Method for modifying flexible loop region of nitrile hydratase and application of method

Abstract

The invention belongs to the technical field of green chemistry, and particularly relates to a method for modifying a flexible ring region of nitrile hydratase and application of the method. The specific method comprises the following steps: carrying out double mutation on nitrile hydratase sites alpha D38 and alpha I152, or carrying out double mutation on nitrile hydratase sites alpha E157 and beta G197, or carrying out quadruple mutation on the nitrile hydratase sites alpha D38, alpha I152, alpha E157 and beta G197, so as to construct the nitrile hydratase mutant with improved thermal stability. The thermal stability of the nitrile hydratase modified by mutation is greatly improved. The mutant nitrile hydratase can keep the conformation and activity for a longer time, the resistance to external thermal interference is enhanced, and the half-life period for catalyzing an adiponitrile reaction is remarkably prolonged, which is the inherent excellent attribute of the mutant nitrile hydratase as a catalyst, and the mutant nitrile hydratase has important reference significance for research on improvement of the thermal stability of the nitrile hydratase.

Description

Technical Field

[0001] This invention belongs to the field of green chemistry technology, specifically relating to a method for modifying the flexible ring region of nitrile hydratase and its application. Background Technology

[0002] Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme that catalyzes the formation of amide compounds from nitrile compounds. Due to the different metal ions contained in the active site of nitrile hydratases, they are generally classified into iron-type nitrile hydratases (Fe-NHase) and cobalt-type nitrile hydratases (Co-NHase). Most nitrile hydratases have poor thermostability, and Fe-type nitrile hydratases are generally less thermostable than Co-type nitrile hydratases. The nitrile hydratase derived from *Rhodococcus erythropolis* CCM2595 is an Fe-type nitrile hydratase, a tetrameric protease composed of two α-subunits and two β-subunits. It exhibits high specificity and regioselectivity for dinitriles. It can efficiently catalyze the hydration of one cyano group of adiponitrile to form the intermediate 5-cyanopentamide, with the final product being adipamide.

[0003] Amides are core structures in nylon, pharmaceuticals, pesticides, daily chemicals, and solvents. Among many high-value amide compounds, 5-cyanopentamide is a dedicated raw material for the synthesis of 6-aminohexanamide, a key intermediate in the synthesis of the herbicides pyrazosulfuron and caprolactam. The chemical synthesis of 5-cyanopentamide is difficult to separate, highly polluting, and costly. In contrast, the biocatalytic method offers milder reaction conditions, simpler separation, and recyclable catalysts, making it a green and environmentally friendly technology with promising application prospects.

[0004] Transforming the nitrile hydratase gene into *E. coli* allows for the large-scale expression of nitrile hydratase through induction. However, nitrile hydratase exhibits poor thermostability and easily loses activity under harsh environments and high temperatures, a drawback as a biocatalyst. To utilize a green and environmentally friendly biocatalytic method to generate 5-cyanopentamide, it is necessary to improve the thermostability of nitrile hydratase to make industrial production possible. Therefore, this invention combines the regression consensus sequence method and the disulfide bond construction method to perform site mutations in the flexible region of nitrile hydratase, obtaining a mutant nitrile hydratase with improved thermostability and almost no change in catalytic activity. This invention provides important insights into improving enzyme thermostability and enhances the industrial application potential of nitrile hydratase. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method and application for improving thermal stability by modifying the flexible loop region of nitrile hydratase.

[0006] The technical solution of the present invention is as follows: A method for modifying the flexible loop region of nitrile hydratase involves double mutation of αD38 and αI152 at the nitrile hydratase site, or double mutation of αE157 and βG197 at the nitrile hydratase site, or quadruple mutation of αD38, αI152, αE157, and βG197 at the nitrile hydratase site, to construct a nitrile hydratase mutant with improved thermostability. The specific method is as follows: Construction of the αD38P-αI152L nitrile hydratase recombinant plasmid: Using the recombinant plasmid WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. After double digestion of the target sequence and the template plasmid with Bsa I and Pst I as restriction enzyme sites, the target sequence was ligated into the recombinant plasmid βWT-ReNHase-AC-His using T4 ligase, thus constructing αD38P-αI152L-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent *E. coli* to express the mutant nitrile hydratase αD38P-αI152L.

[0007] Construction of the αE157C-βG197C nitrile hydratase recombinant plasmid: Using the recombinant plasmid WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. Sac II and Ahd I were used as restriction enzyme sites for double digestion of the target sequence and the template plasmid. The target sequence was then ligated into the recombinant plasmid WT-ReNHase-AC-His using T4 ligase, thus constructing αE157C-βG197C-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent *E. coli* to express the mutant nitrile hydratase αE157C-βG197C.

[0008] Construction of the αD38P-αI152L-αE157C-βG197C nitrile hydratase recombinant plasmid: Using the recombinant plasmid αD38P-αI152L-WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. Sac II and Ahd I were used as restriction enzyme sites for double digestion of both the target sequence and the template plasmid. The target sequence was then ligated into the recombinant plasmid αD38P-αI152L-WT-ReNHase-AC-His using T4 ligase, thus constructing the nitrile hydratase mutant αD38P-αI152L-αE157C-βG197C-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent *E. coli* to express the mutant nitrile hydratase αD38P-αI152L-αE157C-βG197C (MUT4).

[0009] The DNA sequence of the recombinant plasmid WT-ReNHase-AC-His is shown in SEQ ID NO.1. The amino acid sequence of the wild-type WT nitrile hydratase is shown in SEQ ID NO.2, the amino acid sequence of the mutant nitrile hydratase αD38P-αI152L is shown in SEQ ID NO.3, the amino acid sequence of the mutant nitrile hydratase αE157C-βG197C is shown in SEQ ID NO.4, and the amino acid sequence of the mutant nitrile hydratase MUT4 is shown in SEQ ID NO.5.

[0010] SEQ ID NO.1: SEQ ID NO.2: MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTV MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGGPFPLSRPSESEGRPARVDTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVDLFEGYLEPA SEQ ID NO.3: MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPPGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLSCCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTELASDVEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTV MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGGPFPLSRPSESEGRPARVDTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVDLFEGYLEPA SEQ ID NO.4: MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVCIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTV MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPL SRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGCSVVVDLFEGYLEPA SEQ ID NO.5: MSVTIDHTTENAAPAQAPVSDRAWALFRALDGKGLVPPGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTELASDVCIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAVPQVPTV MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPL SRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGCSVVVDLFEGYLEPA The purified enzyme of the nitrile hydratase mutant obtained by the above method was used as a catalyst, the substrate was a nitrile compound, and the product was an amide compound.

[0011] The beneficial effects of this invention are: (1) The mutant nitrile hydratase has significantly improved its thermal stability. The mutant nitrile hydratase can maintain its conformation and activity for a longer period of time, has enhanced resistance to external thermal disturbances, and significantly prolongs the half-life of catalyzing adiponitrile reactions. This is an inherently excellent property of it as a catalyst, and has important reference value for improving the thermal stability of nitrile hydratase. This improvement makes the nitrile hydratase itself a more robust and adaptable biocatalyst, which is its most fundamental value enhancement.

[0012] (2) This increases the possibility of industrial application of the specific conversion route from adiponitrile to 5-cyanopentamide. It directly adds weight to the industrial feasibility of this green chemical route and provides a theoretical basis for its industrial application. Attached Figure Description

[0013] Figure 1 The half-life curves of wild-type (WT) nitrile hydratase and mutant nitrile hydratase at 35 °C are shown.

[0014] Figure 2 The half-life curves of wild-type (WT) nitrile hydratase and mutant nitrile hydratase at 40 °C are shown.

[0015] Figure 3 The initial enzyme activities of wild-type (WT) nitrile hydratase and mutant nitrile hydratase are shown. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0017] The following steps were used to perform performance testing: 1. Plasmid Transformation: The recombinant plasmid carrying the nitrile hydratase gene was transformed into E. coli Arctic Expression (DE3) competent cells for expression. This operation was performed in a clean bench. ArcticExpress (DE3) competent cells were removed from the -80°C freezer and quickly placed on ice. 50 μL of competent cells were mixed with 3 μL of recombinant plasmid and incubated on ice for 25 min. The cells were then heat-shocked in a 42°C water bath for 45 s, and quickly returned to ice and incubated for 2 min. 700 μL of antibiotic-free LB liquid medium (pH 15) was added to a centrifuge tube, mixed well, and incubated at 37°C, 200 rpm for 60 min. The cells were collected by centrifugation at 5000 rpm for 2 min. 500 μL of the supernatant was removed, the remaining portion was resuspended and mixed thoroughly, and 100 μL of the bacterial culture was spread onto an LB agar plate containing 50 μg / mL Kan. Incubate overnight at 37°C, then pick single colonies for further culture (culture conditions: 2 mL LB liquid medium, 2 μL Kan, shaker at 37°C and 220 rpm). Mix 700 μL of glycerol with 700 μL of the transformed bacteria and store at -80°C; this is the glycerol-producing bacteria.

[0018] 2. Bacterial Culture Collection: Inoculate 100 μL of glycerol-containing bacteria into 6 mL of LB liquid medium containing 50 μg / mL kanamycin and incubate at 37 ℃ and 220 rpm for 4.5–5.5 h in a shaker. Transfer 5 mL of the culture to 500 mL of LB liquid medium and continue incubation under the same conditions for another 4.5–5.5 h in a shaker. After incubation, add 500 μL of 100 mmol / mL IPTG to the medium and incubate overnight at 16 ℃ and 160 rpm for 13–14 h. After induction, centrifuge the bacterial culture at 7000 rpm for 7 min and collect the bacterial cells. Wash twice with phosphate buffer (pH 7.4, 50 mM) under the same conditions, and finally resuspend the bacterial cells in 20 mL of binding buffer (pH 7.4).

[0019] 3. Cell disruption and purification: Cells were disrupted by sonication for 50 min (300 W, 3 s disruption, 7 s interval). The disruption buffer was centrifuged at 12500 r / m for 20 min at 4 ℃. The supernatant was collected and filtered through a 0.22 μm aqueous filter to remove impurities, yielding a crude enzyme solution. The enzyme was purified using an ion-exchange chromatography column in an AKTA pure purification system. The Bradford method was used to determine the enzyme concentration, and the solution was diluted to the desired concentration with PBS buffer.

[0020] 4. Half-life determination: One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of product within 1 min at 35 °C. Before the reaction, a portion of the pure enzyme solution was incubated in water baths at 35 °C and 40 °C for different times. After incubation, the pure enzyme was placed in an ice box and allowed to stand for 25 min. The reaction system consisted of 250 μL of pure enzyme (0.1 mg / mL) mixed with 250 μL of adiponitrile (400 mM), and the reaction was carried out at 35 °C with shaking at 300 r / m for 3 min, with a reaction volume of 500 μL. After the reaction, the reaction was terminated with 500 μL of methanol. Enzyme inactivation was first-order irreversible, following a first-order inactivation model. The half-life of the enzyme was calculated using the half-life formula.

[0021] 5. High-performance liquid chromatography (HPLC) analysis: The sample was filtered through a 0.22 μm filter membrane and analyzed using an Ultimate LP-C18 (5 μm, 4.6 × 250 mm) HPLC column. The concentrations of 5-cyanopentamide and adipamide were detected by UV light. The column temperature was set at 30 ℃, the detection wavelength at 200 nm, the mobile phase was 25 mmol / L phosphoric acid aqueous solution and 100% pure methanol (89%:11%), and the flow rate was 1 mL / min. The contents of 5-cyanopentamide and adipamide were calculated.

[0022] Wild-type (WT) nitrile hydratase served as the control group, while mutant nitrile hydratase served as the experimental group. The thermostability of the nitrile hydratase was determined by measuring the half-life of the wild-type and mutant nitrile hydratases.

[0023] The half-lives of WT and nitrile hydratase mutants at 35 °C and 40 °C are shown in Table 1. As can be seen from the table, the half-lives of WT at 35 °C and 40 °C are 43.19 min and 4.07 min, respectively, indicating poor thermostability. αD38P-αI152L and αE157C-βG197C both exhibited good half-lives, with half-lives at 35 °C being 2.53 times and 2.46 times that of βWT, respectively, and at 40 °C being 2.91 times and 5.73 times that of WT, respectively, demonstrating superior thermostability compared to WT. Through iteration on both, the constructed quadruple mutant MUT4 combines the advantages of αD38P-αI152L and αE157C-βG197C, exhibiting half-lives at 35 °C and 40 °C that significantly exceed those of WT, by 4.34 times and 14.66 times, respectively, demonstrating the best thermostability. Figure 3 It can be seen that the initial enzyme activities of the WT and nitrile hydratase mutants are similar, with no significant difference. This indicates that the mutant nitrile hydratase has disrupted the activity-thermal stability balance, retaining its initial activity despite a significant improvement in thermal stability.

[0024] Table 1. Half-life of WT and nitrile hydratase mutants at 35 °C and 40 °C

Claims

1. A method for modifying the flexible loop region of nitrile hydratase, characterized in that, Double mutations were performed on nitrile hydratase sites αD38 and αI152, or double mutations were performed on nitrile hydratase sites αE157 and βG197, or quadruple mutations were performed on nitrile hydratase sites αD38, αI152, αE157 and βG197 to construct nitrile hydratase mutants with improved thermostability.

2. The method for modifying the flexible loop region of nitrile hydratase according to claim 1, characterized in that, The specific method is as follows: Construction of the αD38P-αI152L nitrile hydratase recombinant plasmid: Using the recombinant plasmid WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. Bsa I and Pst I were used as restriction enzyme sites for double digestion of the target sequence and the template plasmid. The target sequence was then ligated into the recombinant plasmid βWT-ReNHase-AC-His using T4 ligase, thus constructing αD38P-αI152L-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent Escherichia coli to express the mutant nitrile hydratase αD38P-αI152L. Construction of the αE157C-βG197C nitrile hydratase recombinant plasmid: Using the recombinant plasmid WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. Sac II and Ahd I were used as restriction enzyme sites for double digestion of the target sequence and the template plasmid. The target sequence was then ligated into the recombinant plasmid WT-ReNHase-AC-His using T4 ligase, thus constructing αE157C-βG197C-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent Escherichia coli to express the mutant nitrile hydratase αE157C-βG197C. Construction of the αD38P-αI152L-αE157C-βG197C nitrile hydratase recombinant plasmid: Using the recombinant plasmid αD38P-αI152L-WT-ReNHase-AC-His as a template, the target sequence was amplified using overlap extension PCR. Sac II and Ahd I were used as restriction enzyme sites for double digestion of the target sequence and the template plasmid. The target sequence was then ligated into the recombinant plasmid αD38P-αI152L-WT-ReNHase-AC-His using T4 ligase, thus constructing the nitrile hydratase mutant αD38P-αI152L-αE157C-βG197C-WT-ReNHase-AC-His. This recombinant plasmid needs to be introduced into competent Escherichia coli to express the mutant nitrile hydratase αD38P-αI152L-αE157C-βG197C (MUT4).

3. The method for modifying the flexible loop region of nitrile hydratase according to claim 2, characterized in that, The DNA sequence of the recombinant plasmid WT-ReNHase-AC-His is shown in SEQ ID NO.1; the amino acid sequence of the mutant nitrile hydratase αD38P-αI152L is shown in SEQ ID NO.3; the amino acid sequence of the mutant nitrile hydratase αE157C-βG197C is shown in SEQ ID NO.4; and the amino acid sequence of the mutant nitrile hydratase MUT4 is shown in Amino Acid Sequence NO.

5.

4. Using the pure enzyme of the nitrile hydratase mutant obtained by the method of modifying the flexible ring region of nitrile hydratase according to any one of claims 1-3 as a catalyst, the substrate is a nitrile compound and the product is an amide compound.

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