A nitrile hydratase mutant with enhanced activity
By modifying the 177th amino acid of the β subunit of the thermophilic Pseudonocardia nitrile hydratase, an efficient nitrile hydratase mutant was constructed, which solved the problems of low stability and catalytic activity of nitrile hydratase and achieved efficient conversion of acrylonitrile to acrylamide.
Patent Information
- Application Number
- CN202411982861.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2044-12-31
AI Technical Summary
Existing nitrile hydratases have problems such as poor stability, low catalytic activity and a narrow catalytic substrate spectrum, which limit their application in the preparation of amides.
By modifying the amino acids in the substrate channel of the nitrile hydratase from Pseudonocardia thermophila, especially by mutating the 177th amino acid residue isoleucine of the β subunit to other amino acids such as lysine, a nitrile hydratase mutant with higher catalytic activity was constructed.
The catalytic activity of nitrile hydratase was significantly improved, and the enzyme activity of the mutant was increased by more than 1.4 times, enhancing its efficiency in catalyzing the conversion of acrylonitrile to acrylamide.
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Figure CN119752869B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a nitrile hydratase mutant with enhanced activity, in particular to modification of the amino acid motif of a nitrile hydratase substrate channel for use in the preparation of acrylamide, belonging to the technical field of bioengineering. Background Art
[0002] Amides, which carry unique amide groups, possess special physiological functions and potential biological activities, making them a valuable resource for synthesizing a wide range of fine chemicals, polymers, and synthetic intermediates. Numerous synthetic methods have been developed for the preparation of amides. Acrylamide, as an important commercial chemical, is widely used as a raw material for organic synthesis and polymer materials. Because its polymers are water-soluble, they are often used to produce flocculants for water treatment.
[0003] Nitriles are valuable intermediates in synthesis and can be readily obtained through a variety of simple, direct, and cost-effective synthetic methods. Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme that catalyzes the hydration of nitriles into high-value-added amides. It has been widely used in the industrial production of nicotinamide and acrylamide. Currently, bioproduction of amides is gradually replacing traditional chemical methods due to its environmental friendliness, mild reaction conditions, and high safety profile, aligning with the principles of sustainable development and green production.
[0004] Nitrile hydratases are typically composed of two subunits, α and β. Studies have found that most reported prokaryotic nitrile hydratases suffer from poor stability, low catalytic activity, and a narrow substrate spectrum. Numerous studies have attempted to modify these enzymes to improve their properties. However, low catalytic efficiency and poor stability continue to limit the further development and application of nitrile hydratases. Summary of the Invention
[0005] In view of the existing technical difficulties and problems, the present invention aims to improve the catalytic performance by modifying the substrate channel amino acids of nitrile hydratase from Pseudonocardia thermophila.
[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 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, glutamic acid 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 order of connection of genes on the expression vector is 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 present 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, which is as follows:
[0021] The invention uses a nitrile hydratase PtNHase derived from Pseudonocardia thermophila 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; and the mutant is prepared by mutating the isoleucine I at position 177 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 the method uses acrylonitrile as a substrate and utilizes the nitrile hydratase mutant described in the first technical solution or the recombinant cell described in the fourth technical solution to catalyze the production of acrylamide.
[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 discovers that amino acid residue 177 on the β subunit of Pt-NHase may be a key structural domain of nitrile hydratase and play an important role in its catalytic activity. The present invention provides a Pt-NHase mutant, characterized by a mutation of isoleucine at position 177 on the β subunit relative to the wild-type, resulting in mutant I177K. The mutant exhibits significantly improved acrylonitrile catalysis activity, exceeding that of the wild-type by more than 1.4 times. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Initial screening of a mutant library constructed for amino acid residues in the PtNHase substrate channel and enzyme activity determination.
[0027] Figure 2 Rescreening of the mutant library of PtNHase and determination of enzyme activity. DETAILED DESCRIPTION
[0028] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended 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 substrate to 1 μmol 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 are:
[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×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 using the whole-plasmid PCR method.
[0038] First, using the plasmid pET24a(+)-PtNHase WT 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 of LB medium and cultured at 37°C and 200 rpm for 7-8 h. The seed solution was transferred to 100 mL of 2×YT medium at 1% (v / v) and cultured at 37°C and 200 rpm until the 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 values of WT and I177K mutant cells were adjusted using 10 mM KPB (prepared according to K2HPO4:KH2PO4=4:1, pH 7.4) buffer. 600 Adjust the concentration to 0.5, transfer 10 μL to a 1.5 mL centrifuge tube, and place in a 25°C metal bath. Add 490 μL of substrate (100 mM acrylonitrile solution) to the tube, vortex thoroughly, and react at 25°C for 5 minutes. Terminate the reaction by adding 500 μL of 0.1 M H₃PO₄ solution. Pass the reaction solution through a 0.22 μm filter for liquid chromatography 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] The specific implementation method is shown in Example 1, except that a single-point saturation mutation is performed on the wild-type PtNHase, where the isoleucine at position 177 on the β subunit is replaced with 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 mutants are cultured and induced for expression, the cells are adjusted to the same OD of 0.5 according 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 I177N 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 I177Q 684.0±5.1 I177K 735.5±5.5 I177M 583.1±6.0 I177V 550.6±5.3 I177Y 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 above in terms of preferred embodiments, 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 From Pseudonocardia Pseudonocardia thermophila ) as a parent; the parent is composed of an α subunit and a β subunit, the amino acid sequence of the α subunit 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 characterized in that the isoleucine at position 177 of the β subunit is mutated to lysine, and the α subunit remains unchanged.
2. A gene encoding the mutant according to claim 1.
3. A recombinant plasmid carrying the gene according to claim 2.
4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid uses pET-24a(+) as an expression vector.
5. A recombinant cell expressing the mutant according to claim 1, or containing the gene according to claim 2, or transformed with the recombinant plasmid according to claim 3 or 4.
6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses Escherichia coli as a host.
7. The recombinant cell according to claim 6, characterized in that The Escherichia coli is E. coli BL21(DE3).
8. A method for improving the catalytic activity of nitrile hydratase, characterized in that: The method is derived from Pseudonocardia ( Pseudonocardia thermophila ) as a parent; the parent is composed of an α subunit and a β subunit, the amino acid sequence of the α subunit 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 characterized in that isoleucine I at position 177 of the β subunit is mutated to lysine K, and the α subunit remains unchanged.
9. A method for preparing acrylamide, characterized in that: The method uses acrylonitrile as a substrate and utilizes the nitrile hydratase mutant according to claim 1 or the recombinant cell according to any one of claims 5 to 7 to catalyze the production of acrylamide.
10. Use of the nitrile hydratase mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to any one of claims 3 to 4, or the recombinant cell according to any one of claims 5 to 7 in the preparation of acrylamide.