A nitrile hydratase with high activity and thermal stability and its application

Abstract

The present invention discloses a nitrile hydratase with high activity and thermal stability and its application. Pseudonocardia thermophilus JCM3095 Nitrile hydratase ( Pt NHase ) of the α and β subunits, from Aurantimonas manganoxydans Nitrile hydratase ( Am NHase ) were reconstructed for the ancestral enzyme sequence. Among them, ASR_NHase showed the highest enzyme activity, which could reach 1566.82±30.21 U / mg under the optimal conditions. The optimal temperature of the enzyme was 35℃, the optimal pH was 7.5, and the residual activity after incubation at 65℃ for 24 h was 67.5%. It also had catalytic activity for nitrile compounds such as nicotinonitrile, acrylonitrile, benzonitrile, 2-cyanopyrazine, isobutyronitrile, and cinnamonitrile.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a nitrile hydratase with high activity and thermal stability and application thereof. Background Art

[0002] Amide is one of the most common compounds among natural bioactive molecules and organic synthesis intermediates, and has extremely important applications in pharmaceutical chemistry, biochemistry and polymer synthesis. Nicotinamide is a common amide substance in industry, which can directly participate in the metabolism of proteins, fats and carbohydrates as a coenzyme; at the same time, nicotinamide can promote the circulation of the stratum corneum and the metabolism of epidermal cells and prevent the transport of melanin to keratinocytes. Adding an appropriate amount to cosmetics can delay skin aging and brighten the skin tone.

[0003] There are two main methods for synthesizing amide compounds: chemical synthesis and biosynthesis. Traditional industrial synthesis mainly uses chemical synthesis, which has harsh synthesis conditions and requires strong acids and bases, high temperature and high pressure, and the participation of catalysts. There are many by-products, which does not conform to the principles of green economy. With the discovery of nitrile hydratase, the industry has opened the door to the biological synthesis of amide compounds. Compared with chemical synthesis, biosynthesis has the advantages of low energy consumption, mild reaction conditions, few by-products, and simple operation. At present, biosynthesis has completely replaced chemical synthesis in industry for the synthesis of amide compounds.

[0004] Nitrile hydratase is a type of metalloenzyme that acts on carbon-nitrogen triple bonds. Its catalytic active center usually contains a non-corrin cobalt ion or non-porphyrin iron ion, which can convert nitriles into corresponding amide compounds at room temperature and physiological pH, and is widely used in industrial production. Since the hydration reaction releases a large amount of heat, the temperature of the mixture increases during the biotransformation process, resulting in the loss of NHase activity. Most nitrile hydratases will be rapidly inactivated when the temperature exceeds 50°C. Although several natural NHases from Bacillus have excellent thermal stability, their enzyme activity is extremely low. Therefore, it is of great significance to obtain nitrile hydratases with both good thermal stability and high enzyme activity. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a nitrile hydratase with high activity and thermal stability.

[0006] Another object of the present invention is to provide a mutant of the nitrile hydratase having high activity and thermal stability.

[0007] Another object of the present invention is to provide applications of the above-mentioned nitrile hydratase having high activity and thermal stability and its mutants.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A nitrile hydratase with high activity and thermal stability, composed of α subunit and β subunit, which form a protein complex through protein interaction to exert catalytic activity.

[0010] The α subunit is one of α1-α6 and αI167T, wherein the amino acid sequences of α1-α6 are shown in SEQ ID NO.1-6, and the amino acid sequence of αI167T is obtained by mutating the 167th position of the amino acid sequence shown in SEQ ID NO.1 from I to T.

[0011] The β subunit is one of β1 to β7, βH11D, βV42T, βT90D, βQ116E, βS67A, and βE29G, wherein the amino acid sequences of β1 to β7 are as shown in SEQ ID NOs.7 to 13, the amino acid sequence of βH11D is obtained by mutation of H to D at position 11 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βV42T is obtained by mutation of V to T at position 42 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βT90D is obtained by mutation of T to D at position 90 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βQ116E is obtained by mutation of Q to E at position 116 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βS67A is obtained by mutation of S to A at position 67 of the amino acid sequence shown in SEQ ID NO.13, and the amino acid sequence of βE29G is obtained by mutation of S to A at position 67 of the amino acid sequence shown in SEQ ID NO.13. The amino acid sequence shown in NO.13 was obtained by mutation of E to G at position 29.

[0012] Preferably, the combination of the α subunit and the β subunit in the nitrile hydratase having high activity and thermal stability is at least one of the following:

[0013] α1 / β7, α4 / β1, α1 / βH11D, α1 / βV42T, α1 / βT90D, α1 / βQ116E, βS67A, α1 / βE29G, αI167T / βE29G.

[0014] The nitrile hydratase with high activity and thermal stability can produce corresponding amides using nitrile compounds as substrates.

[0015] The nitrile compound includes at least one of nicotinonitrile, benzonitrile, acrylonitrile, cinnamonitrile, 2-cyanopyrazine, isobutyronitrile and 3-cyanopyridine.

[0016] The nucleotide sequence of the coding gene of the above-mentioned nitrile hydratase with high activity and thermal stability is obtained according to the codon coding rule.

[0017] The coding gene consists of a nucleotide sequence encoding an alpha subunit and a nucleotide sequence encoding a beta subunit.

[0018] The nucleotide sequences encoding the α subunit are shown in SEQ ID NOs. 14-19.

[0019] The nucleotide sequences encoding the β subunit are shown in SEQ ID NOs. 20-26.

[0020] The above-mentioned nitrile hydratase with high activity and thermal stability is obtained by integrating the nucleotide sequence encoding the α subunit, the nucleotide sequence encoding the β subunit and the nucleotide sequence of the regulatory protein into an expression vector in the order of β subunit-α subunit-regulatory protein, and then transferring them into Escherichia coli for expression and purification.

[0021] The nucleotide sequence of the regulatory protein is shown in SEQ ID NO.27.

[0022] Application of the above-mentioned nitrile hydratase with high activity and thermal stability in the production of nicotinamide.

[0023] The invention discloses a nitrile hydratase gene with high activity and thermal stability, which consists of a nucleotide sequence encoding an alpha subunit and a nucleotide sequence encoding a beta subunit.

[0024] The α subunit is one of α1-α6 and αI167T, wherein the nucleotide sequences of α1-α6 are shown in SEQ ID NOs.14-19, and the nucleotide sequence of αI167T is obtained by mutating the nucleotide sequence shown in SEQ ID NO.14 to ACC at positions 499-501.

[0025] The β subunit is one of β1 to β7, βH11D, βV42T, βT90D, βQ116E, βS67A, and βE29G, wherein the nucleotide sequences of β1 to β7 are as shown in SEQ ID NOs. 20 to 26, the nucleotide sequence of βH11D is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 31 to 33 to GAT, the nucleotide sequence of βV42T is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 124 to 126 to ACC, the nucleotide sequence of βT90D is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 268 to 270 to GAT, the nucleotide sequence of βQ116E is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 346 to 348 to GAA, and the nucleotide sequence of βS67A is obtained by SEQ ID NO. The nucleotide sequence of βE29G is obtained by mutation of positions 199 to 201 of the nucleotide sequence shown in SEQ ID NO.26 to GCC, and the nucleotide sequence of βE29G is obtained by mutation of positions 85 to 87 of the nucleotide sequence shown in SEQ ID NO.26 to GGT.

[0026] Preferably, the combination of the α subunit and the β subunit in the nitrile hydratase gene with high activity and thermal stability is at least one of the following:

[0027] α1 / β7, α4 / β1, α1 / βH11D, α1 / βV42T, α1 / βT90D, α1 / βQ116E, α1 / βS67A, α1 / βE29G, αI167T / βE29G.

[0028] Application of the above-mentioned nitrile hydratase gene with high activity and thermal stability in the production of nicotinamide.

[0029] A recombinant expression vector comprises the nitrile hydratase gene with high activity and thermal stability.

[0030] A recombinant expression strain comprises the above-mentioned nitrile hydratase gene with high activity and thermal stability.

[0031] A method for preparing amide substances comprises the following steps:

[0032] The nitrile compounds are used as substrates and the nitrile hydratase with high activity and thermal stability is used to catalyze the reaction to obtain amide substances.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] (1) The specific enzyme activity of the nitrile hydratase in the present invention is 1566.82 ± 30.21 U / mg at 35°C and pH 7.4, which is higher than that of most nitrile hydratases, and the enzyme activity is maintained at more than 80% of the maximum activity in the range of 35-45°C, and when the temperature rises to 55°C, it can still maintain more than 50% of the maximum activity. It is beneficial to be applied to the industrial production of nicotinamide, improve catalytic efficiency, and reduce production costs.

[0035] (2) The nitrile hydratase of the present invention exhibits excellent heat resistance, with a residual activity of 67.5% after incubation at 65°C for 24 h. Compared with other nitrile hydratases, the stability is significantly improved.

[0036] (3) The specific enzyme activities of the nitrile hydratase mutants βH11D, βV42T, βT90D, βQ116E and αI167T / βE29G provided by the present invention at 25°C were increased by 24.6%, 13.8%, 12.9%, 23.26% and 17.85% respectively compared with the wild type. The residual activity of the mutant βS67A whole cells incubated in 2 M nicotinamide solution for 30 min was 33.48%, while the residual activity of the wild type was 21.75%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the SDS-PAGE electrophoresis analysis result of the ancestral nitrile hydratase; A: NHase_α1β7; B: NHase_α2β2; C: NHase_α3β3; D: NHase_α4β1; E: NHase_α5β6; F: NHase_ α6β4, and each lane is M: protein marker; 1: total protein; 2: soluble protein; 3: flow-through peak; 4: 10% Buffer B eluent; 5: 20% Buffer B eluent; 6: 30% Buffer B eluent; 7: 70% Buffer B eluent; 8: 100% Buffer B eluent.

[0038] Figure 2 This is a graph showing the results of the relative enzyme activity test on nicotinonitrile catalyzed by each ancestral enzyme.

[0039] Figure 3 This is the ASR_NHase enzymatic property detection diagram, where a: optimal pH; b: optimal temperature; c: thermal stability at 65°C; d: substrate spectrum; e: product tolerance.

[0040] Figure 4 The figure shows the relative enzymatic activity results of the wild type and mutants on nicotinonitrile, where WT is defined as 100%.

[0041] Figure 5The figure shows the tolerance of the wild type and mutants to 2 M nicotinamide solution. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0043] If the specific experimental conditions are not specified in the following embodiments, the conventional experimental conditions or the experimental conditions recommended by the reagent company are usually followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0044] The whole gene synthesis in the embodiment of the present invention was completed by Suzhou Genewise Biotechnology Co., Ltd.

[0045] The reagents used in the embodiments of the present invention are as follows:

[0046] LB seed medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl.

[0047] Binding buffer (Buffer A): 20 mM Tris, 0.5 M NaCl, pH adjusted to 7.5 with HCl.

[0048] Elution buffer (Buffer B): 20 mM Tris, 0.5 M NaCl, 250 mM imidazole, pH adjusted to 8.0 with HCl.

[0049] Example 1

[0050] Nitrile hydratase is a type of metalloenzyme that acts on carbon-nitrogen triple bonds. It consists of two parts, an α subunit and a β subunit. The two parts interact with each other to form a protein complex to exert catalytic activity (refer to the literature Nitrile Hydratases: From Industrial Application to Acetamiprid and ThiaclopridDegradation. [J]. Journal of agricultural and food chemistry, 2021, 69 (36): 10440-10449. DOI: 10.1021 / acs.jafc.1c03496.). It can convert nitriles into corresponding amide compounds at room temperature and physiological pH. However, the existing nitrile hydratase cannot take into account both high enzyme activity and thermal stability. In order to obtain a new heat-resistant nitrile hydratase with high enzyme activity, the present invention performs the following enzyme mining process:

[0051] (1) Through literature search, nitrile hydratases with high thermal stability or high enzyme activity were selected from Pseudonocardia thermophilus JCM3095Nitrile hydratase ( Pt NHase), the specific enzyme activity of which was 311.0 U / mg and Aurantimonas manganoxydans Nitrile hydratase ( Am The half-life of the enzyme at 50°C is 1.5 h.

[0052] (2) Pt The α subunit sequence (NCBI: WP_073455624.1) and β subunit sequence (NCBI: WP_073455623.1) of NHase and Am The NHase β subunit sequence (NCBI: WP_009208458.1) was used as a probe and the online website FireProt ASR Ancestral enzyme sequence reconstruction was performed, and default settings were selected throughout the reconstruction process.

[0053] (3) The phylogenetic tree of nitrile hydratase α and β subunits was obtained through the fully automatic analysis of the website. Among them, nodes 82, 96, 105, 125, 146 and 157 were selected in the α subunit and numbered as α1 to α6 (amino acid sequences are shown in SEQ ID: NO.1 to 6, and nucleotide sequences are shown in SEQ ID: NO.14 to 19); nodes 151, 167, 191, 225, 217 and 263 were selected in the β subunit and numbered as β1 to β6 (amino acid sequences are shown in SEQ ID: NO.7 to 12, and nucleotide sequences are shown in SEQ ID: NO.20 to 25). Am In the reconstructed sequence with NHase as the probe, the most original ancestral node was selected, numbered β7 (the amino acid sequence is shown in SEQ ID: NO.13, and the nucleotide sequence is shown in SEQ ID: NO.26). The 6 α sequences and 7 β sequences obtained were combined, and each combination was modeled from scratch using AlphaFold3. Autodock vina was used for molecular docking with the substrate 3-cyanopyridine. According to the scores, the combinations of ASR_α1β7, ASR_α2β2, ASR_α3β3, ASR_α4β1, ASR_α5β6, and ASR_α6β4 were selected for the next step of synthesis and characterization. The regulatory protein was derived from Pt NHase (nucleotide sequence is shown in SEQ ID: NO.27).

[0054] Example 2 Construction and expression of recombinant plasmid

[0055] The sequences ASR_α1β7, ASR_α2β2, ASR_α3β3, ASR_α4β1, ASR_α5β6 and ASR_α6β4 obtained by reconstructing the ancestral enzyme sequence in Example 1 were connected in the order of β subunit-α subunit-regulatory protein, and a 6×His affinity purification tag was added after the β subunit. The two gene segments were connected with the DNA sequence TGAAAGGAGATATAGATATG. Codon optimization was performed according to the codon preference of Escherichia coli. The expression vector selected was pET-24a(+), and the gene insertion position selected was N Ⅰ and XOt Ⅰ Two restriction sites, the resulting recombinant plasmid was transformed into E. coli BL21(DE3), select positive clones to extract plasmids and sequence them to obtain recombinant strains.

[0056] Example 3 Purification of recombinant protein

[0057] (1) Induction culture

[0058] The recombinant strain was inoculated into 10 mL LB medium (final concentration of kanamycin was 50 μg / mL) and cultured at 37°C and 200 rpm for 16 h to obtain seed solution. The seed solution was measured by OD 600 =0.1 was transferred to 100 mL LB medium and cultured at 37°C and 200 rpm until OD 600 To 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.2 mM and 0.1 g / L CoCl2·6H2O, change the culture temperature to 25℃, and induce expression for 12-16 h.

[0059] After fermentation, the bacterial precipitate was collected by centrifugation at 7800 rpm for 7 min, washed with water, resuspended with 10 mL Buffer A solution, placed on an ice-water mixture for ultrasonic disruption, and the disrupted liquid was centrifuged at 4°C and 12000 rpm for 30 min to obtain the supernatant; the sample was taken for SDS-PAGE electrophoresis, the concentration of the concentrated gel was 4%, the concentration of the separation gel was 12%, the sample and the loading buffer were mixed in a ratio of 4:1, and the sample was electrophoresed in a boiling water bath for 5 min. The initial voltage of the electrophoresis instrument was set to 60 V, and the voltage was increased to 120 V when the sample moved to the separation gel until the electrophoresis was terminated when the sample moved to the bottom of the electrophoresis tank.

[0060] (2) Ni affinity chromatography

[0061] The collected supernatant was purified by nickel column affinity chromatography using an ATKA protein purifier. 10%, 20%, and 30% Buffer B were used to remove impurities with poor affinity, and 70% Buffer B was used to elute the ancestral nitrile hydratase to obtain pure enzyme solutions of NHase_α1β7, NHase_α2β2, NHase_α3β3, NHase_α4β1, NHase_α5β6, and NHase_α6β4. Finally, 100% Buffer B was used to elute all strongly bound proteins. The purity of the target protein in the eluate was analyzed by SDS-PAGE electrophoresis. Since the role of the regulatory protein is to help the apoenzyme bind to metal ions and form the correct conformation, and it does not serve as a component of the enzyme molecule to exert activity, two bands of the α subunit and the β subunit should appear after purification. The detection is as follows: Figure 1 As shown, after purification, the protein band is single and the purification quality is high.

[0062] Example 4 Determination of Enzymatic Properties

[0063] (1) Enzyme activity determination

[0064] Enzyme activity of nitrile hydratase (U): Unit enzyme activity is defined as the amount of enzyme required to catalyze nicotinonitrile to produce 1 μmol nicotinamide per minute at 25°C.

[0065] Specific enzyme activity of nitrile hydratase (U / mg): the enzyme activity per mg of nitrile hydratase.

[0066] Dilute the pure enzyme to 0.05 mg / mL with 100 mM KPB (potassium phosphate buffer, pH 7.5) solution, take 10 μL to a 1.5 mL centrifuge tube, and place it in a 25°C metal bath. Add 490 μL of substrate (nicotinonitrile concentration is 200 mM, prepared with potassium phosphate buffer, pH = 7.5) to the centrifuge tube, vortex thoroughly, react at 25°C for 10 min, then add 500 μL of pure acetonitrile solution to terminate, and filter through a 0.22 μm filter membrane.

[0067] Liquid phase detection method: The mobile phase composition was acetonitrile: water = 1:2 (v / v), the flow rate was 0.6 mL / min, the detection wavelength was 215 nm, the column temperature was 40°C, and the amount of nicotinamide produced in the reaction system was determined. The specific enzyme activities of NHase_α1β7, NHase_α2β2, NHase_α3β3, NHase_α4β1, NHase_α5β6 and NHase_α6β4 were 1212 U / mg, 29.22 U / mg, 53.02 U / mg, 581.28 U / mg, 25.59 U / mg and 384.23 U / mg, respectively. Among them, NHase_α1β7 had the highest enzyme activity, so it was selected as the research object in subsequent experiments and renamed ASR_NHase.

[0068] (2) Determination of optimum temperature

[0069] At pH = 7.5, nitrile hydratase and its substrate are mixed, and the enzyme activity in the range of 20°C to 80°C is determined by referring to the method in step (1). The enzyme activity at the optimum temperature is taken as 100%, and the relative enzyme activity of nitrile hydratase at different temperatures is calculated.

[0070] The results are as follows Figure 3 As shown in Figure a, with the increase of temperature, the activity of ASR_NHase showed a trend of first increasing and then decreasing, and showed the highest enzyme activity at 35°C, reaching 1566.82 ± 30.21 U / mg, and showed more than 80% of the highest activity in the range of 35-45°C. When the temperature rose to 55°C, it could still maintain more than 50% of the highest activity.

[0071] (3) Optimal pH determination

[0072] A 200 mM nicotinonitrile solution was prepared using 100 mM potassium phosphate buffer solution with different pH gradients (pH 5.0-8.5), and the enzyme activity was determined at 25°C according to the method in step (1). The highest enzyme activity was defined as 100%, and the relative enzyme activity of nitrile hydratase under each pH condition was calculated.

[0073] The results are as follows Figure 3 As shown in Figure b, the optimal reaction pH of ASR_NHase is 7.5, at which the enzyme activity is 1372.21 ± 27.52 U / mg, and more than 70% of the highest enzyme activity is shown in the range of pH 7.0 to 9.0. In addition, the enzyme activity of ASR_NHase changes slowly in an alkaline environment, but under acidic conditions, the activity of ASR_NHase changes greatly, and the relative enzyme activity at pH 5.0 is less than 1%.

[0074] (4) Thermal stability determination

[0075] The metal bath temperature was set at 65°C, and samples were taken after 0, 6, 12, 18 and 24 h of insulation, and the residual enzyme activity was determined. The sample treated for 0 h (enzyme without heat treatment) was used as the control, and its enzyme activity was defined as 100%. The relative enzyme activities of the remaining samples were calculated in turn, and the thermal stability of the pure enzyme at 65°C was analyzed.

[0076] The results are as follows Figure 3 As shown in Figure c, the ancestral enzyme ASR_NHase exhibited excellent thermal stability, with a residual activity of 67.5% after incubation at 65°C for 24 h.

[0077] (5) Product tolerance test

[0078] Prepare 0.25 M, 0.5 M, 1 M and 2 M nicotinamide solutions, take 10 μL of 0.05 mg / mL ASR_NHase pure enzyme solution or OD 600 =1 were added to a 1.5 mL centrifuge tube in advance and placed in a 25°C constant temperature metal bath for preheating for 5 min; 250 μL of a certain concentration of nicotinamide was added to the reaction group, and an equal volume of 10 mM KPB buffer was added to the control group for 30 min; 250 μL of 50 mM nicotinonitrile was added to react for 10 min, and 500 μL of acetonitrile was used as a terminator. After filtration through a 0.22 μm organic filter membrane, the substrate reduction was detected by high performance liquid chromatography, the corresponding enzyme activity was calculated, and the product (nicotinamide) tolerance of pure enzyme or cells was compared.

[0079] The results are as follows Figure 3 As shown in Figure d, the residual enzyme activity of the ancestral enzyme ASR_NHase after treatment with 0.2, 0.5, 1 and 2 M nicotinonitrile was 82.4%, 54.3%, 28.4% and almost no activity, respectively.

[0080] (6) Substrate spectrum determination

[0081] 10 μL of 0.1 mg / mL ASR_NHase pure enzyme solution was added to 490 μL of 200 mM acrylonitrile, 50 mM benzonitrile, 100 mM 2-cyanopyrazine, 200 mM isobutyronitrile, 100 mM n-valeronitrile, 5 mM cinnamonitrile and 200 mM 3-cyanopyridine solutions, respectively, to obtain a reaction system. The reaction system was reacted at 25°C for 10 min, and then the reaction was terminated with 500 μL acetonitrile to obtain a reaction solution. The supernatant of the reaction solution was passed through a 0.22 μm membrane as a sample for liquid phase determination to detect the specific enzyme activity of nitrile hydratase.

[0082] The results are as follows Figure 3As shown in Figure e, except for n-valeronitrile, ASR_NHase has high catalytic activity towards other nitriles. The specific enzyme activities for benzonitrile, acrylonitrile, cinnamonitrile, 2-cyanopyrazine, isobutyronitrile and 3-cyanopyridine are 4146 U / mg, 2449 U / mg, 925 U / mg, 3000.44 U / mg, 685 U / mg and 1212 U / mg, respectively.

[0083] Example 5 Preparation of ASR_NHase mutants

[0084] In order to further improve the enzymatic activity of ASR_NHase, mutation sites were designed to obtain ASR_NHase mutants. Using plasmid pET24a(+)-ASR_NHase as a template, the mutation sites βH11D, βV42T, βT90D, βQ116E and βS67A were designed on primers, and plasmids with mutated base sequences were amplified by whole-plasmid PCR. For the mutant αI167T / βE29G, the ASR_NHase gene containing the mutation site and the pET-24a(+) backbone were amplified by PCR, and the primer sequences used are shown in Table 1.

[0085] The reaction conditions for PCR amplification were as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 68°C for 1 min 20 s; 30 cycles in total; (3) annealing at 68°C for 10 min and incubation at 12°C.

[0086] The products of whole plasmid PCR need to be Dpn I digestion enzyme to remove the original template plasmid. The digestion system is to add 5 μL Fast Digest Buffer and 2.5 μL DpnI digestion enzyme to every 50 μL PCR product. Place in a 37℃ constant temperature metal bath for 1 h. Then, the digested PCR product is transformed E.coli Top10 , spread on LB medium plates containing 50 mg / L kanamycin, and culture inverted at 37°C overnight.

[0087] A single colony was picked and inoculated into 10 mL LB medium and cultured overnight at 37°C and 200 rpm. The recombinant plasmid was obtained using the TIANGEN plasmid extraction kit and sequenced and verified by Beijing Qingke Biotechnology Co., Ltd., and finally the recombinant plasmid was obtained.

[0088] Table 1 Introduced mutations and primers

[0089]

[0090] Example 6 Expression and determination of wild-type and mutant ASR_NHase

[0091] After obtaining the mutant recombinant plasmid, expression and protein purification were performed according to the methods of Examples 2 and 3, and finally pure enzyme solutions of mutants βH11D, βV42T, βT90D, βQ116E and αI167T / βE29G were obtained. The enzyme activity was then determined according to the method of Example 4. The results are as follows: Figure 4 and Figure 5 shown.

[0092] The calculation results of the specific enzyme activities of WT (unmutated ASR_NHase) and mutants are shown in Figure 4 As shown, the enzyme activity of WT was defined as 100%, and the specific enzyme activities of mutants βH11D, βV42T, βT90D, βQ116E and αI167T / βE29G at 25°C were increased by 24.6%, 13.8%, 12.9%, 23.26% and 17.85% compared with WT, respectively.

[0093] The calculation results of enzyme activity and product tolerance of WT and mutant βS67A are shown in Figure 5 As shown, the enzyme activity of mutant βS67A whole cells was comparable to that of WT, and the residual activity of the mutant after incubation in 2 M nicotinamide solution for 30 min was 33.48%, while the residual activity of WT was 21.75%.

[0094] In summary, the nitrile hydratase provided by the present invention through reconstruction of the ancestral enzyme sequence has excellent thermal stability and enzyme activity. The specific enzyme activity under the optimal conditions can reach 1566.82±30.21 U / mg, and the residual activity after incubation at 65°C for 24 h is 67.5%. In addition, the nitrile hydratase in the present invention has high catalytic activity for a variety of nitriles, and the activity of the mutant nitrile hydratase is further improved on the basis of the wild type, with a maximum increase of 24.6%. It is beneficial to be applied to the industrial production of nicotinamide, improve catalytic efficiency, and reduce production costs.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A nitrile hydratase with high activity and thermal stability, characterized in that: It is composed of α subunit and β subunit, which form a protein complex through protein interaction to exert catalytic activity; The α subunit is one of α1 and αI167T, wherein the amino acid sequence of α1 is as shown in SEQ ID NO.1, and the amino acid sequence of αI167T is obtained by mutating the 167th position of the amino acid sequence shown in SEQ ID NO.1 from I to T; The β subunit is one of β7, βH11D, βV42T, βT90D, βQ116E, βS67A, and βE29G, wherein the amino acid sequence of β7 is as shown in SEQ ID NO.13, the amino acid sequence of βH11D is obtained by mutation of H to D at position 11 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βV42T is obtained by mutation of V to T at position 42 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βT90D is obtained by mutation of T to D at position 90 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βQ116E is obtained by mutation of Q to E at position 116 of the amino acid sequence shown in SEQ ID NO.13, the amino acid sequence of βS67A is obtained by mutation of S to A at position 67 of the amino acid sequence shown in SEQ ID NO.13, and the amino acid sequence of βE29G is obtained by mutation of E to G at position 29 of the amino acid sequence shown in SEQ ID NO.13; The combination of the α subunit and the β subunit in the nitrile hydratase with high activity and thermal stability is at least one of the following: α1 / β7, α1 / βH11D, α1 / βV42T, α1 / βT90D, α1 / βQ116E, α1 / βS67A, αI167T / βE29G.

2. The nitrile hydratase with high activity and thermal stability according to claim 1, characterized in that: The nitrile hydratase with high activity and thermal stability can produce corresponding amides using nitrile compounds as substrates; The nitrile compound includes at least one of nicotinonitrile, benzonitrile, acrylonitrile, cinnamonitrile, 2-cyanopyrazine, isobutyronitrile and 3-cyanopyridine.

3. The nitrile hydratase with high activity and thermal stability according to claim 1, characterized in that: The nucleotide sequence of the coding gene of the nitrile hydratase with high activity and thermal stability is obtained according to the codon coding rule; The coding gene is composed of a nucleotide sequence encoding an α1 subunit and a nucleotide sequence encoding a β7 subunit; The nucleotide sequence encoding the α1 subunit is shown in SEQ ID NO.14; The nucleotide sequence encoding the β7 subunit is shown in SEQ ID NO.

26.

4. Use of the nitrile hydratase with high activity and thermal stability according to any one of claims 1 to 3 in the production of nicotinamide.

5. A nitrile hydratase gene with high activity and thermal stability, characterized in that: It is composed of a nucleotide sequence encoding an α subunit and a nucleotide sequence encoding a β subunit; The α subunit is one of α1 and αI167T, wherein the nucleotide sequence of α1 is as shown in SEQ ID NO. 14, and the nucleotide sequence of αI167T is obtained by mutating the nucleotide sequence shown in SEQ ID NO. 14 to ACC at positions 499 to 501; The β subunit is one of β7, βH11D, βV42T, βT90D, βQ116E, βS67A, and βE29G, wherein the nucleotide sequence of β7 is as shown in SEQ ID NO. 26, the nucleotide sequence of βH11D is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 31 to 33 to GAT, the nucleotide sequence of βV42T is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 124 to 126 to ACC, the nucleotide sequence of βT90D is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 268 to 270 to GAT, the nucleotide sequence of βQ116E is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 346 to 348 to GAA, the nucleotide sequence of βS67A is obtained by mutating the nucleotide sequence of SEQ ID NO. 26 at positions 199 to 201 to GCC, and the nucleotide sequence of βE29G is obtained by SEQ ID NO. The nucleotide sequence shown in NO.26 has mutated from position 85 to 87 to GGT; The combination of the α subunit and the β subunit in the nitrile hydratase gene with high activity and thermal stability is at least one of the following: α1 / β7, α1 / βH11D, α1 / βV42T, α1 / βT90D, α1 / βQ116E, α1 / βS67A, αI167T / βE29G.

6. Use of the nitrile hydratase gene with high activity and thermal stability as claimed in claim 5 in the production of nicotinamide.

7. A recombinant expression vector, characterized in that: The invention comprises the nitrile hydratase gene with high activity and thermal stability as claimed in claim 5.

8. A recombinant expression strain, characterized in that: The invention comprises the nitrile hydratase gene with high activity and thermal stability as claimed in claim 5.

9. A method for preparing amide substances, characterized in that The steps include: The nitrile compound is used as a substrate and the nitrile hydratase with high activity and thermal stability as claimed in any one of claims 1 to 3 is used to catalyze the reaction to obtain an amide substance.

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