A nitrile hydratase mutant and use thereof

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a nitrile hydratase mutant and application thereof, wherein the mutant is obtained by single mutation or multiple mutations of positions 22, 42, 63, 73, 105, 131, 145 and 163 of the amino acid sequence shown in SEQ ID NO. 1. The nitrile hydratase mutant of the application can be expressed in a high-density fermentation host bacterium in a heterologous manner, can improve the substrate catalytic concentration to 1000 g / L, is applied to catalytic synthesis of 4-acetyl-2-methylbenzamide, is green, environmentally friendly, non-polluting, has high catalytic efficiency and high product conversion rate.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and relates to a nitrile hydratase mutant and its application, specifically to a nitrile hydratase mutant and its application in the catalytic synthesis of 4-acetyl-2-methylbenzamide. Background Technology

[0002] Fluralaner, developed by Nissan Chemical Industries, Ltd. of Japan, is a broad-spectrum isoxazoline insecticide and the first marketed novel isoxazole-based, highly effective insecticide. The successful development of fluralaner pioneered a new type of GABA-gated chloride channel interferon, primarily acting by interfering with chloride channels of γ-aminobutyric acid (GABA) receptors and glutamate receptors in the invertebrate nervous system, similar to the target mechanisms of cyclopentadiene, phenylpyrazole, and macrolide insecticides. Fluralaner is a safe and effective broad-spectrum antibody-mediated ectoparasitic drug, exhibiting good insecticidal activity against ticks, fleas, lice, hemiptera, and dipterans. Its toxicity is higher than or comparable to commonly used insecticides, and it shows no significant cross-resistance with existing insecticides, even exhibiting good insecticidal activity against some resistant pests. 4-Acetyl-2-methylbenzamide is an indispensable intermediate fragment in the synthesis of fluralaner.

[0003] The traditional synthesis of 4-acetyl-2-methylbenzamide uses 4-acetyl-2-methylbenzonitrile as a raw material, which is obtained through nitrile hydrolysis, chlorination, and amination. This method involves many reaction steps, generates a large amount of wastewater during hydrolysis, has a low yield, and the raw materials are expensive.

[0004] .

[0005] Therefore, it is necessary to develop a method for preparing 4-acetyl-2-methylbenzamide with mild process conditions and high purity and yield to meet the requirements of industrial production. Biocatalysis offers advantages such as mild reaction conditions, environmental friendliness, and no heavy metal pollution. 4-acetyl-2-methylbenzonitrile can be converted to 4-acetyl-2-methylbenzamide under the catalysis of nitrile hydratase.

[0006] .

[0007] Although nitrile hydratases have wide applications, no nitrile hydratase catalyzing 4-acetyl-2-methylbenzamide was found in the literature. Therefore, the enzymatic synthesis of 4-acetyl-2-methylbenzamide presents significant technical challenges. Summary of the Invention

[0008] To address the aforementioned problems in the existing technology, this application provides a nitrile hydratase mutant and its application, which can be heterologously expressed in high-density fermentation host bacteria, exhibiting high catalytic efficiency and high product conversion rate.

[0009] To address the above problems, the present invention provides the following technical solution: Firstly, a nitrile hydratase mutant is obtained by making single or multiple mutations at positions 22, 42, 63, 73, 105, 131, 145, and 163 based on the amino acid sequence shown in SEQ ID NO.1.

[0010] In one embodiment of this application, the nitrile hydratase is named NHaseA, derived from... Burkholderiales bacterium .

[0011] In one embodiment of this application, the nitrile hydratase mutant is based on the amino acid sequence shown in SEQ ID NO.1 and includes one or more amino acid mutations selected from the following sites: S22A, F42P, M63G, Y73K, K105E, Q131P, G145A, P163G.

[0012] In one embodiment of this application, the nitrile hydratase mutant is obtained by combining mutations of S22A, F42P, M63G, Y73K, K105E, Q131P, G145A and P163G based on the amino acid sequence shown in SEQ ID NO.1, and is named NHaseA_M, with its amino acid sequence shown in SEQ ID NO.2.

[0013] In one embodiment of this application, the serine at position 22 of S22A (SEQ ID NO.1) is mutated to alanine, and named NHaseA_S22A.

[0014] In one embodiment of this application, the phenylalanine at position 42 of F42P, i.e., SEQ ID NO.1, is mutated to proline and named NHaseA_F42P.

[0015] In one embodiment of this application, the methionine at position 63 of M63G (SEQ ID NO.1) is mutated to glycine and named NHaseA_M63G.

[0016] In one embodiment of this application, the tyrosine at position 73 of Y73K (SEQ ID NO.1) is mutated to lysine and named NHaseA_Y73K.

[0017] In one embodiment of this application, the lysine at position 105 of K105E (SEQ ID NO.1) is mutated to glutamic acid and named NHaseA_K105E.

[0018] In one embodiment of this application, Q131P, i.e., the glutamine at position 131 of SEQ ID NO.1, is mutated to proline and named NHaseA_Q131P.

[0019] In one embodiment of this application, the glycine at position 145 of G145A (SEQ ID NO.1) is mutated to alanine and named NHaseA_G145A.

[0020] In one embodiment of this application, the proline at position 163 of SEQ ID NO.1 in P163G is mutated to glycine, and named NHaseA_P163G.

[0021] In one embodiment of this application, NHaseA_M, i.e., SEQ ID NO.1, is mutated as follows: serine at position 22 is mutated to alanine, phenylalanine at position 42 is mutated to proline, methionine at position 63 is mutated to glycine, tyrosine at position 73 is mutated to lysine, lysine at position 105 is mutated to glutamic acid, glutamine at position 131 is mutated to proline, glycine at position 145 is mutated to alanine, and proline at position 163 is mutated to glycine.

[0022] In one embodiment of this application, the fusion protein obtained by attaching a tag to the protein end defined by the nitrile hydratase mutant is also within the scope of protection of this application.

[0023] Secondly, this application provides a gene encoding a nitrile hydratase mutant.

[0024] In one embodiment of this application, the nucleotide sequence of the gene encoding NHaseA is shown in SEQ ID NO.3.

[0025] In one embodiment of this application, the nucleotide sequence of the gene encoding NHaseA_M is shown in SEQ ID NO.4.

[0026] Thirdly, this application provides a recombinant vector for the encoding gene of a nitrile hydratase mutant.

[0027] In one embodiment of this application, the recombinant vector is constructed as follows: the nitrile hydratase mutant gene is inserted into the pRSFDuet-1 vector. Nde I / Xho A recombinant vector containing a nitrile hydratase mutant gene was constructed between the I sites.

[0028] Fourthly, this application provides a recombinant genetically engineered bacterium containing a gene encoding a nitrile hydratase mutant.

[0029] In one embodiment of this application, recombinant genetically engineered bacteria are prepared by transferring a constructed recombinant vector into a host bacterium to obtain recombinant genetically engineered bacteria.

[0030] In one embodiment of this application, the host bacteria include, but are not limited to, various conventional engineered bacteria in the art.

[0031] In one embodiment of this application, the host bacterium may be E. coli BL21.

[0032] Fifthly, this application provides the use of nitrile hydratase mutants in the catalytic synthesis of 4-acetyl-2-methylbenzamide from 4-acetyl-2-methylbenzonitrile.

[0033] The bioactive nitrile hydratase mutant of this application can be used in the form of engineered bacterial wet cells, crude enzyme solution, or purified enzyme solution. Furthermore, the nitrile hydratase mutant of this invention can also be prepared into an immobilized enzyme using immobilization methods known in the art.

[0034] In one embodiment of this application, wet bacterial cells obtained by inducing expression of recombinant genetically engineered bacteria with nitrile hydratase mutant are used as catalysts, and 4-acetyl-2-methylbenzonitrile is used as a substrate to carry out a transformation reaction at 30-60°C to obtain 4-acetyl-2-methylbenzoamide.

[0035] In one embodiment of this application, the catalytic synthesis route is as follows: .

[0036] In one embodiment of this application, obtaining wet bacterial cells includes the following steps: activating recombinant engineered bacteria, transferring them to an induction medium, adding an inducer for induction culture, centrifuging and collecting the bacterial cells, and resuspending the bacterial cells in a buffer solution.

[0037] In one embodiment of this application, the reaction temperature is 40-45°C.

[0038] In one embodiment of this application, the pH value of the reaction is 7.0-9.0.

[0039] In one embodiment of this application, the pH value of the reaction is 8.0.

[0040] In one embodiment of this application, the final concentration of the added inducer is 0.001-10 g / L, and the induction time is 4-60 h.

[0041] In one embodiment of this application, the final concentration of the added inducer is 0.02-1 g / L, and the induction time is 4-50 h.

[0042] In one embodiment of this application, the weight ratio of 4-acetyl-2-methylbenzonitrile to the catalyst is 100-300:1.

[0043] In one embodiment of this application, the weight ratio of 4-acetyl-2-methylbenzonitrile to the catalyst is 200:1.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The nitrile hydratase mutant of this application can be heterologously expressed in high-density fermentation host bacteria, which can increase the substrate catalytic concentration to 1000 g / L, effectively solving the problem of substrate inhibition of enzyme activity, improving production efficiency, reducing substrate loss, and promoting the application of biotechnology industry.

[0045] (2) The nitrile hydratase mutant provided in this application can catalyze the synthesis of 4-acetyl-2-methylbenzamide in a short time and with high efficiency. Moreover, the bio-enzyme catalysis method is green, environmentally friendly and pollution-free, and is more suitable for green industrial processing and production.

[0046] (3) The nitrile hydratase mutant provided in this application has high catalytic efficiency and high product conversion rate. Detailed Implementation

[0047] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0048] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. The range defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range.

[0049] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; and the remainder is weight / weight percentage.

[0050] The present invention will be further described below with reference to specific embodiments. Molecular biology experimental methods not specifically described in the following embodiments can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0053] Example 1: Preparation of recombinant bacteria expressing nitrile hydratase NHase A and its mutants The dual-gene expression vector pRSFDuet-1 (disclosed in Han Guangwei. Co-expression and immunogenicity of Clostridium perfringens α, β_1, β_2, ε toxin proteins [D]. Chinese Academy of Agricultural Sciences, 2014) was selected. Nde I / Xho I site insertion Burkholderiales bacterium The nucleotide sequence of the nitrile hydratase (NHaseA) encoding gene or the nucleotide sequence of the mutant encoding gene is used. When the inserted sequence is the nucleotide sequence of the nitrile hydratase (NHaseA) encoding gene, the recombinant expression plasmid pRSFDuet-1-NHaseA is obtained. The recombinant plasmid is induced and expressed in *E. coli* BL21(DE3) (when the fermentation broth OD...). 600 When the expression level reaches 0.3-0.8, an inducer is added to induce expression (the final concentration of the inducer is 0.02-1 g / L, and the induction time is 4-50 h), resulting in BL21-pRSFDuet-1-NHaseA. The amino acid sequence of the nitrile hydratase NHaseA is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.3.

[0054] The PCR amplification technique is as follows: PCR amplification of genes Using plasmids or the E. coli BL21(DE3) genome as templates, PCR amplification was performed using the 2×PhantaR Max Master Mix kit from Nanjing Novizan Biotechnology Co., Ltd. to obtain the coding gene fragments of cysQ, eda, nusA, and NHaseA and its mutants. The reaction procedure was: 95℃, 1 min; 68℃, 2 min; 72℃, 2 min, for 30 cycles. The PCR reaction system is shown in Table 1.

[0055] Table 1. PCR system ; Digestion reaction of restriction endonucleases The plasmid vectors were digested overnight at 30°C using restriction endonucleases from TaKaRa. The composition of the three dual-enzyme digestion systems is shown in Table 2.

[0056] Table 2. Double enzyme digestion reaction system ; Connection reaction The linearized plasmid and the target gene fragment were ligated using the CloneEZR recombinant cloning kit from Nanjing Genscript Biotech Co., Ltd. The reaction system is shown in Table 3. The ligation conditions were 22℃ for 30 min and 4℃ for 5 min.

[0057] Table 3 Connection System ; The ligation solution was transferred into E. coli BL21(DE3) competent cells. Single colonies were picked from plates containing kanamycin (50 mg / L) and inoculated into LB medium containing the same concentration of kanamycin. The cells were incubated overnight at 37°C and 200 rpm.

[0058] PCR amplification of the coding gene of the site-directed mutant of NHaseA: Rapid mutation was performed using PCR amplification technology with unmutated strain pRSFDuet-1-NHaseA as template DNA. The NHaseA mutant is based on the amino acid sequence shown in SEQ ID NO.1 and contains one or more amino acid mutations selected from the following sites: S22A, F42P, M63G, Y73K, K105E, Q131P, G145A, P163G.

[0059] The primers for site-directed mutagenesis of S22A are: Forward primer: 5'-CAACAACgctCTGAGCTATAAACCGGTGTTTCATG-3'; Reverse primer: 5'-AGCTCAGagcGTTGTTGGTATGCGGCAC-3'.

[0060] The primers for site-directed mutagenesis of F42P are: Forward primer: 5'-TATAGCCTGCTGcctCTGGCGGCGGATCGCCTG-3'; Reverse primer: 5'-AGaggCAGCAGGCTATACGCATGATGTTCCCA-3'.

[0061] The primers for the M63G site-directed mutagenesis are: Forward primer: 5'-ATTGAACGCggtGATGCGCGCCATTATTTTGC-3'; Reverse primer: 5'-GCATCaccGCGTTCAATCGCATGGCGCAGTTC-3'.

[0062] The primers for site-directed mutagenesis of Y73K are: Forward primer: 5'-GAGCCCGaagTATGATCGCATTGTGATTGGCG-3'; Reverse primer: 5'- GATCATActtCGGGCTCGCAAAATAATGGCGC-3'.

[0063] The primers for site-directed mutagenesis of K105E are: Forward primer: 5'-AGAATTTgaaCTGGCGCGCCCGTATAGCAGCG-3'; Reverse primer: 5'-GCGCCAGttcAAATTCTTTGCCCAGATAGCGT-3'.

[0064] The primers for site-directed mutagenesis of Q131P are: Forward primer: 5'-AACGCGTGcctGTGAAAGATGAATATGTGCCGGG-3'; Reverse primer: 5'-TTTCACaggCACGCGTTCGCCCACTTCAAACG-3'.

[0065] The primers for site-directed mutagenesis of G145A are: Forward primer: 5'-gcaTATCTGCGCGGCAAACAGGGCGTGATTCT-3'; Reverse primer: 5'-TTGCCGCGCAGATAtgcCGGCGCGCGAATATGGCC-3'.

[0066] The primers for site-directed mutagenesis of P163G are: Forward primer: 5'-TAAATGGggtTTTCCGGATAGCGCGGGCCATG-3'; Reverse primer: 5'-CCGGAAAaccCCATTTATGGGTGGTGCGATGC-3'.

[0067] The mutant protein, obtained by combined mutation of S22A, F42P, M63G, Y73K, K105E, Q131P, G145A, and P163G, is named NHaseA_M, and its amino acid sequence is shown in SEQ ID NO.2. The nucleotide sequence of the nucleic acid molecule encoding the mutant protein is shown in SEQ ID NO.4.

[0068] Example 2: Obtaining whole-cell fermentation products of nitrile hydratase NHase A and its mutants The recombinant expression strains containing nitrile hydratase NHaseA and its mutants prepared in Example 1 were plated onto LB agar plates containing 50 µg / L kanamycin (NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar 20 g / L) and incubated at 37°C for 12 h. The next day, single colonies were selected from the plates and transferred to shake tubes containing 5 mL of LB liquid medium (containing 50 µg / L kanamycin) and cultured at 37°C and 200 rpm for 12 h as seed culture. The seed culture was then transferred at a 1% (v:v) inoculation rate to 100 mL of TB medium (yeast extract 25 g / L, peptone 15 g / L, NaCl 10 g / L, glucose 2 g / L, lactose 0.5 g / L, containing 50 µg / L kanamycin). The medium was then incubated at 37°C and 200 rpm with shaking. After 2 hours, adjust the temperature to 25°C and continue culturing for 20-22 hours.

[0069] Collect the fermentation broth and freeze-centrifuge (4℃, 7000 rpm, 6 min). Resuspend the broth in a buffer (1g of wet bacterial sludge re-dissolved in 5mL buffer) as the whole cell product and store it at 4℃ for later use.

[0070] Example 3 Enzyme activity assay of nitrile hydratase NHase A and its mutants The method for determining the enzyme activity of nitrile hydratase NHase A and its mutants was as follows: 0.1 g of 4-acetyl-2-methylbenzonitrile and 0.2 mg of wet bacterial sludge were added to a 1.5 mL enzyme-catalyzed reaction system, and the pH was adjusted to 7.5 with 100 mM phosphate buffer. The reaction conditions were 30℃ and 200 rpm; sampling times were 0 min, 20 min, and 30 min. Sample preparation: the sample was diluted with 50% acetonitrile aqueous solution, followed by a 100-fold dilution of the extract, and then analyzed by LC. Enzyme activity (U) was defined as the amount of enzyme required to convert 1 µmol of 4-acetyl-2-methylbenzoamide within 1 minute. The relative enzyme activities of other mutant enzymes were calculated using the wild-type enzyme activity as 100%.

[0071] Example 4 Comparison of catalytic efficiency of nitrile hydratase NHase A and its mutants The calculation methods for Km and kcat are as follows: the concentration of 4-acetyl-2-methylbenzonitrile substrate is in the range of 0.00001-50mM, 10μg of purification enzyme (NHaseA and its mutant) is added, the initial reaction rate is detected and calculated, and the corresponding km and kcat are calculated by fitting the Michaelis-Menten equation.

[0072] The changes in km and kcat of nitrile hydratase NHaseA and its mutants were compared, and the catalytic efficiency (kcat / km) was calculated (Table 4). The results showed that the km of the mutant NHaseA_S22A_F42P_M63G_Y73K_K105E_Q131P_G145A_P163G (NHaseA_M) was about 1 / 1500th of that of NHaseA, indicating a higher affinity for the substrate; the kcat of NHaseA_M was about 5000 times that of NHaseA, indicating a faster catalytic rate; and the kcat / km of NHaseA_M was about 7 million times that of NHaseA. The introduction of the mutant greatly improved the catalytic efficiency of NHaseA.

[0073] Table 4 Kinetic parameters of nitrile hydratase NHase A and its mutants ; Example 5: Relative enzyme activity of nitrile hydratase NHase A and its mutants The enzyme activities of NHase A and its mutants were measured, and the relative enzyme activities of the mutants were calculated with the NHase A activity as 100%. The enzyme activity detection protocol was the same as above. Enzyme activity detection showed that the relative enzyme activity of the mutant NHase A_S22A_F42P_M63G_Y73K_K105E_Q131P_G145A_P163G (NHase A_M) was 6892.98 times that of NHase A, which greatly improved the catalytic ability of nitrile hydratase.

[0074] Table 5. Relative enzyme activities of nitrile hydratase NHase A and its mutants ; Example 6 Biocatalytic synthesis of 4-acetyl-2-methylbenzamide from 4-acetyl-2-methylbenzonitrile The biocatalytic synthesis of 4-acetyl-2-methylbenzonitrile from 4-acetyl-2-methylbenzonitrile was carried out using a reaction system consisting of 1000 g of 4-acetyl-2-methylbenzonitrile, a pH 8.0 buffer environment, and 5 g of whole-cell bacterial sludge. After 12 h of reaction at 40 °C, the yields of NHaseA and its mutant are shown in Table 6. NHaseA_S22A_F42P_M63G_Y73K_K105E_Q131P_G145A_P163G (NHaseA_M) catalyzed the conversion of 1000 g of 4-acetyl-2-methylbenzonitrile into 99.8% of 4-acetyl-2-methylbenzoamide in 5 g of whole-cell bacterial sludge after 12 h. Clearly, the mutant NHaseA_M significantly improved the conversion rate of 4-acetyl-2-methylbenzonitrile, increasing it by 129.7 times compared to the wild type.

[0075] Table 6. Conversion rates of 1000 g of 4-acetyl-2-methylbenzonitrile catalyzed by nitrile hydratase NHase A and its mutants ; The present application has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application.

Claims

1. A nitrile hydratase mutant, characterized in that, The nitrile hydratase mutant is obtained by making single or multiple mutations at positions 22, 42, 63, 73, 105, 131, 145, and 163 based on the amino acid sequence shown in SEQ ID NO.

1.

2. The nitrile hydratase mutant according to claim 1, characterized in that, The nitrile hydratase mutant is based on the amino acid sequence shown in SEQ ID NO.1 and contains one or more amino acid mutations selected from the following sites: S22A, F42P, M63G, Y73K, K105E, Q131P, G145A, P163G.

3. The nitrile hydratase mutant according to claim 2, characterized in that, The nitrile hydratase mutant was obtained by combining mutations of S22A, F42P, M63G, Y73K, K105E, Q131P, G145A and P163G based on the amino acid sequence shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

4. A gene encoding a nitrile hydratase mutant as described in any one of claims 1-3.

5. A recombinant vector containing the gene of claim 4.

6. A recombinant genetically engineered bacterium containing the gene of claim 4.

7. The use of the nitrile hydratase mutant according to any one of claims 1-3 in the catalytic synthesis of 4-acetyl-2-methylbenzamide from 4-acetyl-2-methylbenzonitrile.

8. The application according to claim 7, characterized in that, Using wet bacterial cells obtained by inducing expression of recombinant genetically engineered bacteria with nitrile hydratase mutant as a catalyst, and 4-acetyl-2-methylbenzonitrile as a substrate, a transformation reaction was carried out at 30-60℃ to obtain 4-acetyl-2-methylbenzoamide.

9. The application according to claim 8, characterized in that, The pH value of the reaction is 7.0-9.

0.

10. The application according to claim 8, characterized in that, The weight ratio of 4-acetyl-2-methylbenzonitrile to the catalyst is 100-300:1.