Nitrile hydratase mutant and application thereof

Abstract

The invention provides a nitrile hydratase mutant and application thereof. The nitrile hydratase mutant is obtained by mutation of amino acid residues on a flexible ring structural domain of the wild type nitrile hydratase and comprises any one or a combination of mutations 1-3: mutation 1: polar amino acid mutation on a flexible ring between secondary structural domains S1 and S2 of a beta subunit of the wild type nitrile hydratase, and mutation 2: polar amino acid mutation on a flexible ring between the secondary structural domains S1 and S2 of the beta subunit of the wild type nitrile hydratase, and mutation 3: polar amino acid mutation on a flexible ring between the secondary structural domains S1 and S2 of the beta subunit of the wild type nitrile hydratase; the 152-site amino acid site of the beta subunit is mutated; mutation 2: polar amino acid mutation on a flexible ring between secondary structural domains H6 and H7 located at the starting end of the beta subunit of the wild nitrile hydratase, and mutation of the 110-site amino acid site of the beta subunit; mutation 3: a hydrophobic amino acid located on an initial flexible ring L1 of a beta subunit of the wild nitrile hydratase is mutated, and a 17-site amino acid site of the beta subunit is mutated. The nitrile hydratase mutant provided by the invention singly or plays a synergistic regulation effect, has certain enzyme activity, and has good thermal stability and tolerance.

Description

[0001] This application is a divisional application based on the invention patent application with application number 202311054507.0, application date August 21, 2023, applicants being Tsinghua University and Beijing Yanwei Technology Co., Ltd., and invention name being "Nitrile hydratase mutants and their applications". Technical Field

[0002] The invention belongs to the fields of protein engineering and genetic engineering, and relates to a nitrile hydratase mutant, a genetic engineering bacterium containing the mutant and application thereof. Background Art

[0003] Amide compounds are important organic synthesis intermediates and chemicals, and are widely used in the fields of medicine, pesticides, feed, food, environmental protection, oil production, materials, etc. The corresponding amide compounds can be obtained from nitrile compounds through hydration reaction, which is also one of the important methods for preparing such compounds in industry. Nitrile hydratase (NHase, EC 4.2.1.84) is a multi-subunit enzyme that can convert nitrile compounds into corresponding amide substances through hydration. The use of nitrile hydratase catalyst to catalyze the hydration of acrylonitrile and nicotinonitrile to produce acrylamide and nicotinamide is one of the most successful cases of biotechnology replacing chemical methods for industrial production of bulk chemicals.

[0004] Nitrile hydratase is usually composed of two subunits, α and β (or referred to as α and β subunits), and is distributed in a variety of microorganisms such as 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 research on the production of acrylamide from acrylonitrile catalyzed by nitrile hydratase has a history of several 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), Rhodococcus 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).

[0005] In the process of catalyzing the hydration of acrylonitrile, nitrile hydratase catalysts often face the problem of sudden temperature rise caused by the exothermic effect of the hydration reaction, which leads to enzyme inactivation and cell rupture, resulting in reduced economic benefits. In addition, high product concentrations have a greater toxic effect on cells, which will increase the inactivation of the catalyst. Therefore, the construction of a new generation of catalysts with high stability and high product tolerance is an important requirement for the industrial production of amide compounds. Most wild-type nitrile hydratases have problems such as poor stability, poor product tolerance, and insufficient catalytic activity. In order to further improve the production performance of nitrile hydratase, researchers have conducted in-depth research from 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.).

[0006] With the rapid development of bioinformatics technology and protein crystallography, computer simulation and protein crystal structure are increasingly used to guide protein engineering research, thus developing a variety of protein engineering methods such as semi-rational design and rational design. Although the crystal structures of some nitrile hydratase have been resolved, the catalytic mechanism of nitrile hydratase is relatively complex and there is still no conclusion, which increases the difficulty of rational design and semi-rational design. Therefore, the progress of nitrile hydratase modification is relatively slow. In order to meet industrial needs, how to quickly and efficiently obtain new industrial catalysts with improved catalytic performance has become a hot spot and difficulty in research. This is mainly because the catalytic mechanism of nitrile hydratase is relatively complex, which increases the difficulty of rational design and semi-rational design. Therefore, the progress of nitrile hydratase modification is relatively slow. Summary of the invention

[0007] Therefore, the purpose of the present 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 has certain enzyme activity alone or exerts a synergistic regulatory effect, and has better thermal stability and tolerance.

[0008] 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 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:

[0009] 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;

[0010] 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;

[0011] Mutation 3: A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase β subunit.

[0012] 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 erythronicus, and Nocardia.

[0013] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant satisfies at least one of the following conditions:

[0014] 1) In mutation 1, the amino acid position 152 of the β subunit mutated;

[0015] 2) In mutation 2, the amino acid position 110 of the β subunit mutated;

[0016] 3) In mutation 3, a mutation occurred at the amino acid position 17 of the β subunit.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 3.

[0021] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 1 and mutation 2

[0022] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes any combination of mutation 2 and mutation 3.

[0023] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant includes a combination of mutation 1, mutation 2 and mutation 3.

[0024] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant comprises at least one of an amino acid residue at position 167 of the β subunit mutated to serine, an amino acid residue at position 174 of the α subunit mutated to leucine, and an amino acid residue at position 178 of the α subunit mutated to glutamate.

[0025] 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.

[0026] 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.

[0027] 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 / α178E、β110R / β152S / β17D、β17E / β110K / β152S / β16 7S / 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.

[0028] In a second aspect, the embodiments of the present application provide an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the nitrile hydratase mutant of the first aspect.

[0029] In a third aspect, an embodiment of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0030] According to an embodiment of one aspect of the present application, the expression vector is a plasmid vector.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] According to an embodiment of one aspect of the present application, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0035] In a fourth aspect, the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.

[0036] 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.

[0037] 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.

[0038] According to an embodiment of one aspect of the present application, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamon nitrile, benzyl cyanide or p-hydroxyphenyl cyanide.

[0039] According to an embodiment of one aspect of the present application, the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

[0040] The technical solution of this application has at least the following advantages:

[0041] 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 hydratase 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 hydratase from different sources, which can further improve the thermal stability and product tolerance of nitrile hydratase, and has a certain activity.

[0042] The embodiments of the present application can provide a nitrile hydratase mutant having thermal stability, product tolerance, or catalytic activity. The nitrile hydratase mutant is very useful in the production of amide compounds.

[0043] The embodiments of the present application can further provide 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 embodiments of the present application can further provide a method for preparing an amide compound, which utilizes the nitrile hydratase extracted from the culture of the transformant and its production, and uses the aforementioned nitrile hydratase or uses the culture or a processed product of the culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the technical solution of the present application will be described in detail below with reference to the accompanying drawings:

[0045] Figure 1 A schematic diagram of the spatial positions of mutation sites 1-3 according to the examples of the present application based on the spatial conformation of the nitrile hydratase of Rhodococcus rubrum is shown.

[0046] Figure 2 The graph shows the substrate consumption effect of the wild-type nitrile hydratase and the mutant M133 nitrile hydratase of the example of the present application.

[0047] Figure 3 The residual activity effect diagram of the wild-type nitrile hydratase and the mutant M133 nitrile hydratase of the example of the present application is shown. DETAILED DESCRIPTION

[0048] 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.

[0049] 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, the equalization conversions and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or manufacturers. If the manufacturers are 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 given 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 to highlight the purport of the present application.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally 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 the present invention should be understood to include the inevitable systematic errors in industrial production.

[0051] In all discussions herein, the standard single letter codes for amino acids are used. Standard substitution notation is also used, i.e., β110A means that the amino acid residue at position 110 from the N-terminal end of the β subunit is replaced by alanine (A); or α174L means that the amino acid residue at position 174 from the N-terminal end of the α subunit is replaced by leucine (L).

[0052] In the paragraphs of this document where different amino acids at specific positions are separated by the / symbol, the " / " symbol means "and" or "or". For example, β17S / β110R / β152S / βS141K / β143K / β144E / β215C / α133C may indicate that the above mutations are all present in nitrile hydratase.

[0053] In the text paragraphs separated by the / symbol for different amino acids at specific positions, and included in brackets, the / symbol means "or", and the () symbol indicates the 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.

[0054] In the paragraphs herein separated by the / symbol at different positions, the " / " symbol means "and" such that Y51 / N55 is Y51 and N55.

[0055] The term "wild type" refers to a gene or gene product that is separated 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 term "modified", "mutant" or "variant" refers to a gene or gene product that displays 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 codon (ATG) for methionine at the relevant position in a polynucleotide encoding a mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art.

[0056] Wild-type nitrile hydratase

[0057] Nitrile hydratase (NHase) is a metal-dependent enzyme with a non-heme iron atom or cobalt atom in the active center. It can be divided into two types: Fe-type and Co-type. Currently known nitrile hydratases all have two subunits, α and β (or a and b subunits), which exist in the form of heteropolymers. The amino acid residues in the active center are relatively conservative (-C(S / T)LCSC(T / Y)-). This study selected Co-type nitrile hydratase as the research object.

[0058] Specifically, the inventors selected a variety of wild-type nitrile hydratases from Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus or Nocardia.

[0059] Nitrile hydratase mutant

[0060] 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, and obtained a series of mutant strains with improved catalytic performance, which were significantly improved compared with the parent strain in terms of stability and product tolerance; 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.

[0061] 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.

[0062] The purpose of this application is achieved through the following technical solutions:

[0063] 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 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:

[0064] 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;

[0065] 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;

[0066] Mutation 3: A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase β subunit.

[0067] According to the embodiments of the present application, the positions of the secondary domains S1 and S2, the secondary domains H6 and H7, and the initial flexible loop L1 can be as follows: Figure 1 shown.

[0068] Based on the existing wild-type nitrile hydratase, the applicant obtained a number of beneficial mutants through structural analysis and rational design, and further found that these beneficial mutations are concentrated in the specific primary structure (mutation of amino acid species) and / or secondary structure domain of the wild-type nitrile hydratase.

[0069] The distribution of these beneficial mutation sites in the spatial structure was further discovered. Based on the structure, the inventors 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, analyzed the interaction force between substrate and enzyme molecules, determined the amino acid sites that may affect the catalytic performance of the enzyme, and combined with multiple sequence alignment analysis to lock the candidate amino acid residues, and conducted directional 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.

[0070] 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, the present application investigated the regulatory effects of the obtained beneficial mutation sites on nitrile hydratases from other sources.

[0071] 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 of the above mutations or any combination thereof based on the amino acid sequence of the above wild-type nitrile hydratase.

[0072] In some optional embodiments, the wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus, and Nocardia.

[0073] 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 erythronicus or Nocardia.

[0074] Specifically, the wild-type nitrile hydratase has a sequence selected from the group consisting of:

[0075] 1) Wild-type nitrile hydratase of Rhodococcus ruber TH, the amino acid sequence of its β subunit is shown in SEQ ID NO: 1, the amino acid sequence of its α subunit is shown in SEQ ID NO: 2, and the structural gene 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.

[0076] SEQ ID NO:1

[0077] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFRESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSRK FDPAEIEKAIERLHEPHSLALPGAEPSFSLGDKVKVKNMNPLGHTRCPKYVRNKIGEIVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISA

[0078] SEQ ID NO:2

[0079] MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV;

[0080] 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.

[0081] 3) Nitrile hydratase from Rhodococcus rhodochrous M8, the GenBank accession number of its β subunit is AAT79339.1, and the α subunit is AAT79340.1, the entire contents of which are incorporated herein by reference.

[0082] 4) Nitrile hydratase from Rhodococcus pyridinivorans, whose β subunit has a UPI number of Q2UZQ6 and α subunit has a UPI number of Q2UZQ5 in the UniProt Archive database, the entire contents of which are incorporated herein by reference.

[0083] 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.

[0084] 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.

[0085] In some optional embodiments, the nitrile hydratase mutant satisfies at least one of the following conditions:

[0086] 1) In mutation 1, the amino acid position 152 of the β subunit mutated;

[0087] 2) In mutation 2, the amino acid position 110 of the β subunit mutated;

[0088] 3) In mutation 3, a mutation occurred at the amino acid position 17 of the β subunit.

[0089] According to the embodiments of the present application, the mutation of the amino acid site 152 of the β subunit can be understood as: the 152th amino acid site of the β subunit from the starting N-terminus mutates. The mutation of the amino acid site 110 of the β subunit can be understood as: the 110th amino acid site of the β subunit from the starting N-terminus mutates. The mutation of the amino acid site 17 of the β subunit can be understood as: the 17th amino acid site of the β subunit from the starting N-terminus mutates. The above mutations can exist alone or in any combination.

[0090] 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.

[0091] According to the embodiments of the present application, it can be understood that the amino acid residue at the amino acid position 152 of the β subunit mutates to any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G.

[0092] 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.

[0093] According to the embodiments of the present application, it can be understood that the amino acid residue at amino acid position 110 mutates to any one of 110A, 110R, 110S, 110T, 110V, 110F, 110L, 110I, and 110K.

[0094] 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.

[0095] According to the embodiments of the present application, it can be understood that the amino acid residue at the 17th amino acid position of the β subunit from the starting N-terminus mutates to any one of 17A, 17R, 17E, 17D, 17S, 17C, 17K, and 17H.

[0096] In some optional embodiments, the nitrile hydratase mutant comprises any combination of mutation 1 and mutation 2. For example, mutants M26-M30.

[0097] In some alternative embodiments, the nitrile hydratase mutant comprises any combination of mutation 1 and mutation 3. Exemplarily, mutants M48-M54.

[0098] In some alternative embodiments, the nitrile hydratase mutant comprises any combination of mutation 2 and mutation 3. Exemplarily, mutants M39-M47.

[0099] In some alternative embodiments, the nitrile hydratase mutant comprises a combination of mutation 1, mutation 2 and mutation 3. Exemplarily, mutants M56-M69.

[0100] According to an embodiment of one aspect of the present application, the nitrile hydratase mutant comprises at least one of an amino acid residue at position 167 of the β subunit mutated to serine, an amino acid residue at position 174 of the α subunit mutated to leucine, and an amino acid residue at position 178 of the α subunit mutated to glutamate.

[0101] It can be understood that: the 167th amino acid from the starting N-terminus of the β subunit is β167S; the 174th amino acid from the starting N-terminus of the α subunit is α174L; the 178th amino acid from the starting N-terminus of the α subunit is α178E. The above mutations can exist alone and be combined with any combination of mutations in mutations 1 to 3. The above mutations can exist in any combination and be combined with any combination of mutations in mutations 1 to 3.

[0102] On the basis of the above mutants, the nitrile hydratase mutant further comprises any one or a combination of asparagine at position 167 of the β subunit mutated to serine, glycine at position 174 of the α subunit mutated to leucine, and aspartic acid at position 178 of the α subunit mutated to glutamic acid. The above mutations of this embodiment can exist in any combination and be combined with any of the above embodiments and mutations comprising any combination of mutations 1 to mutation 3.

[0103] In some optional embodiments, 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.

[0104] It can be understood that: the 141st amino acid from the starting N-terminus of the β subunit is β141K; the 143rd amino acid from the starting N-terminus of the β subunit is β143K; the 144th amino acid from the starting N-terminus of the β subunit is β144E; the 215th amino acid from the starting N-terminus of the β subunit is β215C; the 133rd amino acid from the starting N-terminus of the α subunit is α133C. The above mutations are simultaneously present in the domain mutations. It can be combined with the mutations of other embodiments at will.

[0105] On the basis of the above mutant or wild type, salt bridge modification and disulfide bond are further introduced, and the nitrile hydratase mutant comprises: a combination of mutation of serine at position 141 of the β subunit to lysine, mutation of serine at position 143 to lysine and mutation of leucine at position 144 to glutamate, a combination of mutation of aspartic acid at position 215 to cysteine ​​and mutation of proline at position 133 of the α subunit to cysteine, and any combination of any mutant of the above embodiment and coexistence.

[0106] 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.

[0107] It can be understood that the 17th amino acid of the β subunit from the starting 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 starting 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 starting N-terminus can be any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G. The nitrile hydratase mutant of the embodiment of the present application includes any one of the above three mutations, or a random combination of any one of them.

[0108] 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, β110R / β152C, β110K / β152T, β110K / β152E 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 / α178E, β110R / 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.

[0109] It can be understood that: compared with the wild-type nitrile hydratase, or compared with the mutant nitrile hydratase in the related art, the sites of the nitrile hydratase include mutations in three of the sites, and the 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 starting N-terminus of the β subunit is serine, the 110th amino acid from the starting N-terminus of the β subunit is arginine; the 152nd amino acid from the starting N-terminus of the β subunit is cysteine.

[0110] Catalytic activity of nitrile hydratase

[0111] Nitrile hydratase activity refers to the activity of catalyzing the hydration of nitrile compounds to produce amide compounds. The substrate (nitrile compound) is reacted with nitrile hydratase under certain conditions, and the consumption of the substrate and the increase of the product are measured in a unit time to calculate the activity of nitrile hydratase. As a substrate, any nitrile compound can be used as long as the nitrile hydratase reacts, and acrylonitrile and nicotinonitrile can be selected. The reaction conditions are the general conditions of the hydration reaction, as long as the nitrile hydratase can be guaranteed to have catalytic activity, and the consumption of the substrate and the increase of the product can be detected and quantitatively analyzed by HPLC and GC.

[0112] The general formula of nitrile compounds is shown in (1):

[0113] R-CN(1)

[0114] 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.

[0115] The general formula of amide compounds is shown in (2):

[0116] R-CONH2(2)

[0117] 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.

[0118] According to research, the wild-type nitrile hydratase and mutant nitrile hydratase described in the present application have catalytic activity.

[0119] 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.

[0120] 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.

[0121] Recombinant vector and transformant of nitrile hydratase mutant

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

[0123] According to the embodiments of the present application, the selected expression vector can stably exist in various hosts of prokaryotic or eukaryotic cells and can replicate autonomously, such as conventional plasmids (pET series), shuttle vectors pNV18.1, phage or viral vectors, etc. in the art.

[0124] In some alternative embodiments, the expression vector is a plasmid vector.

[0125] 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.

[0126] According to the embodiment 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 by molecular biological operations 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 embodiment of the present application, the coding gene of the nitrile hydratase mutant of the embodiment of the present application can be constructed into a recombinant expression plasmid.

[0127] 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.

[0128] 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 by the calcium chloride method or electroporation transformation method.

[0129] In some optional embodiments, the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

[0130] In some optional embodiments, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0131] In a fourth aspect, the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.

[0132] Depending on the host cell, the nucleotide sequence encoding the nitrile hydratase mutant can be constructed into different types of recombinant vectors, or directly integrated into the chromosome of the host bacteria. As the vector used, plasmid DNA, bacteriophage DNA, reverse transcription transposon DNA, artificial chromosome DNA, etc. can be listed. Taking Escherichia coli and Rhodococcus rubrum as examples, PET series vectors and PNV series vectors can be selected.

[0133] 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.

[0134] 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 a method using calcium ions and an electroporation method.

[0135] 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.

[0136] Catalyst and method for preparing amide compound

[0137] In some alternative embodiments, the catalyst is a whole cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst.

[0138] 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 ultrasonic crushing or high-pressure homogenization crushing and centrifugation of the whole cell, and also includes pure enzymes obtained by protein purification. The immobilized enzyme catalyst is to select different immobilized carriers and immobilize the free protein catalyst to obtain different forms of immobilized nitrile hydratase mutants.

[0139] 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;

[0140] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamon nitrile, benzyl cyanide or p-hydroxyphenyl cyanide.

[0141] In some optional embodiments, the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

[0142] The following examples describe the disclosure of the present application in more detail, and these examples are intended for illustrative purposes only, as various modifications and variations within the scope of the disclosure of the present application 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 can be used directly without further processing, and the instruments used in the examples are commercially available.

[0143] Example

[0144] Example 1 Sequences of nitrile hydratase mutants

[0145] 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 shown in Table 1:

[0146] Table 1

[0147]

[0148]

[0149]

[0150] Example 2 Construction of recombinant expression vector of nitrile hydratase

[0151] In Escherichia coli cells, plasmid pET-28a was used as an expression vector. The inventors designed Nco I and Xho I restriction sites at both ends of the nucleotide sequence encoding nitrile hydratase, and used restriction endonucleases Nco I and Xho I to perform double digestion on the plasmid vector and the nucleotide sequence of the nitrile hydratase in this embodiment. The gene fragments after digestion were recovered by nucleic acid electrophoresis (1.0% agarose) and a kit, and then the target gene fragments after digestion were connected to the plasmid vector after digestion.

[0152] 10μL ligation system includes:

[0153] 1 μL 10×T4 DNA ligase Buffer (Takara);

[0154] 2.5 μL target gene fragment;

[0155] 2.5 μL plasmid fragment;

[0156] 1 μL T4 DNA ligase;

[0157] 3 μL ddH2O;

[0158] The cells were connected overnight at 16°C and transformed into BL21 (DE3) competent cells. Single clones were selected for sequencing verification, and the recombinant plasmid with correct sequencing was extracted to obtain a recombinant expression vector containing the nitrile hydratase encoding gene.

[0159] Example 3 Construction of nitrile hydratase mutants

[0160] The present application adopts the whole plasmid amplification method to construct a recombinant plasmid containing a mutant gene of nitrile hydratase. The upstream and downstream primers are designed for the desired mutant site, and the whole plasmid amplification is performed using PrimeSTAR HS DNA Polymerase (Takara Company) with plasmid pET28a-NH as a template. The recombinant plasmid containing a mutant sequence encoding nitrile hydratase is amplified by PCR.

[0161] The 20 μL PCR reaction system includes:

[0162] 1 μL pET28a-NH plasmid template (about 220 ng / μL);

[0163] 10 μL 2× PrimeSTAR HS DNA polymerase;

[0164] 1.0 μL forward primer (10 μM);

[0165] 1.0 μL reverse primer (10 μM);

[0166] 7 μL ddH2O.

[0167] 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. Due to space limitations, it is not described here one by one, but those skilled in the art should know that designing primers based on known sequences and obtaining target products are conventional technical means of those skilled in the art.

[0168] After the PCR stock solution was digested with Dpn I enzyme to remove the template sequence, it was transformed into BL21 (DE3) competent cells using the heat shock method, and spread on LB plates containing kanamycin (50 μg / mL) and placed in an incubator at 37°C for inversion culture for about 12 hours. Single clones were selected for sequencing verification. After the sequencing was correct, 20% (V / V) glycerol was used to preserve the bacteria and stored in a refrigerator at -70°C.

[0169] Example 4 Construction of nitrile hydratase combination mutants

[0170] The nitrile hydratase combination mutants are obtained by multiple rounds of site-directed mutagenesis. After obtaining a single point mutant, a recombinant plasmid containing the mutation site is used as a template, upstream and downstream primers of the mutation site are designed, and whole-plasmid PCR amplification is performed. A recombinant plasmid containing a gene sequence encoding the nitrile hydratase combination mutants is amplified by PCR.

[0171] The forward primer can use specific primers constructed for different mutants, which are not described here due to space limitations, but 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.

[0172] After the PCR stock solution was digested with Dpn I enzyme to remove the template sequence, it was transformed into BL21 (DE3) competent cells using the heat shock method, and spread on LB plates containing kanamycin (50 μg / mL) and placed in an incubator at 37°C for about 12 hours. Single clones were picked for sequencing verification.

[0173] Example 5 Construction of Escherichia coli genetically engineered bacteria for nitrile hydratase and its mutants and preparation of catalysts

[0174] The genetically engineered E. coli prepared in Examples 3 and 4 were coated on LB plates containing kanamycin (50 μg / mL) and cultured overnight at 37°C. Single colonies were picked and transferred to LB liquid culture containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. The samples were sent for sequencing, and the correct clones were stored in a -70°C refrigerator to obtain genetically engineered bacteria with E. coli as the host.

[0175] The genetically engineered bacteria containing the coding sequence of nitrile hydratase and its mutants are inoculated into LB liquid medium containing kanamycin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent is deionized water, pH=7.0), placed in a test tube containing LB medium (4 mL, containing kanamycin at a final concentration of 50 μg / mL) in a 37° C. shaker, and cultured at a speed of 200 rpm for 10 to 12 hours to obtain a seed solution.

[0176] 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, and the induction temperature is 16-37°C. The optional IPTG concentration is 0.2mM, the optional cobalt ion concentration is 0.2mM, and the optional induction temperature is 16°C. After induction for 24 hours under the optional conditions, the cells are collected by centrifugation to obtain the Escherichia coli cell catalyst of nitrile hydratase, which is stored in a -70°C refrigerator.

[0177] Weigh 10g of frozen wet cells, add them to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mMNaCl, 10mM imidazole, 375μL / L mercaptoethanol), the final concentration is 10g / L, place them at room temperature to melt, filter and remove the blocky cells that are not completely dissolved. Then use a high-pressure homogenizer to crush them, the pressure is controlled at 700-800bar, and a low-temperature circulation device is used to cool down during the crushing process, and the crushing cycle is repeated 2-3 times. The collected crushing liquid is centrifuged, the supernatant is collected, and a free nitrile hydratase catalyst is obtained.

[0178] Weigh an appropriate amount of immobilized amino resin material, balance it with potassium phosphate buffer (100mM, pH 7.0), add the treated tree carrier to the buffer, the ratio of carrier to solution is 1 / 5 (w / v), then add glutaraldehyde solution (50% V / V) to a final concentration of 2%. After activation in an oscillator (16°C, 200rpm) for 2-3h, wash the activated carrier with deionized water to remove residual glutaraldehyde. Weigh an appropriate amount of activated immobilized carrier and place it in the buffer, add enzyme solution, the ratio of carrier to enzyme solution is still 1 / 5 (w / v). Place it in a constant temperature shaker or oscillator (16°C, 200rpm) for immobilization for 8h. Then wash the immobilized enzyme with buffer to remove residual enzyme solution, and then store it in a 4°C refrigerator for later use.

[0179] Example 6 Construction of the Rhodococcus system of nitrile hydratase mutants and preparation of catalysts

[0180] The recombinant expression vectors prepared in Examples 3 and 4 were used as templates to design universal primers for nitrile hydratase. The universal primer sequence contained the homologous fragments of the optimized 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 was based on the suicide plasmid pYsacB and added with homologous arm sequences of about 1000 bp at both ends of the upstream and downstream of the nitrile hydratase gene. The obtained nitrile hydratase mutant gene was connected to the suicide plasmid pYsacB after enzyme digestion by the Gibson Assembly seamless cloning kit.

[0181] The 20 μL ligation system includes:

[0182] 4 μL target gene;

[0183] 6 μL suicide plasmid vector;

[0184] 10 μL ligation reagent;

[0185] Connect at 55°C for 50 minutes, transfer the connection solution to Top 10 competent cells, pick up single clone sequencing verification, extract the recombinant suicide plasmid, use the electroporation method to transform the successfully constructed recombinant suicide plasmid into the competent cells of Rhodococcus, spread it on the plate culture medium containing antibiotics after recovery culture, and culture it at 20-37°C; then pick a single colony, and verify it by colony PCR 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 the seed culture medium without antibiotics, cultured at 20-37°C for 12h, diluted 100 times, and 200μL is spread on the plate containing 100g / L sucrose, cultured at 28°C, and after a single colony grows, colony PCR is performed to verify that the nitrile hydratase gene sequence is integrated into the genome, and sent to the sequencing company, and the strain with correct sequencing is stored in a -70°C refrigerator for standby use.

[0186] 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, the seed culture medium was inoculated 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 hours, the cells were collected by centrifugation to obtain the nitrile hydratase Rhodococcus cell catalyst.

[0187] Weigh 10g of frozen wet cells, add them to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mMNaCl, 10mM imidazole, 375μL / L mercaptoethanol), the final concentration is 10g / L, place them at room temperature to melt, filter and remove the blocky cells that are not completely dissolved. Then use a high-pressure homogenizer to crush them, the pressure is controlled at 1200-1500bar, and a low-temperature circulation device is used to cool down during the crushing process, and the crushing cycle is repeated 2-3 times. The collected crushing liquid is centrifuged, the supernatant is collected, and a free nitrile hydratase catalyst is obtained.

[0188] Weigh an appropriate amount of immobilized carrier, balance it with potassium phosphate buffer (100mM, pH 7.0), add the treated tree carrier to the buffer, the ratio of carrier to solution is 1 / 5 (w / v), then add glutaraldehyde solution (50%) to a final concentration of 2% V / V. After activation in an oscillator (16°C, 200rpm) for 2-3h, wash the activated carrier with deionized water to remove residual glutaraldehyde. Weigh an appropriate amount of activated immobilized carrier and place it in the buffer, add enzyme solution, the ratio of carrier to enzyme solution is still 1 / 5 (w / v). Place it in a constant temperature shaker or oscillator (16°C, 200rpm) for immobilization for 8h. Then wash the immobilized enzyme with buffer to remove residual enzyme solution, and then store it in a 4°C refrigerator for later use.

[0189] Example 7 Nitrile Hydrate Activity Determination Method

[0190] The method for determining the activity of nitrile hydratase on the substrate acrylonitrile is as follows: Take 50-100 μL of catalyst (cells, free enzyme, immobilized enzyme) and add it to a centrifuge tube, add pure water to make up to 4.5 mL, and place it in a 28°C water bath for 10 minutes to stabilize the temperature. Add 100 μL of acrylonitrile, mix well and react for 5 minutes, and add 200 μL of 3 mol / L hydrochloric acid to terminate the reaction. Take 1 mL of the reaction solution and centrifuge it at 13000×g for 2 minutes, take 500 μL of the supernatant and mix it with 500 μL of the internal standard solution (40 g / L acetamide), and perform gas chromatography analysis. Determine the area ratio of acrylamide to acetamide, determine the concentration of the product acrylamide by the internal standard method, and calculate the activity.

[0191] The amount of product generated was detected by gas chromatography, and the detection conditions were as follows: Thermo Fisher Trace 1300 gas chromatograph; Abel Bonded AB-I NOWAX column (inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm); FID detector. Column temperature, injection port temperature, and detector temperature were 19°C, 26°C, and 26°C, respectively; the carrier gas was nitrogen, constant pressure mode, partial pressure 10 8 kPa; injection volume was 1 μL, split injection, split ratio was 30:1.

[0192] The activity of the nitrile hydratase is calculated as follows:

[0193]

[0194] K is the internal standard constant, and its value is 0.6001; Cac is the acetamide concentration of 40 g / L; Vtotal is the total volume, and its value is 5 mL; V is the actual volume of the catalyst added; t is the reaction time; MW is acrylamide, and its molecular weight is 71; U is the total enzyme activity, and the unit is μmol acrylamide / (min·mL bacterial solution).

[0195] 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.

[0196] After testing, the substrate consumption rate of mutant M133 was as follows Figure 2 As shown, compared with the wild-type nitrile hydratase, it still has good catalytic ability and can catalyze the reaction of the substrate.

[0197] Example 8 Evaluation of the thermal stability and product tolerance of nitrile hydratase

[0198] The nitrile hydratase catalysts obtained in Examples 5 and 6 were resuspended in deionized water (10 g / L), placed in a constant temperature water bath at 55° C., and samples were taken out at intervals of 2 h. The residual activity was determined according to the method in Example 7, and the inactivation curve of the catalyst was plotted. The enzyme inactivation half-life was obtained by linear fitting. The higher the half-life, the better the stability, which was used to measure the thermal stability of nitrile hydratase.

[0199] 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, and 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 the inactivation curve of the catalyst was drawn. The enzyme inactivation half-life was linearly fitted. The higher the half-life, the better the stability, which was used to measure the product tolerance of nitrile hydratase.

[0200] The residual activity of mutant M133 was tested. Figure 3 As shown, compared with the wild-type nitrile hydratase, it still has better activity.

[0201] Example 9 Results of stability and tolerance determination of nitrile hydratase and its mutants

[0202] 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 nitrile hydratase mutants obtained 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.

[0203] Table 2

[0204]

[0205]

[0206] In Table 2, +: 5.1-6.0 times increase, ++: 6.1-8.0 times increase; +++: 8.1-12.0 times increase.

[0207] Example 10 Regulatory effects of key amino acid sites on nitrile hydratases from different sources

[0208] 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 located at similar positions in other nitrile hydratases were determined by multiple sequence alignment, and the mutations 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 in the UniProt Archive database, and P21219 for its α subunit. 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.

[0209] 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.

[0210] The nitrile hydratase derived from Rhodococcus sp. has a UPI number of Q59785 for its β subunit and Q59786 for its α subunit in the UniProtArchive database.

[0211] 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 UniProtArchive database.

[0212] The obtained mutant nitrile hydratase has enzyme activity, and the thermal stability and product tolerance are improved, indicating that these mutation sites have a beneficial regulatory effect on the thermal stability and product tolerance of nitrile hydratase from different sources. The results are shown in Table 3.

[0213] For example, β110R / β152D / β17D indicates the simultaneous existence of amino acid sites 110, 152, and 17 in the β subunit. Other descriptions in this way also indicate the simultaneous existence.

[0214] In Table 3, ++: 6.1-8.0 times higher; +++: 8.1-12.0 times higher

[0215]

[0216]

[0217]

[0218] .

Claims

1. A nitrile hydratase mutant, wherein Based on the wild-type nitrile hydratase, the nitrile hydratase mutant is a nitrile hydratase in which the amino acid residues on the flexible loop domain of the wild-type nitrile hydratase are mutated, comprising mutation 2, Mutation 2 includes: a polar amino acid mutation on a flexible loop between the secondary domains H6 and H7 at the starting end of the wild-type nitrile hydratase β subunit, and a mutation at the amino acid site 110 of the β subunit; the amino acid residue at the amino acid site 110 mutates to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine.

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, wherein The nitrile hydratase mutant comprises at least one of mutation 1 and mutation 3: 1) Mutation 1: a polar amino acid mutation on the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase β subunit; a mutation occurs at the amino acid position 152 of the β subunit; 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) Mutation 3: a hydrophobic amino acid mutation located on the starting flexible loop L1 of the wild-type nitrile hydratase β subunit; a mutation occurs at the amino acid site 17 of the β subunit; optionally, the amino acid residue at the amino acid site 17 mutates to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.

4. The nitrile hydratase mutant according to claim 3, wherein The nitrile hydratase mutant satisfies at least one of the following conditions: The nitrile hydratase mutant includes any combination of mutation 1 and mutation 2; The nitrile hydratase mutant includes any combination of mutation 2 and mutation 3; The nitrile hydratase mutant comprises a combination of mutation 1, mutation 2 and mutation 3.

5. The nitrile hydratase mutant according to claim 3 or 4, wherein The nitrile hydratase mutant comprises at least one of the following: the amino acid residue at the amino acid position 167 of the β subunit is mutated to serine, the amino acid residue at the amino acid position 174 of the α subunit is mutated to leucine, and the amino acid residue at the amino acid position 178 of the α subunit is mutated to glutamic acid.

6. The nitrile hydratase mutant according to claim 3 or 4, wherein The nitrile hydratase mutant comprises: a structural domain mutation, which includes: the amino acid residue at the 141st amino acid position of the β subunit mutates to lysine, the amino acid residue at the 143rd amino acid position mutates to lysine, the amino acid residue at the 144th amino acid position mutates to glutamic acid, the amino acid residue at the 215th amino acid position mutates to cysteine, and the amino acid residue at the 133rd amino acid position of the α subunit mutates to cysteine.

7. The nitrile hydratase mutant according to claim 5, wherein The nitrile hydratase mutant comprises: a structural domain mutation, which includes: the amino acid residue at the 141st amino acid position of the β subunit mutates to lysine, the amino acid residue at the 143rd amino acid position mutates to lysine, the amino acid residue at the 144th amino acid position mutates to glutamic acid, the amino acid residue at the 215th amino acid position mutates to cysteine, and the amino acid residue at the 133rd amino acid position of the α subunit mutates to cysteine.

8. The nitrile hydratase mutant according to claim 1, wherein The nitrile hydratase mutants include β17S / β110R / β152C, β17R / β110R / β152D, β17S / β110K / 152C, β17S / β110R / β152C, β17S / β110R / β152C, β110K / β152T, β110K / β152D, β110R / β152T , β110K / β17E, β110K / β17C, β110K / β17D, β152S / β17H, β152S / β17E, β110K / β15 2S / β17E, β17A / β110K / β152G / α174L / α178E, β17E / β110R / β152S / α174L / α178E, β17S / β110V / β152S / βS141K / β143K / β144E, β17D / β110K / β152V / α174L / α178E, β110R / β152S / β17D, β17E / β110K / β152S / β167S / α174L / α178E, β17E / β110K / β152S / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E, β17R / β110S / β152Q / β167S / α174L / α178E / β2 Any one of 15C / α133C / / βS141K / β143K / β144E, 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.

11. The expression vector according to claim 10, wherein The expression vector is a plasmid vector, and the plasmid vector includes any one of the pET series, shuttle vectors, phage and virus vectors.

12. A host cell, wherein Comprising the nucleic acid molecule of claim 9 or the expression vector of claim 10 or 11.

13. The host cell according to claim 12, wherein The host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

14. A catalyst, wherein The invention comprises the nitrile hydratase mutant according to any one of claims 1 to 8.

15. 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 14 to catalyze a hydration reaction of a nitrile compound to obtain an amide compound.

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