Deacetylase mutants and their use in the synthesis of clofencet

Abstract

This invention belongs to the fields of genetic engineering and enzymology, specifically relating to deacetylase mutants and their application in the synthesis of clethodim. The deacetylase mutants are obtained by single-point or multi-point mutations at positions 272, 92, 203, 304, 305, 306, and 270 of the amino acid sequence shown in SEQ ID NO.1, resulting in mutants with enhanced specificity for the substrate chloroacetamide. These mutants can catalyze the synthesis of chloroamines from chloroacetamide for subsequent clethodim production. Compared to the wild type, the relative enzyme activities of mutants Y305E, Y305E / G304L, and Y305E / H306F are increased by 7.8 times, 9.6 times, and 10.3 times, respectively. Specifically, 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. When using the deacetylase mutant provided by this invention to synthesize chloroamines, the reaction conditions are mild, environmentally friendly, and the reaction time is short, making it promising for large-scale production of chloroamines and subsequent products.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzymology, specifically relating to deacetylases and their application in the synthesis of clethodim. Background Technology

[0002] O-3-chloro-2-propenylhydroxylamine (abbreviated as chloroamine) is an important pesticide intermediate, mainly used in the production of cyclohexenone herbicides, such as clethodim, cycloxydim, and tralkoxydim. These herbicides are post-emergence selective herbicides, commonly used to control grassy weeds and free-growing cereal crops in broadleaf fields. Cyclohexenone herbicides inhibit the activity of plant meristems and are widely used to control annual and perennial grasses in various broadleaf crops such as soybeans, cotton, and flax, and are highly safe for broadleaf crops. They are widely used due to their high efficiency, low toxicity, and good selectivity.

[0003] Currently, industrial production methods for chloroamines include the acetone oxime ether hydrolysis method, the phthalimide hydrolysis method, and the oxime method. However, chemical methods for producing chloroamines generally suffer from significant pollution or a high amount of impurities, which is generally not conducive to industrial-scale promotion.

[0004] Biocatalysis is increasingly being used in the synthesis of organic compounds. Its advantages of high efficiency, specificity, and mildness can overcome the drawbacks of chemical methods for producing 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] To address the need for highly efficient enzymes for the catalytic production of chloroamines in existing technologies, this invention provides a deacetylase mutant and its application in the synthesis of clethodim. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a deacetylase mutant, which is obtained by single-point 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] Further, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 according to one of the following mutation methods:

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

[0009] (2) The glutamic acid at position 272 is mutated into lysine, arginine or histidine;

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

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

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

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

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

[0015] (8) Tyrosine at position 305 is mutated into glutamic acid, and glycine at position 304 is mutated into leucine.

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

[0017] Secondly, the present invention provides the encoding gene of the above-mentioned deacetylases mutant.

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

[0019] Fourthly, the present invention provides a genetically engineered bacterium containing the above-mentioned encoding gene.

[0020] Fifthly, the present invention provides the application of the above-mentioned deacetylase mutant or the above-mentioned genetically engineered bacteria in the catalytic synthesis of chloroamine from chloroacetamide.

[0021] In a sixth aspect, the present invention provides the application 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 chloroamine, comprising: using chloroacetamide as a substrate, and using the crude enzyme solution, pure enzyme or immobilized enzyme of the above-mentioned deacetylase mutant, or the wet cell or crude enzyme solution of the above-mentioned genetically engineered bacteria as a catalyst to form a reaction system, and reacting to obtain chloroamine.

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

[0024] Using the mutant provided by this invention as a catalyst, chloroacetamide can be efficiently catalyzed to chloroamine for subsequent production of clethodim. The reaction time is short, the reaction conversion rate is high, and the environmental pollution is small, which is conducive to the large-scale production of chloroamine and clethodim.

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

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

[0027] Through experiments on the effect of catalyst dosage on substrate conversion rate, this invention found that the optimal catalyst concentration is 25 g / L. Above 25 g / L, the rate of increase in conversion rate slows down or does not change significantly.

[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 this invention, it was found that adding zinc ions during the catalytic reaction can effectively enhance the activity of deacetylase. Specifically, 0.25 mM and 0.5 mM zinc ions both have an enhancing effect on the activity of deacetylase, while increasing the concentration of zinc ions to 0.75 mM will inhibit the activity of deacetylase.

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

[0032] This invention rationally modifies the key site of the deacetylase hHDAC10 that determines substrate specificity, constructing a mutant with enhanced specificity for the substrate chloroacetamide. This mutant can catalyze the synthesis of chloroamines from chloroacetamide. Compared to the wild type, the relative enzyme activities of mutants Y305E, Y305E / G304L, and Y305E / H306F are increased by 7.8 times, 9.6 times, and 10.3 times, respectively. Specifically, 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. When using the deacetylase mutants provided by this invention to synthesize chloroamines, the reaction conditions are mild, environmentally friendly, and the reaction time is short, showing promising application prospects for large-scale production of chloroamines and subsequent products. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the reaction in which deacetylase catalyzes the synthesis of chloroamine from chloroacetamide. Deacetylase represents a deacetylase.

[0034] Figure 2 This is a schematic diagram comparing the structures of chloroacetamide and acetylpolyamine compounds; where the acetylpolyamine compound is N... 8 - Acetylspermine, acetylputrescine.

[0035] Figure 3 This is a schematic diagram showing the relative enzyme activities of different mutants catalyzing the synthesis of chloroamines.

[0036] Figure 4 This is a schematic diagram of the amino acids surrounding -Cl in chloroacetamide when a deacetylase binds to a substrate molecule. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0038] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0040] The culture medium used in the embodiments of this invention consists of:

[0041] LB liquid medium (g / L): peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent: water, pH 7.0, autoclaved at 115℃ 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 12g / L, yeast extract 24g / L, K2HPO4·3H2O 12.54g / L, KH2PO4 2.31g / L, glycerol 5g / L, solvent: water, pH 7.2±0.2, autoclaved at 115℃ for 30min.

[0044] 50 mg / mL kanamycin (Kan) stock solution: Weigh 0.5 g of kanamycin and place it in a clean bench for UV sterilization for 15-20 min. Dissolve it in 10 mL of sterile water, filter it through a 0.22 μm filter membrane for sterilization, dispense it, and store it at -20℃ for later use.

[0045] 0.1M Isopropyl-β-D-thiogalactopyranoside (IPTG) stock solution: Weigh 1.2g of IPTG, place it in a clean bench for UV sterilization for about 15 minutes, dissolve it in 50mL of sterile water, filter it through a 0.22μm filter membrane for sterilization, dispense it, and store it in a -20℃ refrigerator for later use.

[0046] The schematic diagram of the reaction between the wild-type deacetylase and the deacetylase mutant catalyzing the synthesis of chloroamine from chloroacetamide in this invention is shown below. Figure 1 As shown.

[0047] In this invention, the amino acid sequence of the wild-type deacetylase is shown in SEQ ID NO.1, the nucleotide sequence of the TAT solubilizing 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 deacetylases, construction of engineered bacteria, and detection of enzyme activity.

[0055] 1. Screening of wild-type deacetylases

[0056] Through screening using literature reports, databases, and bioinformatics methods, deacetylases and amidases catalyzing similar substrates were identified on the Brenda website. Next, the enzymatic properties and catalytic reaction formulas of the corresponding deacetylases were found in the UniProt database to identify the optimal deacetylase hHDAC10. Optimization was performed based on the codon bias of *E. coli*, and EcoRI, XhoI, His tags, and terminators were introduced into the sequence published in GenBank. The wild-type deacetylase hHDAC10 gene was synthesized by Nanjing GenScript Biotech Co., Ltd.

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

[0058] The wild-type gene (hHDAC10 gene) obtained in step 1 was ligated to plasmid pET28a(+) to construct a recombinant plasmid, which was then transformed into E. coli. The transformation steps for constructing wild-type recombinant engineered bacteria using BL21(DE3) competent cells were as follows: BL21(DE3) competent E. coli cells were thawed on ice for 2 min, recombinant plasmids were added, the tube was gently tapped to mix, and the cells were placed on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 90 s, incubated on ice for 2 min, and then LB liquid medium without antibiotics was added. The cells were then thawed at 37°C for 45-60 min, centrifuged, and a portion of the supernatant was discarded. The cells were then plated on LB solid medium containing 50 mg / mL kanamycin and cultured at 37°C for 12 h. Single colonies were picked and inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 180 rpm at 37°C for 7-8 h. The bacteria were then preserved and sequenced by Hangzhou Qingke Biotechnology Co., Ltd. The successfully sequenced wild-type recombinant engineered bacteria were preserved in glycerol tubes to construct the wild-type recombinant engineered bacteria E. coli BL21(DE3)-pET28a(+)-hHDAC10.

[0059] 3. Determination of substrate activity of wild-type deacetylase recombinant engineered bacteria.

[0060] (1) Preparation of wet bacterial cells

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

[0062] (2) Substrate conversion ability test

[0063] In a 25 mL reaction flask, 1x PBS Buffer (pH 7.2-7.6), 25 g / L wet bacterial cells, and a final concentration of 20 mM chloroacetamide were added to form a 10 mL reaction system. The mixture was reacted for 16 h at 35 °C and 600 rpm in a water bath equipped with a magnetic stirrer. 200 μL of the reaction solution was then boiled for 10 min to terminate the reaction. Subsequently, the mixture was centrifuged at 12000 rpm for 1 min. 100 μL of the supernatant was collected, diluted with methanol to 1 mL, and then 250 μL of ultrapure water was added and thoroughly mixed. The mixture was filtered through a 0.22 μm organic membrane. The filtrate was analyzed using high-performance liquid chromatography (HPLC) to detect the peak areas of the chloroacetamide substrate and the chloroamine product, and the content was calculated based on their respective standard curves.

[0064] Substrate standard curve: Accurately weigh chloroacetamide powder and dissolve it in methanol to prepare a 50 mM substrate solution. Dilute with methanol to 10 mM, 20 mM, 30 mM, and 40 mM, and detect the peak area using high-performance liquid chromatography (HPLC). Plot the substrate concentration on the x-axis and the peak area on the y-axis to construct a standard curve, obtaining the substrate standard curve y(mAU*min) = 4.97x(mM) + 6.161.

[0065] Product standard curve: Accurately weigh the chloroamine standard solution, dissolve it in methanol to prepare a 50 mM product solution, and dilute it with methanol to 10 mM, 20 mM, 30 mM, and 40 mM. Detect the peak area using high performance liquid chromatography (HPLC). Plot the product concentration on the x-axis and the peak area on the y-axis to construct the standard curve, obtaining the product standard curve y(mAU*min) = 1.74x(mM) + 6.26.

[0066] High-performance liquid chromatography (HPLC) determination conditions: Thermo Fisher Scientific HPLC instrument with a C18 column was used. The column temperature was 40℃, and the UV absorbance was 210nm. The mobile phase (pH adjusted to 2.0 with phosphoric acid) was methanol:water = 3:7 (v / v) with 5mL of NaOH added. The substrate and product were analyzed simultaneously by HPLC. The substrate peaked at approximately 7.9 min, and the product peaked at approximately 4.1 min.

[0067] Inclusion bodies are high-density, insoluble protein particles enclosed in a membrane that form when exogenous genes are expressed in prokaryotic cells, especially during efficient expression in *E. coli*. To address the issue of excessive inclusion bodies in enzyme production, a polypeptide tag (TAT lysosome tag, nucleotide sequence as shown in SEQ ID NO.2) was inserted at the front of a thrombin site sequence (nucleotide sequence as shown in SEQ ID NO.3) and fused to the N-terminus of the target gene, resulting in the recombinant vector pET28a(+)-TAT-hHDAC10. This vector was transformed into *E. coli* BL21(DE3) host cells to construct the recombinant engineered bacterium *E. coli* BL21(DE3)-pET28a(+)-TAT-hHDAC10 for subsequent experiments.

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

[0069] Example 2: Preparation of crude deacetylases and method for detecting enzyme activity

[0070] 1. Preparation of wet mycelium

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

[0072] 2. Preparation of crude enzyme solution

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

[0074] 3. Enzyme activity detection

[0075] Enzyme activity assay conditions: In a 25 mL reaction flask, add 1x PBS Buffer (pH 7.2-7.6), the crude enzyme solution added is 25 mg / L based on protein content, and the substrate chloroacetamide at a final concentration of 20 mM to form a 10 mL reaction system. In a water bath equipped with a magnetic stirrer, react at 35 °C and 600 rpm for 16 h. Take 200 μL of the reaction solution and boil at high temperature for 10 min to terminate the reaction. Then centrifuge at 12000 rpm for 1 min, take 100 μL of the supernatant, dilute with methanol to 1 mL, add 250 μL of ultrapure water and mix thoroughly. Filter through a 0.22 μm organic membrane, and use the filtrate to determine the substrate and product concentrations using the high-performance liquid chromatography (HPLC) procedure and conditions described in Example 1. The relative enzyme activity assay results of wild-type deacetylase are shown in Table 2.

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

[0077] Relative enzyme activity is defined as follows: the original enzyme activity is defined as 100%, and the amount of enzyme that can convert 1 micromolar of substrate (μmol) per minute is the relative enzyme activity compared to the original 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 shown in SEQ ID NO.1) is determined by two factors: firstly, the glutamate at position 272 can react with acetylpolyamine compounds, such as N... 8 - Acetylspermine and acetylputremine form specific NH2 electrostatic interactions; secondly, hHDAC10 has a unique 3 10 The helix restricts the active site, making it suitable for long and narrow substrates. Chloroacetamides and acetylpolyamines have been observed to be long and narrow substrates, consistent with their specificity. However, as... Figure 2 As shown, the -NH2 group in acetylpolyamine compounds forms a specific electrostatic interaction with E272, which corresponds to -Cl in chloroacetamides. Therefore, based on charge interactions, the following mutation occurs:

[0080] First round of mutation: Mutate glutamic acid at position 272 into positively charged lysine, arginine, or histidine.

[0081] The plasmid pET28a(+)-TAT-hHDAC10 of the wild-type recombinant engineered bacterium E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 was extracted and amplified by PCR using primers E272K-F / R, E272R-F / R, and E272H-F / R listed in Table 1. The PCR product was added to 1 μL of DpnI and digested on a shaker at 37°C for at least 2 h. 10 μL of the digested PCR product was added to 100 μL of E. coli BL21(DE3) competent cell suspension (on ice), incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then rapidly cooled on ice for 2 min. 600 μL of LB liquid medium was added to the tube, and the mixture was incubated at 37°C and 180 rpm for 45–60 min. 100 μL of bacterial culture was plated onto LB agar plates containing 50 mg / mL kanamycin. After the culture was completely absorbed, the plates were incubated upside down at 37°C for 12 h. Single colonies were then picked and inoculated into LB liquid agar containing 50 mg / mL kanamycin. The plates were incubated at 37°C for 7-8 h at 180 rpm, centrifuged at 12000 rpm for 10 min, and the bacterial cells were collected. Plasmids were extracted and sequenced by Hangzhou Qingke Biotechnology Co., Ltd. The engineered bacteria corresponding to the correctly sequenced plasmids were screened, and their relative enzyme activities were tested using the method described in Example 2. The results are shown in Table 2. The relative enzyme activities of mutants E272K, E272R, and E272H were 2.88-fold, 2.02-fold, and 1.93-fold higher than those of the wild type, respectively. PCR reaction system: 2×PhantaMax 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℃ for 5 min; (95℃ for 15 s, 60℃ for 15 s, 72℃ for 4 min) for 32 cycles; 72℃ for 5 min.

[0083] Table 1. Primers used for the mutant genes

[0084]

[0085]

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

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

[0088] Using the primers in Table 1, the same procedures as in the first round of mutation were followed, such as... Figure 4 As shown in the deacetylase hHDAC10, the following amino acids surrounding the substrate chloroacetamide-Cl are mutated with alanine: 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, yielding E22A, G143A, Y305A, D265A, D172A, H174A, W203A, H134A, H135A, F202A, C145A, G303A, and I25A. The relative enzyme activity was tested using the method in 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 the alanine mutation.

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

[0090]

[0091] The third round of mutations: Alanine scanning showed a significant increase in enzyme activity at Y305A. Saturation mutations were then performed at site Y305, and the results are shown in Table 3. Y305E showed the greatest increase in enzyme activity. Next, sites where mutations to alanine significantly increased or decreased enzyme activity after mutating other amino acids surrounding the substrate chloroacetamide-Cl were used as templates for saturation double mutations with mutant Y305E, selecting site Y305. Saturation double mutations were performed at sites D270, H306, and G304 within the spatial range.

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

[0093]

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

[0095] The relative enzyme activity was tested using the method in Example 2, and 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 increased, with mutant Y305E / H306F showing the greatest increase in enzyme activity.

[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 engineered 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 the dominant mutant

[0101] Crude enzyme solutions were prepared from the wild-type E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 constructed in Example 1, and 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 dominant mutants screened in Example 3, respectively, according to the method in Example 2.

[0102] Using chloroacetamide as a substrate, with concentration gradients of 0.2, 0.4, 0.8, 1.5, and 2 mM, crude enzyme solution with a final concentration of 0.225 mg / mL (based on protein content) was added in a volume of 800 μL. The reaction mixture was reacted at 35 °C in a metal bath at 1000 rpm. The peak areas of the substrate chloroacetamide and the product chloroamine were detected by high-performance liquid chromatography (HPLC). The final substrate concentration and the reaction rate for different substrate concentration gradients were calculated. m and V max The Michaelis-Menten equation was generated using nonlinear fitting via software Origin, and k was used. cat equals V max / Enzyme concentration. Catalytic efficiency (kJ) of mutant Y305E. cat / K mAPP The catalytic efficiency (kJ / kF) of Y305E / H306F is 3.2 times higher than that of the wild type. cat / K mAPP It is 4.2 times higher than the wild type.

[0103] Example 5: Catalyzing different concentrations of substrate using different concentrations of wet bacterial cells.

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

[0105] Wet cells of E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 were prepared using the method described in Example 1. In a 25 mL reaction flask, 1x PBS Buffer (pH 7.2-7.6) was added at concentrations of 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, and 50 g / L, respectively. A 10 mL reaction system was prepared with a final concentration of 30 mM chloroacetamide. The reaction was carried out in a water bath equipped with a magnetic stirrer at 35 °C and 600 rpm for 16 h. 200 μL of the reaction solution was then boiled at high temperature for 10 min to terminate the reaction. After centrifugation at 12000 rpm for 1 min, 100 μL of the supernatant was taken, diluted with methanol to 1 mL, and then 250 μL of ultrapure water was added and mixed thoroughly. The mixture was then passed through a 0.22 μm organic membrane. The concentrations of the substrate chloroacetamide and the product chloroamine were determined 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 bacterial cells is 25 g / L. Above 25 g / L, the rate of increase in conversion rate slows down or does not change much.

[0106] Under the same conditions, the concentration of the prepared wet bacterial cells was changed to 25 g / L, and the substrate concentration was changed to 20 mM, 30 mM, 40 mM, and 80 mM. Other operations were the same, and 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] Using the wet bacterial cells prepared by the recombinant engineered bacteria Y305E / H306F mutant according to the method in Example 2 as a catalyst, the concentration of wet bacterial cells was changed to 25 g / L, and the substrate concentration was changed to 20 mM, 30 mM, 40 mM, and 80 mM. Other operations were the same as in step 1, and the substrate conversion rates were 100%, 98.5%, 89.1%, and 43.7%, respectively.

[0109] Example 6: Yield determination of different wet bacterial cell catalytic substrates

[0110] The wild-type E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10 wet cells constructed in Example 1, the recombinant engineered E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E wet cells corresponding to the superior mutants screened in Example 3, and E. coli Wet cells of BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / H306F and E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10-Y305E / G304L were added to a 25 mL reaction flask. 1xPBS Buffer (pH 7.2-7.6) was added, with the wet cell addition amount being 25 g / L. A 10 mL reaction system was prepared with a final concentration of 20 mM of chloroacetamide. The reaction was carried out in a water bath equipped with a magnetic stirrer at 35 °C and 600 rpm for 16 h. 200 μL of the reaction solution was then boiled at high temperature for 10 min to terminate the reaction. After centrifugation at 12000 rpm for 1 min, 100 μL of the supernatant of the reaction solution was taken, diluted with methanol to 1 mL, and then 250 μL of ultrapure water was added and mixed thoroughly. The mixture was passed through a 0.22 μm organic membrane. The concentration of the chloroamine product was detected by high performance liquid chromatography as described in Example 1. The product concentration was calculated based on the product standard curve. 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 different wet bacterial cell catalytic substrates

[0112]

[0113] Example 7: Effect of Metal Ions on the Catalytic Effect of Wet Bacterial Cells

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

[0115] Wild-type E. coli BL21(DE3)-pET28a-TAT-hHDAC10 wet cells constructed in Example 1 were added to a 25 mL reaction flask with 1x PBS. A 10 mL reaction system was prepared with a buffer (pH 7.2-7.6), 25 g / L of wet bacterial cells, and a final concentration of 20 mM chloroacetamide. Then, 0.5 mM of CuCl2, ZnCl2, H4MnO8P2, CoCl2, MgSO4, and FeCl2 metal reagents were added. The reaction was carried out in a water bath equipped with a magnetic stirrer at 35 °C and 600 rpm for 16 h. 200 μL of the reaction solution was taken and boiled for 10 min to terminate the reaction. The mixture was then centrifuged at 12000 rpm for 1 min. 100 μL of the supernatant was taken, diluted with methanol to 1 mL, and then 250 μL of ultrapure water was added and thoroughly mixed. The mixture was filtered through a 0.22 μm organic membrane. The concentrations of the substrate chloroacetamide and the product chloroamine were determined by high-performance liquid chromatography (HPLC) as described in Example 1. The relative enzyme activity was tested using the method described in Example 2, and the results are shown in Table 6. 2+ Mn 2+ Co 2 + Mg 2+ Fe 2+ The ions had virtually no promoting effect on E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10, and even had an inhibitory effect, while Zn 2+ It promotes the catalytic ability of wet bacteria.

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

[0117]

[0118]

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

[0120] Step 1 shows that ZnCl2 promotes the growth of E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10, altering the ZnCl2 content. 2+To determine the concentration, a 10 mL reaction system was constructed using the above-mentioned wild-type strain, and then 0.25 mM, 0.5 mM, and 0.75 mM ZnCl2 were added. The experimental steps in step 1 were repeated. The relative enzyme activity was tested using the method in Example 2, and the results are shown in Table 7. It can be seen that 0.25 mM and 0.5 mM zinc ions promoted the catalytic activity of E. coli BL21(DE3)-pET28a(+)-TAT-hHDAC10, while 0.75 mM zinc ions inhibited it. Among these, 0.25 mM zinc ions showed the best effect, increasing the relative enzyme activity by 2.3 times.

[0121] Table 7. Relative enzyme activity at different zinc ion concentrations.

[0122]

Claims

1. A deacetylase mutant, 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 methods: (1) Tyrosine at position 305 is mutated to glutamic acid; (2) Tyrosine at position 305 is mutated into glutamic acid, and alanine at position 92 is mutated into methionine or leucine; (3) Tyrosine at position 305 is mutated into glutamic acid, and glutamic acid at position 272 is mutated into lysine, arginine, or histidine; (4) Tyrosine at position 305 is mutated into glutamic acid, and tryptophan at position 203 is mutated into cysteine ​​or glycine; (5) Tyrosine at position 305 is mutated to glutamic acid, and aspartic acid at position 270 is mutated to methionine, serine, or tyrosine. (6) Tyrosine at position 305 is mutated to glutamic acid and histidine at position 306 is mutated to phenylalanine; (7) Tyrosine at position 305 is mutated into glutamic acid, and glycine at position 304 is mutated into leucine.

2. The encoding gene of the deacetylase mutant as described in claim 1.

3. A recombinant vector, characterized in that, It includes the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that, It includes the coding gene as described in claim 2.

5. The application of the deacetylase mutant as described in claim 1, or the genetically engineered bacteria as described in claim 4, in the catalytic synthesis of chloroamines from chloroacetamide.

6. A method for producing chloroamine, characterized in that, include: Chloroamine is obtained by reacting a reaction system consisting of chloroacetamide as a substrate and crude enzyme solution, pure enzyme, or immobilized enzyme of the deacetylase mutant as described in claim 1, or wet cell or crude enzyme solution of the genetically engineered bacteria as described in claim 4 as a catalyst.

7. The method according to claim 6, characterized in that, The 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.

8. The method according to claim 6, characterized in that, 0-0.5 mM zinc ions are added to the reaction system.

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