CN114317507A

CN114317507A - Nitrile hydratase mutant and application thereof

Info

Publication number: CN114317507A
Application number: CN202111448620.8A
Authority: CN
Inventors: 于慧敏; 李付龙
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-12
Also published as: WO2023098453A1

Abstract

The invention provides nitrile hydratase mutants and applications thereof. The invention provides a nitrile hydratase mutant or fragment thereof comprising the following mutations compared to the wild-type nitrile hydratase: mutation I: a hydrophobic amino acid mutation in a domain located between the secondary domains alpha helix-1 and alpha helix-2 at the beginning of the wild-type nitrile hydratase beta subunit; mutation II: from the active center residue-C (S/T) LCSC (T/Y)

Polar amino acid mutations on random coil domains within the domain. The nitrile hydratase combination mutant provided by the invention can exert a synergistic regulation effect on the basis of the improved nitrile hydratase, has better thermal stability and tolerance, and further remarkably improves the activity.

Description

Nitrile hydratase mutant and application thereof

Technical Field

The invention belongs to the field of enzyme engineering and genetic engineering, and relates to a nitrile hydratase mutant, a genetic engineering bacterium containing the mutant and application of the nitrile hydratase mutant.

Background

Nitrile hydratase is a kind of metalloenzyme which can catalyze nitrile substance hydration reaction to convert nitrile substance into amide compound. The method for producing acrylamide and nicotinamide by catalyzing acrylonitrile and nicotinonitrile hydration by using nitrile hydratase or nitrile hydratase-containing microorganisms is one of the most successful cases of industrial biotechnology replacing chemical methods for producing bulk chemicals. Compared with a chemical method, the biocatalysis method has the advantages of mild reaction conditions, low energy consumption, simplicity in operation, high conversion rate, high product concentration, high product purity and the like, and is a mainstream method for industrial production of acrylamide at present.

Nitrile hydratases are generally composed of two subunits, alpha and beta (or called a and b subunits), and are distributed in various microorganisms such as Rhodococcus, Nocardia, Brevibacterium, Mycobacterium, and Pseudomonas. Among them, Rhodococcus rhodochrous J1(Nagasawa T, et al applied Microbiology and Biotechnology, 1993,40 (2-3): 189) and Rhodococcus rhodochrous Rruber TH (MaY, et al Bioresource Technology, 2010,101 (1): 285-291) and genetically engineered bacteria thereof have been successfully applied to the industrial production of acrylamide.

The acrylonitrile hydration reaction process is strongly exothermic, and the concentration of the later-stage product is high, which easily leads to the rapid inactivation of nitrile hydratase, so that the construction of a biocatalyst with high activity and high stability is an important requirement for the industrial production of acrylamide. Most of wild nitrile hydratases reported at present have the problems of poor catalytic stability, insufficient catalytic activity and the like. Many studies have been made to improve the stability of nitrile hydratase by constructing strategies such as salt bridges, disulfide bridges, linkers, or fusion subunits (Jianao S, et al. applied Microbiology and Biotechnology, 2020,104 (3): 1001 and 1012.), but reports on improvement of both nitrile hydratase activity and stability have been rare. Mainly because the catalytic mechanism of nitrile hydratase is more complex, the difficulty of rational design and semi-rational design is increased, so the improvement of nitrile hydratase is more slow. In order to meet industrial requirements, how to quickly and efficiently obtain a novel industrial catalyst with improved catalytic performance becomes a hotspot and a difficulty of research.

The university of Qinghua reported nitrile hydratase derived from Rhodococcus erythropolis for the first time, and identified its gene sequence and amino acid sequence (patent No. CN 200910076710.1); based on a molecular simulation result, the 141 th serine, 143 th serine and 144 th leucine of a nitrile hydratase beta subunit are replaced, so that the number of salt bridges is increased, and the heat resistance, the acrylamide tolerance and the ultrasonic tolerance of nitrile hydratase are remarkably improved (the patent number is CN201110415465. X); the thermal stability and acrylamide tolerance of nitrile hydratase are further improved by introducing a disulfide bond between the alpha and beta subunits (patent No.: CN 201710456875.6).

However, the catalytic activity of the above-mentioned modified nitrile hydratase mutant still needs to be improved. Rational design and modification aiming at the improvement of the catalytic activity of the nitrile hydratase have important significance for the industrial production of the amide compounds.

Disclosure of Invention

Therefore, the present invention is directed to a nitrile hydratase mutant based on the prior art. Compared with the prior art, the nitrile hydratase mutant has better thermal stability and tolerance and better activity. The invention also provides application of the nitrile hydratase mutant, in particular application of the nitrile hydratase mutant in catalyzing and synthesizing a plurality of amide compounds with important application values, including acrylamide and nicotinamide.

The invention concept of the invention is as follows: the inventor constructs a nitrile hydratase mutant library from wild nitrile hydratase through rational design of enzyme molecules, obtains a series of mutant strains with improved catalytic activity, and obviously improves the activity compared with that of a parent; on the basis, the inventor introduces a beneficial mutation site into the improved nitrile hydratase with improved stability and tolerance, and unexpectedly obtains the nitrile hydratase combination mutant provided by the invention, which can further exert a synergistic regulation effect on the basis of the improved nitrile hydratase, and has better thermal stability and tolerance and obviously improved activity.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the invention provides a nitrile hydratase mutant or fragment thereof comprising the following mutations compared to the wild-type nitrile hydratase:

mutation I: a hydrophobic amino acid mutation in a domain located between the secondary domains alpha helix-1 and alpha helix-2 at the beginning of the wild-type nitrile hydratase beta subunit;

mutation II: distance-active catalytic residue-C (S/T) LCSC (T/Y)

Polar amino acid mutations on random coil domains within the domain.

The nitrile hydratase mutant or fragment thereof according to the invention, wherein the wild-type nitrile hydratase is derived from Rhodococcus rhodochrous, Rhodococcus pyridinivorans, Rhodococcus ruber or Nocardia.

Specifically, the wild-type nitrile hydratase has a sequence selected from the group consisting of:

nitrile hydratase of wild-type Rhodococcus ruber (Rhodococcus ruber TH), the amino acid sequence of the β -subunit of which is as shown in SEQ ID NO: 1, the amino acid sequence of the alpha subunit is shown as SEQ ID NO: 2, the expression related structural gene and regulatory gene sequences are described in Chinese patent No. ZL200910076710.1, which is incorporated herein by reference in its entirety. Rhodococcus ruber is sometimes also translated as Rhodococcus ruber.

Nitrile hydratase derived from Rhodococcus rhodochrous J1(Rhodococcus rhodochrous J1) having a β subunit UPI number P21220 and an α subunit P21219 in the Uniprot Archive database, the entire contents of which are incorporated herein by reference.

The nitrile hydratase enzyme derived from Rhodococcus rhodochrous M8(Rhodococcus rhodochrous M8) having GenBank accession No. AAT79339.1 for the beta subunit and AAT79340.1 for the alpha subunit, which is incorporated by reference herein in its entirety.

Nitrile hydratase derived from Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) having a UPI number Q2UZQ6 for the beta subunit and Q2UZQ5 for the alpha subunit in the UniProt Archive database, which is incorporated herein by reference in its entirety.

Nitrile hydratase derived from Rhodococcus (Rhodococcus sp.) having a UPI number for the beta subunit of Q59785 and an alpha subunit of Q59786 in the UniProt Archive database, the entire contents of which are incorporated herein by reference.

A nitrile hydratase derived from Nocardia sp.jbrs having a UPI of the β subunit Q8GE66 and an α subunit Q8GE67 in the UniProt Archive database, the entire contents of which are incorporated herein by reference.

The nitrile hydratase mutant or fragment thereof according to the invention, wherein in mutation I the domain comprises fragment 1: -G (M/I) Sw; preferably, the mutation is a methionine, isoleucine or tryptophan mutation therein; more preferably, the methionine is mutated to cysteine, aspartic acid, serine, alanine, valine, or

The isoleucine is mutated into cysteine, aspartic acid, serine, alanine and valine; and/or

The tryptophan is mutated to alanine, arginine, glutamic acid, aspartic acid, serine, threonine, asparagine, cysteine, valine, glutamine, phenylalanine, tyrosine, proline, glycine, leucine, isoleucine, methionine, lysine or histidine.

The nitrile hydratase mutant or fragment thereof according to the invention, wherein in mutation II the domain comprises at least one fragment selected from the group consisting of:

fragment 2: - (S/T) (S/T) (S/A) (E/D) (I/L/V/M/T) -;

fragment 3: -G (Y/F) (A/S/T) (G/S) (E/R) (Q/H) (A/G) (H/E) -;

fragment 4: -H (D/G) TGGMTGY-;

fragment 5: k (N/S) MNPL (G/E) HTR-.

Optionally, the mutation is selected from one or more of:

in fragment 2, one or more threonine is mutated to serine and/or one or more serine is mutated to glycine and/or aspartic acid or glutamic acid is mutated to threonine.

In the fragment 3, tyrosine is mutated into threonine or serine and/or glutamine is mutated into asparagine and/or threonine is mutated into serine and/or glutamic acid is mutated into aspartic acid;

in the fragment 4, aspartic acid is mutated into cysteine or valine and/or threonine is mutated into alanine and/or tyrosine is mutated into threonine or serine;

in segment 5, one or more asparagine mutations to alanine and/or threonine mutations to serine and/or arginine mutations to valine and/or lysine mutations to cysteine;

the nitrile hydratase mutant or fragment thereof according to the invention, wherein the mutant or fragment thereof further comprises mutation III and/or mutation IV:

mutation III: an introduced salt bridge in the domain between the secondary domains alpha helix-7 and beta helix-1 located at the start of the wild-type nitrile hydratase beta subunit; simultaneously introducing a disulfide bond into a structural domain between a secondary structure alpha helix-10 and a secondary structure beta helix-4 of a wild-type nitrile hydratase beta subunit;

preferably, in mutation III, the domain introducing the salt bridge comprises fragment 6: -SFSLG-; more preferably, the mutation is to mutate serine in the amino acid sequence into lysine and leucine in the amino acid sequence into glutamic acid;

and/or the presence of a gas in the gas,

in the mutation III, the disulfide bond-introduced domain includes fragment 7: -GNGKD-, fragment 8: -VADP-; more preferably, the mutation is to change aspartic acid and proline in the amino acid sequence to cysteine;

and/or

Mutation Iv: a polar amino acid mutation in the secondary structure beta helix-2 domain of the wild-type nitrile hydratase beta subunit; preferably, it comprises fragment 9: -rnkit; more preferably, the mutation Iv is a mutation of asparagine to serine.

In a second aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding said nitrile hydratase mutant or fragment thereof;

preferably, the nucleotide sequence is obtained by base substitution on the basis of the sequence of the wild-type nitrile hydratase.

In a third aspect of the invention, there is provided an expression vector comprising the isolated nucleic acid molecule of the second aspect of the invention. The expression vector selected by the present invention can stably exist in various hosts of prokaryotic or eukaryotic cells and can be autonomously replicated, such as conventional plasmids (pET series), shuttle vectors pNV18.1, phage or virus vectors and the like in the field, and the preferred vectors are pET-28a and pNV18.1.

In a specific embodiment, a nucleotide sequence of wild nitrile hydratase is inserted into pET-28a or pNV18.1 through molecular biological operations such as enzyme digestion, ligation and the like to construct recombinant expression plasmids which are respectively named as pET28a-Nh and pNV18.1-Nh; the coding gene of the nitrile hydratase mutant is constructed into recombinant expression plasmids which are respectively named as pET28a-Nh_mutant，pNv18.1-Nh_mutant。

In a fourth aspect of the invention there is provided a host cell comprising an isolated nucleic acid molecule or expression vector as described in the second and third aspects of the invention. The host cell is selected from Escherichia coli, Rhodococcus, Nocardia, Corynebacterium propionate, Bacillus subtilis, and Corynebacterium glutamicum. Rhodococcus ruber and Escherichia coli are preferred in the present invention. The isolated nucleic acid molecule of the second aspect of the invention is inserted directly into the chromosome of the host bacterium, or the expression vector is introduced into the host bacterium by calcium chloride or electroporation.

A fifth aspect of the present invention provides a catalyst comprising the nitrile hydratase mutant or fragment thereof according to the first aspect of the invention and a process for producing the same. The catalyst comprises three forms of a whole-cell catalyst, a free protein catalyst and an immobilized enzyme catalyst. The whole cell catalyst is a whole cell obtained by enrichment culture and induced expression of target protein of the host cell constructed by the fourth aspect of the invention; the free protein catalyst is crude enzyme liquid obtained by crushing whole cells by ultrasonic crushing or high-pressure homogenization and centrifuging, and also comprises pure enzyme obtained by a protein purification means. The immobilized enzyme catalyst is different immobilized carriers, and the free protein catalyst is immobilized, so that immobilized nitrile hydratase mutants with different forms are obtained.

The sixth aspect of the present invention provides the use of the nitrile hydratase mutant described in the first aspect of the invention in catalyzing the hydration of nitrile compounds including acrylonitrile, nicotinonitrile, 2-cyanopyrazine, cinnamonitrile, phenylacetonitrile, p-hydroxyphenylacetonitrile, and the like, to prepare amide compounds including acrylamide, nicotinamide, pyrazinamide, cinnamamide, phenylacetamide, and p-hydroxyphenylacetamide.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the beneficial mutation sites of the inventor obviously improve the activity of nitrile hydratase; beneficial mutation sites are further introduced into the improved nitrile hydratase with improved stability and tolerance, and the nitrile hydratase combination mutant provided by the invention is obtained unexpectedly, can exert a synergistic regulation effect on the basis of the improved nitrile hydratase, and further remarkably improves the activity while having better thermal stability and tolerance.

Drawings

In order to more clearly illustrate the technical solution of the embodiment of the present invention, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings:

FIG. 1 shows a model of the three-dimensional structure of a wild-type nitrile hydratase derived from Rhodococcus ruber of the invention, wherein FIG. 1A shows the three-dimensional crystal structure of the wild-type nitrile hydratase and its catalytic active center and substrate channels; FIG. 1B shows the molecular binding pattern of the substrate to wild-type nitrile hydratase.

FIG. 2 shows the secondary domain characteristics of wild-type nitrile hydratase derived from Rhodococcus ruber of the invention, wherein FIG. 2A is the secondary domain characteristics of the alpha subunit of wild-type nitrile hydratase and FIG. 2B is the secondary domain characteristics of the beta subunit of wild-type nitrile hydratase.

FIG. 3 shows the secondary domain characteristics of the alpha subunit of wild-type nitrile hydratase from different sources according to the invention.

FIG. 4 shows the secondary domain characteristics of the beta subunit of wild-type nitrile hydratase from different sources according to the invention.

Detailed Description

Features and exemplary embodiments of various aspects of the present invention will be described in detail below. The embodiments of the present invention are described as examples of the present invention, and the present invention is not limited to the embodiments described below.

Any equivalent modifications and substitutions to the embodiments described below are within the scope of the present invention for those skilled in the art. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments are not indicated by manufacturers, and are conventional products which can be purchased commercially. In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps that are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all units used in the present specification are international standard units, and numerical values and numerical ranges appearing in the present invention should be understood to include systematic errors inevitable in industrial production.

Definition of

In all discussions herein, the standard one-letter code for amino acids is used. Standard substitution notation is also used, i.e. Q β 45R means that Q at position 45 from the N-terminus of the β subunit is replaced by R; or Q α 42R means that Q at position 42 from the N-terminus from the α subunit is replaced by R.

In this paragraph, where the different amino acids at a particular position are separated by/symbol,/symbol means "or". For example, Q87R/K means Q87R or Q87K.

In this paragraph, where different amino acids at a particular position are separated by/symbol and included in parentheses, where/symbol means "or" () symbol indicates an amino acid at the same position. For example, (Y/F) (A/S/T) means that the two amino acid residues can be YA, YS, YT, FA, FS or FT.

In the paragraphs herein separated by/symbols at different positions,/symbol means "and" such that Y51/N55 are Y51 and N55.

The term "wild-type" refers to a gene or gene product that is isolated from a naturally occurring source. Wild-type genes are the most commonly observed genes in a population, and are therefore arbitrarily designed as "normal" or "wild-type" forms of genes. Conversely, the terms "modified," "mutant" or "variant" refer to a gene or gene product that exhibits a modification (e.g., substitution, truncation, or insertion), post-translational modification, and/or a functional characteristic (e.g., altered characteristic) of sequence as compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art.

Wild-type nitrile hydratase

Nitrile hydratase (NHase) is a metal-dependent enzyme having an active center containing a nonheme iron atom or a cobalt atom and is classified into Fe-type and Co-type. The nitrile hydratases known so far have both alpha and beta subunits (or so-called a and b subunits) in the form of heteromultimers, and the amino acid residues in the active center are relatively conserved (-C (S/T) LCSC (T/Y) -). Co-type nitrile hydratase was selected as the subject of the study.

Specifically, the inventors selected various wild-type nitrile hydratases derived from Rhodococcus rhodochrous, Rhodococcus pyridinivorans, Rhodococcus ruber or Nocardia.

Nitrile hydratase mutant

On the basis of the existing wild-type nitrile hydratase, the applicant obtained a number of useful mutants by structural analysis and rational design. It was further found that these advantageous mutations were concentrated in a specific secondary domain of the wild-type nitrile hydratase. The applicants first analyzed the crystal structure of nitrile hydratase derived from Rhodococcus ruber (FIG. 1A), determined the amino acid residues of the active center thereof, including (-CTLCCY), by structural analysis, and analyzed the secondary domain. On the basis of the structure, the combination state of the substrate and the enzyme molecule is simulated by utilizing molecular docking, the combination process of the substrate and the enzyme molecule is researched through molecular dynamics simulation, the interaction force between the substrate and the enzyme molecule is analyzed, and the amino acid sites and the spatial structure position thereof which influence the possibly influenced enzyme catalytic activity are determined. And then based on the remodeling of the substrate binding pocket, increasing the volume of the pocket, designing a mutant, carrying out activity measurement, and screening out a plurality of mutants with improved catalytic performance, wherein the result shows that the obtained beneficial mutation sites are all concentrated in a specific secondary structure domain of wild nitrile hydratase.

And then, taking nitrile hydratase derived from Rhodococcus ruber as a template, obtaining structural models of nitrile hydratases from different sources through homologous modeling, and finding that specific secondary domains in the nitrile hydratases have high conservation through structural comparison, so that the invention also considers the regulation and control effects of the obtained beneficial mutation sites on nitrile hydratases from other sources. These nitrile hydratases include:

nitrile hydratase derived from Rhodococcus ruber (Rhodococcus ruber TH) and having an amino acid sequence of the β subunit of SEQ ID NO: 1, the amino acid sequence of the alpha subunit is shown as SEQ ID NO: 2, the expression related structural gene and regulatory gene sequences are described in Chinese patent No. ZL200910076710.1, which is incorporated herein by reference in its entirety.

Further, the inventors have found that, on the basis of the above mutation, if salt bridge modification and disulfide bond are further introduced, a mutant having more excellent catalytic performance is obtained by combining the mutations.

The nitrile hydratase mutant of the invention or fragment thereof comprises the following mutations compared with the wild-type nitrile hydratase:

mutation II: from the active center residue-C (S/T) LCSC (T/Y)

Polar amino acid mutations on random coil domains within the domain.

fragment 2: - (S/T) (S/T) (S/A) (E/D) (I/L/V/M/T) -;

fragment 3: -G (Y/F) (A/S/T) (G/S) (E/R) (Q/H) (A/G) (H/E) -;

fragment 4: -H (D/G) TGGMTGY-;

fragment 5: k (N/S) MNPL (G/E) HTR-.

Optionally, the mutation is selected from one or more of:

mutation III: an introduced salt bridge in the domain between the secondary domains alpha helix-7 and beta helix-1 located at the start of the wild-type nitrile hydratase beta subunit; simultaneously introducing a disulfide bond into a structural domain between a secondary structure alpha helix-10 and a secondary structure beta helix-4 of a wild-type nitrile hydratase b subunit;

preferably, in mutation III, the domain introducing the salt bridge comprises fragment 6: -SFSLG-; more preferably, the mutation is a mutation of serine to lysine and/or leucine to glutamic acid;

and/or the presence of a gas in the gas,

and/or

The nitrile hydratase of the invention also includes a protein having a nitrile hydratase activity, which has an amino acid sequence obtained by deleting, substituting or adding 1 or more, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 2 amino acids in the amino acid sequence of the nitrile hydratase of the invention.

Determination of the catalytic Activity of nitrile hydratase

The nitrile hydratase activity refers to an activity of catalyzing hydration of a nitrile compound to produce an amide compound. A substrate (nitrile compound) is reacted with nitrile hydratase under conditions such that the nitrile hydratase activity is calculated by measuring the amount of substrate consumed and the amount of product increased per unit time. As the substrate, any nitrile compound can be used as long as nitrile hydratase is reacted, and acrylonitrile and nicotinonitrile are preferable. The reaction conditions are general conditions for hydration reaction, as long as catalytic activity of nitrile hydratase is ensured, and the amount of substrate consumed and the amount of product increased can be detected and quantitatively analyzed by HPLC and GC.

The nitrile compound has a general formula shown in (1):

R-CN (1)

here, the R group is an optionally substituted linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, an optionally substituted cycloalkyl or aryl group having 3 to 18 carbon atoms, or an optionally substituted saturated or unsaturated heterocyclic group.

The general formula of the amide compound is shown as (2):

R-CONH₂ (2)

3. Recombinant vector and transformant for nitrile hydratase mutant

The nucleotide sequence encoding the nitrile hydratase mutant can be constructed into different types of recombinant vectors according to the host cell, or can be directly integrated into the chromosome of the host bacterium. Examples of the vector to be used include plasmid DNA, phage DNA, retrotransposon DNA, and artificial chromosome DNA. As examples of Escherichia coli and Rhodococcus ruber, PET series vectors and PNV series vectors are preferable.

The host to be used for the transformant of the present invention is not particularly limited as long as it can express the target nitrile hydratase after introducing the above-mentioned recombinant vector or nucleotide sequence encoding a nitrile hydratase mutant, and bacteria such as Rhodococcus and Escherichia coli, yeast, animal cells, insect cells, plant cells, and the like can be used.

The method for introducing a recombinant vector into a bacterium is not particularly limited as long as it is a method for introducing a DNA into a bacterium. Examples thereof include a method using calcium ions and an electroporation method.

The method for incorporating the nucleotide sequence encoding the nitrile hydratase mutant into the bacterium is not particularly limited as long as it is a method of introducing DNA into the bacterium. Examples thereof include the use of homologous recombination and gene editing.

Examples

Example 1 sequence of nitrile hydratase mutant

On the basis of wild-type nitrile hydratase derived from Rhodococcus ruber, the inventors designed a mutant in which the amino acid sequence of the β subunit of the wild-type nitrile hydratase derived from Rhodococcus ruber is as shown in SEQ ID NO: 1, the amino acid sequence of the alpha subunit is shown as SEQ ID NO: 2, said mutants are detailed in table 1:

TABLE 1

Mutant 1: on the basis of the wild-type nitrile hydratase, M.beta.45C (1) in fragment 1 was present, indicating that methionine in fragment 1 was mutated to cysteine.

Example 2 construction of recombinant expression vector for nitrile hydratase

The inventor designs BamH I and EcoR I restriction sites at two ends of a nucleotide sequence for coding nitrile hydratase, uses plasmid pET-28a as a vector, performs double restriction on the plasmid and the nucleotide sequence of the nitrile hydratase in the embodiment by using endonucleases BamH I and EcoR I, recovers a gene fragment after restriction by using nucleic acid electrophoresis (1.0% agarose) and a kit, and then connects the target gene fragment after restriction to the plasmid vector after restriction.

The 20. mu.L ligation system included:

2. mu.L of 10 XT 4 DNA ligase Buffer (Takara Co.);

5 mu L of target gene fragment;

5 μ L of plasmid fragment;

2 μ L T4 DNA ligase;

8μL ddH₂O；

connecting at 16 mu for overnight, transforming into DH5 alpha competent cells, selecting monoclonals for sequencing verification, extracting recombinant plasmids with correct sequencing, obtaining recombinant expression vectors containing nitrile hydratase encoding genes, and using the recombinant expression vectors as female parents for subsequent transformation.

EXAMPLE 3 construction of nitrile hydratase mutant

The present invention adopts the whole plasmid PCR method to construct the recombinant plasmid containing nitrile hydratase mutant gene, firstly designing the upstream and downstream primers containing the mutant site, using plasmid pET28a-NH as the template, using PrimeSTAR HS DNA Polymerase (Takara company) to perform the whole plasmid amplification. A recombinant plasmid containing a mutation in the gene sequence encoding nitrile hydratase was amplified by PCR.

20 μ L of PCR reaction system included:

1 μ L of pET28a-NH plasmid template (approximately 100 ng/. mu.L);

10 μ L of 2 XPrimeSTAR HS DNA polymerase;

1.5. mu.L of forward primer (10. mu.M);

1.5 μ L reverse primer (10 μ M);

6μL ddH₂O。

the forward primer is a specific primer used in the construction process of different mutants. The reverse primer is a specific primer used in the construction process of different mutants. This is not repeated at length, but those skilled in the art will know that designing primers based on known sequences and obtaining the desired product are routine technical means for those skilled in the art.

The conditions for the PCR reaction were as follows:

(1) pre-denaturation at 98 ℃ for 1 min;

(2) denaturation at 98 ℃ for 30 s;

(3) annealing the primer at Tm-5 ℃ for 10 s;

(4) extending for 7min at 72 ℃;

the above steps (2) - (4) are performed for 30 cycles in total, and finally, the extension is performed for 10min at 72 ℃.

After removing the template sequence by DpnI enzymatic digestion, the PCR stock solution was transformed into Ecoli top 10 competent cells by heat shock method, spread on LB plate containing kanamycin (50. mu.g/mL), and placed in 37 ℃ incubator for inverted culture for about 12 h. And (4) selecting the monoclonals for sequencing verification, preserving bacteria by using 20% (V/V) glycerol after the sequencing is correct, and storing in a refrigerator at-70 ℃.

EXAMPLE 4 construction of nitrile hydratase combination mutants

The nitrile hydratase combination mutant is obtained by a mode of multi-round fixed point mutation, after a single point mutant is obtained, recombinant plasmids containing the mutant points are used as templates, upstream and downstream primers of the mutant points are designed, full plasmid PCR amplification is carried out, and the recombinant plasmids containing the gene sequences of the coding nitrile hydratase combination mutant are amplified through PCR.

20 μ L of PCR reaction system included:

mu.L of the parental/plasmid template containing a single mutation site (about 100 ng/. mu.L);

10μL 2×PrimeSTAR HS DNA Polymerase；

1.5. mu.L of forward primer (10. mu.M);

1.5 μ L reverse primer (10 μ M);

6μL ddH₂O。

the forward primer can use specific primers constructed for different mutants, which are not described in detail herein, but those skilled in the art should know that designing primers based on known sequences and obtaining the desired product are routine technical means for those skilled in the art.

The conditions for the PCR reaction were as follows:

(1) pre-denaturation at 98 ℃ for 1 min;

(2) denaturation at 98 ℃ for 30 s;

(3) (Tm-5 for primer) DEG C for 10 s;

(4) extending for 7min at 72 ℃;

Example 5 construction of Escherichia coli Gene engineering bacteria for nitrile hydratase and its mutant and preparation of catalyst

The recombinant expression vectors prepared in examples 3 and 4 were transformed into competent cells e.coli BL21(DE3) by heat shock, coated with LB plates containing kanamycin (50 μ g/mL), cultured overnight at 37 ℃, then single colonies were picked up and transferred to LB liquid cultures containing 50 μ g/mL kanamycin, cultured for 12 hours at 37 ℃, sampled for sequencing, and the correct clones were stored in a-70 ℃ refrigerator, to thereby obtain genetically engineered bacteria in which escherichia coli was the host.

The genetic engineering bacteria containing the nitrile hydratase and the coding sequence of the mutant thereof are inoculated into LB liquid medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent deionized water, pH 7.0) containing kanamycin resistance, and the mixture is placed in a test tube (4mL, containing kanamycin with the final concentration of 50 mug/mL) containing LB medium, and cultured in a shaker at 37 ℃ for 10-12 h at the rotating speed of 200rpm, so as to obtain seed liquid.

The seed solution in the tube was transferred to a shake flask (100mL containing kanamycin at a final concentration of 50. mu.g/mL) containing LB medium in a clean bench. And (3) placing the LB culture medium containing the seed liquid into a shaking table at 37 ℃, and culturing for 2-3h at the rotating speed of 200 rpm. OD of the culture solution₆₀₀When the value reaches 0.6-0.8, IPTG and Co 0.1-0.8mM are added to the mixture to obtain a final concentration of 0.1-0.8mM²⁺Carrying out induced expression by ions at the induction temperature of 16-37 ℃. A preferred concentration of IPTG is 0.2mM, a preferred concentration of cobalt ions is 0.2mM, and a preferred induction temperature is 16 ℃. After 24h of induction under the preferred conditions, the cells were collected by centrifugation to obtain the E.coli cell catalyst for nitrile hydratase, and stored in a refrigerator at-70 ℃.

10g of the cryopreserved wet cells were weighed, added to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mM NaCl, 10mM imidazole, 375. mu.L/L mercaptoethanol) to a final concentration of 10g/L, left to melt at room temperature, and the incompletely dissolved clumped cells were removed by filtration. And then crushing by using a high-pressure homogenizer, controlling the pressure at 700-800bar, and cooling by using low-temperature circulating equipment in the crushing process for circulating crushing for 2-3 times. The collected disrupted solution was centrifuged, and the supernatant was collected to obtain a free nitrile hydratase catalyst.

The immobilized amino resin material was weighed in an appropriate amount, equilibrated with potassium phosphate buffer (100mM, pH 7.0), the treated tree carrier was added to the buffer at a carrier to solution ratio of 1/5(w/V), followed by addition of glutaraldehyde solution (50% V/V) to a final concentration of 2%. After activation in a shaker (16 ℃, 200rpm) for 2-3h, the activated support was washed with deionized water to remove residual glutaraldehyde. Weighing a proper amount of the activated immobilization carrier, placing the immobilization carrier in a buffer solution, and adding an enzyme solution, wherein the ratio of the carrier to the enzyme solution is still 1/5 (w/v). The resulting mixture was immobilized on a constant temperature shaker or shaker (16 ℃ C., 200rpm) for 8 hours. The immobilized enzyme was then washed with a buffer to remove the residual enzyme solution, and then stored in a refrigerator at 4 ℃ for later use.

EXAMPLE 6 construction of Rhodococcus System for nitrile hydratase mutant and preparation of catalyst

Using the recombinant expression vectors prepared in examples 3 and 4 as templates, respectively, universal primers for nitrile hydratase were designed, the sequences of which contained the homologous fragment of the optimized version of suicide plasmid pYsacB and the nitrile hydratase gene fragment, and the nitrile hydratase gene sequence containing the mutation site was amplified by PCR. The optimized suicide plasmid pYsacB1 is formed by adding homologous arm sequences of about 1000bp at both upstream and downstream ends of a nitrile hydratase gene on the basis of the suicide plasmid pYsacB.

Homologous recombination double-exchange experimental process

1) Using methylase deficient Escherichia coli E.coli Trans110 as host, constructing and extracting suicide plasmid pS18mobsacBopt-amiE, transforming R.ruber TH, coating on a plate culture medium containing 50 mug/mL spectinomycin after recovery culture, and culturing at 28 ℃ for 3 days to grow colonies.

2) Single colonies were picked and colony PCR verified (one primer on the genome, upstream of the upstream homology arm; one primer on the plasmid, downstream of the downstream homology arm), ensures integration of the suicide plasmid into the correct position.

3) And inoculating the colony which is successfully subjected to single exchange into a seed culture medium without antibiotics, culturing at 28 ℃ and 200rpm for 1 day, diluting by 10-100 times, coating 200 mu L of the colony on a plate containing 100g/L of sucrose, and culturing at 28 ℃ for 2-3 days to grow the colony.

4) Single colonies were picked and subjected to colony PCR (primers on the genome, one above the upstream homology arm and one below the downstream homology arm) to verify whether gene knock-out or back mutation occurred.

The conditions for the PCR reaction were as follows:

(1) pre-denaturation at 98 ℃ for 1 min;

(2) denaturation at 98 ℃ for 30 s;

(3) (Tm-5 for primer) DEG C for 10 s;

(4) extending for 2min at 72 ℃;

Connecting the obtained nitrile hydratase mutant gene with the digested suicide plasmid pYsacB by a Gibson Assembly seamless cloning kit,

10 μ L of the ligation system included:

2 μ L of the target gene;

3 μ L of suicide plasmid vector;

5 μ L ligation reagent;

connecting at 50 ℃ for 30min, converting the connecting solution into Top 10 competent cells, picking up monoclonal sequencing verification, extracting recombinant suicide plasmids, transforming the successfully constructed recombinant suicide plasmids into the competent cells of R.ruber TH9 of Rhodococcus by an electrical transformation method, coating the competent cells on a plate culture medium containing 50 mug/mL spectinomycin after recovery culture, and culturing at 20-37 ℃; then selecting a single colony, carrying out colony PCR verification, and verifying that the suicide plasmid is integrated into the correct position in the genome of the rhodococcus; inoculating the colony which is successfully subjected to single exchange into a seed culture medium without antibiotics, culturing for 12h at 20-37 ℃, diluting by 100 times, coating 200 mu L of the colony on a plate containing 100g/L of sucrose, culturing at 28 ℃, carrying out colony PCR after the single colony grows out, verifying that the nitrile hydratase gene sequence is integrated on the genome, sending the genome to a sequencing company, and storing the correctly sequenced strain in a refrigerator at-70 ℃ for later use.

The conditions for the PCR reaction were as follows:

(1) pre-denaturation at 98 ℃ for 1 min;

(2) denaturation at 98 ℃ for 30 s;

(3) (Tm-5 for primer) DEG C for 10 s;

(4) extending for 3min at 72 ℃;

Inoculating the constructed Rhodococcus containing the coding sequence of the nitrile hydratase mutant into a seed culture medium, and culturing until the thallus OD₆₀₀Up to about 30. At this time, the initial OD is pressed₆₀₀Inoculating the seed culture medium into 50mL fermentation medium with the inoculum size of 3.0For the inducible expression of nitrile hydratase, a final concentration of 0.08mM C0 was added to the fermentation medium²⁺And after 48 hours of induction expression, centrifuging and collecting cells to obtain the nitrile hydratase rhodococcus cell catalyst.

10g of the cryopreserved wet cells were weighed, added to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mM NaCl, 10mM imidazole, 375. mu.L/L mercaptoethanol) to a final concentration of 10g/L, left to melt at room temperature, and the incompletely dissolved clumped cells were removed by filtration. And then crushing by using a high-pressure homogenizer, controlling the pressure at 1200-1500bar, and cooling by using low-temperature circulating equipment in the crushing process for circulating crushing for 2-3 times. The collected disrupted solution was centrifuged, and the supernatant was collected to obtain a free nitrile hydratase catalyst.

The appropriate amount of the immobilization carrier was weighed, equilibrated with potassium phosphate buffer (100mM, pH 7.0), and the treated tree carrier was added to the buffer at a carrier to solution ratio of 1/5(w/V), followed by addition of glutaraldehyde solution (50%) to a final concentration of 2% V/V. After activation in a shaker (16 ℃, 200rpm) for 2-3h, the activated support was washed with deionized water to remove residual glutaraldehyde. Weighing a proper amount of the activated immobilization carrier, placing the immobilization carrier in a buffer solution, and adding an enzyme solution, wherein the ratio of the carrier to the enzyme solution is still 1/5 (w/v). The resulting mixture was immobilized on a constant temperature shaker or shaker (16 ℃ C., 200rpm) for 8 hours. The immobilized enzyme was then washed with a buffer to remove the residual enzyme solution, and then stored in a refrigerator at 4 ℃ for later use.

Example 7 nitrile hydratase Activity assay method

The nitrile hydratase activity on the substrate acrylonitrile was determined as follows: 50-100. mu.L of a catalyst (cells, free enzyme, immobilized enzyme) was put into a centrifuge tube, 10mMPBS (pH 7.0) was added to make up to 4.5mL, and the mixture was left in a 28 ℃ water bath for 10 minutes to stabilize the temperature. Add 200. mu.L acrylonitrile, mix well and react for 5 minutes, add 200. mu.L 3mol/L hydrochloric acid to terminate the reaction. 13000 Xg of 1mL of the reaction solution was centrifuged for 2 minutes, and 500. mu.L of the supernatant and 500. mu.L of the internal standard solution (40g/L of acetamide) were mixed and analyzed by gas chromatography. And (3) measuring the area ratio of acrylamide to acetamide, measuring the concentration of the product acrylamide by using an internal standard method, and calculating the activity.

Detecting the amount of product formed by gas phase under the following conditions: the gas chromatographic conditions were as follows: a American Saimei fly Trace 1300 gas chromatograph; abel BondedAB-I NOWAX chromatography column (0.25 mm ID, 30m length, 0.25 μm membrane thickness); FID detector. The column temperature, the injection port temperature and the detector temperature are respectively 19 ℃, 26 ℃ and 26 ℃; the carrier gas is nitrogen, the constant pressure mode is adopted, and the partial pressure is 10⁸kPa; the sample injection volume is 1 mu L, and the split sample injection is carried out, wherein the split ratio is 50: 1.

The nitrile hydratase activity was calculated as follows:

k is an internal standard constant with a value of 0.6001; c_acThe concentration of acetamide is 40 g/L; v total is the total volume, and the numerical value is 5 mL; v is the actual catalyst volume added; t is the reaction time; MW is acrylamide, molecular weight 71; u is total enzyme activity and the unit is mu mol acrylamide/(min. mL bacterial liquid).

The enzyme activity (U) is defined as: under the above reaction conditions, the amount of enzyme required to catalyze 1. mu. mol of the substrate per minute is one enzyme activity unit, and is represented by U.

Example 8 evaluation of nitrile hydratase thermostability and tolerability

The nitrile hydratase catalysts obtained in examples 5 and 6 were resuspended in 10mM PBS buffer (pH 7.0, 10g/L), allowed to stand still in a thermostatic water bath at 50 ℃ and samples were taken at 15-minute intervals, the residual activity was measured as described in example 7, and an inactivation curve was plotted to determine the half-life of the catalyst according to a linear fit, and the higher the half-life value, the better the stability, and the thermal stability of nitrile hydratase was evaluated.

Similarly, the nitrile hydratase catalysts obtained in examples 5 and 6 were resuspended in 10mM PBS buffer (pH 7.0, 10g/L), 10mL of each of the resuspended catalysts were placed in 50mL triangular flasks, acrylamide was added to a final concentration of 40% (v/v), the residual activity was measured after standing in a 30 ℃ constant temperature water bath for 1h, and the relative activity value was calculated from the initial activity, and the product tolerance of nitrile hydratase was evaluated by indicating that the higher the relative activity value was, the better the tolerance was.

Example 9 determination of the Activity of nitrile hydratase mutants

The mutants constructed as in examples 3 and 4 were tested for activity according to the method of example 7, and the results are shown in Table 2, and the catalytic activities of mutants 1-6, 8-12, 15-19, 21-27, 29-31, 33-36, 38-40 and mutant 61 were increased by a factor of 1.0-2.0 after the designed mutation site was introduced into Nitrile Hydratase (NH); mutants 7, 13, 14, 20, 28, 32, 37, 41, 42-44, 49, 59, 61, 64 and mutant 66 had a fold increase in catalytic activity of 2.0-4.0; the catalytic activity of mutants 45-48, 52-58, 63 and 69 is improved by 3.0-6.0 times; the catalytic activity of mutants 50, 51, 60, 65, 67, 68, 70-73, 75 and 79-82 is improved by 6.0-8.0 times, and the catalytic activity of mutants 74 and 76-78 is improved by 8.0-10.0 times. Through structure comparison and substrate binding pocket analysis, the pocket volume increase and the change of polar environment of the beneficial mutant are beneficial to the binding of the substrate and the enzyme, and are the main reasons for improving the catalytic activity of the mutant.

TABLE 2

+: 1.0-2.0 times, increased by ++: the improvement is 2.0 to 4.0 times; +++: the improvement is 3.0 to 6.0 times;

++++: 6.0-8.0 times higher, and ++++: the improvement is 8.0 to 10.0 times.

EXAMPLE 10 nitrile hydratase and its mutant stability and tolerance assay results

The mutants introduced with the salt bridge or the disulfide bond in the example 9 are subjected to stability and tolerance determination according to the method in the example 8, wherein the stability can be improved to 2.2 times of that of the wild type when the salt bridge is introduced, and the tolerance can be improved to 1.6 times of that of the wild type; the stability can be improved to 2.8 times of that of the wild type when the disulfide bond is introduced, and the tolerance is improved to 2.0 times of that of the wild type; when the salt bridge and the disulfide bond are introduced at the same time, the stability can be improved to 4.2 times that of the wild type, and the tolerance is improved to 5.0 times that of the wild type.

After the designed mutation site is introduced into nitrile hydratase, the obtained beneficial mutant not only has improved activity, but also has improved thermal stability and tolerance, which indicates that the synergistic regulation and control effect is exerted by combined mutation on the basis of salt bridge and disulfide bond modification, and the thermal stability and tolerance are improved while the activity is higher.

EXAMPLE 11 Regulation of other nitrile hydratase enzymes at Key amino acid sites

The beneficial mutation sites obtained as in example 1 are located partly around the active pocket and these amino acids are functionally closely related to the catalytic function of nitrile hydratase, whereas at structural positions these amino acids are conserved in similar positions in nitrile hydratases of different origin, as shown in FIGS. 3 and 4. The present inventors examined the regulatory effect of the beneficial mutation site as described in example 1 on nitrile hydratase from other sources (Table 3). Amino acid residues at similar positions in other nitrile hydratases were first identified by multiple sequence alignment and mutation and performance assays were performed with reference to the mutants designed in examples 1-10. Nitrile hydratase derived from Rhodococcus rhodochrous J1(Rhodococcus rhodochrous J1) having a UPI number P21220 for the beta subunit and P21219 for the alpha subunit in the Uniprot Archive database. Nitrile hydratase derived from Rhodococcus rhodochrous M8(Rhodococcus rhodochrous M8) having GenBank accession No. AAT79339.1 for the beta subunit and AAT79340.1 for the alpha subunit.

Nitrile hydratase derived from Rhodococcus pyridinivorans RP (Rhodococcus pyridinivorans) having a UPI number Q2UZQ6 for the beta subunit and Q2UZQ5 for the alpha subunit in the UniProt Archive database.

Nitrile hydratase derived from Rhodococcus R.sp (Rhodococcus sp.) having a UPI number of Q59785 for the β subunit and Q59786 for the α subunit in the UniProt Archive database.

A nitrile hydratase derived from Nocardia JBR (Nocardia sp. JBRs) having a UPI of the β subunit Q8GE66 and an α subunit Q8GE67 in the UniProt Archive database.

The catalytic activity and the stability of the obtained mutant nitrile hydratase are improved simultaneously, which indicates that the mutant sites have a synergistic regulation effect on the activities of nitrile hydratases from different sources.

Sequence listing

<110> Qinghua university

<120> nitrile hydratase mutant and use thereof

<130> 21NI2506

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 229

<212> PRT

<213> Rhodococcus ruber TH

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Met Asp Gly Ile His Asp Thr Gly Gly Met Thr Gly Tyr Gly Pro Val

1 5 10 15

Pro Tyr Gln Lys Asp Glu Pro Phe Phe His Tyr Glu Trp Glu Gly Arg

20 25 30

Thr Leu Ser Ile Leu Thr Trp Met His Leu Lys Gly Met Ser Trp Trp

35 40 45

Asp Lys Ser Arg Phe Phe Arg Glu Ser Met Gly Asn Glu Asn Tyr Val

50 55 60

Asn Glu Ile Arg Asn Ser Tyr Tyr Thr His Trp Leu Ser Ala Ala Glu

65 70 75 80

Arg Ile Leu Val Ala Asp Lys Ile Ile Thr Glu Glu Glu Arg Lys His

85 90 95

Arg Val Gln Glu Ile Leu Glu Gly Arg Tyr Thr Asp Arg Asn Pro Ser

100 105 110

Arg Lys Phe Asp Pro Ala Glu Ile Glu Lys Ala Ile Glu Arg Leu His

115 120 125

Glu Pro His Ser Leu Ala Leu Pro Gly Ala Glu Pro Ser Phe Ser Leu

130 135 140

Gly Asp Lys Val Lys Val Lys Asn Met Asn Pro Leu Gly His Thr Arg

145 150 155 160

Cys Pro Lys Tyr Val Arg Asn Lys Ile Gly Glu Ile Val Thr Ser His

165 170 175

Gly Cys Gln Ile Tyr Pro Glu Ser Ser Ser Ala Gly Leu Gly Asp Asp

180 185 190

Pro Arg Pro Leu Tyr Thr Val Ala Phe Ser Ala Gln Glu Leu Trp Gly

195 200 205

Asp Asp Gly Asn Gly Lys Asp Val Val Cys Val Asp Leu Trp Glu Pro

210 215 220

Tyr Leu Ile Ser Ala

225

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<213> Rhodococcus ruber TH

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Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys

1 5 10 15

Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala

20 25 30

Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly

35 40 45

Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys

50 55 60

Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala

65 70 75 80

Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr

85 90 95

His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val

100 105 110

Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg

115 120 125

Val Val Ala Asp Cys Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp

130 135 140

Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile

145 150 155 160

Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser

165 170 175

Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val

180 185 190

Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val

195 200

Claims

1. A nitrile hydratase mutant or fragment thereof, wherein the mutant or fragment comprises at least one of the following mutations compared to the wild-type nitrile hydratase:

mutation II: from the active center residue-C (S/T) LCSC (T/Y)

Polar amino acid mutations on random coil domains within the domain.

2. A nitrile hydratase mutant or fragment thereof according to claim 1 wherein the wild-type nitrile hydratase is derived from Rhodococcus rhodochrous, Rhodococcus pyridinivorans, Rhodococcus ruber or Nocardia.

3. A nitrile hydratase mutant or fragment thereof according to claim 1 or 2 wherein in mutation I the domain comprises fragment 1: -G (M/I) SW-;

preferably, the mutation is a methionine, isoleucine or tryptophan mutation therein; more preferably, the methionine is mutated to cysteine, aspartic acid, serine, alanine, valine, or

4. A nitrile hydratase mutant or fragment thereof according to any one of claims 1 to 3 wherein in mutation II the domain comprises at least one fragment selected from the group consisting of:

fragment 2: - (S/T) (S/T) (S/A) (E/D) (I/L/V/M/T) -;

fragment 3: -G (Y/F) (A/S/T) (G/S) (E/R) (Q/H) (A/G) (H/E) -;

fragment 4: -H (D/G) TGGMTGY-;

fragment 5: -K (N/S) MNPL (G/E) HTR-;

optionally, the mutation is selected from one or more of:

in fragment 2, one or more threonine is mutated to serine; and/or

One or more serines are mutated to glycine; and/or

Aspartic acid or glutamic acid is mutated to threonine;

in fragment 3, tyrosine is mutated to threonine or serine; and/or

Glutamine mutated to asparagine; and/or

Threonine to serine; and/or

Glutamic acid to aspartic acid;

in fragment 4, aspartic acid is mutated to cysteine or valine; and/or

Threonine mutant alanine; and/or

Tyrosine is mutated to threonine or serine;

in fragment 5, one or more asparagine is mutated to alanine; and/or

Threonine to serine; and/or

Arginine to valine; and/or

Lysine is mutated to cysteine.

5. A nitrile hydratase mutant or fragment thereof according to any one of claims 1 to 4 wherein the mutant or fragment thereof further comprises mutation III and/or mutation IV:

and/or the presence of a gas in the gas,

and/or

Mutation IV: a polar amino acid mutation in the alpha helix-2 domain of the secondary structure of the wild-type nitrile hydratase beta subunit; preferably, it comprises fragment 9: -rnkit; more preferably, the mutation IV is a mutation of asparagine to serine.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a nitrile hydratase mutant or fragment thereof according to any one of claims 1 to 5.

7. An expression vector comprising the isolated nucleic acid molecule of claim 6;

preferably, the expression vector is a plasmid vector, preferably a pET series, a shuttle vector, a phage or a viral vector;

more preferably, the expression vector is pET-28a or pNV18.1.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the expression vector of claim 7;

preferably, the host cell is selected from the group consisting of E.coli, Rhodococcus, Nocardia, Corynebacterium propionate, Bacillus subtilis, and Corynebacterium glutamicum;

more preferably, the host cell is rhodococcus ruber or/and e.coli BL21(DE 3).

9. A catalyst comprising a nitrile hydratase mutant or fragment thereof according to any one of claims 1 to 5;

preferably, the catalyst is a whole cell catalyst, a free protein catalyst or an immobilized enzyme catalyst.

10. Use of the catalyst according to claim 9 for the preparation of amides.

11. A method for preparing amide compounds, which comprises catalyzing nitrile compounds to perform hydration reaction by using the catalyst of claim 9 to obtain amide compounds;

preferably, the nitrile compound is selected from acrylonitrile, nicotinonitrile, 2-cyanopyrazine, cinnamonitrile, phenylacetonitrile or p-hydroxyphenylacetonitrile;

the amide substance is selected from acrylamide, nicotinamide, pyrazinamide, cinnamide, phenylacetamide or p-hydroxyphenylacetamide.