Nitrile hydratase mutant and use thereof

Abstract

A nitrile hydratase mutant and the use thereof. The nitrile hydratase mutant is a wild-type nitrile hydratase having a mutation of an amino acid residue in the flexible loop domain, and comprises any one of or a combination of mutations 1-3, wherein mutation 1 is a polar amino acid mutation located in the flexible loop between secondary domains S1 and S2 of the β subunit of the wild-type nitrile hydratase; mutation 2 is a polar amino acid mutation located in the flexible loop between secondary domains H6 and H7 at the starting end of the β subunit of the wild-type nitrile hydratase; and mutation 3 is a hydrophobic amino acid mutation in starting flexible loop L1 of the β subunit of the wild-type nitrile hydratase. On the basis of the wild-type nitrile hydratase, the nitrile hydratase mutant can comprise any one of or a combination of the mutations described above, which can exert regulatory effects alone or synergistically, has a certain enzyme activity, and has good thermal stability and tolerance.

Description

Nitrile hydratase mutant and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311054507.0, filed on August 21, 2023, entitled “Nitrile Hydratase Mutants and Applications Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The invention belongs to the fields of protein engineering and genetic engineering, and relates to a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant and applications thereof. Background Art

[0004] Amide compounds are important organic synthesis intermediates and chemicals, widely used in pharmaceuticals, pesticides, feed, food, environmental protection, oil recovery, materials, and other fields. Hydration of nitrile compounds to obtain corresponding amide compounds is an important method for the industrial preparation of these compounds. Nitrile hydratase (NHase, EC 4.2.1.84) is a multi-subunit enzyme that converts nitrile compounds into the corresponding amides through hydration. The use of NHase catalysts to catalyze the hydration of acrylonitrile and nicotinonitrile to produce acrylamide and nicotinamide is one of the most successful examples of biotechnology replacing chemical methods in the industrial production of bulk chemicals.

[0005] Nitrile hydratase is typically composed of two subunits, α and β (also known as α and β subunits), and is found in a variety of microorganisms, including Rhodococcus, Nocardia, Brevibacterium, and Pseudomonas. As early as 1980, Japanese scholar Asano first discovered this type of enzyme in Rhodococcus rhodochrous J1 and defined it as nitrile hydratase (Asano Y, et al. Agricultural and Biological Chemistry, 1982, 46(5):1165-1174). The use of nitrile hydratase to catalyze the production of acrylamide from acrylonitrile has been studied for decades, and the production strains used have also gone through three generations. Among them, Rhodococcus rhodochrous J1 (Nagasawa T, et al. Applied Microbiology and Biotechnology, 1993, 40(2-3):189-195), R. ruber TH and their genetically engineered strains are widely used in the industrial production of acrylamide (Ma Y, et al. Bioresource Technology, 2010, 101(1):285-291).

[0006] During the catalytic hydration of acrylonitrile, nitrile hydratase catalysts often face the problem of a sudden temperature rise caused by the exothermic effect of the hydration reaction, leading to enzyme inactivation and cell rupture, resulting in reduced economic benefits. Furthermore, high product concentrations can be highly toxic to cells, further deactivating the catalyst. Therefore, developing a new generation of catalysts with high stability and high product tolerance is a key requirement for the industrial production of amide compounds. However, most wild-type nitrile hydratases suffer from poor stability, product tolerance, and insufficient catalytic activity. In order to further improve the production performance of nitrile hydratase, researchers have conducted in-depth research on multiple aspects at the enzyme molecular level, such as the design of salt bridges and disulfide bonds and the introduction of molecular chaperones, and improved the stability of nitrile hydratase (Liu Y, et al. Applied Biochemistry and Biotechnology, 2016, 180(1): 3-9; Ma Y, et al. Journal of Industrial Microbiology & Biotechnology, 2012, 39(10): 1421-1430; Tian Y, et al. Journal of Microbiology and Biotechnology, 2016, 26(2): 337-346; Jiao S, et al. Applied Microbiology and Biotechnology, 2020, 104(3): 1001-1012.).

[0007] With the rapid development of bioinformatics and protein crystallography, computer simulations and protein crystal structures are increasingly being used to guide protein engineering research, leading to the development of various protein engineering approaches, such as semi-rational design and rational design. Although the crystal structures of some nitrile hydratases have been resolved, the catalytic mechanism of nitrile hydratase is relatively complex and remains inconclusive, increasing the difficulty of rational and semi-rational design. As a result, progress in modifying nitrile hydratase has been relatively slow. To meet industrial needs, how to quickly and efficiently obtain new industrial catalysts with improved catalytic performance has become a research hotspot and difficulty. This is mainly due to the relatively complex catalytic mechanism of nitrile hydratase, which increases the difficulty of rational and semi-rational design. Therefore, progress in modifying nitrile hydratase has been relatively slow.

[0008] Summary of the Invention

[0009] Therefore, the purpose of this application is to provide a nitrile hydratase mutant based on the prior art. The nitrile hydratase mutant of the present application can contain the above mutations on the basis of wild-type nitrile hydratase, which can exert a synergistic regulatory effect alone or in combination, have a certain enzyme activity, and have better thermal stability and tolerance.

[0010] In a first aspect, the embodiments of the present application provide a nitrile hydratase mutant, based on a wild-type nitrile hydratase, wherein the nitrile hydratase mutant is a nitrile hydratase mutant in which an amino acid residue on the flexible loop domain of the wild-type nitrile hydratase is mutated, comprising any one or a combination of mutations 1-3:

[0011] Mutation 1: a polar amino acid mutation located in the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase β subunit;

[0012] Mutation 2: a polar amino acid mutation in the flexible loop between the secondary domains H6 and H7 at the start end of the wild-type nitrile hydratase β subunit;

[0013] Mutation 3: A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase β subunit.

[0014] According to an embodiment of one aspect of the present application, the wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus rubrum, and Nocardia sp.

[0015] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant satisfies at least one of the following conditions:

[0016] 1) In mutation 1, the amino acid position 152 of the β subunit is mutated;

[0017] 2) In mutation 2, the amino acid position 110 of the β subunit is mutated;

[0018] 3) In mutation 3, the amino acid position 17 of the β subunit mutated.

[0019] According to an embodiment of one aspect of the present application, the amino acid residue at amino acid position 152 is mutated to any one of cysteine, threonine, glutamic acid, aspartic acid, valine, serine, glycine, and glutamine.

[0020] According to an embodiment of one aspect of the present application, the amino acid residue at amino acid position 110 is mutated to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine.

[0021] According to an embodiment of one aspect of the present application, the amino acid residue at amino acid position 17 is mutated to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.

[0022] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 3.

[0023] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 2.

[0024] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 2 and mutation 3.

[0025] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes mutation 1, mutation 2 and mutation 3.

[0026] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant comprises at least one of a mutation of the amino acid residue at amino acid position 167 of the β subunit to serine, a mutation of the amino acid residue at amino acid position 174 of the α subunit to leucine, and a mutation of the amino acid residue at amino acid position 178 of the α subunit to glutamate.

[0027] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant comprises: a domain mutation, which includes: the amino acid residue at the amino acid position 141 of the β subunit mutates to lysine, the amino acid residue at the amino acid position 143 mutates to lysine, the amino acid residue at the amino acid position 144 mutates to glutamate, the amino acid residue at the amino acid position 215 mutates to cysteine, and the amino acid residue at the amino acid position 133 of the α subunit mutates to cysteine.

[0028] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes at least one of 17A / 17R / 17E / 17D / 17S / 17C / 17K / 17H, 110A / 110R / 110S / 110T / 110V / 110F / 110L / 110I / 110K, and 152C / 152A / 152T / 152E / 152D / 152V / 152S / 152Q / 152G.

[0029] According to an embodiment of one aspect of the present application, β17S / β110R / β152C, β17R / β110R / β152D, β17S / β110K / 152C, β17S / β110 R / β152C, β17S / β110R / β152C, β110K / β152T, β110K / β152D, β110R / β152T, β110K / β17E, β 110K / β17C, β110K / β17D, β152S / β17H, β152S / β17E, β110K / β152S / β17E, β17A / β110K / β1 52G / α174L / α178E, β17E / β110R / β152S / α174L / α178E, β17S / β110V / β152S / βS141K / β143K / β144E、β17D / β110K / β152V / α174L / α178Eβ110R / β152S / β17D、β17E / β110K / β152S / β167 S / α174L / α178E, β17E / β110K / β152S / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E, β17R / β110S / β152Q / β167S / α174L / α178E / β215C / α133C / / βS141K / β143K / β144E, β17D / / β110F / β152C / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E.

[0030] In a second aspect, the embodiments of the present application provide an isolated nucleic acid molecule comprising a nucleotide sequence encoding the nitrile hydratase mutant according to the first aspect.

[0031] In a third aspect, an embodiment of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0032] According to an embodiment of one aspect of the present application, the expression vector is a plasmid vector.

[0033] According to an embodiment of one aspect of the present application, the expression vector is a pET series, a shuttle vector, a phage or a viral vector.

[0034] In a third aspect, an embodiment of the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0035] According to an embodiment of one aspect of the present application, the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

[0036] According to an embodiment of one aspect of the present application, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0037] In a fourth aspect, the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.

[0038] According to an embodiment of one aspect of the present application, the catalyst is a whole-cell catalyst, a free protein catalyst or an immobilized enzyme catalyst.

[0039] In a fifth aspect, an embodiment of the present application provides a method for preparing an amide compound, the method comprising using the catalyst of the first aspect or the fourth aspect to catalyze a hydration reaction of a nitrile compound to obtain an amide compound.

[0040] According to an embodiment of one aspect of the present application, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamononitrile, benzyl cyanide or p-hydroxyphenyl cyanide.

[0041] According to an embodiment of one aspect of the present application, the amide substance is selected from acrylamide, nicotinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

[0042] The technical solution of this application has at least the following advantages:

[0043] The nitrile hydratase mutant of the embodiment of the present application has the above-mentioned mutation site, which significantly improves the thermal stability and product tolerance of the nitrile hydratase; the above-mentioned mutation site is further introduced into nitrile hydratases from different sources, and it is unexpectedly obtained that the nitrile hydratase mutation site of the present application also exerts a regulatory effect in nitrile hydratases from different sources, which can further improve the thermal stability and product tolerance of nitrile hydratase and has a certain activity.

[0044] The embodiments of the present application can provide nitrile hydratase mutants with thermal stability, product tolerance, or catalytic activity. Nitrile hydratase mutants are very useful in the production of amide compounds.

[0045] The present invention further provides a nucleic acid molecule encoding the mutant nitrile hydratase, an expression vector comprising the nucleic acid molecule encoding the mutant nitrile hydratase, and a transformant comprising the expression vector. The present invention further provides a method for preparing an amide compound, which utilizes the nitrile hydratase extracted from the culture of the transformant, the method for producing the amide compound, and methods using the nitrile hydratase, the culture, or a processed product thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the technical solutions of the present application will be described in detail below with reference to the accompanying drawings:

[0047] FIG1 shows a schematic diagram of the spatial positions of mutations 1-3 according to an embodiment of the present application based on the spatial conformation of nitrile hydratase from Rhodococcus rubrum.

[0048] FIG2 shows a graph showing the substrate consumption effects of the wild-type nitrile hydratase and the mutant M133 nitrile hydratase of the examples of the present application.

[0049] FIG3 shows the residual activity of wild-type nitrile hydratase and mutant M133 nitrile hydratase according to an example of the present application. DETAILED DESCRIPTION

[0050] The features and exemplary embodiments of various aspects of the present application will be described in detail below. The specific embodiments listed in the present invention are only examples of the present application, and the present invention is not limited to the specific embodiments described below.

[0051] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should all be encompassed within the scope of the present application. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of ​​the present application.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0053] In all discussions herein, the standard single-letter code for amino acids is used. Standard substitution notation is also used, i.e., β110A means that the amino acid residue at position 110 from the N-terminus of the β subunit is substituted with alanine (A); or α174L means that the amino acid residue at position 174 from the N-terminus of the α subunit is substituted with leucine (L).

[0054] In the paragraphs herein where different amino acids at specific positions are separated by a / symbol, the " / " symbol means "and" or "or." For example, β17S / β110R / β152S / βS141K / β143K / β144E / β215C / α133C may indicate that all of the above mutations are present in nitrile hydratase.

[0055] In the text paragraphs for different amino acids at specific positions separated by the / symbol and enclosed in brackets, the / symbol means "or" and the () symbol indicates amino acids 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.

[0056] In the present paragraphs separated by the / symbol at different positions, the “ / ” symbol means “and” so that Y51 / N55 is Y51 and N55.

[0057] The term "wild type" refers to a gene or gene product that has been isolated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modification (e.g., substitution, truncation, or insertion), post-translational modification, and / or functional properties (e.g., altered properties) compared to a 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 a wild-type gene or gene product. Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced by arginine (R) by replacing the methionine codon (ATG) with the arginine codon (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art.

[0058] Wild-type nitrile hydratase

[0059] Nitrile hydratase (NHase) is a metal-dependent enzyme with a non-heme iron or cobalt atom in its active center. It can be divided into two types: Fe-type and Co-type. Currently known nitrile hydratases all exist as heteromeric multimers consisting of two subunits, α and β (also known as a and b subunits), with relatively conserved amino acid residues in the active center (-C(S / T)LCSC(T / Y)-). This study selected Co-type nitrile hydratases as the research object. Specifically, the inventors selected various wild-type nitrile hydratases from Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus rubrum, and Nocardia sp.

[0060] Nitrile hydratase mutant

[0061] The technical concept of the present application is as follows: starting from the wild-type nitrile hydratase, the inventors analyzed the crystal structure information of the enzyme, locked the key regulatory amino acid residues through structural analysis, molecular dynamics simulation and enzyme molecular sequence alignment, and constructed a nitrile hydratase mutant library through targeted design, obtaining a series of mutant strains with improved catalytic performance, which were significantly improved in stability and product tolerance compared to the parent strain; on this basis, the inventors introduced beneficial mutation sites into nitrile hydratases from different sources, and unexpectedly obtained that the mutation sites of the nitrile hydratase of the present application also exerted a regulatory effect in nitrile hydratases from different sources, which can further improve the thermal stability and product tolerance of nitrile hydratase.

[0062] Taking nitrile hydratase molecules as the research object, through structure-function relationship research, the interaction between substrate and enzyme molecules was analyzed, and the enzyme molecules were reconstructed to improve their stability and product tolerance at the molecular level.

[0063] The purpose of this application is achieved through the following technical solutions:

[0064] In a first aspect, the embodiments of the present application provide a nitrile hydratase mutant, based on a wild-type nitrile hydratase, wherein the nitrile hydratase mutant is a nitrile hydratase mutant in which an amino acid residue on the flexible loop domain of the wild-type nitrile hydratase is mutated, comprising any one or a combination of mutations 1-3:

[0065] Mutation 1: a polar amino acid mutation located in the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase β subunit;

[0066] Mutation 2: a polar amino acid mutation in the flexible loop between the secondary domains H6 and H7 at the start end of the wild-type nitrile hydratase β subunit;

[0067] Mutation 3: A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase β subunit.

[0068] According to an embodiment of the present application, the positions of the secondary structural domains S1 and S2, the secondary structural domains H6 and H7, and the initial flexible loop L1 can be as shown in FIG1 .

[0069] Based on the existing wild-type nitrile hydratase, the applicant obtained multiple beneficial mutants through structural analysis and rational design. It was further discovered that these beneficial mutations are concentrated in specific primary structures (mutations in amino acid species) and / or secondary structural domains of the wild-type nitrile hydratase.

[0070] The inventors further discovered the distribution of these beneficial mutation sites in the spatial structure. Based on the structure, they used molecular docking to simulate the binding state of substrate and enzyme molecules, and studied the binding process of substrate and enzyme molecules through molecular dynamics simulation. They analyzed the interaction force between substrate and enzyme molecules, identified amino acid sites that may affect the catalytic performance of the enzyme, and combined multiple sequence alignment analysis to lock in candidate amino acid residues. They also conducted targeted design based on structural position and evolutionary conservation analysis, constructed a mutant library, and screened out multiple mutants with improved stability and product tolerance through catalytic performance evaluation.

[0071] Using nitrile hydratases from Rhodococcus rubrum TH, Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinophilus, and Nocardia as templates, structural models of nitrile hydratases from different sources were obtained through homology modeling. Structural comparison revealed that the primary structures and specific secondary domains in these nitrile hydratases were highly conserved. Therefore, this application investigated the regulatory effects of the obtained beneficial mutation sites on nitrile hydratases from other sources.

[0072] As an example, based on the nitrile hydratase of Rhodococcus ruber TH, the amino acid sequence of its β subunit is shown in SEQ ID NO: 1, and the amino acid sequence of the α subunit is shown in SEQ ID NO: 2; the nitrile hydratase mutant of the present application comprises any one or any combination of the above mutations based on the amino acid sequence of the above wild-type nitrile hydratase.

[0073] In some optional embodiments, the wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus rubrum, and Nocardia sp.

[0074] According to the embodiments of the present application, the wild-type nitrile hydratase may be derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus rubrum or Nocardia sp.

[0075] Specifically, the wild-type nitrile hydratase has a sequence selected from the group consisting of:

[0076] 1) Wild-type nitrile hydratase from Rhodococcus ruber TH, the amino acid sequence of its β subunit is shown in SEQ ID NO: 1, and the amino acid sequence of its α subunit is shown in SEQ ID NO: 2. The structural and regulatory gene sequences expressing the relevant amino acid sequences are detailed in Chinese invention patent No. ZL200910076710.1, the entire contents of which are incorporated herein by reference. Rhodococcus ruber is sometimes also translated as "red coccus."

[0077] 2) Nitrile hydratase from Rhodococcus rhodochrous J1, the UPI number of its β subunit in the UniProt Archive database is P21220, and the UPI number of its α subunit is P21219, the entire contents of which are incorporated herein by reference.

[0078] 3) Nitrile hydratase from Rhodococcus rhodochrous M8, the GenBank accession numbers of its β subunit are AAT79339.1 and α subunit are AAT79340.1, the entire contents of which are incorporated herein by reference.

[0079] 4) Nitrile hydratase from Rhodococcus pyridinivorans, the UPI number of its β subunit in the UniProt Archive database is Q2UZQ6, and the UPI number of its α subunit is Q2UZQ5, the entire content of which is incorporated herein by reference.

[0080] 5) Nitrile hydratase from Rhodococcus sp., the UPI number of its β subunit in the UniProt Archive database is Q59785, and the UPI number of its α subunit is Q59786, the entire contents of which are incorporated herein by reference.

[0081] 6) Nitrile hydratase from Nocardia sp. JBRs, the UPI number of its β subunit in the UniProt Archive database is Q8GE66, and the UPI number of its α subunit is Q8GE67, the entire contents of which are incorporated herein by reference.

[0082] In some optional embodiments, the nitrile hydratase mutant satisfies at least one of the following conditions:

[0083] 1) In mutation 1, the amino acid position 152 of the β subunit is mutated;

[0084] 2) In mutation 2, the amino acid position 110 of the β subunit is mutated;

[0085] 3) In mutation 3, the amino acid position 17 of the β subunit mutated.

[0086] According to the embodiments of the present application, a mutation at amino acid position 152 of the β subunit can be understood as a mutation at amino acid position 152 from the N-terminus of the β subunit. A mutation at amino acid position 110 of the β subunit can be understood as a mutation at amino acid position 110 from the N-terminus of the β subunit. A mutation at amino acid position 17 of the β subunit can be understood as a mutation at amino acid position 17 from the N-terminus of the β subunit. The above mutations may exist alone or in any combination.

[0087] In some optional embodiments, the amino acid residue at amino acid position 152 is mutated to any one of cysteine, threonine, glutamic acid, aspartic acid, valine, serine, glycine, and glutamine.

[0088] According to the embodiments of the present application, it can be understood that the amino acid residue at amino acid position 152 of the β subunit is mutated to any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G.

[0089] In some optional embodiments, the amino acid residue at amino acid position 110 is mutated to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine.

[0090] According to the embodiments of the present application, it can be understood that the amino acid residue at amino acid position 110 is mutated to any one of 110A, 110R, 110S, 110T, 110V, 110F, 110L, 110I, and 110K.

[0091] In some optional embodiments, the amino acid residue at amino acid position 17 is mutated to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.

[0092] According to the embodiments of the present application, it can be understood that the amino acid residue at the 17th amino acid position at the starting N-terminus of the β subunit is mutated to any one of 17A, 17R, 17E, 17D, 17S, 17C, 17K, and 17H.

[0093] In some alternative embodiments, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 2. For example, mutants M26-M30.

[0094] In some alternative embodiments, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 3. For example, mutants M48-M54.

[0095] In some alternative embodiments, the nitrile hydratase mutant includes any combination of mutation 2 and mutation 3. For example, mutants M39-M47.

[0096] In some optional embodiments, the nitrile hydratase mutants include mutation 1, mutation 2, and mutation 3. For example, mutants M56-M69.

[0097] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant comprises at least one of a mutation of the amino acid residue at amino acid position 167 of the β subunit to serine, a mutation of the amino acid residue at amino acid position 174 of the α subunit to leucine, and a mutation of the amino acid residue at amino acid position 178 of the α subunit to glutamate.

[0098] This can be understood as follows: the 167th amino acid from the N-terminus of the β subunit is β167S; the 174th amino acid from the N-terminus of the α subunit is α174L; and the 178th amino acid from the N-terminus of the α subunit is α178E. The above mutations can exist alone or in combination with any combination of mutations from Mutation 1 to Mutation 3. The above mutations can exist in any combination and in combination with any combination of mutations from Mutation 1 to Mutation 3.

[0099] Based on the above mutants, the nitrile hydratase mutant further comprises any one or a combination of asparagine at position 167 of the β subunit to serine, glycine at position 174 of the α subunit to leucine, and aspartic acid at position 178 of the α subunit to glutamate. The above mutations of this embodiment can be present in any combination and combined with any of the above embodiments and mutations comprising any combination of mutations 1 to 3.

[0100] In some optional embodiments, the nitrile hydratase mutant comprises: a domain mutation, which includes: the amino acid residue at amino acid position 141 of the β subunit mutates to lysine, the amino acid residue at amino acid position 143 mutates to lysine, the amino acid residue at amino acid position 144 mutates to glutamate, the amino acid residue at amino acid position 215 mutates to cysteine, and the amino acid residue at amino acid position 133 of the α subunit mutates to cysteine.

[0101] This can be understood as follows: the 141st amino acid from the N-terminus of the β subunit is β141K; the 143rd amino acid from the N-terminus of the β subunit is β143K; the 144th amino acid from the N-terminus of the β subunit is β144E; the 215th amino acid from the N-terminus of the β subunit is β215C; and the 133rd amino acid from the N-terminus of the α subunit is α133C. The above mutations are simultaneously present in this domain mutation and can be combined with any of the mutations in other embodiments.

[0102] On the basis of the above mutant or wild type, salt bridge modification and disulfide bond are further introduced. The nitrile hydratase mutant comprises: a combination of serine at position 141 of the β subunit mutated to lysine, serine at position 143 mutated to lysine, and leucine at position 144 mutated to glutamate, aspartic acid at position 215 mutated to cysteine, and proline at position 133 of the α subunit mutated to cysteine, and any combination of any mutants of the above embodiments and co-existing.

[0103] In some optional embodiments, the nitrile hydratase mutant includes at least one of 17A / 17R / 17E / 17D / 17S / 17C / 17K / 17H, 110A / 110R / 110S / 110T / 110V / 110F / 110L / 110I / 110K, and 152C / 152A / 152T / 152E / 152D / 152V / 152S / 152Q / 152G.

[0104] It can be understood that the 17th amino acid of the β subunit from the N-terminus can be any one of 17A, 17R, 17E, 17D, 17S, 17C, 17K, and 17H, and the 110th amino acid of the β subunit from the N-terminus can be any one of 110A, 110R, 110S, 110T, 110V, 110F, 110L, 110I, and 110K. The 152nd amino acid of the β subunit from the N-terminus can be any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G. The nitrile hydratase mutants of the embodiments of the present application include any one of the above three mutations, or a random combination of any one of them.

[0105] In some optional embodiments, β17S / β110R / β152C, β17R / β110R / β152D, β17S / β110K / 152C, β17S / β110R / β152C, β17S / β110R / β152C, β110K / β152T, β110K / β152D, β110R / β152T, β110K / β17E, β110R / β152D, β110R / β152T, β110K / β152E, β110R / β152C, β17S / β110R / β152C, β110K / β152T, β110K / β152 0K / β17C, β110K / β17D, β152S / β17H, β152S / β17E, β110K / β152S / β17E, β17A / β110K / β152 G / α174L / α178E, β17E / β110R / β152S / α174L / α178E, β17S / β110V / β152S / βS141K / β143K / β144E, β17D / β110K / β152V / α174L / α178Eβ110R / β152S / β17D, β17E / β110K / β152S / β167S / α174L / α178E、β17E / β110K / β152S / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / Any one of β144E, β17R / β110S / β152Q / β167S / α174L / α178E / β215C / α133C / / βS141K / β143K / β144E, β17D / / β110F / β152C / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E.

[0106] It can be understood that, compared with the wild-type nitrile hydratase or the mutant nitrile hydratase in the related art, the sites of the nitrile hydratase include mutations at three of the sites, and specific forms of the mutations include: 17S / 110R / 152C, 17R / 110R / 152D, 17S / 110K / 152C, 17S / 110R / 152C, 17S / 110R / 152C, etc. For example, 17S / 110R / 152C can be understood as: the 17th amino acid from the N-terminus of the β subunit is serine, the 110th amino acid from the N-terminus of the β subunit is arginine, and the 152nd amino acid from the N-terminus of the β subunit is cysteine.

[0107] Catalytic activity of nitrile hydratase

[0108] Nitrile hydratase activity refers to the activity of catalyzing the hydration of nitrile compounds to produce amide compounds. Substrate (nitrile compound) and nitrile hydratase are reacted under certain conditions, and the consumption of substrate and the increase of product are measured in unit time to calculate the activity of nitrile hydratase. As substrate, as long as nitrile hydratase reacts, no matter what nitrile compound can be used, acrylonitrile and nicotinonitrile can be selected. Reaction conditions are the universal conditions of hydration reaction. As long as it is possible to ensure that nitrile hydratase has catalytic activity, the consumption of substrate and the increase of product can be detected and quantitatively analyzed by HPLC and GC.

[0109] The general formula of nitrile compounds is shown in (1): R-CN (1)

[0110] 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.

[0111] The general formula of amide compounds is shown in (2): R-CONH2 (2)

[0112] 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.

[0113] After research, it was found that the wild-type nitrile hydratase and mutant nitrile hydratase described in this application have catalytic activity.

[0114] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the nitrile hydratase mutant according to the first aspect.

[0115] According to an embodiment of the present application, the nucleic acid molecule comprises a nucleotide sequence encoding the nitrile hydratase mutant. The nucleotide sequence may be obtained by base mutation based on the sequence of the wild-type nitrile hydratase.

[0116] Recombinant vector and transformant of nitrile hydratase mutant

[0117] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0118] According to the embodiments of the present application, the selected expression vector can stably exist and autonomously replicate in various hosts of prokaryotic or eukaryotic cells, such as conventional plasmids (pET series), shuttle vector pNV18.1, phage or viral vectors in the art.

[0119] In some alternative embodiments, the expression vector is a plasmid vector.

[0120] In some optional embodiments, the expression vector is a pET series, a shuttle vector, a phage or a viral vector; optionally, the expression vector is pET-28a or pNV18.1.

[0121] According to the examples of the present application, the nucleotide sequence of the wild-type nitrile hydratase is inserted into an expression vector such as pET-28a or pNV18.1 through molecular biological manipulations such as enzyme digestion and ligation to construct a recombinant expression plasmid, which is named pET28a-Nh and pNV18.1-Nh, respectively. According to the examples of the present application, the encoding gene of the nitrile hydratase mutant of the example of the present application can be constructed into the recombinant expression plasmid.

[0122] In a third aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0123] According to the embodiments of the present application, the isolated nucleic acid molecule described in the second aspect of the present application can be directly inserted into the chromosome of the host bacteria, or the expression vector of the third aspect can be introduced into the host bacteria using the calcium chloride method or electroporation transformation method.

[0124] In some alternative embodiments, the host cell comprises any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

[0125] In some optional embodiments, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0126] In a fourth aspect, the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.

[0127] Depending on the host cell, the nucleotide sequence encoding the nitrile hydratase mutant can be constructed into various types of recombinant vectors or directly integrated into the host bacterial chromosome. Examples of vectors include plasmid DNA, phage DNA, retrotransposon DNA, and artificial chromosome DNA. For Escherichia coli and Rhodococcus rubrum, PET and PNV series vectors are suitable.

[0128] The host that can be used for the transformant of the present application is not particularly limited as long as it can express the target nitrile hydratase after the introduction of the above-mentioned recombinant vector or the nucleotide sequence encoding the nitrile hydratase mutant. For example, bacteria such as Rhodococcus and Escherichia coli, yeast, animal cells, insect cells, plant cells, etc. can be used.

[0129] The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include methods using calcium ions and electroporation.

[0130] The method for integrating the nucleotide sequence encoding the nitrile hydratase mutant into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include homologous recombination and gene editing.

[0131] Catalyst and method for preparing amide compound

[0132] In some alternative embodiments, the catalyst is a whole-cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst.

[0133] According to the embodiments of the present application, the whole cell catalyst refers to the whole cell obtained after enrichment culture and induced expression of the target protein by the host cell constructed by the fourth aspect of the present application. The free protein catalyst is a crude enzyme solution obtained by ultrasonication or high-pressure homogenization of the whole cell and centrifugation, and also includes pure enzyme obtained by protein purification. The immobilized enzyme catalyst is to select different immobilization carriers and immobilize the free protein catalyst to obtain different forms of immobilized nitrile hydratase mutants.

[0134] In a fifth aspect, the present application provides a method for preparing an amide compound, the method comprising using the catalyst of the first aspect or the fourth aspect to catalyze a hydration reaction of a nitrile compound to obtain an amide compound;

[0135] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamonitrile, benzyl cyanide or p-hydroxyphenyl cyanide.

[0136] In some optional embodiments, the amide substance is selected from acrylamide, nicotinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

[0137] The following examples further describe the present disclosure. These examples are intended for illustrative purposes only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. All reagents used in the examples are commercially available or synthesized according to conventional methods and used without further processing. All instruments used in the examples are commercially available.

[0138] Example

[0139] Example 1 Sequences of Nitrile Hydratase Mutants

[0140] Based on the wild-type nitrile hydratase (WT) from Rhodococcus rubrum, the following mutants were designed, wherein the amino acid sequence of the β subunit of the wild-type nitrile hydratase from Rhodococcus rubrum is shown in SEQ ID NO: 1, and the amino acid sequence of the α subunit is shown in SEQ ID NO: 2. The mutants are detailed in Table 1:

[0141] Table 1

[0142] Example 2 Construction of recombinant expression vector for nitrile hydratase

[0143] In Escherichia coli cells, using plasmid pET-28a as an expression vector, the inventors designed Nco I and Xho I restriction sites at both ends of the nucleotide sequence encoding nitrile hydratase. The plasmid vector and the nucleotide sequence of the nitrile hydratase in this example were double-digested using restriction endonucleases Nco I and Xho I. The gene fragments after enzyme digestion were recovered using nucleic acid electrophoresis (1.0% agarose) and a kit. The target gene fragments were then ligated to the plasmid vector after enzyme digestion.

[0144] The 10 μL ligation system includes:

[0145] 1 μL 10× T4 DNA ligase Buffer (Takara);

[0146] 2.5 μL target gene fragment;

[0147] 2.5 μL plasmid fragment;

[0148] 1 μL T4 DNA ligase;

[0149] 3 μL ddH2O;

[0150] The cells were ligated overnight at 16°C and transformed into BL21 (DE3) competent cells. Single clones were picked for sequencing verification, and the recombinant plasmids with correct sequencing results were extracted to obtain a recombinant expression vector containing the nitrile hydratase encoding gene.

[0151] Example 3 Construction of Nitrile Hydratase Mutants

[0152] This application uses a full plasmid amplification method to construct a recombinant plasmid containing a mutant gene for nitrile hydratase. Upstream and downstream primers are designed for the desired mutant site, and full plasmid amplification is performed using PrimeSTAR HS DNA Polymerase (Takara) using plasmid pET28a-NH as a template. A recombinant plasmid containing a mutant sequence encoding the nitrile hydratase gene is amplified by PCR.

[0153] A 20 μL PCR reaction system includes:

[0154] 1 μL pET28a-NH plasmid template (approximately 220 ng / μL);

[0155] 10 μL 2× PrimeSTAR HS DNA polymerase;

[0156] 1.0 μL forward primer (10 μM);

[0157] 1.0 μL reverse primer (10 μM);

[0158] 7 μL ddH2O.

[0159] The forward primers are specific primers used in the construction process of different mutants. The reverse primers are specific primers used in the construction process of different mutants. Due to space limitations, they are not described in detail here, but those skilled in the art will be aware that designing primers based on known sequences and obtaining the desired product are routine techniques within the skill of the art.

[0160] After digesting the PCR stock with Dpn I to remove the template sequence, the cells were transformed into BL21(DE3) competent cells using the heat shock method. Plates were plated onto LB plates containing kanamycin (50 μg / mL) and incubated upside down in a 37°C incubator for approximately 12 hours. Single clones were selected for sequencing verification. Once sequencing was confirmed, the clones were maintained in 20% (v / v) glycerol and stored in a refrigerator at -70°C.

[0161] Example 4 Construction of Nitrile Hydratase Combination Mutants

[0162] The nitrile hydratase combination mutants were obtained through multiple rounds of site-directed mutagenesis. After obtaining a single point mutant, a recombinant plasmid containing the mutation site was used as a template, upstream and downstream primers of the mutation site were designed, and whole-plasmid PCR amplification was performed. The recombinant plasmid containing the gene sequence encoding the nitrile hydratase combination mutants was amplified by PCR.

[0163] The forward primer can use specific primers constructed for different mutants, which are not described here due to space limitations. However, those skilled in the art should know that designing primers based on known sequences and obtaining target products are routine technical means for those skilled in the art.

[0164] After digesting the PCR product with Dpn I to remove the template sequence, the product was transformed into BL21(DE3) competent cells using the heat shock method. The plate was spread onto LB plates containing kanamycin (50 μg / mL) and incubated upside down in a 37°C incubator for approximately 12 hours. Single clones were selected for sequencing verification.

[0165] Example 5 Construction of Escherichia coli genetically engineered bacteria producing nitrile hydratase and its mutants and preparation of catalysts

[0166] The genetically engineered E. coli prepared in Examples 3 and 4 was spread on an LB plate containing kanamycin (50 μg / mL) and cultured overnight at 37°C. A single colony was picked and transferred to an LB liquid culture containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. The sample was sent for sequencing, and the correct clone was stored in a -70°C refrigerator to obtain a genetically engineered bacterium with E. coli as the host.

[0167] The genetically engineered bacteria containing the coding sequence of nitrile hydratase and its mutants were inoculated into LB liquid medium containing kanamycin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent: deionized water, pH = 7.0), and placed in a test tube containing LB medium (4 mL, containing kanamycin at a final concentration of 50 μg / mL) in a shaker at 37° C. and cultured at 200 rpm for 10 to 12 hours to obtain a seed solution.

[0168] In a clean bench, transfer the seed solution in the test tube to a shake flask containing LB medium (100 mL, containing kanamycin at a final concentration of 50 μg / mL). Place the LB medium containing the seed solution in a 37°C shaker and culture at 200 rpm for 2 to 3 hours. 600 When the value reaches 0.6-0.8, add IPTG with a final concentration of 0.1-0.8mM and 0.1-0.8mM Co 2+ The expression is induced by ions at a temperature of 16-37°C. The optional IPTG concentration is 0.2 mM, the optional cobalt ion concentration is 0.2 mM, and the optional induction temperature is 16°C. After 24 hours of induction under the optional conditions, the cells are collected by centrifugation to obtain the E. coli cell catalyst of nitrile hydratase, which is then stored in a -70°C refrigerator.

[0169] 10g of frozen wet cells were weighed and added to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mM NaCl, 10mM imidazole, 375μL / L mercaptoethanol) to a final concentration of 10g / L. The cells were allowed to thaw at room temperature and filtered to remove any incompletely dissolved bulk cells. The cells were then crushed using a high-pressure homogenizer with a pressure control of 700-800bar. During the crushing process, a low-temperature circulation device was used to cool the cells, and the cells were crushed 2-3 times. The collected crushed liquid was centrifuged, and the supernatant was collected to obtain a free nitrile hydratase catalyst.

[0170] Weigh an appropriate amount of immobilized amino resin material and equilibrate it with potassium phosphate buffer (100 mM, pH 7.0). Add the treated resin support to the buffer at a 1 / 5 (w / v) ratio of support to solution. Then, add glutaraldehyde solution (50% v / v) to a final concentration of 2%. Activate the support in a shaker (16°C, 200 rpm) for 2-3 hours. Rinse the activated support with deionized water to remove any residual glutaraldehyde. Weigh an appropriate amount of activated immobilized support and place it in the buffer. Add the enzyme solution, maintaining a 1 / 5 (w / v) ratio of support to enzyme solution. Immobilize the support in a thermostatic shaker or shaker (16°C, 200 rpm) for 8 hours. Rinse the immobilized enzyme with buffer to remove any residual enzyme solution and store in a refrigerator at 4°C until ready for use.

[0171] Example 6 Construction of Rhodococcus system of nitrile hydratase mutant and preparation of catalyst

[0172] Using the recombinant expression vectors prepared in Examples 3 and 4 as templates, universal primers for nitrile hydratase were designed. The universal primer sequences contained homologous fragments of the optimized suicide plasmid pYsacB and a nitrile hydratase gene fragment. The nitrile hydratase gene sequence containing the mutation site was amplified by PCR. The optimized suicide plasmid pYsacB1 was prepared by adding about 1000bp of homology arm sequences at both ends of the nitrile hydratase gene on the basis of the suicide plasmid pYsacB. The obtained nitrile hydratase mutant gene was ligated with the enzyme-digested suicide plasmid pYsacB using the Gibson Assembly seamless cloning kit.

[0173] The 20 μL ligation system includes:

[0174] 4 μL target gene;

[0175] 6 μL suicide plasmid vector;

[0176] 10 μL ligation reagent;

[0177] Ligate at 55°C for 50 minutes, transfer the ligation solution to Top 10 competent cells, and after single clone sequencing verification, extract the recombinant suicide plasmid. The successfully constructed recombinant suicide plasmid is transformed into competent cells of Rhodococcus by electroporation. After recovery culture, it is spread on a plate medium containing antibiotics and cultured at 20-37°C. Then, a single colony is picked and colony PCR verification is performed to verify that the suicide plasmid is integrated into the correct position in the Rhodococcus genome. The colony with successful single exchange is inoculated into a seed medium without antibiotics, cultured at 20-37°C for 12 hours, diluted 100 times, and 200 μL is spread on a plate containing 100 g / L sucrose and cultured at 28°C. After a single colony grows, colony PCR is performed to verify that the nitrile hydratase gene sequence is integrated into the genome. The strain is sent to a sequencing company and stored in a -70°C refrigerator for future use.

[0178] The Rhodococcus bacteria containing the coding sequence of the nitrile hydratase mutant constructed above were inoculated into the seed culture medium and cultured until the bacterial OD 600 Reach about 30. At this time, press the initial OD 600 =3.0 inoculation volume to inoculate the seed culture medium into 50 mL of fermentation medium. For the induction expression of nitrile hydratase, a final concentration of 0.08 mM Co was added to the fermentation medium. 2+ After inducing expression for 48 h, the cells were collected by centrifugation to obtain the Rhodococcus cell catalyst for nitrile hydratase.

[0179] 10g of frozen wet cells were weighed and added to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mM NaCl, 10mM imidazole, 375μL / L mercaptoethanol) to a final concentration of 10g / L. The cells were placed at room temperature to melt and filtered to remove any incompletely dissolved bulk cells. The cells were then crushed using a high-pressure homogenizer with a pressure control of 1200-1500bar. A low-temperature circulation device was used to cool the cells during the crushing process, and the cells were crushed 2-3 times. The collected crushed liquid was centrifuged and the supernatant was collected to obtain a free nitrile hydratase catalyst.

[0180] Weigh an appropriate amount of immobilized support and equilibrate with potassium phosphate buffer (100 mM, pH 7.0). Add the treated support to the buffer at a 1 / 5 (w / v) ratio of support to solution. Then, add glutaraldehyde solution (50%) to a final concentration of 2% v / v. Activate the support in a shaker (16°C, 200 rpm) for 2-3 hours. Rinse the activated support with deionized water to remove any residual glutaraldehyde. Weigh an appropriate amount of activated immobilized support and place it in the buffer. Add the enzyme solution, maintaining a 1 / 5 (w / v) ratio. Immobilize the support in a thermostatic shaker or shaker (16°C, 200 rpm) for 8 hours. Rinse the immobilized enzyme with buffer to remove any residual enzyme solution and store in a refrigerator at 4°C until ready for use.

[0181] Example 7 Nitrile Hydratase Activity Assay Method

[0182] The activity of nitrile hydratase on the substrate acrylonitrile is determined as follows: 50-100 μL of catalyst (cells, free enzyme, immobilized enzyme) is added to a centrifuge tube, and the volume is made up to 4.5 mL with pure water. The tube is placed in a 28°C water bath for 10 minutes to stabilize the temperature. 100 μL of acrylonitrile is added, mixed, and reacted for 5 minutes. The reaction is terminated by adding 200 μL of 3 mol / L hydrochloric acid. 1 mL of the reaction solution is centrifuged at 13,000 × g for 2 minutes, and 500 μL of the supernatant is mixed with 500 μL of an internal standard solution (40 g / L acetamide) and analyzed by gas chromatography. The area ratio of acrylamide to acetamide is determined, and the concentration of the product acrylamide is determined using the internal standard method to calculate the activity.

[0183] The product production was determined by gas chromatography using the following conditions: Gas chromatography analysis was performed using a Thermo Fisher Trace 1300 gas chromatograph, an Abel Bonded AB-I NOWAX column (0.25 mm inner diameter, 30 m length, 0.25 μm film thickness), and an FID detector. Column, inlet, and detector temperatures were 19°C, 26°C, and 26°C, respectively. The carrier gas was nitrogen, operating in constant pressure mode with a partial pressure of 10 8 kPa; injection volume was 1 μL, split injection was performed, and the split ratio was 30:1.

[0184] The activity of the nitrile hydratase is calculated as follows:

[0185] K is the internal standard constant, and its value is 0.6001; C ac is the acetamide concentration of 40 g / L; Vtotal is the total volume, which is 5 mL; V is the actual volume of catalyst added; t is the reaction time; MW is acrylamide, with a molecular weight of 71; U is the total enzyme activity, the unit is μmol acrylamide / (min·mL bacterial solution).

[0186] The enzyme activity (U) is defined as: under the above reaction conditions, the amount of enzyme required to catalyze 1 μmol of substrate per minute is one enzyme activity unit, represented by U.

[0187] After testing, the substrate consumption rate of the mutant M133 is shown in FIG2 . Compared with the wild-type nitrile hydratase, the mutant M133 still has good catalytic ability and can catalyze the substrate reaction.

[0188] Example 8 Evaluation of Nitrile Hydratase Thermal Stability and Product Tolerance

[0189] The nitrile hydratase catalysts obtained in Examples 5 and 6 were resuspended in deionized water (10 g / L) and placed in a constant temperature water bath at 55° C. Samples were taken out at 2 h intervals. The residual activity was determined according to the method in Example 7, and a catalyst inactivation curve was plotted. The enzyme inactivation half-life was calculated by linear fitting. A higher half-life indicates better stability, which is used to measure the thermal stability of nitrile hydratase.

[0190] Similarly, the nitrile hydratase catalysts obtained in Examples 5 and 6 were resuspended in deionized water (10 g / L), and an appropriate amount of each catalyst was placed in a 10-mL centrifuge tube. Acrylamide was added to a final concentration of 50% (v / v). After standing still in a constant temperature water bath at 28° C. for 2 h, the residual activity of the catalyst was measured, and a catalyst inactivation curve was drawn. The enzyme inactivation half-life was calculated by linear fitting. The higher the half-life, the better the stability, which was used to measure the product tolerance of the nitrile hydratase.

[0191] After testing, the residual activity of the mutant M133 is shown in FIG3 , which still has better activity than the wild-type nitrile hydratase.

[0192] Example 9 Results of Stability and Tolerance Determination of Nitrile Hydratase and Its Mutants

[0193] The mutants constructed in Examples 3 and 4 were subjected to stability and tolerance tests according to the method of Example 8. The results showed that the thermal stability and tolerance of the obtained nitrile hydratase mutants were improved to varying degrees. The product tolerance of mutants M1-M35 increased by 5.1-6.0 times, and the thermal stability of mutants M1-M35 increased by 5.1-6.0 times; the product tolerance of mutants M36-M119, M121-M125, M127-M130, M133 and M134 increased by 6.1-8.0 times; the thermal stability of mutants M36-M119, M121-M125, M127-M130, M133 and M134 increased by 6.1-8.0 times; the product tolerance of mutants M120, M126, M131, M132, M135-M139 increased by 8.1-12.0 times.

[0194] Table 2

[0195] In Table 2, +: increased by 5.1-6.0 times, ++: increased by 6.1-8.0 times; +++: increased by 8.1-12.0 times.

[0196] Example 10 Regulatory Effects of Key Amino Acid Sites on Nitrile Hydratases from Different Sources

[0197] The present application studies the regulatory effect of the beneficial mutation sites described in Example 1 on nitrile hydratases from other sources (Table 1). First, the amino acid residues at similar positions in other nitrile hydratases were determined by multiple sequence alignment, and mutation and performance measurements were performed with reference to the mutants designed in Examples 1-9. The nitrile hydratase derived from Rhodococcus rhodochrous J1 has a UPI number of P21220 for its β subunit and P21219 for its α subunit in the UniProt Archive database. The nitrile hydratase derived from Rhodococcus rhodochrous M8 has a GenBank accession number of AAT79339.1 for its β subunit and AAT79340.1 for its α subunit.

[0198] The nitrile hydratase from Rhodococcus pyridinivorans RP has a UPI number of Q2UZQ6 for its β subunit and Q2UZQ5 for its α subunit in the UniProt Archive database.

[0199] The nitrile hydratase is derived from Rhodococcus sp., and the UPI number of its β subunit in the UniProt Archive database is Q59785, and the UPI number of its α subunit is Q59786.

[0200] The nitrile hydratase derived from Nocardia sp. JBRs has a UPI number of Q8GE66 for its β subunit and Q8GE67 for its α subunit in the UniProt Archive database.

[0201] The obtained mutant nitrile hydratases had enzymatic activity and improved thermostability and product tolerance, indicating that these mutation sites had a beneficial regulatory effect on the thermostability and product tolerance of nitrile hydratases from different sources. The results are shown in Table 3.

[0202] For example, β110R / β152D / β17D indicates the simultaneous presence of amino acid sites 110, 152, and 17 in the β subunit. Other descriptions in this way also indicate simultaneous presence.

[0203] In Table 3, ++: 6.1-8.0 times improvement; +++: 8.1-12.0 times improvement.

[0204]

[0205]

[0206]

[0207]

Claims

1. A nitrile hydratase mutant, wherein Based on the wild-type nitrile hydratase, the mutant or fragment thereof is a mutant in which the amino acid residues on the flexible loop domain of the wild-type nitrile hydratase are mutated, comprising any one or a combination of mutations 1-3: Mutation 1: a polar amino acid mutation located in the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase β subunit; Mutation 2: a polar amino acid mutation in a flexible loop between the secondary domains H6 and H7 at the start end of the β subunit of the wild-type nitrile hydratase; Mutation 3: A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase β subunit.

2. The nitrile hydratase mutant according to claim 1, wherein The wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus, and Nocardia.

3. The nitrile hydratase mutant according to claim 1 or 2, wherein The nitrile hydratase mutant satisfies at least one of the following conditions: 1) In mutation 1, the amino acid position 152 of the β subunit is mutated, and optionally, the amino acid residue at the amino acid position 152 is mutated to any one of cysteine, threonine, glutamic acid, aspartic acid, valine, serine, glycine, and glutamine; 2) In mutation 2, the amino acid position 110 of the β subunit is mutated, and optionally, the amino acid residue at the amino acid position 110 is mutated to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine; 3) In mutation 3, the amino acid position 17 of the β subunit mutated; Optionally, the amino acid residue at the amino acid position 17 is mutated to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.

4. The nitrile hydratase mutant according to any one of claims 1 to 3, wherein The nitrile hydratase mutant comprises any combination of mutation 1 and mutation 3; and / or, The nitrile hydratase mutant comprises any combination of mutation 1 and mutation 2; and / or, The nitrile hydratase mutant comprises a combination of mutation 1, mutation 2 and mutation 3.

5. The nitrile hydratase mutant according to any one of claims 1 to 4, wherein The nitrile hydratase mutant includes any combination of mutation 2 and mutation 3.

6. The nitrile hydratase mutant according to any one of claims 1 to 5, wherein The mutant or its fragment comprises at least one of the following: the amino acid residue at amino acid position 167 of the β subunit is mutated to serine, the amino acid residue at amino acid position 174 of the α subunit is mutated to leucine, and the amino acid residue at amino acid position 178 of the α subunit is mutated to glutamic acid.

7. The nitrile hydratase mutant according to any one of claims 1 to 6, wherein The mutant or its fragment comprises: a domain mutation, which includes: the amino acid residue at the amino acid position 141 of the β subunit mutates to lysine, the amino acid residue at the amino acid position 143 mutates to lysine, the amino acid residue at the amino acid position 144 mutates to glutamic acid, the amino acid residue at the amino acid position 215 mutates to cysteine, and the amino acid residue at the amino acid position 133 of the α subunit mutates to cysteine.

8. The nitrile hydratase mutant according to claims 1-7, wherein The mutant or fragment thereof includes at least one of 17A / 17R / 17E / 17D / 17S / 17C / 17K / 17H, 110A / 110R / 110S / 110T / 110V / 110F / 110L / 110I / 110K, 152C / 152A / 152T / 152E / 152D / 152V / 152S / 152Q / 152G; Alternatively, β17S / β110R / β152C, β17R / β110R / β152D, β17S / β110K / 152C, β17S / β110R / β152C, β 17S / β110R / β152C, β110K / β152T, β110K / β152D, β110R / β152T, β110K / β17E, β110K / β17 C. β110K / β17D, β152S / β17H, β152S / β17E, β110K / β152S / β17E, β17A / β110K / β152G / α17 4L / α178E, β17E / β110R / β152S / α174L / α178E, β17S / β110V / β152S / βS141K / β143K / β144 E. β17D / β110K / β152V / α174L / α178Eβ110R / β152S / β17D, β17E / β110K / β152S / β167S / α1 74L / α178E, β17E / β110K / β152S / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β1 Any one of β17R / β110S / β152Q / β167S / α174L / α178E / β215C / α133C / / βS141K / β143K / β144E and β17D / / β110F / β152C / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E.

9. An isolated nucleic acid molecule, wherein The nucleic acid molecule comprises a nucleotide sequence encoding the nitrile hydratase mutant according to any one of claims 1 to 8.

10. An expression vector, wherein: Comprising the nucleic acid molecule of claim 9; Optionally, the expression vector is a plasmid vector, which may be a pET series, a shuttle vector, a phage or a viral vector; Optionally, the expression vector is pET-28a or pNV18.

1.

11. A host cell, wherein Comprising the nucleic acid molecule of claim 9 or the expression vector of claim 10; Optionally, the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum; Optionally, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21.

12. A catalyst, wherein A nitrile hydratase mutant comprising any one of claims 1 to 8; Optionally, the catalyst is a whole cell catalyst, a free protein catalyst or an immobilized enzyme catalyst.

13. A method for preparing an amide compound, wherein: The method comprises using the nitrile hydratase mutant according to any one of claims 1 to 8 or the catalyst according to claim 12 to catalyze a hydration reaction of a nitrile compound to obtain an amide compound; Optionally, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamon nitrile, benzyl cyanide or p-hydroxyphenyl cyanide; Optionally, the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

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