A method for modifying nitrile hydratase to regulate the regioselectivity of nitrile compounds and its application

Abstract

The present invention belongs to the technical field of green chemistry, and particularly relates to a method for modifying nitrile hydratase to regulate the regioselectivity of nitrile compounds and its application. The construction method is to perform site-directed mutagenesis on the key amino acids located in the substrate channel and binding pocket of nitrile hydratase to construct a pure enzyme of nitrile hydratase mutant. The newly constructed pure enzyme of nitrile hydratase mutant can be applied to regulate the regioselectivity of the catalytic reaction of nitrile compounds. Compared with before the modification, the regioselectivity of the enzyme-catalyzed reaction is biased towards the intermediate 5-cyanovaleramide, significantly inhibiting the formation of the by-product adipamide, achieving the enrichment of 5-cyanovaleramide, and having important reference significance for the regulation of the regioselectivity of the catalytic reaction and the synthesis research of the intermediate, which is beneficial to enhancing the industrial application value of nitrile hydratase.

Description

Technical Field

[0001] The present invention belongs to the technical field of green chemistry, and specifically relates to a method for modifying nitrile hydratase to regulate the regioselectivity of nitrile compounds and its application. The method for modifying nitrile hydratase can regulate the regioselectivity of the catalytic reaction of nitrile hydratase on nitrile compounds and achieve the enrichment of intermediate products. Background Art

[0002] Amide is an important chemical product and also an important raw material for synthesizing chemical products such as organic reaction intermediates, stabilizers, and plasticizers. It has been widely used in fields such as organic synthesis, chemical production, food and feed, and healthcare. At the same time, its application in fine chemical synthesis gives it extremely high chemical added value. In the synthesis process of amide, chemical synthesis was dominant in the past. However, the harsh reaction conditions and the use of polluting catalysts have limited the chemical synthesis of amide. With the proposal of the green chemistry concept of energy conservation, environmental protection, and sustainable development, biocatalytic synthesis represented by nitrile hydratase has gradually been advocated as a synthesis process route for amide and is considered an effective alternative to chemical synthesis.

[0003] Nitrile hydratase (EC4.2.1.84, Nitrile Hydratase, abbreviated as NHase) is a type of metalloenzyme among nitrile-converting enzymes. According to the types of metal ions in its active center, it is divided into Fe-type nitrile hydratase and Co-type nitrile hydratase. The above metal ions cooperate with the α subunit and the β subunit to form an active nitrile hydratase together. The earliest nitrile hydratase was discovered in bacteria. Researchers found that this enzyme can catalyze nitrile compounds to form amides, with great research value and industrial application potential. Therefore, more and more nitrile hydratases have been discovered in different types of bacteria and fungi. With the continuous in-depth research on this enzyme, its industrial application has become increasingly mature. Currently, the industrial synthesis of acrylamide and nicotinamide using nitrile hydratase as a biocatalyst has been realized, effectively replacing chemical synthesis.

[0004] In the related research of organic synthesis and synthetic biology, the synthesis of various catalytic reaction intermediate products is a hot and difficult point in current scientific research and industrial applications. At the same time, it has been found that nitrile hydratases from different sources show different regioselectivities for catalyzing dinitrile and trinitrile compounds, and there are significant differences in the amide products generated. Regioselectivity refers to when the substrate in a catalytic reaction has multiple groups, preferentially reacting with a certain same functional group at different positions within the molecule, and other groups react subsequently or are not reacted. The most representative reaction is the catalytic synthesis of adiponitrile to the intermediate product 5-cyanovaleramide.

[0005] 5-Cyanopentanamide (5-CVAM) is a fine chemical intermediate with high added value, which is used in the synthesis of the new herbicide Azafenidin. Additionally, it is also a precursor for the synthesis of the chemical intermediate 6-aminohexanamide. Since 5-cyanopentanamide is a monoamide intermediate product in which only one end of the cyano group is catalytically generated during the catalytic reaction of adiponitrile, traditional chemical catalysis not only has the defects of harsh reaction conditions and huge catalyst consumption, but also it is difficult to enrich this monoamide product. Therefore, people have turned their attention to biocatalysis. In previous studies, by screening and excavating new strains, nitrile hydratase strains with good regioselectivity for 5-cyanopentanamide can be obtained, but they often face defects such as too long screening cycles, extremely difficult cultivation, and low catalytic efficiency, making it difficult to meet the requirements of industrial applications. With the booming development of crystal analysis and bioinformatics technology, people have a deeper understanding of the reaction mechanism of nitrile hydratase catalyzing nitrile compounds, and at the same time, there have been significant breakthroughs in the application and modification of nitrile hydratase. By heterologously expressing and molecularly modifying nitrile hydratase, not only can engineering strains that are easy to cultivate at high density and have industrial application prospects be obtained, but even the properties such as the regioselectivity of the enzyme can be regulated at the molecular level.

[0006] In the early stage of the research of this invention, a Fe-type nitrile hydratase was discovered, and a nitrile hydratase with high efficiency in catalyzing adiponitrile to generate 5-cyanopentanamide was constructed through heterologous expression and modification optimization. However, it was found in the research that after the nitrile hydratase catalyzed the cyano group at one end of adiponitrile to generate 5-cyanopentanamide, the cyano group at the other end would continue to react to generate the diamide product - adipamide (ADM), resulting in serious waste of the intermediate product 5-cyanopentanamide and making it impossible to enrich. Therefore, in this invention, point mutations were made to the key amino acids located in the active pocket and substrate channel of the nitrile hydratase, and a mutant with excellent regioselectivity for 5-cyanopentanamide was obtained, and the enrichment of the monoamide intermediate product was achieved. This invention has important reference significance for the regulation of the regioselectivity of the catalytic reaction and the synthesis research of the intermediate product, which is beneficial to enhancing the industrial application value of nitrile hydratase. Summary of the Invention

[0007] This invention provides a method for modifying nitrile hydratase and its application, which can greatly improve the regioselectivity for the monoamide intermediate product and achieve the enrichment of the monoamide intermediate product by making point mutations to some sites in the binding pocket and substrate channel of nitrile hydratase.

[0008] Technical solution of this invention:

[0009] A method for modifying nitrile hydratase to regulate the regioselectivity of nitrile compounds. The modification method is to perform site-directed mutagenesis on sites Y37, M40, and Y72 of the nitrile hydratase recombinant plasmid βWT-ReNHase-AC-His to construct a nitrile hydratase mutant with excellent regioselectivity for monoamide products. The specific method is as follows: Using the nitrile hydratase recombinant plasmid βWT-ReNHase-AC-His as a template, using Pst I and Sac I as restriction enzyme sites, gene splicing by overlap extension (SOE) PCR is used to amplify the target sequence. After double digestion of the target sequence and the template plasmid, T4 ligase is used to ligate the target sequence to βWT-ReNHase-AC-His to achieve the construction of recombinant plasmids of mutants Y37P-ReNHase, M40F-ReNHase, and Y72A-ReNHase.

[0010] The above three constructed recombinant plasmids are respectively transformed into ArcticExpress(DE3) Escherichia coli competent cells by heat shock at 42°C. After overnight culture, single colonies are picked for culture, and then IPTG is added for low-temperature induction culture. After the induction is completed, the bacterial solution is centrifuged, washed with PB buffer, resuspended, and sonicated. The supernatant after centrifugation is filtered to remove impurities, and protein purification is carried out using AKTA Pure to obtain pure nitrile hydratase mutants Y37P-ReNHase, M40F-ReNHase, and Y72A-ReNHase.

[0011] The amino acid sequence of the template nitrile hydratase βWT-ReNHase-AC-His is shown in SEQ ID NO.1, the amino acid sequence of mutant Y37P-ReNHase is shown in SEQ ID NO.2, the amino acid sequence of mutant M40F-ReNHase is shown in SEQ ID NO.3, and the amino acid sequence of mutant Y72A-ReNHase is shown in SEQ ID NO.4.

[0012] SEQ ID NO.1:

[0013] MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTVMDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLSRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0014] SEQ ID NO.2:

[0015] MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTVMDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPPSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLSRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0016] SEQ ID NO.3:

[0017] MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTVMDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLFFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLSRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0018] SEQ ID NO.4:

[0019] MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTVMDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPAYERYVIGVAALMVEKGILTQEELESLAGGPFPLSRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0020] The application of the above-prepared pure nitrile hydratase mutant in the reaction of catalyzing nitrile compounds is specifically manifested in the catalytic reaction of nitrile compounds. The obtained pure nitrile hydratase mutant is used as a catalyst for the catalytic reaction.

[0021] The nitrile compound described is an aliphatic nitrile compound.

[0022] The catalytic reaction of the nitrile compound includes catalyzing adiponitrile to synthesize 5-cyanovaleramide and by-product adipamide.

[0023] Advantages of the present invention:

[0024] (1) The nitrile hydratase modified by point mutation can significantly regulate the regioselectivity of the catalytic reaction of adiponitrile, making the regioselectivity shift from originally favoring adipamide to favoring 5-cyanovaleramide, effectively avoiding the further conversion and waste of 5-cyanovaleramide during the catalytic reaction of adiponitrile, and having important reference significance for the research on the regulation of regioselectivity and the synthesis of intermediate products in the catalytic reaction;

[0025] (2) The mutant constructed in the present invention can achieve the enrichment of the intermediate product 5-cyanovaleramide, enhance the industrial application value of nitrile hydratase, and provide a theoretical basis for its industrial application. Description of the drawings

[0026] Figure 1 For βWT-ReNHase-AC-His and three mutants Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase, the concentrations and ratios of the conversion of adiponitrile to 5-cyanovaleramide and adipamide by whole-cell catalysis under a reaction time of 30 min. Each group of experiments was repeated three times.

[0027] Figure 2 For the pure enzymes of βWT-ReNHase-AC-His and three mutants Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase, the concentrations of the conversion of adiponitrile to 5-cyanovaleramide and adipamide at 2 / 5 / 10 / 30 min respectively. Each group of experiments was repeated three times. Detailed implementation manners

[0028] The following further describes the detailed implementation manners of the present invention in combination with the drawings and technical solutions.

[0029] A method for modifying nitrile hydratase to regulate the regioselectivity of nitrile compounds, which performs point mutation modification on the Y37, M40, and Y72 sites of nitrile hydratase, constructs three mutant strains of nitrile hydratase, and obtains pure nitrile hydratase mutants through protein expression; the specific steps are as follows:

[0030] (1) Construction of mutant strains: Using the recombinant plasmid of nitrile hydratase βReNHase-AC-His as a template and cloning vector, with Pst I and Sac I as restriction enzyme sites, the gene sequences at positions Y37, M40, and Y72 on the β subunit were subjected to site-directed mutagenesis using gene splicing by overlap extension PCR (SOE-PCR) technology, while amplifying the target gene sequence. Finally, the target gene sequence and the vector plasmid were double-digested and ligated using T4 ligase to obtain three recombinant plasmids of nitrile hydratase mutants after site-directed mutagenesis. The three constructed recombinant plasmids of nitrile hydratase mutants were respectively transformed into ArcticExpress(DE3) Escherichia coli competent cells by heat shock at 42°C to construct nitrile hydratase mutant strains. To distinguish the three mutant strains, they were named Y37P-ReNHase, M40F-ReNHase, and Y72A-ReNHase respectively, and then the strains were preserved in glycerol with a volume fraction of 30% (v / v) to prepare glycerol stocks.

[0031] (2) Protein expression and purification: Inoculate the glycerol stocks of the three nitrile hydratase mutant strains prepared in a test tube, and then add IPTG for low-temperature overnight induction of protein expression; centrifuge the bacterial solution the next day, wash and resuspend the bacteria with PB buffer to obtain a bacterial suspension. After ultrasonic disruption and centrifugation, the supernatant was filtered through a membrane, and the above three mutant proteins were purified using AKTA pure to obtain the corresponding pure enzymes.

[0032] Among them, the amino acid sequences of the template nitrile hydratase βWT-ReNHase-AC-His and mutants Y37P, M40F, and Y72A are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

[0033] Through control experiments, it was found that the three experimental group mutants after site-directed mutagenesis changed the regioselectivity of nitrile hydratase towards adiponitrile reaction, effectively inhibited the formation of by-product adipamide, and achieved the enrichment of the intermediate 5-cyanovaleramide.

[0034] Example 1 Determination of the products of the whole-cell catalysis of adiponitrile by Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase

[0035] (1) Preparation of bacterial suspension: Take 2 μL of each of the three constructed recombinant plasmids of nitrile hydratase mutants and the control recombinant plasmid βWT-ReNHase-AC-His and add them to 50 μL of competent Escherichia coli Arctic Express (DE3) respectively, and let them stand on ice for 30 min. Then heat shock at 42 °C for 90 seconds and quickly place on ice for 3 min. Add 500 μL of LB liquid medium and activate by shaking at 37 °C and 220 rpm for 1 h. Pipette 100 μL of the activated Escherichia coli into two pre-prepared LB solid media respectively and spread them evenly on the plates. Incubate them in an inverted position on a constant temperature shaker overnight. The next day, a large number of monoclonal colonies grow on the plates. Pick the monoclonal colonies on the above plates and inoculate them into test tubes containing LB liquid medium with 50 μg / mL kanamycin, and culture them by shaking at 37 °C and 220 rpm until the OD 600 value is between 0.6 and 0.8. Save a part of the bacterial liquid in glycerol with a volume ratio of 30% (v / v) to prepare glycerol bacteria and store them in a -80 °C refrigerator; at the same time, take out 300 μL of the remaining bacterial liquid in the test tube and transfer it into 30 mL of LB liquid medium for scale-up culture, and save a part of the bacterial liquid in glycerol with a volume ratio of 30% (v / v) to prepare glycerol bacteria and store them in a -80 °C refrigerator. When the OD 600 value is in the range of 0.6 - 0.8, add IPTG with a volume ratio of 0.2% (v / v) and induce at low temperature at 16 °C and 200 rpm overnight for 18 h. The next day, collect the bacterial liquid at 5000 rpm and 4 °C and pour off the supernatant. Wash twice with PB buffer (i.e., pH = 7, 20 mM) and resuspend the cells with 3 mL of the above PB buffer to obtain a bacterial suspension. Dilute the bacterial suspension with a UV spectrophotometer until the OD 600 = 1.2, and complete the preparation of the bacterial suspension for catalytic reaction;

[0036] (2) Whole-cell catalytic conversion reaction of adiponitrile: Add 250 μL of 100 mM adiponitrile, 50 μL of 20 mM PB buffer (pH = 7) to 4 EP tubes in sequence, and finally add 50 μL of the bacterial suspension prepared in (1) to each tube. Place them in a shaker and carry out the whole-cell catalytic conversion reaction of adiponitrile under the conditions of 200 rpm, 30 °C and light. The reaction concentration of the substrate adiponitrile in the system is 50 mM, and the reaction time is 30 min. Finally, terminate the reaction with 500 μL of methanol;

[0037] (3) Ultra-high performance liquid chromatography (UPLC) analysis: Centrifuge the above four tubes with terminated reactions at 10000 rpm for 10 min, filter them with a 0.22 μm filter membrane and then carry out high performance liquid chromatography detection. The ultra-high performance liquid chromatography uses ACQUITYUPLC CSH TMA C18 1.7μm chromatographic column was used with a mobile phase of 25 mM phosphoric acid aqueous solution and methanol (89:11, vol:vol). The detection wavelength was 200 nm, the column temperature was 25 °C, and the flow rate was 0.3 ml / min. The contents of 5-cyanovaleramide and the by-product adipamide generated in the experimental group and the control group were detected and calculated. As Figure 1 shown, in the amide products of the control group WT-ReNHase-AC-His after 30 min of reaction, the by-product adipamide accounted for more than 90%, and only a very small amount of 5-cyanovaleramide; while in the amide products of the experimental groups Y37P-ReNHase, M40F-ReNHase, and Y72A-ReNHase after 30 min of reaction, almost all were 5-cyanovaleramide, and no by-product adipamide was generated.

[0038] Example 2 Determination of the reaction process of Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase pure enzyme catalyzing adiponitrile

[0039] (1) Preparation of bacterial suspension: In test tubes of LB liquid medium containing 50 μg / mL kanamycin, 50 μL of the glycerol bacteria of the three nitrile hydratase mutants prepared in Example 1 and the control group βWT-ReNHase-AC-His strain were inoculated and cultured with shaking at 37 °C and 220 rpm until the OD 600 value was between 0.6 and 0.8. Then 5 mL of the bacterial solution was taken out and transferred into 500 mL of LB liquid medium for scale-up culture. When the OD 600 value was in the range of 0.6 - 0.8, IPTG with a volume ratio of 0.2% (v / v) was added, and low-temperature induction culture was carried out overnight at 16 °C and 200 rpm for 18 h. The next day, the bacterial solution was recovered at 5000 rpm and 4 °C, and the supernatant was discarded. It was washed twice with PB buffer (i.e., PB solution with pH = 7 and 20 mM), and the cells were resuspended with 20 mL of PB buffer to obtain the mutant Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase and the control group βWT-ReNHase-AC-His bacterial suspensions;

[0040] (2) Cell disruption and enzyme purification: The above-mentioned resuspended bacterial suspension was disrupted by an ultrasonic disruptor at a power of 300 W, disrupting for 1 s and intermittent for 9 s, for a total of 90 cycles. The disrupted suspension was centrifuged at 10000 rpm for 30 min, and then the supernatant was filtered through a 0.22 μm filter membrane. Then, through an AKTA pure protein purification system, ion exchange chromatography was used for enzyme purification to obtain pure enzyme. The concentration of the pure enzyme was determined by a Bradford protein quantification detection kit and diluted to 0.3 mg / mL;

[0041] (3) Process of the conversion reaction catalyzed by pure enzyme: In four EP tubes, 250 μL of 100 mM adiponitrile, 200 μL of 20 mM PB buffer (pH = 7) were added successively. Finally, 50 μL of the pure enzyme dilution of mutant Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase at 0.3 mg / mL and the pure enzyme dilution of the control group βWT-ReNHase-AC-His were added respectively. Then, the tubes were placed in a shaker and the conversion reaction of adiponitrile catalyzed by pure enzyme was carried out under the conditions of 200 rpm, 30 °C and light. The reaction concentration of the substrate adiponitrile in the system was 50 mM. After the reaction for 2 / 5 / 10 / 30 / min, 500 μL of methanol was added to terminate the reaction;

[0042] (4) Ultra-high performance liquid chromatography (UPLC) analysis: The specific operation process was the same as that in step (3) of Example 1. As Figure 2 shown, after the control group's βWT-ReNHase-AC-His completely converted the substrate adiponitrile into 5-cyanovaleramide in 2 min, it further converted 5-cyanovaleramide into the by-product adipamide at an extremely fast rate, resulting in the loss of 5-cyanovaleramide and making it difficult to enrich. However, the three mutants Y37P-ReNHase / M40F-ReNHase / Y72A-ReNHase in the experimental group converted the substrate adiponitrile into 5-cyanovaleramide completely at different times with different catalytic efficiencies, and basically no by-product adipamide was generated. Moreover, the subsequent 5-cyanovaleramide was not converted into adipamide either, enabling the enrichment of 5-cyanovaleramide.

Claims

1. A method for modifying nitrile hydratase for regulating the regioselectivity of nitrile compounds, characterized in that: The transformation method is to perform point mutation on the site Y37, M40 or Y72 of the nitrile hydratase in the nitrile hydratase recombinant plasmid βWT-ReNHase-AC-His, respectively, to construct a nitrile hydratase mutant with excellent regional selectivity for the monoamide product; the specific method is as follows: Using the nitrile hydratase recombinant plasmid βWT-ReNHase-AC-His as a template, using Pst I and Sac I as restriction sites, using gene bridges, PCR amplification of the target sequence, double restriction digestion of the target sequence and the template plasmid, and then using T4 ligase to connect the target sequence to βWT-ReNHase-AC-His to achieve the construction of the recombinant plasmid of mutant Y37P-ReNHase, M40F-ReNHase or Y72A-ReNHase; The three recombinant plasmids constructed above were introduced into ArcticExpress (DE3) Escherichia coli competent cells via 42°C heat shock transformation, and single clones were selected for culture after overnight culture, followed by the addition of IPTG for low-temperature induction culture; after the induction, the bacterial solution was centrifuged, PB buffer was added for washing, the bacterial solution was resuspended and ultrasonically disrupted, the supernatant after centrifugation was filtered through a membrane to remove impurities, and AKTA Pure was used for protein purification to obtain pure enzymes of nitrile hydratase mutants Y37P-ReNHase, M40F-ReNHase or Y72A-ReNHase; The amino acid sequence of the nitrile hydratase in the nitrile hydratase recombinant plasmid βWT-ReNHase-AC-His is shown in SEQ ID NO.1, the amino acid sequence of the mutant Y37P-ReNHase is shown in SEQ ID NO.2, the amino acid sequence of the mutant M40F-ReNHase is shown in SEQ ID NO.3, and the amino acid sequence of the mutant Y72A-ReNHase is shown in SEQ ID NO.4; The regulating the regioselectivity of the nitrile compound is to regulate the regioselectivity of the catalytic reaction of adiponitrile, so that the regioselectivity is changed from being biased towards adipic acid amide to being biased towards 5-cyanovaleramide.

2. Use of a pure nitrile hydratase mutant enzyme obtained by the method for modifying nitrile hydratase for regulating the regioselectivity of nitrile compounds as claimed in claim 1 in catalyzing the reaction of adiponitrile to synthesize 5-cyanovaleramide and the by-product adipamide.

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