme solutions according to the method of Example 2.

[0102] Chloroacetamide was used as substrate, with a concentration gradient of 0.2, 0.4, 0.8, 1.5, and 2 mM. A crude enzyme solution with a final concentration of 0.225 mg / mL based on protein content was added, with a volume of 800 μL. The reaction solution was heated at 35°C and the metal bath was heated at 1000 rpm. The peak areas of substrate chloroacetamide and product chloroamine were detected by high performance liquid chromatography, and the reaction rates of the final substrate concentration and different substrate concentration gradients were calculated. K m and V max Select the Michaelis-Menten equation nonlinear fitting through the software origin, k cat Equal to V max / enzyme concentration. The catalytic efficiency of mutant Y305E (k cat / K mAPP ) was 3.2 times higher than that of the wild type, and the catalytic efficiency of Y305E / H306F (k cat / K mAPP ) was 4.2 times higher than that of the wild type.

[0103] Example 5 Using different concentrations of wet bacteria to catalyze different concentrations of substrates

[0104] 1. Effect of catalyst dosage on substrate conversion rate

[0105] E. coli BL21 (DE3) -pET28a (+) -TAT -hHDAC10 wet cells were prepared by the method of Example 1. 1x PBS Buffer (pH 7.2-7.6) was added to a 25 mL reaction bottle. The amount of wet cells added was 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, and a final concentration of 30 mM substrate chloroacetamide constituted a 10 mL reaction system. In a water bath equipped with a magnetic stirrer, the reaction was carried out at 35 ° C and 600 rpm for 16 h. 200 μL of the reaction solution was boiled at high temperature for 10 min to terminate the reaction. The mixture was centrifuged at 12000 rpm for 1 min. 100 μL of the supernatant of the reaction solution was diluted to 1 mL with methanol, and 250 μL of ultrapure water was added to mix thoroughly. The mixture was passed through a 0.22 μm organic membrane. The filtrate was used to detect the concentrations of substrate chloroacetamide and product chloroamine by high performance liquid chromatography as described in Example 1. The substrate conversion rates were 11.4%, 19.6%, 24.9%, 33.9%, 33.4%, 34.9%, 33.5% and 34.7%, respectively. It can be seen that the optimal concentration of wet cells is 25 g / L. When the concentration exceeds 25 g / L, the conversion rate increases slowly or does not change much.

[0106] Under the same conditions, the concentration of the prepared wet bacteria was changed to 25g / L, the substrate concentration was changed to 20mM, 30mM, 40mM, and 80mM, and other operations were the same. The substrate conversion rates were 38.4%, 26.9%, 18.7%, and 10.1%, respectively.

[0107] 2. Effect of substrate concentration on conversion rate

[0108] The wet bacteria prepared by the recombinant engineering bacteria of mutant Y305E / H306F according to the method of Example 2 were used as catalysts, the concentration of wet bacteria added was changed to 25 g / L, the substrate concentrations were changed to 20 mM, 30 mM, 40 mM, and 80 mM, and the other operations were the same as step 1. The substrate conversion rates were 100%, 98.5%, 89.1%, and 43.7%, respectively.

[0109] Example 6 Determination of the yield of substrates catalyzed by different wet bacteria

[0110] The wild-type E. coli BL21 (DE3) -pET28a (+) -TAT-hHDAC10 wet cells constructed in Example 1, the recombinant engineered bacteria E. coli BL21 (DE3) -pET28a (+) -TAT-hHDAC10-Y305E wet cells corresponding to the superior mutants screened in Example 3, and the E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / H306F wet bacteria and E.coliBL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / G304L wet bacteria were added to a 25 mL reaction bottle with 1xPBSBuffer (pH7.2-7.6). The amount of wet bacteria added was 25 g / L, and the final concentration of 20 mM substrate chloroacetamide constituted a 10 mL reaction system. The reaction was carried out at 35°C and 600 rpm in a water bath equipped with a magnetic stirrer for 16 h. 200 μL of the reaction solution was boiled at high temperature for 10 min to terminate the reaction. The mixture was centrifuged at 12000 rpm for 1 min. 100 μL of the supernatant of the reaction solution was diluted to 1 mL with methanol and then 250 μL of ultrapure water was added to mix thoroughly. The mixture was passed through a 0.22 μm organic membrane. The filtrate was tested for the concentration of the product chloroamine by high performance liquid chromatography as described in Example 1. The product concentration was calculated based on the product standard curve. The yield = 16 h product concentration / theoretical product concentration for complete conversion of 20 mM substrate. The results are shown in Table 5.

[0111] Table 5 Yields of substrates catalyzed by different wet bacteria

[0112]

[0113] Example 7 Effect of metal ions on the catalytic effect of wet bacteria

[0114] 1. Effects of different metal ions on wet bacteria

[0115] The wild-type E. coli BL21 (DE3)-pET28a-TAT-hHDAC10 wet cells constructed in Example 1 were placed in a 25 mL reaction bottle and 1x PBS was added. Buffer (pH7.2-7.6), the amount of wet bacteria added was 25g / L, the final concentration of 20mM substrate chloroacetamide constituted a 10mL reaction system, and then 0.5mM CuCl2, ZnCl2, H4MnO8P2, CoCl2, MgSO4, FeCl2 metal reagents were added, and the reaction was carried out at 35°C and 600rpm in a water bath equipped with a magnetic stirrer for 16h. 200μL of the reaction solution was boiled at high temperature for 10min to terminate the reaction, and then centrifuged at 12000rpm for 1min. 100μL of the supernatant of the reaction solution was diluted to 1mL with methanol and then 250μL of ultrapure water was added to mix thoroughly, and passed through a 0.22μm organic membrane. The filtrate was tested for the concentration of substrate chloroacetamide and product chloroamine by high performance liquid chromatography as described in Example 1. The relative enzyme activity was tested by the method of Example 2, and the results are shown in Table 6. Cu 2+ , Mn 2+ 、Co 2 + Mg 2+ , Fe 2+ Ions have little promoting effect on E.coli BL21(DE3)-pET28a(+)-TAT-hHDAC10, and even have an inhibitory effect, while Zn 2+ It has a promoting effect on the catalytic ability of wet bacteria.

[0116] Table 6 Relative enzyme activities at 0.5 mM of different metal ions

[0117]

[0118]

[0119] 2. Effects of different concentrations of zinc ions on wet bacteria

[0120] From step 1, we can see that ZnCl2 has a promoting effect on E.coli BL21(DE3)-pET28a(+)-TAT-hHDAC10. 2+Concentration, the wild-type strain was constructed into a 10mL reaction system, and then 0.25mM, 0.5mM, and 0.75mM ZnCl2 were added, and the experimental steps of step 1 were repeated. The relative enzyme activity was tested by the method of Example 2, and the results are shown in Table 7. It can be seen that 0.25mM and 0.5mM zinc ions have a promoting effect on the catalytic ability of E.coli BL21(DE3)-pET28a(+)-TAT-hHDAC10, while 0.75mM zinc ions have an inhibitory effect. Among them, 0.25mM zinc ions have the best effect, and the relative enzyme activity is increased by 2.3 times.

[0121] Table 7 Relative enzyme activity at different concentrations of zinc ions

[0122]

Claims

1. A deacetylase mutant, characterized in that The deacetylase mutant is obtained by single-point mutation or multi-point mutation at positions 272, 92, 203, 304, 305, 306 and 270 of the amino acid sequence shown in SEQ ID NO.

1.

2. The deacetylase mutant according to claim 1, characterized in that The mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 according to one of the following mutation forms: (1) Tyrosine at position 305 is mutated to alanine, phenylalanine, leucine, asparagine, glutamic acid or valine; (2) Glutamic acid at position 272 is mutated to lysine, arginine, or histidine; (3) tyrosine at position 305 mutated to glutamic acid, and alanine at position 92 mutated to methionine or leucine; (4) tyrosine at position 305 mutated to glutamic acid, and glutamic acid at position 272 mutated to lysine, arginine, or histidine; (5) tyrosine at position 305 mutated to glutamic acid, and tryptophan at position 203 mutated to cysteine ​​or glycine; (6) Tyrosine at position 305 mutated to glutamic acid, and aspartic acid at position 270 mutated to methionine, serine, or tyrosine; (7) tyrosine at position 305 mutated to glutamic acid, and histidine at position 306 mutated to phenylalanine; (8) The tyrosine at position 305 was mutated to glutamic acid, and the glycine at position 304 was mutated to leucine.

3. The gene encoding the deacetylase mutant according to claim 1.

4. A recombinant vector, characterized in that: Contains the coding gene as described in claim 3.

5. A genetically engineered bacterium, characterized in that: Contains the coding gene as described in claim 3.

6. Use of the deacetylase mutant according to any one of claims 1 or 2, or the genetically engineered bacteria according to claim 5, in catalyzing the synthesis of chloroamines from chloroacetamide.

7. Use of the deacetylase mutant according to any one of claims 1 or 2, or the genetically engineered bacterium according to claim 5 in the production of clethodim.

8. A method for producing chloroamines, characterized in that: include: Chloroacetamide is used as a substrate, and the crude enzyme solution, pure enzyme or immobilized enzyme of the deacetylase mutant according to claim 1, or the wet bacteria or crude enzyme solution of the genetically engineered bacteria according to claim 5 is used as a catalyst to form a reaction system to react and obtain chloroamine.

9. The method according to claim 8, characterized in that The amount of catalyst added in the reaction system is greater than 20 g / L; the substrate concentration in the reaction system is 20 mM to 40 mM.

10. The method according to claim 8, characterized in that 0-0.5 mM zinc ions are added into the reaction system.

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