A nitrile hydratase mutant with improved stability
By mutating specific amino acids in the β subunit of nitrile hydratase from *Pseudomonas thermophilus*, the thermal stability and catalytic efficiency of nitrile hydratase were improved, overcoming the shortcomings of nitrile hydratase in terms of thermal stability and catalytic activity, and achieving a more efficient catalytic effect.
Patent Information
- Application Number
- CN202611470372.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-21
- Publication Date
- 2026-11-06
AI Technical Summary
Existing nitrile hydratases suffer from poor stability, low catalytic activity, and a narrow catalytic substrate spectrum, which limits their application in industrial production.
A nitrile hydratase mutant was constructed by mutating the valine residue at position 4 of the β subunit of the nitrile hydratase derived from Pseudonocardia thermophila to isoleucine, thereby improving its thermal stability.
The mutant exhibits significantly improved thermal stability. After heat treatment at 65°C, the residual enzyme activity of the recombinant whole cells reached 1.3 times that of the wild type, and the catalytic efficiency was increased by 33%.
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Abstract
Description
Technical Field
[0001] This invention relates to a nitrile hydratase mutant with improved stability, belonging to the field of bioengineering technology. Background Technology
[0002] Amides, with their unique amide groups, possess special physiological functions and potential biological activities, making them a valuable resource for synthesizing a large number of fine chemicals, polymers, and synthetic intermediates. Currently, many synthetic methods have been developed for the preparation of amides. Acrylamide, as an important commercial chemical, has a wide range of applications, serving as a raw material for organic synthesis and polymer materials. Because its polymers are water-soluble, it is frequently used to produce flocculants for water treatment.
[0003] Nitriles are valuable intermediates in synthesis, and they can be readily obtained from many simple, direct, and cost-effective synthetic methods. Nitrile hydratase (NHase, EC 4.2.1.84) is a class of metalloenzymes that 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. Currently, biological production technologies for amides are gradually replacing traditional chemical methods due to their advantages of being environmentally friendly, having mild reaction conditions, and high safety, aligning with the concepts of sustainable development and green production.
[0004] Nitrile hydratases are typically composed of α and β subunits. Studies have found that most reported prokaryotic nitrile hydratases suffer from poor stability, low catalytic activity, and a narrow catalytic substrate spectrum. Although many studies have attempted to modify them to improve these properties—for example, the study "Heterologous Expression and Catalytic Process Study of Novel Thermostrophic Nitrile Hydrate Enzymes" disclosed a nitrile hydratase derived from *Caldalkalibacillus thermarum* TA2.A1, which had a half-life of 3 h at 65°C, a specific enzyme activity of 395 U / mg for catalyzing nicotinamide at 30°C, and a whole-cell catalytic reaction rate nearly twice as fast as previously reported, resulting in a 25% increase in nicotinamide yield—the thermostability of nitrile hydratases exhibits strong species and site specificity. Modification strategies targeting a specific source or site are difficult to predict or transfer to enzymes from other sources or with different structural regions. Therefore, the low catalytic efficiency and poor thermostability still limit the further development and application of nitrile hydratases. Summary of the Invention
[0005] To address the existing technical difficulties and problems, this invention addresses the challenges posed by *Pseudomonas thermophilus* (…). Pseudonocardia thermophilaThe amino acids at the subunit interface of the nitrile hydratase in JCM3095 were modified in order to improve its thermal stability.
[0006] This invention provides a nitrile hydratase mutant, wherein the nitrile hydratase comprises an α subunit and a β subunit, 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 formed by mutating valine at position 4 of the β subunit to isoleucine.
[0007] The present invention also provides a gene encoding the mutant.
[0008] In one embodiment, the gene comprises a nucleotide sequence encoding the α subunit as shown in SEQ ID NO.3, and a gene sequence based on the nucleotide sequence encoding the β subunit as shown in SEQ ID NO.4, with the codon encoding valine at position 4 mutated to a codon encoding isoleucine.
[0009] The present invention also provides a recombinant plasmid carrying the said gene.
[0010] In some embodiments, the recombinant plasmid uses the pET series as an expression vector.
[0011] In some embodiments, the recombinant plasmid is expressed using pET-24a(+) as an expression vector.
[0012] In some embodiments, the gene linking sequence on the expression vector is, in order, a gene encoding the β subunit, a gene encoding the α subunit, and a gene encoding the activator protein; the activator protein is a cobalt ion insertion chaperone protein essential for the maturation of nitrile hydratase, responsible for loading the cobalt ion at the catalytic center and the correct folding of the holoenzyme, and is a co-expression protein necessary for obtaining an active holoenzyme through recombinant expression, the nucleotide sequence encoding the protein being shown in SEQ ID NO.5.
[0013] The present invention also provides a mutant expressing the mutant, or a recombinant cell containing the gene or the recombinant plasmid.
[0014] In some embodiments, the host of the recombinant cells comprises prokaryotic or eukaryotic microorganisms.
[0015] In some embodiments, the recombinant cells use Escherichia coli as a host.
[0016] In some embodiments, the *E. coli* is E. coli BL21(DE3).
[0017] This invention provides a recombinant Escherichia coli, using Escherichia coli E. coliUsing BL21(DE3) as the host and pET-24a(+) as the expression vector, the nitrile hydratase mutant was expressed.
[0018] This invention also provides a method for improving the thermostability of nitrile hydratase, derived from... Pseudonocardia thermophila Nitrile hydratase Pt NHase is the parent; the fourth valine in the β subunit is mutated to isoleucine.
[0019] In one embodiment, 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.
[0020] The present invention also provides a method for preparing acrylamide, using acrylonitrile as a substrate and utilizing the nitrile hydratase mutant or the recombinant cell to catalyze the generation of acrylamide.
[0021] In one embodiment, the concentration of acrylonitrile in the reaction system is 20~120 mM.
[0022] In one embodiment, the reaction is carried out at 24-26°C for at least 5 minutes.
[0023] The present invention also provides the use of the nitrile hydratase mutant, or the gene, or the recombinant plasmid, or the recombinant cell in the preparation of acrylamide.
[0024] Beneficial effects: This invention discovers Pt The fourth amino acid residue on the β subunit of NHase is located in a key structural domain at the subunit interface of nitrile hydratase, playing an important role in the thermostability of nitrile hydratase. This invention provides... Pt The -NHase mutant, relative to the wild type, involves a mutation at position 4 of the β subunit, yielding mutant V4I. In a recombinant whole-cell catalytic acrylonitrile system, after heat treatment at 65°C for 30 min on both recombinant whole cells expressing mutant V4I and recombinant whole cells expressing wild-type enzymes, the residual enzyme activity of mutant V4I (306.02 U / mL) was significantly higher than that of the wild type (229.36 U / mL), representing a 1.3-fold increase (33% higher than the wild type). Attached Figure Description
[0025] Figure 1 for Pt Screening of mutant libraries of NHase and determination of residual enzyme activity. Detailed Implementation
[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0027] Test method: (I) Methods for detecting whole-cell enzyme activity Nitrile hydratase activity (U): One unit of enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of amide product from a nitrile substrate per minute at 25°C. Whole-cell enzyme activity (U / mL): The enzyme activity possessed by each milliliter of recombinant whole-cell bacterial suspension with the stated OD value. In this invention, "residual enzyme activity" refers to the whole-cell enzyme activity retained after heat treatment of the recombinant whole cells (bacterial cells).
[0028] (ii) Culture medium LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, kanamycin final concentration 50 μg / mL.
[0029] 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.
[0030] Example 1: Pt Construction of NHase mutants To carry wild type Pt The plasmid pET24a(+)- encodes NHase and the activator protein (the nucleotide sequences encoding the α and β subunits are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the nucleotide sequence encoding the activator protein is shown in SEQ ID NO. 5). Pt NHaseWT (this plasmid is disclosed in "Computational Design of Nitrile Hydratase from") Pseudonocardia thermophila Using JCM3095 for Improved Thermostability as a template, the mutant plasmid pET24a(+)-PtNHase-V4I was constructed using whole-plasmid PCR. The mutant sequence was designed on primers, and the mutated DNA fragment was amplified by PCR. The corresponding primer sequences used for the mutant plasmid are shown in Table 1. The single-point mutant plasmid pET24a(+)-PtNHase-V4I was constructed using whole-plasmid PCR.
[0031] The amplification system is shown in Table 2. The PCR amplification reaction conditions are: 98 ℃ pre-denaturation for 1 min, 98 ℃ denaturation for 10 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 1 min 30 s, and 72 ℃ extension for 5 min, for a total of 30 cycles.
[0032] Table 1 Primer Sequences
[0033] Table 2 PCR amplification system
[0034] Example 2: Pt Catalytic efficiency of wild-type and mutant NHase for acrylonitrile The construct containing Example 1 Pt NHase wild-type WT plasmid pET24a(+)- Pt NHaseWT and recombinant plasmid pET24a(+)- containing mutant V4I Pt NHase - V4I conversion to E. coli BL21(DE3) was cultured by picking a single colony and transferring it to 5 mL of LB medium. The culture was then incubated at 37°C and 200 rpm for 7-8 h. The seed culture was then transferred at 1% (v / v) to 100 mL of 2×YT medium and incubated at 37°C and 200 rpm until OD500 was reached. 600 When the concentration reached 0.6-0.8, isopropyl thiogalactoside (IPTG) at a final concentration of 0.4 mM and CoCl2·6H2O at 0.1 g / L were added. The culture temperature was changed to 25℃, and expression was induced for 16 h. After centrifugation, recombinant whole cells of wild-type WT and mutant V4I were obtained.
[0035] WT and mutant V4I cells were oxidized using 10 mM KPB (prepared at a 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 it on a metal bath at 25°C. Add 490 μL of substrate (100 mM acrylonitrile solution) to the centrifuge tube, vortex thoroughly to mix, and react at 25°C for 5 min. Then, stop the reaction by adding 500 μL of 0.1 M H3PO4 solution. Filter the reaction solution through a 0.22 μm filter membrane and perform liquid chromatography analysis.
[0036] 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℃, and the amount of acrylamide generated in the reaction system was determined.
[0037] The calculated total enzyme activity of WT and mutants is as follows: Figure 1 As shown, the whole-cell enzyme activity of the wild-type enzyme WT before heat treatment was 559.44 U / mL, while that of the mutant V4I was 588.31 U / mL.
[0038] Example 3: Pt Detection of the thermal stability of wild-type and mutant NHase The recombinant bacteria expressing the V4I mutant constructed in Example 2 and the recombinant bacteria expressing the wild-type enzyme were heat-treated at 65°C for 30 min, and the residual enzyme activity in whole cells after heat treatment was measured. The results showed that OD 600 The whole-cell enzyme activity of the wild-type enzyme WT with a strength of 0.5 was 229.36 U / mL, while that of the mutant V4I was 306.02 U / mL.
[0039] The specific steps are as follows: a mutation occurs at the 4th amino acid residue on the β subunit, and the residual enzyme activity of the whole cell lysate of nitrile hydratase is significantly increased after heat treatment at 65°C for 30 min. This indicates that the 4th amino acid residue may play an important role in the key structural domain of the enzyme and in the thermostability of nitrile hydratase.
[0040] Table 3 Comparison of enzyme activities against acrylonitrile between wild-type and mutant strains
[0041] Comparative Example For specific implementation details, please refer to Example 1, the difference being that for the wild type... Pt NHase was used to perform a single-point saturation mutagenesis, replacing the valine at position 4 of the β subunit with 19 other amino acids (mutated to A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, respectively). After the mutants were cultured and induced to express, the cells were adjusted to the same OD value of 0.5 as described in Example 2, and their whole-cell enzyme activity and residual whole-cell enzyme activity after heat treatment at 65°C for 30 min were measured. The results showed that when position 4 was mutated to isoleucine, mutant V4I had the highest whole-cell residual enzyme activity (306.02 U / mL, 1.3 times that of wild type), and the whole-cell residual enzyme activity of the other mutants was lower than that of mutant V4I. Figure 1 ).
[0042] Table 4. Comparison of whole-cell catalytic activity against acrylonitrile between wild-type and mutant strains.
[0043] Note: In Table 4, " / " indicates that the value is not listed. The whole-cell residual enzyme activity of each mutant after heat treatment at 65℃ for 30 min is shown in Table 4. Figure 1 .
[0044] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A nitrile hydratase mutant, characterized in that, The valine at position 4 of the β subunit of the nitrile hydratase parent is mutated to isoleucine; 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.
2. The gene encoding the mutant of claim 1.
3. A recombinant plasmid carrying the gene described in claim 2.
4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmids used the pET series as expression vectors.
5. A recombinant cell expressing the mutant of claim 1, or containing the gene of claim 2, or containing the recombinant plasmid of claim 3 or 4.
6. Recombinant Escherichia coli, characterized in that, by E. coli BL21(DE3) is the host, expressing the mutant described in claim 1.
7. A cell catalyst containing the recombinant Escherichia coli of claim 6.
8. A method for preparing acrylamide, characterized in that, Acrylamide was prepared using acrylonitrile as a substrate and catalyzed by the nitrile hydratase mutant of claim 1 or the recombinant Escherichia coli of claim 6.
9. The method according to claim 8, characterized in that, The concentration of acrylonitrile in the reaction system is 20~120 mM.
10. The use of the nitrile hydratase mutant of claim 1, or the gene of claim 2, or any of the recombinant plasmids of claims 3-4, or the recombinant cell of claim 5, or the recombinant Escherichia coli of claim 6, or the cell catalyst of claim 7 in the preparation of acrylamide.