Nitrile hydratase mutant with improved activity

Abstract

The invention discloses a nitrile hydratase mutant with improved activity, particularly relates to modification of a nitrile hydratase substrate channel amino acid motif for acrylamide preparation, and belongs to the technical field of bioengineering. The invention finds that an amino acid residue at a 177 site on a beta subunit of Pt-NHase may be in a key structural domain of nitrile hydratase and plays an important role in catalytic activity of nitrile hydratase, the invention provides a Pt-NHase mutant, and compared with a wild type, the mutant is obtained by mutating isoleucine at a 177 site on the beta subunit. For acrylonitrile catalysis, the enzyme activity of the mutant is remarkably improved compared with that of a wild type, and is improved by 1.4 times or above.

Description

Technical Field

[0001] The invention relates to a nitrile hydratase mutant with improved activity, in particular to the modification of the amino acid motif of the nitrile hydratase substrate channel for the preparation of acrylamide, and belongs to the technical field of bioengineering. Background Art

[0002] Amide substances carrying unique amide groups have special physiological functions and potential biological activities. They are advantageous resources for synthesizing a large number of fine chemicals, polymers and synthetic intermediates. At present, many synthetic methods have been developed for the preparation of amide substances. Acrylamide, as one of the important commercial chemicals, has a wide range of uses and can be used as a raw material for organic synthesis and polymer materials. Because its polymer is soluble in water, it is often used to produce flocculants for water treatment.

[0003] Nitriles are valuable intermediates in synthesis, and they can be easily obtained from many simple, direct and cost-effective synthetic methods. Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme that can catalyze the conversion of nitrile substances into high-value-added amide compounds through hydration reactions. It has been widely used in the industrial production of nicotinamide and acrylamide. At present, the biological production technology of amides has gradually replaced the traditional chemical method with its advantages of green environmental protection, mild reaction conditions and high safety factor, which is in line with the concept of sustainable development and green production.

[0004] Nitrile hydratase is usually composed of two subunits, α and β. Studies have found that most of the reported nitrile hydratases from prokaryotes have problems such as poor stability, low catalytic activity, and a narrow catalytic substrate spectrum. Many studies have also attempted to modify it to improve its related properties. However, the current low catalytic efficiency and poor stability still limit the further development and application of nitrile hydratase. Summary of the invention

[0005] In view of the existing technical difficulties and problems, the present invention transforms the substrate channel amino acids of nitrile hydratase from Pseudonocardia thermophila in order to improve the catalytic performance.

[0006] The first technical solution provided by the present invention is a nitrile hydratase mutant, wherein the nitrile hydratase comprises an α subunit and a β subunit, as shown below:

[0007] The nitrile hydratase PtNHase derived from Pseudonocardia thermophila is used as a parent; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; the mutant is obtained by replacing the 177th mutation of the β subunit.

[0008] In certain embodiments, the mutant is a mutation of isoleucine I at position 177 of the β subunit to lysine K, asparagine N, histidine H, glutamate E, serine S, glycine G, glutamine Q, methionine M, aspartic acid D, arginine R or threonine T.

[0009] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0010] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0011] In certain embodiments, the recombinant plasmid uses the pET series as an expression vector.

[0012] In certain embodiments, the recombinant plasmid uses pET-24a(+) as an expression vector.

[0013] In certain embodiments, the genes on the expression vector are connected in the order of gene encoding β subunit, gene encoding α subunit, and gene encoding regulatory protein.

[0014] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0015] In certain embodiments, the host of the recombinant cell comprises a prokaryotic or eukaryotic microorganism.

[0016] In certain embodiments, the recombinant cell is hosted by Escherichia coli.

[0017] In certain embodiments, the E. coli is E. coli BL21 (DE3).

[0018] The invention provides a recombinant strain, which expresses the nitrile hydratase mutant.

[0019] In certain embodiments, the recombinant strain uses Escherichia coli BL21 (DE3) as a host and pET-24a (+) as an expression vector.

[0020] The fifth technical solution provided by the present invention is a method for improving the catalytic activity of nitrile hydratase, and the method is as follows:

[0021] The nitrile hydratase PtNHase derived from Pseudonocardia thermophila is used as a parent; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; the mutant is prepared by mutating the 177th isoleucine I of the β subunit to lysine K, asparagine N, histidine H, glutamic acid E, serine S, glycine G, glutamine Q, methionine M, aspartic acid D, arginine R or threonine T.

[0022] The sixth technical solution provided by the present invention is a method for preparing acrylamide, wherein acrylonitrile is used as a substrate and acrylamide is catalyzed by the nitrile hydratase mutant described in the first technical solution or the recombinant cell described in the fourth technical solution.

[0023] The seventh technical solution provided by the present invention is the use of the nitrile hydratase mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant plasmid described in the third technical solution, or the recombinant cell described in the fourth technical solution in the preparation of acrylamide.

[0024] The technical effects of the present invention are as follows:

[0025] The present invention finds that the amino acid residue at position 177 on the β subunit of Pt-NHase may be in the key structural domain of nitrile hydratase and plays an important role in the catalytic activity of nitrile hydratase. The present invention provides a Pt-NHase mutant, wherein the mutant is mutated to the isoleucine at position 177 on the β subunit relative to the wild type, thereby obtaining a mutant I177K. For acrylonitrile catalysis, the enzyme activity of the mutant is significantly improved compared with the wild type, which is increased by more than 1.4 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Preliminary screening of mutant library constructed for amino acid residues of PtNHase substrate channel and enzyme activity determination.

[0027] Figure 2 To screen the mutant library of PtNHase and determine the enzyme activity. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] Test method:

[0030] Enzyme activity detection method

[0031] Enzyme activity of nitrile hydratase (U): Unit enzyme activity is defined as the amount of enzyme required to catalyze the conversion of nitrile substrates to 1 μmol of amide product per minute at 25°C.

[0032] Nitrile hydratase activity (U / mL): the enzyme activity of nitrile hydratase per milliliter.

[0033] The raw materials used in the embodiment:

[0034] 1. LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, kanamycin final concentration 50 μg / mL.

[0035] 2. 2×YT medium: tryptone 16.0 g / L, yeast extract 10.0 g / L, NaCl 5.0 g / L, kanamycin final concentration 50 μg / mL.

[0036] Example 1: Construction of Pt NHase mutants

[0037] Based on the wild-type plasmid pET24a(+)-PtNHase WT of Pt NHase (the plasmid is recorded in Cheng et al., Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability, 2020), the mutant plasmid pET24a(+)-I177K was constructed by the whole plasmid PCR method.

[0038] First, plasmid pET24a(+)-PtNHase WT was used as a template, the mutant sequence was designed on the primers, and the DNA fragment with the base sequence mutation was amplified by PCR. The corresponding primer sequences used for the mutant plasmid are shown in Table 1. The single point mutant plasmid pET24a(+)-I177K was first constructed by the whole plasmid PCR method.

[0039] The amplification system is shown in Table 2 , and the PCR amplification reaction conditions were pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min 30 s, and extension at 72°C for 5 min, for a total of 30 cycles.

[0040] Table 1 Primer sequences

[0041]

[0042] Table 2 PCR amplification system

[0043]

[0044] Example 2: Catalytic efficiency of wild-type and mutant Pt NHase for acrylonitrile

[0045] The wild-type WT plasmid pET24a(+)-PtNHase WT and the reconstructed plasmid I177K of PtNHase were transformed into E.coli BL21(DE3), and a single colony was picked to 5 mL LB medium and cultured at 37°C and 200 rpm for 7-8 h. The seed solution was transferred to 100 mL 2×YT medium at 1% (v / v) and cultured at 37°C and 200 rpm until OD 600 To 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.4mM and 0.1g / L CoCl2·6H2O, change the culture temperature to 25℃, induce expression for 16h, and centrifuge to obtain wild-type WT and mutant cells I177K.

[0046] The OD of WT and I177K mutant cells was adjusted using 10 mM KPB (prepared according to K2HPO4:KH2PO4=4:1, pH 7.4) buffer. 600 Adjust to 0.5, take 10 μL to a 1.5 mL centrifuge tube, and place it in a 25°C metal bath. Add 490 μL of substrate (100 mM acrylonitrile solution) to the centrifuge tube, vortex thoroughly, react at 25°C for 5 minutes, and then add 500 μL of 0.1 M H3PO4 solution to terminate. Pass the reaction solution through a 0.22 μm filter membrane for liquid phase detection.

[0047] Liquid phase detection method: the mobile phase composition is acetonitrile: water = 1:2 (v / v), the flow rate is 0.6 mL / min, the detection wavelength is 215 nm, the column temperature is 40°C, and the amount of acrylamide produced in the reaction system is determined.

[0048] The enzyme activity calculation results of WT and mutants are shown in Figure 1 and 2 As shown, the enzyme activity of the wild enzyme WT is 543.5 U / mL, and the enzyme activity of the mutant I177K is 847.4 U / mL.

[0049] That is, when the amino acid residue at position 177 on the β subunit mutates, the enzymatic activity of nitrile hydratase is significantly improved, indicating that the amino acid residue at position 177 may be in the key structural domain of the enzyme and plays an important role in the catalytic activity of nitrile hydratase.

[0050] Table 3 Comparison of enzyme activity of wild type and mutants towards acrylonitrile

[0051] sample WT I177K Enzyme activity (U / mL) 543.5±5.0 847.4±7.4

[0052] Example 3

[0053] For a specific implementation, see Example 1, except that a single-point saturation mutation is performed on the wild-type PtNHase, and the isoleucine at position 177 on the β subunit is replaced by the other 19 amino acids (mutated to A / C / D / E / F / G / H / F / K / L / M / N / P / Q / R / S / T / V / W / Y, respectively). After the mutant is cultured and induced to express, the cells are adjusted to the same OD of 0.5 with reference to the method in Example 2, and the enzyme activity is measured. The results show that the enzyme activity is higher when position 177 is mutated to lysine ( Figure 2 ).

[0054] Table 4 Comparison of the catalytic activity of wild type and mutant whole cells towards acrylonitrile

[0055] sample Enzyme activity (U / mL) WT 560.5±5.0 I177 649.4±5.0 I177F 537.2±6.8 I177H 697.4±5.0 I177L 480.0±4.9 I177P 499.8±4.2 I177E 578.1±5.5 I177S 616.2±5.0 I177G 628.9±6.3 I177W 539.3±5.2 I177 684.0±5.1 I177K 735.5±5.5 I177M 583.1±6.0 I177V 550.6±5.3 I177 517.4±4.1 I177D 580.2±5.1 I177R 627.5±6.4 I177T 581.65±4.7 I177C 550.6±5.7

[0056] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nitrile hydratase mutant, characterized in that The nitrile hydratase from Pseudonocardiathermophila is used as a parent; the parent comprises an alpha subunit and a beta subunit, the amino acid sequence of the alpha subunit is shown in SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown in SEQ ID NO.2; the mutant is prepared by mutating the isoleucine at position 177 of the beta subunit.

2. The mutant according to claim 1, characterized in that The mutant is a mutant in which the 177th isoleucine I of the β subunit is mutated to lysine K, asparagine N, histidine H, glutamic acid E, serine S, glycine G, glutamine Q, methionine M, aspartic acid D, arginine R or threonine T.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant plasmid carrying the gene according to claim 3.

5. The recombinant plasmid according to claim 4, characterized in that The recombinant plasmid uses the pET series as an expression vector; optionally, the recombinant plasmid uses pET-24a(+) as an expression vector.

6. A recombinant cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant plasmid according to claim 4 or 5.

7. The recombinant cell according to claim 4, characterized in that The recombinant cell uses Escherichia coli as a host; optionally, the Escherichia coli is E. coli BL21 (DE3).

8. A method for improving the catalytic activity of nitrile hydratase, characterized in that: The method comprises the following steps: a nitrile hydratase derived from Pseudonocardia thermophila is used as a parent; the parent comprises an alpha subunit and a beta subunit, the amino acid sequence of the alpha subunit is shown in SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown in SEQ ID NO.2; and the mutant comprises a mutated isoleucine I at position 177 of the beta subunit to lysine K, asparagine N, histidine H, glutamic acid E, serine S, glycine G, glutamine Q, methionine M, aspartic acid D, arginine R or threonine T.

9. A method for preparing acrylamide, characterized in that: The method uses acrylonitrile as a substrate and utilizes the nitrile hydratase mutant of claim 1 or 2 or the recombinant cell of claim 6 or 7 to catalyze the production of acrylamide.

10. Use of the nitrile hydratase mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant plasmid according to claim 4 or 5, or the recombinant cell according to claim 6 or 7 in the preparation of acrylamide.

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