Use of a nitrilase in the selective catalysis of 6-chloronicotinonitrile to 6-chloronicotinic acid and mutants

Abstract

The application discloses application of a nitrilase in selective catalysis of 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid and a mutant, wherein the nitrilase mutant is obtained by mutating alanine at the 55th position of the amino acid shown in SEQ ID NO. 2 into serine. In an optimal system, the hydrolysis activity of the nitrilase to 6-chloronicotinonitrile reaches 450 U / g (WCW), 300 g / L 6-chloronicotinonitrile can be catalyzed to synthesize 6-chloronicotinic acid, the yield reaches 92%, and the catalysis synthesizes 2-chloronicotinamide with a concentration of up to 4.8 g / L; when the nitrilase mutant is used to catalyze 6-chloronicotinonitrile, the activity reaches 580 U / g (WCW), 300 g / L 6-chloronicotinonitrile can be catalyzed to synthesize 6-chloronicotinic acid, the yield reaches 99%, and the hydration activity of 2-chloronicotinonitrile is eliminated, and 2-chloronicotinamide is not generated.

Description

(I) Technical Field

[0001] This invention relates to the application of a nitrile hydrolase derived from *Paraburkholderia graminis* in the selective catalytic synthesis of 6-chloronicotinic acid from 6-chloronicotinamide, as well as mutants and their applications. (II) Background Technology

[0002] Chloronicotinic acid derivatives are important intermediates in pharmaceutical and pesticide products. Their unique pyridine ring structure makes them one of the most widely developed and applied compounds among cyclocyclic compounds. 6-Chloronicotinic acid is an important chemical intermediate, a key intermediate for drugs such as tazarotene, and can be used in the synthesis of novel pesticide products; market demand is increasing year by year. The green and efficient synthesis of 6-chloronicotinic acid is receiving increasing attention, while the required purity is also very high. Currently reported methods for synthesizing 6-chloronicotinic acid are mainly chemical methods, using chlorobenzene as a solvent and 2-chloro-5-methylpyridine as a starting material. Crude 6-chloronicotinic acid is directly oxidized under cobalt acetate catalysis, and the final product is obtained through further recrystallization. However, chemical synthesis methods cause serious environmental pollution, are lengthy, and have low yields, increasingly failing to meet the requirements of advanced industry. Furthermore, the synthesis process involves the generation of 2-chloronicotinic acid, and obtaining high-purity target products requires complex post-processing.

[0003] Nitrile hydrolases, as important industrial enzymes in the field of biocatalysis, possess advantages such as mild reaction conditions, environmental friendliness, and high catalytic efficiency, demonstrating increasingly significant application potential and value in the synthesis of carboxylic acid compounds. Using chloronicotinamide derivatives as substrates, nitrile hydrolases can catalyze the hydrolysis in one step to generate the corresponding chloronicotinic acid derivatives. In this process, the catalytic properties of the nitrile hydrolases, such as activity and substrate selectivity, are crucial to the efficient and high-purity synthesis of the target product. Discovering nitrile hydrolases with high activity and selectivity for 6-chloronicotinamide is of great significance for establishing an industrial enzymatic synthetic route for 6-chloronicotinic acid. (III) Summary of the Invention

[0004] This invention provides the application of a nitrile hydrolase in the selective catalytic synthesis of 6-chloronicotinic acid from 6-chloronicotinamide, along with its mutant and applications. The nitrile hydrolase PgNit from *Paraburkholderia graminis* possesses not only hydrolytic activity for the synthesis of 6-chloronicotinic acid from 6-chloronicotinamide but also nitrile hydration activity for the synthesis of 2-chloronicotinamide from 2-chloronicotinamide. Based on this, the enzyme was modified to obtain the mutant PgNit-A55S, which eliminates the hydration activity for 2-chloronicotinamide, thus solving the problems of poor selectivity in the catalysis of chlorinated substrates by traditional nitrile hydrolases.

[0005] The technical solution adopted in this invention is:

[0006] In a first aspect, the present invention provides the application of a nitrile hydrolase PgNit derived from Burkholderia graminis in the selective catalytic synthesis of 6-chloronicotinic acid from 6-chloronicotinamide, wherein the nucleotide sequence of the nitrile hydrolase PgNit is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

[0007] Preferably, the application involves using recombinant *E. coli* containing the nitrile hydrolase *PgNit* gene shown in SEQ ID NO.1, obtained through fermentation culture, as a catalyst, along with 6-chloronicotinamide and 2-chloronicotinamide as substrates, and a buffer solution with pH 6.0-9.0 as the reaction medium. The reaction is carried out at 25-40°C to obtain a reaction solution containing 6-chloronicotinic acid and 2-chloronicotinamide. The amount of crude enzyme solution added is based on the weight of the wet bacterial cells before disruption, and the amount of wet bacterial cells added is 2-20 g / L, preferably 10 g / L, based on the buffer solution volume. The amounts of 6-chloronicotinamide and 2-chloronicotinamide added are each independently 100-500 g / L, preferably 300 g / L, based on the buffer solution volume.

[0008] Preferably, the buffer solution is a 50 mM, pH 7.0 PB buffer. Preferably, the reaction temperature is 30°C.

[0009] Preferably, the wet cells obtained by fermentation culture of the recombinant Escherichia coli containing the nitrile hydrolase PgNit gene or the crude enzyme solution extracted from the wet cells are prepared as follows: 1) Plate culture: The recombinant Escherichia coli containing the nitrile hydrolase PgNit gene is streaked onto a solid LB medium containing 50 μg / mL kanamycin and activated overnight at 37°C to obtain single colonies; the final concentration composition of the LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, water as solvent, pH 7.0;

[0010] 2) Seed culture: Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 10-12 h to obtain seed culture; the final concentration composition of the LB liquid medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0;

[0011] 3) Fermentation culture: The seed culture was inoculated at a volume concentration of 2% into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm until OD. 600The concentration was 0.6-0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The mixture was induced and cultured at 28℃ and 180 rpm for 10-12 h. The bacterial culture was then centrifuged at 8000 rpm and 4℃ to collect the wet bacterial cells.

[0012] 4) Disruption of wet bacterial cells: The wet bacterial cells are suspended in a buffer solution with a pH of 5.0 to 9.0 and placed in an ultrasonic disruptor. The cells are disrupted at a frequency of 10-50 kHz (preferably 20 kHz) for 5-30 min (preferably 2 min, with a 10 s interval, followed by 10 min of disruption). The disrupted mixture is collected to obtain the crude enzyme solution. The volume of the buffer solution with a pH of 5.0 to 9.0 is 10 mL / g based on the wet weight of the wet bacterial cells.

[0013] Secondly, in order to further enhance the hydrolytic activity of nitrile hydrolase PgNit for 6-chloronicotinonitrile while completely eliminating its hydration activity for 2-chloronicotinonitrile, this invention screened and obtained a nitrile hydrolase mutant PgNit-A55S. The nitrile hydrolase mutant PgNit-A55S was obtained by mutating alanine at position 55 of the amino acid shown in SEQ ID NO.2 to serine. The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0014] Any amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 that involves the deletion, insertion, or substitution of one or more amino acids and possesses the catalytic activity for the synthesis of 6-chloronicotinic acid from 6-chloronicotinamide is still within the scope of protection of this invention.

[0015] This invention also provides the encoding gene of the nitrile hydrolase mutant PgNit-A55S, a recombinant vector, and a recombinant genetically engineered bacterium.

[0016] This invention can be constructed by linking the nucleotide sequences of the nitrile hydrolase PgNit and its mutant PgNit-A55S to various vectors using conventional methods in the art. The recombinant vectors of this invention are not limited, as long as they can maintain replication or autonomously replicate in various host cells of prokaryotic and / or eukaryotic cells. The vectors can be various conventional vectors in the art, such as various plasmids, bacteriophages, or viral vectors, preferably pET-28b(+).

[0017] There are no restrictions on the host cells into which the DNA encoding the nitrile hydrolase PgNit and its mutant PgNit-A55S of the present invention is introduced, as long as a recombinant expression system has been established for it, the recombinant expression vector can stably self-replicate, and the nitrile hydrolase gene of the present invention carried by it can be effectively expressed. Examples include *Escherichia coli*, *Bacillus subtilis*, yeast, actinomycetes, *Aspergillus*, as well as animal cells and higher plant cells. *Escherichia coli* is preferred in this invention, and *E. coli* BL21(DE3) is more preferred.

[0018] In this invention, pET28b(+) was selected as the expression vector for the expression of nitrile hydrolase PgNit and its mutant PgNit-A55S. Specifically, the target gene was inserted between NcoI and XhoI on the plasmid pET28b(+) and transformed into E. coli BL21(DE3) host cells.

[0019] Thirdly, this invention provides the application of the nitrile hydrolase mutant PgNit-A55S in the selective catalytic synthesis of 6-chloronicotinic acid from 6-chloronicotinamide. The application involves using wet cells or crude enzyme solution extracted from wet cells of recombinant *E. coli* containing the nitrile hydrolase mutant PgNit-A55S gene (SEQ ID NO. 3) obtained through fermentation culture as a catalyst, 6-chloronicotinamide and 2-chloronicotinamide as substrates, and a buffer solution with pH 6.0-9.0 as the reaction medium. The reaction is carried out at 25-40°C to obtain a reaction solution containing 6-chloronicotinic acid. The amount of crude enzyme solution added is based on the weight of the wet cells before disruption, and the amount of wet cells added is 2-20 g / L, preferably 10 g / L, based on the buffer solution volume. The amounts of 6-chloronicotinamide and 2-chloronicotinamide added are each independently 100-500 g / L, preferably 300 g / L, based on the buffer solution volume. The buffer solution is preferably a 50 mM, pH 7.0 PB buffer. The preferred reaction temperature is 30°C.

[0020] The wet cells or crude enzyme solution extracted from the wet cells of recombinant Escherichia coli containing the nitrile hydrolase mutant PgNit-A55S gene were prepared by fermentation culture as follows:

[0021] 1) Plate culture: Recombinant Escherichia coli containing the nitrile hydrolase mutant PgNit-A55S gene was streaked onto solid LB medium containing 50 μg / mL kanamycin and activated overnight at 37°C to obtain single colonies; the final concentration of the LB solid medium was: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, water as solvent, pH 7.0;

[0022] 2) Seed culture: Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 10-12 h to obtain seed culture; the final concentration composition of the LB liquid medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0;

[0023] 3) Fermentation culture: The seed culture was inoculated at a volume concentration of 2% into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm until OD. 600 The concentration was 0.6-0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The mixture was induced and cultured at 28℃ and 180 rpm for 10-12 h. The bacterial culture was then centrifuged at 8000 rpm and 4℃ to collect the wet bacterial cells.

[0024] 4) Disruption of wet bacterial cells: The wet bacterial cells are suspended in a buffer solution with a pH of 5.0 to 9.0 and placed in an ultrasonic disruptor. The cells are disrupted at a frequency of 10-50 kHz (preferably 20 kHz) for 5-30 min (preferably 2 min, with a 10 s interval, followed by 10 min of disruption). The disrupted mixture is collected to obtain the crude enzyme solution. The volume of the buffer solution with a pH of 5.0 to 9.0 is 10 mL / g based on the wet weight of the wet bacterial cells.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects: (1) The present invention uses recombinant cells containing the nitrile hydrolase PgNit gene or its broken crude enzyme solution as a catalyst to selectively catalyze 6-chloronicotinamide. Under optimal system conditions, the hydrolytic activity of the nitrile hydrolase PgNit provided by the present invention for 6-chloronicotinamide reaches 450 U / g (WCW), which can catalyze the synthesis of 6-chloronicotinic acid from 300 g / L 6-chloronicotinamide with a yield of 92%. At the same time, it also exhibits hydration activity for 2-chloronicotinamide, and the catalytic synthesis of 2-chloronicotinamide reaches a maximum concentration of 4.8 g / L; (2) The present invention uses recombinant cells containing the nitrile hydrolase mutant PgNit-A55S gene or its broken crude enzyme solution as a catalyst to selectively catalyze 6-chloronicotinamide to synthesize high-purity 6-chloronicotinic acid in one step. The process is simple and the industrial cost is low. Compared with the wild type, the mutant completely eliminates the catalytic activity for 2-chloronicotinamide, and no 2-chloronicotinamide is produced in the process. Under optimal conditions, the present invention uses the nitrile hydrolase mutant PgNit-A55S to catalyze 6-chloronicotinamide, achieving an activity of 580 U / g (WCW). It can catalyze the synthesis of 6-chloronicotinic acid from 300 g / L 6-chloronicotinamide with a yield of 99%, while eliminating the hydration activity of 2-chloronicotinamide and producing no 2-chloronicotinamide. (IV) Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0027] The final concentration composition of the LB solid culture medium of the present invention is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, water as solvent, pH 7.0.

[0028] The final concentration of the LB liquid culture medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, with water as the solvent and pH 7.0.

[0029] The PB buffer solution (50mM, pH 7.0) is prepared as follows: Weigh 2.28g of K2HPO4·H2O and add 200mL of ultrapure water to dissolve it completely; then weigh 1.36g of KH2PO4·H2O into 200mL of ultrapure water, mix the two together, and adjust the pH of the buffer solution to 7.0.

[0030] Example 1: Construction of PgNit recombinant bacteria

[0031] The nitrile hydrolase (PgNit) gene (ACCESSION NO: QCT24552.1, nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) from *Paraburkholderia graminis* was synthesized in its entirety at Beijing Qingke Biotechnology Co., Ltd., ligated to the NcoI and XhoI restriction sites of plasmid pET-28b, transformed into *E. coli* DH5 competent cells, plated on LB agar plates containing a final concentration of 50 μg / ml kanamycin, and incubated overnight at 37°C. Positive transformants were then picked and sequenced for identification. The validated positive clones were inoculated into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C. After plasmid extraction and validation, the recombinant expression vector was transformed into E. coli BL21(DE3) strain to obtain recombinant E. coli BL21(DE3) / pET28b-PgNit, which was cultured overnight at 37°C on LB agar plates containing a final concentration of 50 μg / mL kanamycin. Single clones were picked and inoculated into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C. The bacterial culture was then mixed with 30% glycerol (v / v = 1:1) in glycerol tubes and stored at -80°C.

[0032] SEQ ID NO.1:

[0033] atgggtaaagttgtcaaagccgctgctgttcaattttctccagttctgtacagccgcgaagcaaccgtagcaaaagtcgtacagaagatccacgaactgggtctgaaaggcgtgcaattcgctaccttcccggaaaccgttgtcccgtactacccgtatttc gctgcagttcagactggtatcgagctgctgagcggttccgaacacctgcgtctgctggagcaggcggttactgttcctagcgcggctacggatgcaatcggtaaagctgctcgtgaagcaggtatggttgtatctatcggcgtgaacgagcgcgacggtggcacgctgtacaacacccaactgctgtttgatgccgatggcacgctgattcagcgtcgccgtaaaatcaccccaactcacttcgaacgtatgatttggggtcagggtgatggttctggtctgcgtgcggttgattccgccgtgggccgcattggtcagctggcgtgtttcgaacataacaacccactggcccgttacgcgatgatcgctgatggcgaacaaatccattctgcgatgtatccgggcagcgcttttggcgaaggttttgcgcagcgtatggaaatcaacattcgtcagcacgcactggaatccggcgcgttcgtagtcaacgcaaccgcatggctggatgcggatcagcaggcacaaattatgaaagacaccggctgcggcattggtccaattagcggtggttgtttcaccaccattgtttccccggacggtatgctgatggctgaaccgctgcgctctggtgagggcgaggtcatcgttgacctggactttgcacagatcgatcgtcgtaaaatgctgatggacgctgccggtcattacaaccgtccggaactgctgtctctgatgatcgatcgtacccctaccgcgcatgtacatgaacgtgcgccgcactccctgccggtaagcgacaaagcggacgacgacgtgcgcacccaagcggctgcagtcgcgggttcccgcctcgagatt

[0034] SEQ ID NO.2:

[0035] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYF AAVQTGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQRRRKITPTHFERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQ RMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0036] Example 2: Obtaining the PgNit mutant and constructing genetically engineered bacteria containing the mutant

[0037] 1. Construction of mutant libraries

[0038] Using the recombinant expression vector pET28b-PgNit obtained in Example 1 as a template, the amplified sequence was obtained by error-prone PCR amplification.

[0039] The amplification primers were (5'TAATACGACTCACTATAGGG 3') and (5'TGCTAGTTATTGCTCAGCG 3').

[0040] The amplification system consisted of: 50 μL reaction mixture, 5 μL 10x Taq polymerase buffer, and Mg2+. 2+ (25mM): 2-8μl; Mm 2+ (25mM): 2-8μL; 2.5mM each of dATP, dCTP, dGTP and dTTP; 1μL each of upstream and downstream primers at a concentration of 50μM; DNA template: 1μL; Taq DNA polymerase: 10U; make up the volume with double-distilled water.

[0041] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 1 min, followed by temperature cycling at 95℃ for 10 s, 56℃ for 90 s, and 72℃ for 1 min for a total of 30 cycles, and a final extension at 72℃ for 10 min. The error-prone PCR products were purified using a clean-up kit.

[0042] Using the gene fragment obtained from the above random mutation as primers and the pET28b-PgNit plasmid as a template, PCR was performed using high-fidelity DNA polymerase to obtain a linear full-plasmid PCR product. After the full-plasmid PCR product was detected as positive by agarose gel electrophoresis, it was demethylated using the restriction endonuclease DpnⅠ, purified using a clean-up kit, and transformed into E. coli BL21(DE3) competent cells to obtain recombinant cells.

[0043] 2. Screening of mutants

[0044] (1) Single clones from the mutant library were selected using sterile toothpicks and inoculated into 96-well plates containing 600 μL of LB liquid medium with 50 μg / mL kanamycin. The culture was incubated at 37°C and 180 rpm for 20-22 h to obtain the seed culture. Subsequently, 200 μL of the seed culture was transferred to a new 96-well plate and incubated at 37°C and 180 rpm for 2-3 h to allow OD to develop. 600 Once the pH reaches 0.6-0.8, add IPTG to each well to a final concentration of 0.1 mM and incubate at 28°C and 180 rpm for 12 h. Then, centrifuge the 96-well plate at 4°C and 4000 rpm for 15 min, discard the supernatant, and retain the bacterial cells for later use.

[0045] (2) In step (1), 200 μL of PB buffer (50 mM, pH 7.0) was added to each well of the 96-well plate to resuspend the nitrile hydrolase cells. 6-chloronicotinamide was added to a final concentration of 50 mM. The reaction was carried out at 30 °C and 180 rpm for 5 min. 15 μL of 6 M HCl was added to terminate the reaction. Then, the plate was centrifuged at 4 °C and 4000 rpm for 15 min.

[0046] Add 200 μL of PB buffer (50 mM, pH 7.0) to another 96-well plate to resuspend the nitrile hydrolase cells, 0.5 g / L amidase (from different sources, as long as it is active against 2-chloronicotinamide), to a final concentration of 50 mM 2-chloronicotinamide, and react at 30 °C and 180 rpm for 5 min. Add 15 μL of 6 M HCl to terminate the reaction, and then centrifuge at 4 °C and 4000 rpm for 15 min.

[0047] (3) Take 20 μL of the supernatant after centrifugation and transfer it to a black fluorescent microplate. Add fluorescent reagent (prepared by mixing 0.1 g phthalic acid, 50 μL mercaptoethanol and 20 mL anhydrous ethanol and diluting it tenfold) to each well. Incubate at 37°C for 30 min and measure the fluorescence intensity (412 nm excitation wavelength, 467 nm emission wavelength) using a microplate reader. Screen for positive mutants based on changes in fluorescence intensity.

[0048] 3. Rescreening and verification of mutants

[0049] The positive mutants obtained through the high-throughput screening were further screened and validated. 100 μL of the seed culture of the corresponding positive strain from the 96-well plate was added to 100 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm until OD500. 600 The concentration of the culture solution was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 28℃ and 180 rpm for 10-12 h. The harvested bacterial culture was centrifuged at 8000 rpm and 4℃ to collect the wet cells for later use.

[0050] Weigh 0.1 g of bacterial cells (wet weight) and resuspend them in 10 mL of PB buffer (50 mM, pH 7.0). Incubate at 30 °C for 5 min, then add 50 mM 6-chloronicotinamide and 50 mM 2-chloronicotinamide. React at 180 rpm and 30 °C for 5-10 min. Take 1 mL of the reaction solution, add 10 μL of 6 M HCl to terminate the reaction, then let stand for 1 min and centrifuge at 12000 rpm for 2 min. Collect the supernatant, dilute appropriately, and use the sample for high-performance liquid chromatography (HPLC) analysis. The specific chromatographic equipment and analytical conditions are as follows: column type: Welchrom C18 column (250 mm × 4.6 mm, 5 μm); column operating temperature: 30 °C; UV detection wavelength: 210 nm; mobile phase: 25% acetonitrile and 75% water (containing 0.1% H3PO4); flow rate: 1.0 mL / min.

[0051] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of 6-chloronicotinic acid per minute at 30°C and pH 7.0. One unit of enzyme activity (1U) is defined as the amount of enzyme required to generate 1 μmol of 6-chloronicotinic acid per minute.

[0052] Through the above verification, the PgNit-A55S mutant (denoted as PgNit) was obtained. M The sequence of the mutant is shown in SEQ ID NO.2, where the alanine at position 55 is mutated to serine. The amino acid sequence of the mutant is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3.

[0053] The wild-type PgNit exhibits a hydrolytic activity of 450 U / g (WCW) for 6-chloronicotinonitrile and a hydration activity of 12 U / g (WCW) for 2-chloronicotinonitrile; the mutant A55S exhibits a hydrolytic activity of 580 U / g (WCW) for 6-chloronicotinonitrile and eliminates its hydration activity for 2-chloronicotinonitrile.

[0054] SEQ ID NO.3:

[0055] atgggtaaagttgtcaaagccgctgctgttcaattttctccagttctgtacagccgcgaagcaaccgtagcaaaagtcgtacagaagatccacgaactgggtctgaaaggcgtgcaattcgctaccttcccggaaaccgttgtcccgtactacccgtatttc tctgcagttcagactggtatcgagctgctgagcggttccgaacacctgcgtctgctggagcaggcggttactgttcctagcgcggctacggatgcaatcggtaaagctgctcgtgaagcaggtatggttgtatctatcggcgtgaacgagcgcgacggtggcacgctgtacaacacccaactgctgtttgatgccgatggcacgctgattcagcgtcgccgtaaaatcaccccaactcacttcgaacgtatgatttggggtcagggtgatggttctggtctgcgtgcggttgattccgccgtgggccgcattggtcagctggcgtgtttcgaacataacaacccactggcccgttacgcgatgatcgctgatggcgaacaaatccattctgcgatgtatccgggcagcgcttttggcgaaggttttgcgcagcgtatggaaatcaacattcgtcagcacgcactggaatccggcgcgttcgtagtcaacgcaaccgcatggctggatgcggatcagcaggcacaaattatgaaagacaccggctgcggcattggtccaattagcggtggttgtttcaccaccattgtttccccggacggtatgctgatggctgaaccgctgcgctctggtgagggcgaggtcatcgttgacctggactttgcacagatcgatcgtcgtaaaatgctgatggacgctgccggtcattacaaccgtccggaactgctgtctctgatgatcgatcgtacccctaccgcgcatgtacatgaacgtgcgccgcactccctgccggtaagcgacaaagcggacgacgacgtgcgcacccaagcggctgcagtcgcgggttcccgcctcgagatt

[0056] SEQ ID NO.4:

[0057] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYF SAVQTGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQRRRKITPTHFERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQ RMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0058] Example 3: Preparation of PgNit and its mutant PgNit-A55S catalyst

[0059] 1) Plate culture: Recombinant genetically engineered bacteria containing nitrile hydrolase PgNit and bacteria containing mutant PgNit were cultured separately. M The recombinant genetically engineered bacteria were streaked onto solid LB medium containing 50 μg / mL kanamycin and activated overnight at 37°C to obtain single colonies.

[0060] 2) Seed culture: Inoculate a single colony into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and culture at 37℃ and 180 rpm for 10-12 h to obtain seed culture.

[0061] 3) Fermentation culture: Add kanamycin to a shake flask containing 100 mL of liquid LB medium to a final concentration of 50 μg / mL, and inoculate with 2% (v / v) seed culture. Incubate at 37°C and 180 rpm until OD reaches 50%. 600 The concentration of the culture medium was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 28℃ and 180 rpm for 10-12 h. The harvested bacterial culture was centrifuged at 8000 rpm and 4℃ to collect the wet cells, obtaining PgNit wet cells and PgNit cells respectively. M Wet bacterial cells, ready for use.

[0062] Example 4: PgNit and its mutant PgNit M The reaction process of catalytic chloronicotinamide substrate

[0063] 1. Add the PgNit wet cells and PgNit prepared according to the method in Example 3 to 20 mL of PB buffer (pH 7.0, 50 mM). M10 g / L of wet bacterial cells were reacted with a mixture of 100 g / L 2-chloronicotinamide and 100 g / L 6-chloronicotinamide as substrates at 30 °C, and detected by high-performance liquid chromatography as described in Example 2. The results showed that after 1 h of reaction, PgNit and PgNit... M Both wet bacterial cells and 100 g / L 6-chloronicotinamide can be catalyzed, with a yield exceeding 99%. However, PgNit produces 4.8 g / L 2-chloronicotinamide during the catalytic process, while PgNit... M No 2-chloronicotinamide was produced during the mutant catalysis process.

[0064] 2. Add the PgNit wet cells and PgNit prepared according to the method in Example 3 to 20 mL of PB buffer (pH 7.0, 50 mM). M 10 g / L of wet bacterial cells were used as substrates in a reaction at 30 °C with a mixture of 200 g / L 2-chloronicotinamide and 200 g / L 6-chloronicotinamide. The reaction was analyzed using the high-performance liquid chromatography method described in Example 2. The results showed that after 2 hours of reaction, PgNit catalyzed the synthesis of 6-chloronicotinic acid from 200 g / L 6-chloronicotinamide in a yield of 96%, and produced 4.2 g / L 2-chloronicotinamide during the catalytic process. M The catalytic synthesis of 6-chloronicotinic acid from 200 g / L 6-chloronicotinamide achieved a yield of 99%, with no 2-chloronicotinamide produced during the catalytic process.

[0065] 3. Add the PgNit wet cells and PgNit prepared according to the method in Example 3 to 20 mL of PB buffer (pH 7.0, 50 mM). M 10 g / L of wet bacterial cells were used as substrates in a mixture of 300 g / L 2-chloronicotinamide and 300 g / L 6-chloronicotinamide, and the reaction was carried out at 30 °C. The results were analyzed using the high-performance liquid chromatography method described in Example 2. The results showed that after 4 hours of reaction, PgNit catalyzed the synthesis of 6-chloronicotinic acid from 300 g / L 6-chloronicotinamide in a 92% yield, and 3.9 g / L 2-chloronicotinamide was produced during the catalytic process. M The catalytic synthesis of 6-chloronicotinic acid from 200 g / L 6-chloronicotinamide achieved a yield of 99%, with no 2-chloronicotinamide produced during the catalytic process.

[0066] This invention is not limited to the specific textual description above. Various modifications can be made to this invention within the scope outlined in the claims, and all such modifications are within the scope of this invention.

Claims

1. A nitrile hydrolase mutant, PgNit-A55S, that selectively catalyzes the synthesis of 6-chloronicotinamide from 6-chloronicotinamide, characterized in that, The nitrile hydrolase mutant PgNit-A55S was obtained by mutating alanine at position 55 of the amino acid shown in SEQ ID NO.2 to serine.

2. A recombinant genetically engineered bacterium containing the encoding gene of the nitrile hydrolase mutant PgNit-A55S as described in claim 1.

3. The recombinant genetically engineered bacteria as described in claim 2, characterized in that, The recombinant genetically engineered bacteria are prepared by inserting the target gene into the plasmid pET28b(+). Nco I and Xho Between I, transformation into E. coli Obtained from BL21(DE3) host cells.

4. The application of the nitrile hydrolase mutant PgNit-A55S according to claim 1 in the selective catalytic synthesis of 6-chloronicotinic acid from 6-chloronicotinamide.

5. The application as described in claim 4, characterized in that, The application is as follows: using wet cells or crude enzyme solution extracted from recombinant Escherichia coli containing the nitrile hydrolase mutant PgNit-A55S gene obtained by fermentation culture as a catalyst, 6-chloronicotinamide and 2-chloronicotinamide as substrates, and a buffer solution with pH 6.0-9.0 as the reaction medium, the reaction is carried out at 25-40 ℃ to obtain a reaction solution containing 6-chloronicotinic acid.

6. The application as described in claim 5, characterized in that, The amount of crude enzyme solution added is based on the weight of the wet bacterial cells before lysis, and the amount of wet bacterial cells added is 2-20 g / L based on the volume of the buffer solution; the amounts of 6-chloronicotinonitrile and 2-chloronicotinonitrile added are each 100-500 g / L based on the volume of the buffer solution.

7. The application as described in claim 5, characterized in that, The wet cells or crude enzyme solution extracted from the wet cells of recombinant Escherichia coli containing the nitrile hydrolase mutant PgNit-A55S gene were prepared by fermentation culture as follows: 1) Plate culture: Recombinant Escherichia coli containing the nitrile hydrolase mutant PgNit-A55S gene was streaked onto solid LB medium containing 50 μg / mL kanamycin and activated overnight at 37 °C to obtain single colonies; The final concentration of the LB solid medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, with water as the solvent and pH 7.

0. 2) Seed culture: Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 ℃ and 180 rpm for 10-12 h to obtain seed culture; the final concentration composition of the LB liquid medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0; 3) Fermentation culture: The seed culture was inoculated at a volume concentration of 2% into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 ℃ and 180 rpm until OD. 600 The concentration was 0.6-0.

8. Isopropyl-β-D-thiopyranoside was added to a final concentration of 0.1 mM, and the mixture was induced and cultured at 28 ℃ and 180 rpm for 10-12 h. The bacterial culture was then centrifuged at 8000 rpm and 4 ℃ to collect the wet bacterial cells. 4) Wet cell disruption: Suspend wet cells in a buffer solution with pH 5.0~9.0, place them in an ultrasonic disruptor, and disrupt them at a frequency of 10-50 kHz for 5-30 min. Collect the disrupted mixture to obtain the crude enzyme solution.