Engineered nitrile hydratase and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and genetic engineering, and relates to an engineered nitrile hydratase and its applications. Background Technology

[0002] Nitrile hydratase (NHase, EC 4.2.1.84) is a class of metal-dependent enzymes that catalyze the conversion of nitrile substrates into amide compounds. Nitrile hydratases can be used to catalyze the production of bulk chemicals such as acrylamide and nicotinamide, and have been widely applied in industrial production. Nitrile hydratases typically consist of two subunits, α and β, and are distributed in a variety of microorganisms, with a wide range of sources. The production strains used to catalyze the production of acrylamide from acrylonitrile using nitrile hydratase have also undergone multiple generations.

[0003] During the catalytic hydration of nitrile groups, the catalyst experiences an exothermic effect, leading to a rapid temperature rise and impacting catalyst stability. Furthermore, the concentration of catalytic products can reach up to 50% during production; high product concentrations significantly poison the catalyst, accelerating deactivation and ultimately resulting in a substantial increase in catalyst usage. Therefore, developing a new generation of catalysts with high stability and high product tolerance, while reducing catalyst consumption, is an urgent need in industrial production.

[0004] Currently, most wild-type nitrile hydratases suffer from bottlenecks such as instability, 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] This application provides an engineered nitrile hydratase that incorporates the aforementioned mutations based on the wild-type nitrile hydratase, thereby further enhancing the product tolerance and catalytic activity of the engineered nitrile hydratase while ensuring enzyme activity.

[0006] In a first aspect, embodiments of this application provide an engineered nitrile hydratase based on wild-type nitrile hydratase. The engineered nitrile hydratase includes an amino acid residue at position 85 of the α subunit that is mutated to any one of methionine, arginine, or lysine. The wild-type nitrile hydratase includes 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 Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, Rhodococcus ruber, Rhodococcus sp., and Nocardia sp. JBRs, with Rhodococcus ruber being an option.

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

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

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

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

[0012] 2) Mutation 2 includes: the amino acid residue at position 141 of the β subunit is mutated to lysine; the amino acid residue at position 143 of the β subunit is mutated to lysine; the amino acid residue at position 144 of the β subunit is mutated to glutamic acid; the amino acid residue at position 215 of the β subunit is mutated to cysteine; the amino acid residue at position 133 of the α subunit is mutated to cysteine; the amino acid residue at position 167 of the β subunit is mutated to any one of arginine, lysine, or serine; the amino acid residue at position 174 of the α subunit is mutated to leucine; and the amino acid residue at position 178 of the α subunit is mutated to glutamic acid.

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

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

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

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

[0017] In some alternative embodiments, the engineered nitrile hydratase includes 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] Secondly, embodiments of this application provide an isolated nucleic acid molecule containing a nucleotide sequence encoding an engineered nitrile hydratase as described in the first aspect.

[0078] Thirdly, embodiments of this application provide an expression vector comprising the nucleic acid molecule described in the second aspect.

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

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

[0081] Thirdly, embodiments of this application provide a host cell containing either the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

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

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

[0084] Fourthly, this application provides a catalyst comprising the engineered nitrile hydratase of the first aspect.

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

[0086] Fifthly, embodiments of this application provide a method for preparing amide compounds, the method comprising using a catalyst from the first or 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, nicotinic nitrile, 2,6-difluorobenzonitrile, p-hydroxybenzonitrile, or p-hydroxyphenylacetonitrile.

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

[0089] In some optional embodiments, the nitrile compound is selected from acrylonitrile, nicotinic nitrile, cinnamonitrile, phenylacetonitrile, or p-hydroxyphenylacetonitrile.

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

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

[0092] The engineered nitrile hydratase of this application has a mutation at position 85 of the α subunit, replacing any one of methionine, arginine, or lysine, which significantly improves the stability and product tolerance of the nitrile hydratase during the catalytic process. The engineered nitrile hydratase provided in this application exhibits higher catalytic efficiency in the production of amide compounds and can significantly reduce the cost of catalyst use.

[0093] This application embodiment can further provide a nucleic acid molecule encoding the above-mentioned mutant nitrile hydratase, an expression vector containing the nucleic acid molecule encoding the above-mentioned mutant nitrile hydratase, and a transformant containing the expression vector, each of which has the beneficial effect of engineered nitrile hydratase. This application embodiment can further provide a method for preparing an amide compound, which utilizes the nitrile hydratase extracted from the culture of the transformant and its manufacture, as well as methods using the aforementioned nitrile hydratase or using the culture or a treatment of the culture, also having the beneficial effect of engineered nitrile hydratase. Attached Figure Description

[0094] To more clearly illustrate the technical solutions of the embodiments of the present invention, the technical solutions of this application will be described in detail below with reference to the accompanying drawings:

[0095] Figure 1This diagram illustrates the spatial locations of amino acid mutation sites in the flexible ring domain surrounding the active pocket of the nitrile hydratase based on Rhodococcus rubrum.

[0096] Figure 2 The diagram shows the catalytic activity of wild-type nitrile hydratase and engineered nitrile hydratase according to embodiments of this application.

[0097] Figure 3 The diagram shows the product tolerance of wild-type nitrile hydratase and the engineered nitrile hydratase of the present application. Detailed Implementation

[0098] The features and exemplary embodiments of various aspects of this application will now be described in detail. The specific embodiments listed herein are merely examples, and the invention is not limited to the specific embodiments described below.

[0099] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this application. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of this application should be covered within the scope of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate the invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of this application.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0101] In this application, "multiple" refers to two or more (including two). "Multiple types" or "several kinds" refers to two or more (including two). Correspondingly, "multiple items" is interpreted in the same way.

[0102] Throughout this discussion, standard single-letter codes for amino acids are used. Standard substitution notation is also used, where α85K indicates that the 85th amino acid residue at the N-terminus of the α subunit is replaced by lysine (K); or β17E indicates that the 17th amino acid residue at the N-terminus of the β subunit is replaced by glutamic acid (E). The same applies to others.

[0103] In paragraphs of this text where different amino acids at specific positions are separated by the slash " / ", the slash signifies "and" or "or". For example, β17E / β110K / β152S / β57R / α85M can represent the simultaneous presence of the aforementioned mutations at amino acid positions 17, 110, 152, and 57 in the β subunit and at amino acid position 85 in the α subunit in engineered nitrile hydratase. Other descriptive methods using this approach also indicate simultaneous presence.

[0104] In paragraphs of this text where different amino acids at a specific position are separated by the slash " / ", and enclosed in parentheses, the slash " / " means "or", and the parentheses "()" indicates amino acids at the same position. In paragraphs of this text where different positions are separated by the slash " / ", the slash " / " means "and" so that Y51 / N55 is Y51 and N55.

[0105] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in a "normal" or "wild-type" form. These nitrile hydratases are identified by the fact that they possess altered properties compared to wild-type genes or gene products. Methods for introducing or replacing naturally occurring amino acids are well known in the art. Methods for introducing or replacing 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 its active site. It can be classified into Fe-type and Co-type. Currently known nitrile hydratases all exist as heteropolymers with two subunits, α and β (or a and b subunits), and the amino acid residues in the active site are relatively conserved (-C(S / T)LCSC(T / Y)-). This study selected wild-type Co-type nitrile hydratase as the research subject.

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

[0109] As an example, the amino acid sequence of the α subunit of the nitrile hydratase from 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 this application contains mutations of any one or any combination thereof based on the amino acid sequence of the above-mentioned wild-type nitrile hydratase.

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

[0111] 1) Nitrile hydratase from wild-type Rhodococcus ruber TH, the amino acid sequence of its α subunit is shown in SEQ ID NO:1, and the amino acid sequence of its β subunit is shown in SEQ ID NO:2. The structural and regulatory gene sequences expressing the relevant amino acid sequences are detailed in Chinese Invention Patent No. ZL200910076710.1, the entire contents of which are incorporated herein by reference. Rhodococcus ruber is sometimes also translated as *Rhodococcus ruber*. SEQ ID NO:1:

[0112] MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKS

[0113] WVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWP

[0114] VLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0115] SEQ ID NO:2:

[0116] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFR

[0117] ESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSR

[0118] KFDPAEIEKAIERLHEPHSLALPGAEPSFSLGDKVKVKNMNPLGHTRCPKYVRNKIGE

[0119] IVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISA.

[0120] Alternatively, any one of the following can be selected: Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, Rhodococcus sp., or Nocardia sp. JBRs, with Rhodococcus ruber being a suitable alternative.

[0121] Engineered nitrile hydratase

[0122] The technical concept of this application is as follows: Starting from wild-type nitrile hydratase, the inventors resolved the crystal structure information of the enzyme. Through structural analysis, molecular dynamics simulation and enzyme molecule sequence comparison, they identified the flexible ring domain around the substrate binding pocket. Through targeted design, they discovered key sites that regulate catalytic activity and tolerance, constructed an engineered nitrile hydratase library, and obtained a series of mutant strains with improved catalytic performance. Compared with wild-type nitrile hydratase, the engineered nitrile hydratase has significantly improved catalytic activity and product tolerance.

[0123] The objective of this application is achieved through the following technical solution:

[0124] In a first aspect, embodiments of this application provide an engineered nitrile hydratase based on wild-type nitrile hydratase. The engineered nitrile hydratase includes an amino acid residue at position 85 of the α subunit that is mutated to any one of methionine, arginine, or lysine. The wild-type nitrile hydratase includes an α subunit, and the amino acid sequence of the α subunit is shown in SEQ ID NO:1.

[0125] Throughout the discussion in this paper, the mutation of the amino acid residue at position 85 of the α subunit to any one of methionine, arginine, or lysine can be understood as the substitution of any amino acid residue at position 85 from the N-terminus of the α subunit by methionine, arginine, or lysine.

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

[0127] like Figure 1 As shown in the figure, the flexible loop domain and the 85 site are spatially positioned in the engineered nitrile hydratase.

[0128] Based on existing wild-type nitrile hydratases, the applicant obtained several beneficial mutants through structural analysis and rational design. Furthermore, it was discovered that these beneficial mutations are concentrated in specific primary (amino acid mutations) and / or secondary domains 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 activity is calculated by reacting a substrate (nitrile compound) with nitrile hydratase under specific conditions and measuring the amount of substrate consumed and the amount of product increased per unit time. Any nitrile compound can be used as a substrate, as long as the nitrile hydratase reaction occurs; acrylonitrile and nicotinamide can be selected as substrates. The reaction conditions are general conditions for hydration reactions, as long as they ensure the catalytic activity of the nitrile hydratase is maintained. The amount of substrate consumed and the amount of product increased can be detected and quantitatively analyzed by high-performance liquid chromatography (HPLC) and gas chromatography (GC).

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

[0132] R-CN(1)

[0133] In addition, the R group is a straight-chain or branched alkyl or alkenyl group with 1 to 10 carbon atoms that may be substituted, a cycloalkyl or aryl group with 3 to 18 carbon atoms that may be substituted, or a saturated or unsaturated heterocyclic group that may be substituted.

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

[0135] R-CONH2(2)

[0136] In addition, the R group is a straight-chain or branched alkyl or alkenyl group with 1 to 10 carbon atoms that may be substituted, a cycloalkyl or aryl group with 3 to 18 carbon atoms that may be substituted, or a saturated or unsaturated heterocyclic group that may be substituted.

[0137] Studies have shown that the wild-type nitrile hydratase and the mutant nitrile hydratase described in this application have catalytic activity.

[0138] Nucleic acid molecules

[0139] Secondly, this application provides an isolated nucleic acid molecule containing a nucleotide sequence encoding an engineered nitrile hydratase as described in the first aspect.

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

[0141] Expression vectors and transformants of engineered nitrile hydratase

[0142] Thirdly, this application provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0143] According to the embodiments of this application, the selected expression vector can exist stably in various hosts of prokaryotic or eukaryotic cells and can replicate autonomously, such as conventional plasmids (pET series), shuttle vector pNV18.1, bacteriophage or viral vectors in the art.

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

[0145] In some alternative implementations, the expression vector is a pET series, a shuttle vector, a bacteriophage, or a viral vector; alternatively, the expression vector is pET-28a or pNV18.1.

[0146] According to the embodiments of this application, the nucleotide sequence of wild-type nitrile hydratase is inserted into expression vectors such as pET-28a or pNV18.1 through molecular biology operations such as enzyme digestion and ligation to construct recombinant expression plasmids, named pET28a-Nh and pNV18.1-Nh, respectively. According to the embodiments of this application, the coding gene of the engineered nitrile hydratase of this application can be constructed into recombinant expression plasmids.

[0147] Thirdly, this application provides a host cell containing the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0148] According to the embodiments of this application, the nucleic acid molecules isolated in the second aspect of this application can be directly inserted into the chromosome of the host bacteria, or the expression vector of the third aspect can be introduced into the host bacteria using the calcium chloride method or electroporation transformation method.

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

[0150] In some alternative implementations, the host cell is Rhodococcus rubrum or / and Escherichia coli E.coli BL21(DE3).

[0151] Preparation methods of catalysts and amide compounds

[0152] Fourthly, this 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 this 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 according to the fourth aspect of this application. The free protein catalyst is the crude enzyme solution obtained by ultrasonically disrupting or high-pressure homogenizing the whole cell and then centrifuging, and also includes pure enzymes obtained through protein purification methods. The immobilized enzyme catalyst involves selecting different immobilization carriers and immobilizing the free protein catalyst to obtain different forms of immobilized engineered nitrile hydratases.

[0155] Fifthly, this application provides a method for preparing amide compounds, the method comprising catalyzing a hydration reaction of a nitrile compound using a catalyst from the first or fourth aspect to obtain an amide compound;

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

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

[0158] Example

[0159] The following embodiments describe the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. All reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing. Similarly, the instruments used in the embodiments are commercially available.

[0160] Example 1: Sequence design of engineered nitrile hydratase

[0161] Based on the wild-type nitrile hydratase (WT) derived from Rhodococcus rubrum, the following engineered nitrile hydratase was designed. The amino acid sequence of the α subunit of the wild-type nitrile hydratase derived 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 detailed in Table 1.

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

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

[0170] Using plasmid pET-28a as the expression vector, the inventors introduced BamhI and XhoI restriction sites at both ends of the nucleotide sequence encoding nitrile hydratase. The nucleotide sequence of the plasmid vector and the nitrile hydratase in this embodiment were double-digested using restriction endonucleases NcoI and XhoI. The digested gene fragment was recovered using nucleic acid electrophoresis (1.0% agarose) and a DNA kit. Then, the digested target gene fragment was ligated to the digested plasmid vector.

[0171] 20μL total system:

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

[0173] 3.0 μL of the target gene fragment;

[0174] 3.0 μL plasmid fragment;

[0175] 2 μL T4 DNA ligase;

[0176] 10 μL ddH2O;

[0177] The cells were ligated overnight at 16°C and transformed into E. coli BL21(DE3) competent cells. Single clones were picked and sequenced for verification. 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] This application employs a whole-plasmid amplification method to construct a recombinant plasmid containing a nitrile hydratase mutant gene. Upstream and downstream primers were designed for the desired mutant site. Using plasmid pET28a-Nh as a template, whole-plasmid amplification was performed using high-fidelity DAN polymerase. The recombinant plasmid containing the mutated sequence encoding the nitrile hydratase gene was amplified by PCR.

[0180] The 20 μL PCR reaction system includes:

[0181] 1 μL pET28a-Nh plasmid template (approximately 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 primers are specific primers used in the construction process for different mutants. For example, the primer sequences used for the M1 mutation are: M1-U: CCGGTGAGCAGGCAAAGCAAATTTCGGCGGTCT; M1-D: AGACCGCCGAAATTTGCTTTGCCTGCTCACCGG. The reverse primers are also specific primers used in the construction process for different mutants. Due to space limitations, these will not be elaborated upon here, but those skilled in the art should know that designing primers and obtaining the target product based on known sequences is a conventional technique.

[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 plated on LB agar plates containing kanamycin (50 μg / mL). The plates were then incubated upside down at 37°C for approximately 12 hours. Single clones were picked for sequencing verification. After confirming correct sequencing, the cells were preserved with 20% (v / v) glycerol and stored at -70°C.

[0188] Example 4 Construction of engineered nitrile hydratase

[0189] Engineered nitrile hydratases are obtained through multiple rounds of site-directed mutagenesis. After obtaining a single site-directed mutant, a recombinant plasmid containing that mutation site is used as a template. Upstream and downstream primers for the mutation site are designed, and whole-plasmid PCR is performed. The recombinant plasmid containing the gene sequence encoding the engineered nitrile hydratase is amplified by PCR. Taking the construction of the M17 mutant as an example, it is obtained through two rounds of mutagenesis. First, using the wild-type mutant as a template, mutant M1 is constructed using primers M1-U and M1-D. Then, using M1 as a template, M17-U (sequence:

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

[0191] The mutant M17 was constructed using primers CTTGTCCCACCACGAGTTGCCCTTGAGATGC.

[0192] The forward primers may be specific primers constructed for different mutants, which will not be elaborated here due to space limitations. However, those skilled in the art should know that designing primers and obtaining the target product based on a known sequence is a conventional technique 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 plated on LB agar plates containing kanamycin (50 μg / mL). The plates were then incubated upside down at 37°C for approximately 12 hours. Single clones were picked for sequencing verification, and the results were confirmed to be successful.

[0194] Example 5: Construction of Escherichia coli genetically engineered bacteria containing nitrile hydratase and its mutants, and preparation of catalysts.

[0195] The Escherichia coli genetically engineered bacteria prepared in Examples 3 and 4 were plated on LB agar plates containing kanamycin (50 μg / mL) and incubated overnight at 37°C. Single colonies were picked and transferred to LB liquid culture containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. Samples were sent for sequencing, and the correct clones were stored in a -70°C freezer to obtain Escherichia coli genetically engineered bacteria.

[0196] Genetically engineered bacteria containing the coding sequences of nitrile hydratase and its mutants were inoculated into LB liquid medium containing kanamycin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, deionized water as solvent, pH = 7.0). The culture was then placed in test tubes containing LB medium (4 mL, with a final concentration of 50 μg / mL kanamycin) and cultured at 37°C and 200 rpm for 10–12 h to obtain the seed culture.

[0197] In a clean bench, transfer the seed culture from the test tube to a shake flask containing LB medium (100 mL, with a final concentration of 50 μg / mL kanamycin). Incubate the LB medium containing the seed culture in a shaker at 37°C and 200 rpm for 2–3 hours. The OD of the culture medium is then measured. 600 When the value reaches 0.6–0.8, add IPTG to a final concentration of 0.1–0.8 mM and 0.1–0.8 mM Co. 2+ Expression was induced by ions at temperatures ranging from 16 to 37°C. Optional IPTG concentrations and cobalt ion concentrations were 0.2 mM and 16°C, respectively. After induction for 24 hours under these conditions, cells were collected by centrifugation to obtain the *E. coli* cell catalyst for nitrile hydratase, which was then stored at -70°C.

[0198] Weigh 10g of frozen wet cells and add them to 100mL of buffer A (25mM Tris-HCl, pH 8.0; 300mM NaCl, 10mM imidazole, 375μL / L mercaptoethanol) to a final concentration of 10g / L. Thaw at room temperature and filter to remove any incompletely dissolved clumps of cells. Then, homogenize the cells using a high-pressure homogenizer at 700-800 bar, cooling the homogenate during the process. Cycle the homogenate 2-3 times. Centrifuge the collected homogenate and collect the supernatant to obtain the free nitrile hydratase catalyst.

[0199] Weigh an appropriate amount of immobilized amino resin material and equilibrate it with potassium phosphate buffer (100mM, pH 7.0). Add the treated resin carrier to the buffer, with a carrier-to-solution ratio of 1 / 5 (w / v). Then add glutaraldehyde solution (50% v / v) to a final concentration of 2%. Activate in a shaker (16℃, 200rpm) for 2-3 hours. Wash the activated carrier with deionized water to remove residual glutaraldehyde. Weigh an appropriate amount of the activated immobilized carrier and place it in the buffer. Add enzyme solution, maintaining a carrier-to-enzyme solution ratio of 1 / 5 (w / v). Immobilize in a constant temperature shaker or shaker (16℃, 200rpm) for 8 hours. Then wash the immobilized enzyme with buffer to remove residual enzyme solution, and store at 4℃ for later use.

[0200] Example 6: Construction of an engineered nitrile hydratase system in Rhodococcus and preparation of the catalyst

[0201] Using the recombinant expression vectors prepared in Examples 3 and 4 as templates, universal primers for nitrile hydratase were designed. These universal primer sequences contained homologous fragments of the optimized suicide plasmid pYsacB and the nitrile hydratase gene fragment. The nitrile hydratase gene sequence containing the mutation site was amplified by PCR. The optimized suicide plasmid pYsacB1 was created by adding approximately 1000 bp homologous arms upstream and downstream of the nitrile hydratase gene to the suicide plasmid pYsacB. The obtained nitrile hydratase mutant gene was ligated to the enzyme-digested suicide plasmid pYsacB using the Gibson Assembly seamless cloning kit.

[0202] The 20μL connection system includes:

[0203] 4μL of target gene;

[0204] 6 μL suicide plasmid vector;

[0205] 10 μL ligation reagent;

[0206] Ligation was performed at 55℃ for 60 min. The ligation solution was then transferred to Top 10 competent cells. After single-clone sequencing verification, the recombinant suicide plasmid was extracted. The successfully constructed recombinant suicide plasmid was transformed into competent Rhodococcus cells using electroporation. After resuscitation and culture, the cells were plated on antibiotic-containing agar plates and cultured at 20-37℃. Single colonies were then picked and colony PCR was performed to verify the correct integration of the suicide plasmid into the Rhodococcus genome. The successfully exchanged colonies were inoculated into antibiotic-free seed culture medium and cultured at 20-37℃ for 12 h. After a 100-fold dilution, 200 μL of the culture was plated on agar plates containing 100 g / L sucrose and cultured at 28℃. After single colonies grew, colony PCR was performed to verify the integration of the nitrile hydratase gene sequence into the genome. The colonies were then sent to a sequencing company. Strains with correct sequencing results were stored at -70℃ for later use.

[0207] The constructed Rhodococcus species containing the coding sequence of engineered nitrile hydratase were inoculated into seed culture medium and cultured until the bacterial cell OD reached a certain level. 600 When the concentration reaches approximately 25-30, the cells are inoculated into 50 mL of fermentation medium. For the induction of nitrile hydratase expression, metal ions with a final concentration of 0.08 mM need to be added to the fermentation medium. After inducing expression for 48 h, the cells are collected by centrifugation to obtain the nitrile hydratase Rhodococcus cell catalyst.

[0208] Weigh 10g of frozen wet cells and add them to 100mL of buffer solution, bringing the final concentration to 10g / L. Thaw at room temperature and filter to remove any incompletely dissolved clumps of cells. Then, homogenize the cells using a high-pressure homogenizer at 1200-1500 bar, cooling the homogenate during the process using a low-temperature circulation device. Cycle the homogenate 2-3 times. Centrifuge the collected homogenate and collect the supernatant to obtain free engineered nitrile hydratase, i.e., the catalyst.

[0209] Weigh an appropriate amount of immobilized carrier and equilibrate it with potassium phosphate buffer. Add the treated tree carrier to the buffer at a carrier-to-solution ratio of 1 / 5 (w / v). Then add glutaraldehyde solution (50%) to a final concentration of 2% v / v. After activation in a shaker for 2-3 hours, wash the activated carrier with deionized water to remove residual glutaraldehyde. Weigh an appropriate amount of the activated immobilized carrier and place it in the buffer. Add the enzyme solution, maintaining a carrier-to-enzyme ratio of 1 / 5 (w / v). Immobilize it in a constant-temperature shaker or shaker for 8 hours. Then wash the immobilized enzyme with buffer to remove residual enzyme solution. Finally, store the immobilized enzyme at 4°C for later use. This 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 against acrylonitrile substrate:

[0212] Add 50–100 μL of catalyst (cells, free enzyme, immobilized enzyme) to a centrifuge tube, add pure water to make up to 4.5 mL, and place 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. Terminate the reaction by adding 200 μL of 3 mol / L hydrochloric acid. Centrifuge 1 mL of the reaction solution at 13000 × g for 2 minutes. Mix 500 μL of the supernatant with 500 μL of internal standard solution (40 g / L acetamide) and perform gas chromatography analysis. Determine the area ratio of acrylamide to acetamide, and use the internal standard method to determine the concentration of acrylamide in the product, calculating the activity.

[0213] The amount of product generated was determined by gas chromatography. The detection conditions were as follows: Thermo Fisher Scientific Trace 1300 gas chromatograph; Abel Bonded AB-I NOWAX column (0.25 mm inner diameter, 30 m length, 0.25 μm film thickness); FID detector. Column temperature, injection port temperature, and detector temperature were 19℃, 26℃, and 26℃, respectively; nitrogen was used as the carrier gas, constant pressure mode, partial pressure 10. 8 kPa; injection volume of 1 μL, split injection, split ratio of 30:1.

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

[0215]

[0216] K is the internal standard constant, with a value of 0.6; C ac V represents the acetamide concentration; Vtotal is the total volume, which is 5 mL; Vactual is the actual volume of catalyst added; t is the reaction time; MW is acrylamide, with a molecular weight of 71; U is the total enzyme activity, in μmol acrylamide / (min·mL bacterial culture).

[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). Appropriate amounts of catalyst were placed in 10 mL centrifuge tubes. 3.75 mL of 50% (w / v) acrylamide, 150 μL of acrylonitrile, 580 μL of purified water, and 22.5 μL of bacterial culture were added sequentially to each 10 mL centrifuge tube. The tubes were then placed in a 28°C water bath for reaction. After 1 hour, hydrochloric acid was added to terminate the reaction. The remaining acrylonitrile content was detected using gas chromatography, and the acrylonitrile consumption was calculated. Higher acrylonitrile consumption indicates better product tolerance.

[0220] 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, + indicates a 1.1-5.0-fold increase in tolerance, ++ indicates a 6.1-10.0-fold increase in tolerance, and +++ indicates a 10.1-20.0-fold increase in tolerance.

[0229] The above results indicate that the catalytic activity and tolerance of engineered nitrile hydratases are improved to varying degrees compared with wild-type nitrile hydratases. The catalytic activity of engineered nitrile hydratases is enhanced, increasing by 0.25-1.4 times compared to wild-type nitrile hydratases. Furthermore, along with the increased catalytic activity, the tolerance of the products is also significantly improved. The tolerance of engineered nitrile hydratases M1-M3 and M16-M31 is increased by 1.1-5.0 times; the tolerance of engineered nitrile hydratases M4-M15 and M32-M64 is increased by 6.1-10.0 times; and the tolerance of mutants M65-M96 is increased by 10.1-20.0 times.

[0230] The catalytic activities of engineered nitrile hydratases M30, M60, and M96, as well as wild-type nitrile hydratase, were tested as follows: Figure 2 As shown, engineered nitrile hydratases exhibit superior catalytic activity compared to wild-type nitrile hydratases. The product tolerance of engineered nitrile hydratases M30, M60, and M96, as well as wild-type nitrile hydratases, was tested and found to be as follows: Figure 3As shown, M30, M60, and M96 exhibit better product tolerance compared to wild-type nitrile hydratases.

[0231] It should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. An engineered nitrile hydratase, characterized in that, Based on mutation of wild-type nitrile hydratase, the wild-type nitrile hydratase is derived from Rhodococcus rubrum (… Rhodococcus ruber), The wild-type nitrile hydratase is composed of an α subunit and a β subunit, the amino acid sequence of which is shown in SEQ ID NO: 1; the amino acid sequence of which is shown in SEQ ID NO:

2. The mutation of the process of nitrile is any one of the following types: 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、 β45N / β47R / α85K, β47R / β48N / α85K, β47N / β48N / α85K, β45N / β47R / β48N / α85R、β45N / β47N / β48N / α85R、 β45N / β47R / β48N / α85K、β45N / β47N / β48N / α85K、 β17E / β110K / β152S / β57M / β45N / α85K、 β17E / β110K / β152S / β57M / β47R / α85K、 β17E / β110K / β152S / β57M / β48N / α85K、 β17E / β110K / β152S / β57M / β45N / α85R、 β17E / β110K / β152S / β57M / β47R / α85R、 β17E / β110K / β152S / β57M / β48N / α85R、 β17E / β110K / β152S / β57M / β45N / β47R / α85R、 β17E / β110K / β152S / β57M / β47R / β48N / α85R、 β17E / β110K / β152S / β57M / β47N / β48N / α85R、 β17E / β110K / β152S / β57M / β45N / β47R / α85K、 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 / α85R、 β17E / β110K / β152S / β57M / β45N / β47R / β48N / α85K、 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、 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 / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / α85K, 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 / β45N / β47R / 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 / β45N / β47R / β48N / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85K, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / α85R, 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 / β45N / β47R / 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 / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / β48N / α85R, β17E / β110K / β152S / β57M / β167S / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85R、 β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / α85K, 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 / β45N / β47R / 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 / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47R / β48N / α85K, β17E / β110K / β152S / β57M / β167K / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85K, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / α85R、 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 / β45N / β47R / 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 / β47R / β48N / α85R, β17E / β110K / β152S / β57M / β167R / α174L / α178E / β215C / α133C / βS141K / β143K / β144E / β45N / β47N / β48N / α85R.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nucleotide sequence of the engineered nitrile hydratase according to claim 1.

3. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is a plasmid vector, which includes the pET series or shuttle vectors.

5. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 2 or the expression vector of claim 3 or 4.

6. The host cell according to claim 5, characterized in that, The host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis, or Corynebacterium glutamicum.

7. A catalyst, characterized in that, It contains the engineered nitrile hydratase as described in claim 1.

8. A method for preparing an amide compound, characterized in that, The method includes using the engineered nitrile hydratase of claim 1 or the catalyst of claim 7 to catalyze the hydration reaction of nitrile compounds to obtain amide compounds.

Images