Engineered nitrile hydratase and application thereof

Abstract

The invention provides an engineered nitrile hydratase and application thereof. Based on wild type nitrile hydratase, the engineered nitrile hydratase is characterized in that an amino acid residue located at the 85 site of an alpha subunit is mutated into any one of methionine, arginine and lysine, the wild type nitrile hydratase comprises the alpha subunit, the amino acid sequence of the alpha subunit is as shown in SEQ ID NO: 1, and the amino acid sequence of the alpha subunit is as shown in SEQ ID NO: 2. The engineered nitrile hydratase provided by the embodiment of the invention has good catalytic activity and product tolerance.

Description

Technical Field

[0001] The invention belongs to the fields of protein engineering and gene engineering, and relates to an engineered nitrile hydratase and application thereof. Background Art

[0002] Nitrile hydratase (NHase, EC 4.2.1.84) is a metal-dependent enzyme that catalyzes the conversion of nitrile substrates into amide compounds. Nitrile hydratase can be used to catalyze the production of bulk chemicals such as acrylamide and nicotinamide, and has been widely used in industrial production. Nitrile hydratase is usually composed of two subunits, α and β, and is distributed in a variety of microorganisms. Its sources are wide, and the production strains used to catalyze acrylonitrile to produce acrylamide have also gone through many generations.

[0003] During the catalytic hydration of nitrile groups, the catalyst will cause a sudden rise in temperature due to the exothermic effect of the hydration reaction, which will affect the stability of the catalyst. In addition, the concentration of the catalytic product can reach up to 50% during the production process. The high product concentration has a greater toxic effect on the catalyst, which will accelerate the deactivation of the catalyst and ultimately lead to a significant increase in the amount of catalyst used. Therefore, it is an urgent need for industrial production to construct a new generation of catalysts with high stability and high product tolerance and reduce the amount of catalyst used.

[0004] Currently, most wild-type nitrile hydratases have bottlenecks such as stability, poor product tolerance, and insufficient catalytic activity. Therefore, it is necessary to further improve the performance of nitrile hydratase catalysts. Summary of the invention

[0005] The present application provides an engineered nitrile hydratase, which can include the above mutation on the basis of wild-type nitrile hydratase, and further improve the product tolerance and catalytic activity of the engineered nitrile hydratase while ensuring the enzyme activity.

[0006] In a first aspect, an embodiment of the present application provides an engineered nitrile hydratase, based on a wild-type nitrile hydratase, wherein the engineered nitrile hydratase comprises: an amino acid residue at position 85 of the α subunit is mutated to any one of methionine, arginine, and lysine, wherein the wild-type nitrile hydratase comprises an α subunit, and the amino acid sequence of the α subunit is shown in SEQ ID NO: 1.

[0007] In some optional embodiments, the wild-type nitrile hydratase is derived from any one of Rhodococcusrhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, Rhodococcus ruber, Rhodococcus sp., and Nocardia sp. JBRs, and can be selected from Rhodococcus ruber.

[0008] In some optional embodiments, the wild-type nitrile hydratase includes a β subunit, and the amino acid sequence of the β subunit is as follows

[0009] As shown in SEQ ID NO:2.

[0010] In some optional embodiments, the engineered nitrile hydratase further comprises one or more of the following mutants:

[0011] 1) Mutation 1 includes: the amino acid residue at the amino acid position 17 of the β subunit mutates to glutamic acid; the amino acid residue at the amino acid position 110 of the β subunit mutates to lysine; the amino acid residue at the amino acid position 152 of the β subunit mutates to serine; the amino acid residue at the amino acid position 57 of the β subunit mutates to lysine or methionine.

[0012] 2) Mutation 2 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 of the β subunit mutates to lysine; the amino acid residue at the amino acid position 144 of the β subunit mutates to glutamic acid; the amino acid residue at the amino acid position 215 of the β subunit mutates to cysteine; the amino acid residue at the amino acid position 133 of the α subunit mutates to cysteine; the amino acid residue at the amino acid position 167 of the β subunit mutates to any one of arginine, lysine or serine; the amino acid residue at the amino acid position 174 of the α subunit mutates to leucine; the amino acid residue at the amino acid position 178 of the α subunit mutates to glutamic acid.

[0013] 3) Mutation 3 includes: any one or more of the amino acid site at position 45 of the β subunit mutates to asparagine, the amino acid site at position 47 of the β subunit mutates to arginine or asparagine, and the amino acid site at position 48 of the β subunit mutates to asparagine.

[0014] In some alternative embodiments, the engineered nitrile hydratase comprises any combination of mutation 1 and mutation 2.

[0015] In some optional embodiments, the engineered nitrile hydratase comprises any combination of mutation 2 and mutation 3;

[0016] In some alternative embodiments, the engineered nitrile hydratase comprises a combination of mutation 1, mutation 2, and mutation 3.

[0017] In some optional embodiments, the engineered nitrile hydratase comprises any one of the following mutations:

[0018] α85K, α85R, α85M,

[0019] β17E / β110K / β152S / β57R / α85M, β17E / β110K / β152S / β57R / α85R,

[0020] β17E / β110K / β152S / β57R / α85K, β17E / β110K / β152S / β57M / α85M,

[0021] β17E / β110K / β152S / β57M / α85R, β17E / β110K / β152S / β57M / α85K,

[0022] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K,

[0023] β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K,

[0024] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R,

[0025] β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R,

[0026] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85M,

[0027] β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85M,

[0028] β45N / α85K, β47R / α85K, β48N / α85K, β45N / α85R, β47R / α85R,

[0029] β48N / α85R、β45N / β47R / α85R、β47R / β48N / α85R、β47N / β48N / α85R、

[0030] β47R / β48N / α85K, β47N / β48N / α85K, β45N / β47R / α85K,

[0031] β45N / β47R / β48N / α85R、

[0032] β45N / β47N / β48N / α85K、

[0033] β45N / β47N / β48N / α85R、

[0034] β45N / β47R / β48N / α85K、

[0035] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K,

[0036] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K,

[0037] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / α85K,

[0038] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K,

[0039] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K,

[0040] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R、

[0041] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R、

[0042] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / α85R,

[0043] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R,

[0044] β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R、

[0045] β17E / β110K / β152S / β57M / β47R / α85K、

[0046] β17E / β110K / β152S / β57M / β48N / α85K、

[0047] β17E / β110K / β152S / β57M / β47R / α85R、

[0048] β17E / β110K / β152S / β57M / β48N / α85R、

[0049] β17E / β110K / β152S / β57M / β47N / β48N / α85R、

[0050] β17E / β110K / β152S / β57M / β47R / β48N / α85K、

[0051] β17E / β110K / β152S / β57M / β47N / β48N / α85K、

[0052] β17E / β110K / β152S / β57M / β45N / β47R / β48N / α85R、

[0053] β17E / β110K / β152S / β57M / β45N / β47N / β48N / α85K、

[0054] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K,

[0055] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R,

[0056] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K,

[0057] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R,

[0058] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K,

[0059] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R、

[0060] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K、

[0061] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K,

[0062] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K,

[0063] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K,

[0064] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R、

[0065] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R,

[0066] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R、

[0067] β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R、

[0068] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K、

[0069] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K,

[0070] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K,

[0071] β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K,

[0072] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R、

[0073] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R,

[0074] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R,

[0075] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R,

[0076] β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85R.

[0077] In a second aspect, the embodiments of the present application provide an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the engineered nitrile hydratase of the first aspect.

[0078] In a third aspect, an embodiment of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0079] In some optional embodiments, the expression vector is a plasmid vector.

[0080] In some optional embodiments, the expression vector is a pET series, a shuttle vector, a phage or a viral vector.

[0081] 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.

[0082] In some optional embodiments, the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

[0083] In some optional embodiments, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0084] In a fourth aspect, the present application provides a catalyst comprising the engineered nitrile hydratase of the first aspect.

[0085] In some optional embodiments, the catalyst is a whole cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst.

[0086] 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.

[0087] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinonitrile, 2,6-difluorobenzonitrile, p-hydroxybenzonitrile or p-hydroxyphenylacetonitrile.

[0088] In some optional embodiments, the amide substance is selected from acrylamide, nicotinamide, 2,6-difluorobenzamide, p-hydroxybenzamide or p-hydroxyphenylacetamide.

[0089] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamonitrile, benzyl cyanide or p-hydroxyphenyl cyanide.

[0090] In some optional embodiments, the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.

[0091] The technical solution of this application has at least the following advantages:

[0092] The engineered nitrile hydratase of the embodiment of the present application, the amino acid residue at position 85 of the α subunit is mutated to any one of methionine, arginine, and lysine, which significantly improves the stability and product tolerance of the nitrile hydratase during the catalytic process. The engineered nitrile hydratase provided in the embodiment of the present application has higher catalytic efficiency in the production of amide compounds, which can significantly reduce the use cost of the catalyst.

[0093] 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, each of which has the beneficial effects of engineered nitrile hydratase. The embodiments of the present application can further provide a method for preparing amide compounds, which utilizes the nitrile hydratase extracted from the culture of the transformant and its preparation, as well as the use of the aforementioned nitrile hydratase or the use of the culture or the processed product of the culture, which also has the beneficial effects of engineered nitrile hydratase. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] 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:

[0095] Figure 1A schematic diagram of the spatial location of the sites where amino acids in the flexible ring domain surrounding the active pocket according to the embodiments of the present application are mutated is shown based on the spatial conformation of the nitrile hydratase of Rhodococcus rubrum.

[0096] Figure 2 The catalytic activity diagram of the wild-type nitrile hydratase and the engineered nitrile hydratase of the examples of the present application is shown.

[0097] Figure 3 The product tolerance graph of the wild-type nitrile hydratase and the engineered nitrile hydratase of the examples of the present application is shown. DETAILED DESCRIPTION

[0098] 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.

[0099] For those skilled in the art, any equivalent modifications and substitutions to the following embodiments 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.

[0100] 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.

[0101] The term "multiple" in this application refers to more than two (including two). The term "multiple" or "several" in this application refers to more than two (including two). Accordingly, the term "multiple" is also interpreted in the same manner as above.

[0102] In all discussions herein, standard single-letter codes for amino acids are used. Standard substitution notation is also used, i.e., α85K means that the amino acid residue at position 85 from the N-terminal end of the α subunit is replaced by lysine (K); or β17E means that the amino acid residue at position 17 from the N-terminal end of the β subunit is replaced by glutamic acid (E). The same applies to the others.

[0103] In the paragraphs of this text where different amino acids at specific positions are separated by the / symbol, the " / " symbol means "and" or "or". For example, β17E / β110K / β152S / β57R / α85M can represent the situation where the above mutations at amino acid positions 17, 110, 152, 57 in the β subunit, and amino acid position 85 in the α subunit coexist in the engineered nitrile hydratase, and other descriptions in this way also represent the situation where they coexist.

[0104] In the text paragraphs separated by / symbol for different amino acids at specific positions, and included in brackets, the / symbol means "or", and the () symbol indicates the amino acid at the same position. In the text paragraphs separated by / symbol at different positions, the " / " symbol means "and" so that Y51 / N55 is Y51 and N55.

[0105] The term "wild-type" refers to a gene or gene product that is 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 the gene. These nitrile hydratases are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art.

[0106] Wild-type nitrile hydratase

[0107] Nitrile hydratase (NHase) is a metal-dependent enzyme with a non-heme iron or cobalt atom in the active center, which 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, and the amino acid residues in the active center are relatively conservative (-C(S / T)LCSC(T / Y)-). In this study, the wild-type Co-type nitrile hydratase was selected as the research object.

[0108] Specifically, the inventors selected the wild-type nitrile hydratase from Rhodococcus ruber.

[0109] As an example, the amino acid sequence of the α subunit of the nitrile hydratase of Rhodococcus ruber TH is shown in SEQ ID NO: 1, and the amino acid sequence of the β subunit is shown in SEQ ID NO: 2; the engineered nitrile hydratase of the present application comprises any one or any combination of the above mutations based on the amino acid sequence of the wild-type nitrile hydratase.

[0110] Specifically, the wild-type nitrile hydratase has a sequence selected from the group consisting of:

[0111] 1) Wild-type nitrile hydratase of Rhodococcus ruber TH, the amino acid sequence of its α subunit is shown in SEQ ID NO: 1, the amino acid sequence of its β 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. SEQ ID NO: 1:

[0112] MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKS

[0113] WVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWP

[0114] VLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0115] SEQ ID NO:2:

[0116] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFR

[0117] ESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSR

[0118] KFDPAEIEKAIERLHEPHSLALPGAEPSFSLGDKVKVKNMNPLGHTRCPKYVRNKIGE

[0119] IVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISA.

[0120] In addition, any one of Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, Rhodococcus sp., and Nocardia sp. JBRs may be selected, and Rhodococcus ruber may be selected.

[0121] Engineered nitrile hydratase

[0122] 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 flexible ring domain around the substrate binding pocket through structural analysis, molecular dynamics simulation and enzyme molecular sequence alignment, discovered the key sites for regulating catalytic activity and tolerance through directed design, constructed an engineered nitrile hydratase library, and obtained a series of mutants with improved catalytic performance. Compared with the wild-type nitrile hydratase, the catalytic activity and product tolerance of the engineered nitrile hydratase are significantly improved.

[0123] The purpose of this application is achieved through the following technical solutions:

[0124] In a first aspect, an embodiment of the present application provides an engineered nitrile hydratase, based on a wild-type nitrile hydratase, wherein the engineered nitrile hydratase comprises: an amino acid residue at position 85 of the α subunit is mutated to any one of methionine, arginine, and lysine, wherein the wild-type nitrile hydratase comprises an α subunit, and the amino acid sequence of the α subunit is shown in SEQ ID NO: 1.

[0125] In all discussions in this article, the mutation of the amino acid residue at position 85 of the α subunit to any one of methionine, arginine, and lysine can be understood as the 85th amino acid residue from the N-terminus of the α subunit being replaced by any one of methionine, arginine, and lysine.

[0126] According to the embodiments of the present application, the amino acid residue at position 85 of the α subunit is located in the flexible ring domain around the active pocket of the wild-type nitrile hydratase. Studies have shown that the amino acid coating at this position can improve the product tolerance and catalytic activity of the engineered nitrile hydratase.

[0127] like Figure 1 As shown, the figure shows the spatial structure positions of the flexible loop domain and the 85 position in the engineered nitrile hydratase.

[0128] 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.

[0129] Catalytic activity of nitrile hydratase

[0130] 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 the nitrile compound can be selected as acrylonitrile and nicotinonitrile. The reaction conditions are the general conditions of the hydration reaction, as long as the nitrile hydratase can be guaranteed to have catalytic activity, the consumption of the substrate and the increase of the product can be detected and quantitatively analyzed by high performance liquid chromatography (HPLC) and gas chromatography (GC).

[0131] The general formula of nitrile compounds is shown in (1):

[0132] R-CN(1)

[0133] Furthermore, 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.

[0134] The general formula of amide compounds is shown in (2):

[0135] R-CONH2(2)

[0136] Furthermore, 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.

[0137] According to research, the wild-type nitrile hydratase and mutant nitrile hydratase described in the present application have catalytic activity.

[0138] Nucleic acid molecules

[0139] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the engineered nitrile hydratase of the first aspect.

[0140] According to an embodiment of the present application, the nucleic acid molecule comprises a nucleotide sequence encoding an engineered nitrile hydratase. The nucleotide sequence may be based on the sequence of a wild-type nitrile hydratase. According to an embodiment of the present application, the nucleic acid molecule comprises a nucleotide sequence encoding an engineered nitrile hydratase. The nucleotide sequence may be obtained by base mutation or enzyme digestion based on the sequence of a wild-type nitrile hydratase.

[0141] Expression vector and transformant of engineered nitrile hydratase

[0142] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0143] 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.

[0144] In some alternative embodiments, the expression vector is a plasmid vector.

[0145] 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.

[0146] 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 engineered nitrile hydratase of the embodiment of the present application can be constructed into a recombinant expression plasmid.

[0147] 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.

[0148] According to the embodiments of the present application, the isolated nucleic acid molecule of 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.

[0149] In some optional embodiments, the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

[0150] In some optional embodiments, the host cell is Rhodococcus erythrocytes and / or Escherichia coli BL21 (DE3).

[0151] Catalyst and method for preparing amide compound

[0152] In a fourth aspect, the present application provides a catalyst comprising the engineered nitrile hydratase of the first aspect.

[0153] In some alternative embodiments, the catalyst is a whole cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst.

[0154] 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 or high-pressure homogenization of the whole cell and centrifugation, and also includes pure enzymes 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 engineered nitrile hydratase.

[0155] 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;

[0156] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinonitrile, 2,6-difluorobenzonitrile, p-hydroxybenzonitrile or p-hydroxyphenylacetonitrile.

[0157] In some optional embodiments, the amide substance is selected from acrylamide, nicotinamide, 2,6-difluorobenzamide, p-hydroxybenzamide or p-hydroxyphenylacetamide.

[0158] Example

[0159] 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.

[0160] Example 1 Sequence Design of Engineered Nitrile Hydratase

[0161] Based on the wild-type nitrile hydratase (WT) from Rhodococcus rubrum, the following engineered nitrile hydratase was designed. The amino acid sequence of the α subunit of the wild-type nitrile hydratase from Rhodococcus rubrum is shown in SEQ ID NO: 1, and the amino acid sequence of the β subunit is shown in SEQ ID NO: 2. The engineered nitrile hydratase is shown in Table 1:

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] Example 2 Construction of recombinant expression vector of nitrile hydratase

[0170] Using plasmid pET-28a as an expression vector, the inventors introduced BamhI and Xho I restriction sites at both ends of the nucleotide sequence encoding nitrile hydratase, 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 example, used nucleic acid electrophoresis (1.0% agarose) and a DNA kit to recover the gene fragment after digestion, and then connected the target gene fragment after digestion with the plasmid vector after digestion.

[0171] 20μL total system:

[0172] 2 μL 10×T4 DNA ligase Buffer (Takara);

[0173] 3.0 μL target gene fragment;

[0174] 3.0 μL plasmid fragment;

[0175] 2 μL T4 DNA ligase;

[0176] 10 μL ddH2O;

[0177] The cells were connected overnight at 16°C and transformed into competent E. coli BL21 (DE3) 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 wild-type nitrile hydratase encoding gene.

[0178] Example 3 Construction of engineered nitrile hydratase

[0179] The present application adopts the whole plasmid amplification method to construct a recombinant plasmid containing a mutant gene of nitrile hydratase. Upstream and downstream primers are designed for the desired mutant site, and the plasmid pET28a-Nh is used as a template, and a high-fidelity DNA polymerase is used for whole plasmid amplification. The recombinant plasmid containing a mutant sequence encoding nitrile hydratase is amplified by PCR.

[0180] The 20 μL PCR reaction system includes:

[0181] 1 μL pET28a-Nh plasmid template (about 100 ng / μL);

[0182] 10 μL 2× PrimeSTAR HSDNA polymerase;

[0183] 1.5 μL forward primer (10 μM);

[0184] 1.5 μL reverse primer (10 μM);

[0185] 6 μL ddH2O.

[0186] The forward primer is a specific primer used in the process of constructing different mutants, such as the primer sequence used for the M1 mutation: M1-U: CCGGTGAGCAGGCAAAGCAAATTTCGGCGGTCT; M1-D: AGACCGCCGAAATTTGCTTTGCCTGCTCACCGG. The reverse primer is a specific primer used in the process of constructing 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 for those skilled in the art.

[0187] After the PCR stock solution was digested with Dpn I enzyme to remove the template sequence, it was transformed into E. coli BL21 (DE3) competent cells using the heat shock method, and spread on LB plates containing kanamycin (50 μg / mL) and placed in a 37°C incubator for inverted culture for about 12 hours. Single clones were picked 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.

[0188] Example 4 Construction of engineered nitrile hydratase

[0189] The engineered nitrile hydratase was obtained by multiple rounds of site-directed mutagenesis. After obtaining a single point mutant, the recombinant plasmid containing the mutation site was used as a template to design upstream and downstream primers of the mutation site, and the whole plasmid PCR was performed to amplify the recombinant plasmid containing the gene sequence encoding the engineered nitrile hydratase by PCR. Taking the construction of the M17 mutant as an example, it was obtained by two rounds of mutation. First, the wild type was used as a template, and the mutant M1 was constructed using M1-U and M1-D primers. Then, M1 was used as a template to select M17-U (sequence:

[0190] GCATCTCAAGGGCAACTCGTGGTGGGACAAG) and M17-D (sequence:

[0191] CTTGTCCCACCACGAGTTGCCCTTGAGATGC) primers were used to construct mutant M17.

[0192] 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.

[0193] 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 picked for sequencing verification, which was verified to be successful.

[0194] Example 5 Construction of Escherichia coli genetically engineered bacteria for nitrile hydratase and its mutants and preparation of catalysts

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] Example 6 Construction of the Rhodococcus system for engineering nitrile hydratase and preparation of catalyst

[0201] 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.

[0202] The 20 μL ligation system includes:

[0203] 4 μL target gene;

[0204] 6 μL suicide plasmid vector;

[0205] 10 μL ligation reagent;

[0206] Connect at 55°C for 60 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.

[0207] The Rhodococcus bacteria containing the coding sequence of the engineered nitrile hydratase constructed above were inoculated into the seed culture medium and cultured until the bacterial OD 600 When the concentration reaches about 25-30, it is then inoculated into 50 mL of fermentation medium. For the induced expression of nitrile hydratase, metal ions with a final concentration of 0.08 mM need to be added to the fermentation medium. After induced expression for 48 hours, the cells are collected by centrifugation to obtain the nitrile hydratase Rhodococcus cell catalyst.

[0208] Weigh 10g of frozen wet cells, add them to 100mL of buffer, the final concentration is 10g / L, place them at room temperature to melt, and filter to remove the incompletely dissolved lumps of bacteria. Then use a high-pressure homogenizer to crush them, the pressure is controlled at 1200-1500bar, and use a low-temperature circulation device to cool down during the crushing process, and the crushing cycle is repeated 2-3 times. The collected crushed liquid is centrifuged, and the supernatant is collected to obtain free engineered nitrile hydratase, i.e., the catalyst.

[0209] Weigh an appropriate amount of immobilized carrier, balance it with potassium phosphate buffer, add the treated tree carrier to the buffer, the ratio of carrier to solution is 1 / 5 (w / v), and then add glutaraldehyde solution (50%) to a final concentration of 2% V / V. After activation in an oscillator for 2-3 hours, use deionized water to wash the activated carrier to remove residual glutaraldehyde. Weigh an appropriate amount of activated immobilized carrier and place it in a buffer, add enzyme solution, and the ratio of carrier to enzyme solution is still 1 / 5 (w / v). Place it in a constant temperature shaker or oscillator for immobilization for 8 hours. Then wash the immobilized enzyme with buffer to remove residual enzyme solution, and then store the immobilized enzyme in a 4°C refrigerator for standby use. The immobilized enzyme includes the engineered nitrile hydratase constructed above.

[0210] Performance testing of engineered nitrile hydratase

[0211] (1) Method for determining the catalytic activity of engineered nitrile hydratase on substrate acrylonitrile:

[0212] 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 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 using the internal standard method, and calculate the activity.

[0213] 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.

[0214] The activity of the nitrile hydratase is calculated as follows:

[0215]

[0216] K is the internal standard constant, and its value is 0.6; C ac is the acetamide concentration; 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).

[0217] 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, which can also be understood as catalytic activity.

[0218] (2) Product tolerance test of engineered nitrile hydratase:

[0219] The engineered nitrile hydratase prepared above was resuspended in deionized water (10 g / L), and an appropriate amount of each catalyst was placed in a 10-mL centrifuge tube. 3.75 mL of 50% (w / v) acrylamide, 150 μL of acrylonitrile, 580 μL of purified water, and 22.5 μL of bacterial liquid were added to the 10 mL centrifuge tube in sequence, and the tube was placed in a 28° C. water bath for reaction. After 1 hour of reaction, hydrochloric acid was added to terminate the reaction. The residual content of acrylonitrile was detected by gas chromatography, and the consumption of acrylonitrile was calculated. The higher the consumption of acrylonitrile, the better the tolerance of the product.

[0220] The results of the catalytic activity and product tolerance of the engineered nitrile hydratase are shown in Table 2.

[0221] Table 2

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] In Table 2, +: tolerance increased by 1.1-5.0 times, ++: tolerance increased by 6.1-10.0 times; +++: tolerance increased by 10.1-20.0 times.

[0229] The above results show that the catalytic activity and tolerance of the engineered nitrile hydratase are improved to different extents compared with the wild-type nitrile hydratase. The catalytic activity of the engineered nitrile hydratase is improved by 0.25-1.4 times compared with the wild-type nitrile hydratase, and while the catalytic activity is improved, the product tolerance is also significantly improved. The tolerance of the engineered nitrile hydratase M1-M3, M16-M31 is improved by 1.1-5.0 times; the tolerance of the engineered nitrile hydratase M4-M15 and M32-M64 is improved by 6.1-10.0 times; the tolerance of the mutant M65-M96 is improved by 10.1-20.0 times.

[0230] The catalytic activities of engineered nitrile hydratases M30, M60 and M96 and wild-type nitrile hydratase were tested. Figure 2 As shown in Figure 2, the engineered nitrile hydratase has better catalytic activity than the wild-type nitrile hydratase. After testing, the engineered nitrile hydratase M30, M60 and M96 and the wild-type nitrile hydratase have product tolerances such as Figure 3As shown, M30, M60 and M96 have better product tolerance than the wild-type nitrile hydratase.

[0231] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some of the technical features characterized by them can be replaced by equivalents, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An engineered nitrile hydratase, characterized in that Based on the wild-type nitrile hydratase, the engineered nitrile hydratase includes: the amino acid residue at position 85 of the α subunit is mutated to any one of methionine, arginine, and lysine, wherein the wild-type nitrile hydratase includes the α subunit, and the amino acid sequence of the α subunit is shown in SEQ ID NO:

1.

2. The engineered nitrile hydratase according to claim 1, characterized in that The wild-type nitrile hydratase is derived from Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8 Any one of Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, Rhodococcus ruber, Rhodococcus sp., and Nocardia sp. JBRs.

3. The engineered nitrile hydratase according to claim 1 or 2, characterized in that The wild-type nitrile hydratase includes a β subunit, and the amino acid sequence of the β subunit is shown in SEQ ID NO:

2.

4. The engineered nitrile hydratase according to claim 3, characterized in that The engineered nitrile hydratase further comprises one or more of the following mutations: 1) Mutation 1 includes: the amino acid residue at the amino acid position 17 of the β subunit is mutated to glutamic acid; the amino acid residue at the amino acid position 110 of the β subunit is mutated to lysine; the amino acid residue at the amino acid position 152 of the β subunit is mutated to serine; the amino acid residue at the amino acid position 57 of the β subunit is mutated to lysine or methionine; 2) Mutation 2 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 of the β subunit mutates to lysine; the amino acid residue at the amino acid position 144 of the β subunit mutates to glutamic acid; the amino acid residue at the amino acid position 215 of the β subunit mutates to cysteine; the amino acid residue at the amino acid position 133 of the α subunit mutates to cysteine; the amino acid residue at the amino acid position 167 of the β subunit mutates to any one of arginine, lysine or serine; the amino acid residue at the amino acid position 174 of the α subunit mutates to leucine; the amino acid residue at the amino acid position 178 of the α subunit mutates to glutamic acid; 3) Mutation 3 includes: any one or more of the amino acid site at position 45 of the β subunit mutates to asparagine, the amino acid site at position 47 of the β subunit mutates to arginine or asparagine, and the amino acid site at position 48 of the β subunit mutates to asparagine.

5. The engineered nitrile hydratase according to claim 4, characterized in that The engineered nitrile hydratase satisfies one or more of the following conditions: 1) The engineered nitrile hydratase comprises any combination of mutation 1 and mutation 2; 2) the engineered nitrile hydratase comprises any combination of mutation 2 and mutation 3; 3) The engineered nitrile hydratase comprises a combination of mutation 1, mutation 2 and mutation 3.

6. The engineered nitrile hydratase according to claim 1, characterized in that The engineered nitrile hydratase comprises any one of the following mutations: α85K, α85R, α85M, β17E / β110K / β152S / β57R / α85M, β17E / β110K / β152S / β57R / α85R, β17E / β110K / β152S / β57R / α85K, β17E / β110K / β152S / β57M / α85M, β17E / β110K / β152S / β57M / α85R, β17E / β110K / β152S / β57M / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K, β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R、 β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R、 β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85M、 β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85M、 β45N / α85K, β47R / α85K, β48N / α85K, β45N / α85R, β47R / α85R, β48N / α85R、β45N / β47R / α85R、β47R / β48N / α85R、β47N / β48N / α85R、 β47R / β48N / α85K, β47N / β48N / α85K, β45N / β47R / α85K, β45N / β47R / β48N / α85R、 β45N / β47N / β48N / α85K、 β45N / β47N / β48N / α85R、 β45N / β47R / β48N / α85K、 β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R、 β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R、 β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / α85R, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R, β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R、 β17E / β110K / β152S / β57M / β47R / α85K、 β17E / β110K / β152S / β57M / β48N / α85K、 β17E / β110K / β152S / β57M / β47R / α85R、 β17E / β110K / β152S / β57M / β48N / α85R、 β17E / β110K / β152S / β57M / β47N / β48N / α85R、 β17E / β110K / β152S / β57M / β47R / β48N / α85K、 β17E / β110K / β152S / β57M / β47N / β48N / α85K、 β17E / β110K / β152S / β57M / β45N / β47R / β48N / α85R、 β17E / β110K / β152S / β57M / β45N / β47N / β48N / α85K、 β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R, β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K, β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85K, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / α85R、 β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K、 β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R、 β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R、 β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R、 β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85K、 β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85K, β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85K, β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85K, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / / β47R / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β48N / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47R / β48N / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β47N / β48N / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85R.

7. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the engineered nitrile hydratase of any one of claims 1 to 6.

8. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 7.

9. The expression vector according to claim 8, characterized in that The expression vector is a plasmid vector, and the plasmid vector includes any one of the pET series, shuttle vectors, phage and virus vectors.

10. A host cell, characterized in that Comprising the nucleic acid molecule of claim 7 or the expression vector of claim 8 or 9.

11. The host cell according to claim 10, characterized in that The host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.

12. A catalyst, characterized in that The invention comprises the engineered nitrile hydratase according to any one of claims 1 to 6.

13. A method for preparing an amide compound, characterized in that: The method comprises using the engineered nitrile hydratase according to any one of claims 1 to 6 or the catalyst according to claim 12 to catalyze a hydration reaction of a nitrile compound to obtain an amide compound.

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