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WO2025039280A1 - 腈水合酶突变体及其应用 - Google Patents

腈水合酶突变体及其应用 Download PDF

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Publication number
WO2025039280A1
WO2025039280A1 PCT/CN2023/115259 CN2023115259W WO2025039280A1 WO 2025039280 A1 WO2025039280 A1 WO 2025039280A1 CN 2023115259 W CN2023115259 W CN 2023115259W WO 2025039280 A1 WO2025039280 A1 WO 2025039280A1
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amino acid
nitrile hydratase
mutation
subunit
mutant
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English (en)
French (fr)
Inventor
于慧敏
李付龙
梁有向
陈博
王苗苗
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Beijing Evolyzer Co Ltd
Tsinghua University
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Beijing Evolyzer Co Ltd
Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

  • the invention belongs to the fields of protein engineering and genetic engineering, and relates to a nitrile hydratase mutant, a genetic engineering bacterium containing the mutant and application thereof.
  • Amide compounds are important organic synthesis intermediates and chemicals, and are widely used in the fields of medicine, pesticides, feed, food, environmental protection, oil production, materials, etc.
  • the corresponding amide compounds can be obtained from nitrile compounds through hydration reaction, which is also one of the important methods for preparing such compounds in industry.
  • Nitrile hydratase (NHase, EC 4.2.1.84) is a class of multi-subunit enzymes that can convert nitrile compounds into corresponding amide substances through hydration.
  • nitrile hydratase catalyst to catalyze the hydration of acrylonitrile and nicotinonitrile to produce acrylamide and nicotinamide is one of the most successful cases of biotechnology replacing chemical methods for the industrial production of bulk chemicals.
  • Nitrile hydratase is usually composed of two subunits, ⁇ and ⁇ (or called ⁇ and ⁇ subunits), and is distributed in many microorganisms such as Rhodococcus, Nocardia, Brevibacterium and Pseudomonas. As early as 1980, Japanese scholar Asano first discovered this type of enzyme in Rhodococcus rhodochrous J1 and defined it as nitrile hydratase (Asano Y, et al. Agricultural and Biological Chemistry, 1982, 46 (5): 1165-1174.).
  • Rhodococcus rhodochrous J1 Naagasawa T, et al. Applied Microbiology and Biotechnology, 1993, 40(2-3):189-195
  • R. ruber TH R. ruber TH
  • their genetically engineered bacteria are widely used in the industrial production of acrylamide (Ma Y, et al. Bioresource Technology, 2010, 101(1):285-291).
  • nitrile hydratase catalysts In the process of catalyzing the hydration of acrylonitrile, nitrile hydratase catalysts often face the problem of sudden temperature rise caused by the exothermic effect of the hydration reaction, which leads to enzyme inactivation and cell rupture, resulting in reduced economic benefits. In addition, the toxic effect on cells is greater at high product concentrations, which will increase the inactivation of the catalyst. Therefore, the construction of a new generation of catalysts with high stability and high product tolerance is an important demand for the industrial production of amide compounds. Most wild-type nitrile hydratases have problems such as poor stability, poor product tolerance, and insufficient catalytic activity.
  • nitrile hydratase In order to further improve the production performance of nitrile hydratase, researchers have conducted in-depth research from multiple aspects such as the design of salt bridges and disulfide bonds and the introduction of molecular chaperones at the enzyme molecular level, and improved the stability of nitrile hydratase (Liu Y, et al. Applied Biochemistry and Biotechnology, 2016, 180 (1): 3-9; Ma Y, et al. Journal of Industrial Microbiology & Biotechnology, 2012, 39 (10): 1421-1430; Tian Y, et al. Journal of Microbiology and Biotechnology, 2016, 26 (2): 337-346; Jiao S, et al. Applied Microbiology and Biotechnology, 2020, 104 (3): 1001-1012.).
  • the purpose of the present application is to provide a nitrile hydratase mutant based on the prior art.
  • the nitrile hydratase mutant of the present application can contain the above mutations on the basis of wild-type nitrile hydratase, which has certain enzyme activity alone or exerts a synergistic regulatory effect, and has better thermal stability and tolerance.
  • the embodiments of the present application provide a nitrile hydratase mutant, based on a wild-type nitrile hydratase, wherein the nitrile hydratase mutant is a mutated amino acid residue on the flexible loop domain of the wild-type nitrile hydratase, comprising any one or a combination of mutations 1-3:
  • Mutation 1 a polar amino acid mutation located in the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation 2 a polar amino acid mutation in a flexible loop between the secondary domains H6 and H7 at the start end of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation 3 A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase ⁇ subunit.
  • the wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus, and Nocardia.
  • the nitrile hydratase mutant satisfies at least one of the following conditions:
  • the amino acid residue at amino acid position 152 is mutated to any one of cysteine, threonine, glutamic acid, aspartic acid, valine, serine, glycine, and glutamine.
  • the amino acid residue at amino acid position 110 mutates to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine.
  • the amino acid residue at amino acid position 17 is mutated to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.
  • the nitrile hydratase mutant includes any combination of mutation 1 and mutation 3.
  • the nitrile hydratase mutant includes any combination of mutation 1 and mutation 2
  • the nitrile hydratase mutant includes any combination of mutation 2 and mutation 3.
  • the nitrile hydratase mutant includes mutation 1, mutation 2 and mutation 3.
  • the nitrile hydratase mutant comprises at least one of an amino acid residue at position 167 of the ⁇ subunit mutated to serine, an amino acid residue at position 174 of the ⁇ subunit mutated to leucine, and an amino acid residue at position 178 of the ⁇ subunit mutated to glutamate.
  • the nitrile hydratase mutant comprises: a domain mutation, which includes: the amino acid residue at the amino acid position 141 of the ⁇ subunit mutates to lysine, the amino acid residue at the amino acid position 143 mutates to lysine, the amino acid residue at the amino acid position 144 mutates to glutamate, the amino acid residue at the amino acid position 215 mutates to cysteine, and the amino acid residue at the amino acid position 133 of the ⁇ subunit mutates to cysteine.
  • the nitrile hydratase mutant includes at least one of 17A/17R/17E/17D/17S/17C/17K/17H, 110A/110R/110S/110T/110V/110F/110L/110I/110K, and 152C/152A/152T/152E/152D/152V/152S/152Q/152G.
  • the embodiments of the present application provide an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the nitrile hydratase mutant of the first aspect.
  • an embodiment of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
  • the expression vector is a plasmid vector.
  • the expression vector is a pET series, a shuttle vector, a phage or a viral vector.
  • 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.
  • the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.
  • the host cell is Rhodococcus erythrocytes and/or Escherichia coli BL21 (DE3).
  • the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.
  • the catalyst is a whole-cell catalyst, a free protein catalyst or an immobilized enzyme catalyst.
  • 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.
  • the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamon nitrile, benzyl cyanide or p-hydroxyphenyl cyanide.
  • the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.
  • the nitrile hydratase mutant of the embodiment of the present application has the above-mentioned mutation site, which significantly improves the thermal stability and product tolerance of the nitrile hydratase; the above-mentioned mutation site is further introduced into nitrile hydratases from different sources, and it is unexpectedly obtained that the nitrile hydratase mutation site of the present application also exerts a regulatory effect in nitrile hydratases from different sources, which can further improve the thermal stability and product tolerance of the nitrile hydratase, and has a certain activity.
  • the embodiments of the present application can provide a nitrile hydratase mutant having thermal stability, product tolerance, or catalytic activity.
  • the nitrile hydratase mutant is very useful in the production of amide compounds.
  • the embodiments of the present application can further provide a nucleic acid molecule encoding the mutant nitrile hydratase, an expression vector comprising the nucleic acid molecule encoding the mutant nitrile hydratase, and a transformant comprising the expression vector.
  • the embodiments of the present application can further provide a method for preparing an amide compound, which utilizes the nitrile hydratase extracted from the culture of the transformant and its production, and uses the aforementioned nitrile hydratase or uses the culture or a processed product of the culture.
  • FIG1 shows a schematic diagram of the spatial positions of mutation sites 1-3 according to an embodiment of the present application based on the spatial conformation of nitrile hydratase from Rhodococcus rubrum.
  • FIG. 2 shows a graph showing the substrate consumption effects of the wild-type nitrile hydratase and the mutant M133 nitrile hydratase of the examples of the present application.
  • FIG3 shows the residual activity effect diagram of the wild-type nitrile hydratase and the mutant M133 nitrile hydratase of the example of the present application.
  • the / symbol means "or"
  • the () symbol indicates the amino acids at the same position.
  • (Y/F)(A/S/T) means that the two amino acid residues can be YA, YS, YT, FA, FS or FT.
  • wild type refers to a gene or gene product that is separated from a naturally occurring source.
  • a wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal” or "wild-type” form of a gene.
  • modified refers to a gene or gene product that displays sequence modification (e.g., substitution, truncation or insertion), post-translational modification and/or functional properties (e.g., altered properties) compared to a wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to a wild-type gene or gene product.
  • methionine (M) can be replaced by arginine (R) by replacing the codon (ATG) of methionine at the relevant position in the polynucleotide encoding the mutant monomer.
  • Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art.
  • Nitrile hydratase is a metal-dependent enzyme with a non-heme iron atom or cobalt atom in the active center, which can be divided into two types: Fe-type and Co-type.
  • nitrile hydratases all have two subunits, ⁇ and ⁇ (or called 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)-).
  • This study selected Co-type nitrile hydratase as the research object. Specifically, the inventors selected a variety of wild-type nitrile hydratases from Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus rubrum or Nocardia.
  • the technical concept of the present application is as follows: starting from the wild-type nitrile hydratase, the inventors analyzed the crystal structure information of the enzyme, locked the key regulatory amino acid residues through structural analysis, molecular dynamics simulation and enzyme molecular sequence alignment, and constructed a nitrile hydratase mutant library through targeted design, and obtained a series of mutant strains with improved catalytic performance, which were significantly improved in stability and product tolerance compared with the parent strain; on this basis, the inventors introduced beneficial mutation sites into nitrile hydratases from different sources, and unexpectedly obtained that the mutation sites of the nitrile hydratase of the present application also exerted a regulatory effect in nitrile hydratases from different sources, which can further improve the thermal stability and product tolerance of nitrile hydratase.
  • nitrile hydratase molecules as the research object, through structure-function relationship research, the interaction between substrate and enzyme molecules was analyzed, and the enzyme molecules were reconstructed to improve their stability and product tolerance at the molecular level.
  • the embodiments of the present application provide a nitrile hydratase mutant, based on a wild-type nitrile hydratase, wherein the nitrile hydratase mutant is a mutated amino acid residue on the flexible loop domain of the wild-type nitrile hydratase, comprising any one or a combination of mutations 1-3:
  • Mutation 1 a polar amino acid mutation located in the flexible loop between the secondary domains S1 and S2 of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation 2 a polar amino acid mutation in a flexible loop between the secondary domains H6 and H7 at the start end of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation 3 A hydrophobic amino acid mutation located on the initial flexible loop L1 of the wild-type nitrile hydratase ⁇ subunit.
  • the positions of the secondary structural domains S1 and S2, the secondary structural domains H6 and H7, and the initial flexible loop L1 may be as shown in FIG. 1 .
  • 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.
  • the inventors used molecular docking to simulate the binding state of substrate and enzyme molecules, and studied the binding process of substrate and enzyme molecules through molecular dynamics simulation, analyzed the interaction force between substrate and enzyme molecules, determined the amino acid sites that may affect the catalytic performance of the enzyme, and combined with multiple sequence alignment analysis to lock the candidate amino acid residues, and conducted directional design based on structural position and evolutionary conservation analysis, constructed a mutant library, and screened out multiple mutants with improved stability and product tolerance through catalytic performance evaluation.
  • the amino acid sequence of its ⁇ subunit is shown in SEQ ID NO: 1
  • the amino acid sequence of the ⁇ subunit is shown in SEQ ID NO: 2
  • the nitrile hydratase mutant of the present application comprises any one of the above mutations or any combination thereof based on the amino acid sequence of the above-mentioned wild-type nitrile hydratase.
  • the wild-type nitrile hydratase is derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus, and Nocardia.
  • the wild-type nitrile hydratase may be derived from any one of Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythronicus or Nocardia.
  • the wild-type nitrile hydratase has a sequence selected from the group consisting of:
  • Rhodococcus ruber TH The nitrile hydratase of wild-type Rhodococcus ruber TH, the amino acid sequence of its ⁇ subunit is shown in SEQ ID NO: 1, the amino acid sequence of its ⁇ 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.
  • Nitrile hydratase from Rhodococcus rhodochrous J1 whose UPI number in the UniProt Archive database is P21220 for the ⁇ subunit and P21219 for the ⁇ subunit, the entire contents of which are incorporated herein by reference.
  • Nitrile hydratase from Rhodococcus rhodochrous M8 the GenBank accession numbers of its ⁇ subunit are AAT79339.1, and ⁇ subunit is AAT79340.1, the entire contents of which are incorporated herein by reference.
  • Nitrile hydratase from Rhodococcus pyridinivorans whose UPI number in the UniProt Archive database is Q2UZQ6 for the ⁇ subunit and Q2UZQ5 for the ⁇ subunit, the entire contents of which are incorporated herein by reference.
  • Nitrile hydratase from Rhodococcus sp. whose UPI number in the UniProt Archive database is Q59785 for the ⁇ subunit and Q59786 for the ⁇ subunit, the entire contents of which are incorporated herein by reference.
  • Nitrile hydratase from Nocardia sp. JBRs whose UPI number in the UniProt Archive database is Q8GE66 for the ⁇ subunit and Q8GE67 for the ⁇ subunit, the entire contents of which are incorporated herein by reference.
  • the nitrile hydratase mutant satisfies at least one of the following conditions:
  • the mutation of the amino acid site 152 of the ⁇ subunit can be understood as: the 152th amino acid site of the ⁇ subunit from the starting N-terminus mutates.
  • the mutation of the amino acid site 110 of the ⁇ subunit can be understood as: the 110th amino acid site of the ⁇ subunit from the starting N-terminus mutates.
  • the mutation of the amino acid site 17 of the ⁇ subunit can be understood as: the 17th amino acid site of the ⁇ subunit from the starting N-terminus mutates.
  • the above mutations can exist alone or in any combination.
  • the amino acid residue at amino acid position 152 is mutated to any one of cysteine, threonine, glutamic acid, aspartic acid, valine, serine, glycine, and glutamine.
  • the amino acid residue at the amino acid position 152 of the ⁇ subunit mutates to any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G.
  • the amino acid residue at amino acid position 110 is mutated to any one of alanine, arginine, serine, threonine, valine, phenylalanine, leucine, isoleucine, lysine, and histidine.
  • amino acid residue at amino acid position 110 mutates to any one of 110A, 110R, 110S, 110T, 110V, 110F, 110L, 110I, and 110K.
  • the amino acid residue at amino acid position 17 is mutated to any one of alanine, arginine, glutamic acid, aspartic acid, serine, cysteine, lysine, and histidine.
  • the amino acid residue at the 17th amino acid position of the ⁇ subunit from the starting N-terminus mutates to any one of 17A, 17R, 17E, 17D, 17S, 17C, 17K, and 17H.
  • the nitrile hydratase mutant comprises any combination of mutation 1 and mutation 2.
  • mutants M26-M30 For example, mutants M26-M30.
  • the nitrile hydratase mutant comprises any combination of mutation 1 and mutation 3.
  • the nitrile hydratase mutant comprises any combination of mutation 2 and mutation 3.
  • the nitrile hydratase mutants include mutation 1, mutation 2, and mutation 3.
  • the nitrile hydratase mutant comprises at least one of an amino acid residue at position 167 of the ⁇ subunit mutated to serine, an amino acid residue at position 174 of the ⁇ subunit mutated to leucine, and an amino acid residue at position 178 of the ⁇ subunit mutated to glutamate.
  • the 167th amino acid from the starting N-terminus of the ⁇ subunit is ⁇ 167S
  • the 174th amino acid from the starting N-terminus of the ⁇ subunit is ⁇ 174L
  • the 178th amino acid from the starting N-terminus of the ⁇ subunit is ⁇ 178E.
  • the above mutations can exist alone and be combined with any combination of mutations in mutations 1 to 3.
  • the above mutations can exist in any combination and be combined with any combination of mutations in mutations 1 to 3.
  • the nitrile hydratase mutant further comprises any one or a combination of asparagine at position 167 of the ⁇ subunit mutated to serine, glycine at position 174 of the ⁇ subunit mutated to leucine, and aspartic acid at position 178 of the ⁇ subunit mutated to glutamic acid.
  • the above mutations of this embodiment can exist in any combination and be combined with any of the above embodiments and mutations comprising any combination of mutations 1 to mutation 3.
  • the nitrile hydratase mutant comprises: a domain mutation, which includes: the amino acid residue at the amino acid position 141 of the ⁇ subunit mutates to lysine, the amino acid residue at the amino acid position 143 mutates to lysine, the amino acid residue at the amino acid position 144 mutates to glutamate, the amino acid residue at the amino acid position 215 mutates to cysteine, and the amino acid residue at the amino acid position 133 of the ⁇ subunit mutates to cysteine.
  • the 141st amino acid at the N-terminal end of the ⁇ subunit is ⁇ 141K
  • the 143rd amino acid at the N-terminal end of the ⁇ subunit is ⁇ 143K
  • the 144th amino acid at the N-terminal end of the ⁇ subunit is ⁇ 144E
  • the 215th amino acid from the starting N-terminus is ⁇ 215C
  • the 133rd amino acid from the starting N-terminus of the ⁇ subunit is ⁇ 133C.
  • the above mutations are also present in the domain mutations. They can be combined with mutations in other embodiments at will.
  • the nitrile hydratase mutant comprises: a combination of mutation of serine at position 141 of the ⁇ subunit to lysine, mutation of serine at position 143 to lysine and mutation of leucine at position 144 to glutamate, a combination of mutation of aspartic acid at position 215 to cysteine and mutation of proline at position 133 of the ⁇ subunit to cysteine, and any combination of any mutant of the above embodiment and coexistence.
  • the nitrile hydratase mutant includes at least one of 17A/17R/17E/17D/17S/17C/17K/17H, 110A/110R/110S/110T/110V/110F/110L/110I/110K, and 152C/152A/152T/152E/152D/152V/152S/152Q/152G.
  • the 17th amino acid of the ⁇ subunit from the starting N-terminus can be any one of 17A, 17R, 17E, 17D, 17S, 17C, 17K, and 17H
  • the 110th amino acid of the ⁇ subunit from the starting N-terminus can be any one of 110A, 110R, 110S, 110T, 110V, 110F, 110L, 110I, and 110K
  • the 152nd amino acid of the ⁇ subunit from the starting N-terminus can be any one of 152C, 152T, 152E, 152D, 152V, 152S, 152Q, and 152G.
  • the nitrile hydratase mutant of the embodiment of the present application includes any one of the above three mutations, or a random combination of any one of them.
  • the sites of the nitrile hydratase include mutations in three of the sites, and the specific forms of the mutations include: 17S/110R/152C, 17R/110R/152D, 17S/110K/152C, 17S/110R/152C, 17S/110R/152C, etc.
  • 17S/110R/152C can be understood as: the 17th amino acid from the starting N-terminus of the ⁇ subunit is serine, the 110th amino acid from the starting N-terminus of the ⁇ subunit is arginine; the 152nd amino acid from the starting N-terminus of the ⁇ subunit is cysteine.
  • Nitrile hydratase activity refers to the activity of catalyzing the hydration of nitrile compounds to produce amide compounds.
  • the substrate nitrile compound
  • the substrate 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.
  • any nitrile compound can be used as long as the nitrile hydratase reacts, and acrylonitrile and nicotinonitrile can be selected.
  • the reaction conditions are the general conditions of the hydration reaction, as long as the nitrile hydratase can be guaranteed to have catalytic activity, and the consumption of the substrate and the increase of the product can be detected and quantitatively analyzed by HPLC and GC.
  • the R group is an optionally substituted linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, an optionally substituted cycloalkyl or aryl group having 3 to 18 carbon atoms, or an optionally substituted saturated or unsaturated heterocyclic group.
  • the R group is an optionally substituted linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, an optionally substituted cycloalkyl or aryl group having 3 to 18 carbon atoms, or an optionally substituted saturated or unsaturated heterocyclic group.
  • the wild-type nitrile hydratase and mutant nitrile hydratase described in the present application have catalytic activity.
  • the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the nitrile hydratase mutant of the first aspect.
  • the nucleic acid molecule comprises a nucleotide sequence encoding the nitrile hydratase mutant.
  • the nucleotide sequence may be obtained by base mutation based on the sequence of the wild-type nitrile hydratase.
  • the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
  • 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.
  • pET series conventional plasmids
  • shuttle vectors pNV18.1 phage or viral vectors, etc. in the art.
  • 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.
  • 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.
  • the coding gene of the nitrile hydratase mutant of the embodiment of the present application can be constructed into a recombinant expression plasmid.
  • the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.
  • the isolated nucleic acid molecule described in the second aspect of the present application can be directly inserted into the chromosome of the host bacteria, or the expression vector of the third aspect can be introduced into the host bacteria by the calcium chloride method or electroporation transformation method.
  • the host cell includes any one of Escherichia coli, Rhodococcus, Nocardia, Bacillus subtilis or Corynebacterium glutamicum.
  • the host cell is Rhodococcus ruber and/or Escherichia coli BL21 (DE3).
  • the present application provides a catalyst comprising the nitrile hydratase mutant of the first aspect.
  • the nucleotide sequence encoding the nitrile hydratase mutant can be constructed into different types of recombinant vectors, or directly integrated into the chromosome of the host bacteria.
  • vector used plasmid DNA, bacteriophage DNA, reverse transcription transposon DNA, artificial chromosome DNA, etc. can be listed. Taking Escherichia coli and Rhodococcus rubrum as examples, PET series vectors and PNV series vectors can be selected.
  • the host that can be used for the transformant of the present application is not particularly limited as long as it can express the target nitrile hydratase after the introduction of the above-mentioned recombinant vector or the nucleotide sequence encoding the nitrile hydratase mutant.
  • bacteria such as Rhodococcus and Escherichia coli, yeast, animal cells, insect cells, plant cells, etc. can be used.
  • the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include a method using calcium ions and an electroporation method.
  • the method for integrating the nucleotide sequence encoding the nitrile hydratase mutant into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include homologous recombination and gene editing.
  • the catalyst is a whole cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst.
  • the whole cell catalyst refers to the whole cell obtained after enrichment culture and induced expression of the target protein by the host cell constructed by the fourth aspect of the present application.
  • the free protein catalyst is a crude enzyme solution obtained by ultrasonic crushing or high-pressure homogenization crushing and centrifugation of the whole cell, and also includes pure enzymes obtained by protein purification.
  • the immobilized enzyme catalyst is to select different immobilized carriers and immobilize the free protein catalyst to obtain different forms of immobilized nitrile hydratase mutants.
  • 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;
  • the nitrile compound is selected from acrylonitrile, nicotinonitrile, cinnamon nitrile, benzyl acetonitrile or p-hydroxyphenyl acetonitrile.
  • the amide substance is selected from acrylamide, niacinamide, cinnamamide, phenylacetamide or p-hydroxyphenylacetamide.
  • the inventors designed Nco I 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 enzyme digestion on the plasmid vector and the nucleotide sequence of nitrile hydratase in this embodiment, used nucleic acid electrophoresis (1.0% agarose) and a kit to recover the gene fragments after enzyme digestion, and then connected the target gene fragments after enzyme digestion to the plasmid vector after enzyme digestion.
  • 10 ⁇ L ligation system includes:
  • the cells were connected overnight at 16°C and transformed into BL21 (DE3) competent cells. Single clones were selected for sequencing verification, and the recombinant plasmid with correct sequencing was extracted to obtain a recombinant expression vector containing the nitrile hydratase encoding gene.
  • This application uses the whole plasmid amplification method to construct a recombinant plasmid containing a mutant gene of nitrile hydratase.
  • the upstream and downstream primers are designed for the desired mutant site, and the whole plasmid amplification is performed using PrimeSTAR HS DNA Polymerase (Takara) with the plasmid pET28a-NH as a template.
  • the recombinant plasmid containing the mutated sequence of the gene encoding nitrile hydratase is amplified by PCR.
  • the 20 ⁇ L PCR reaction system includes:
  • the forward primer is a specific primer used in the construction process for different mutants.
  • the reverse primer is a specific primer used in the construction process for different mutants. Due to space limitations, this is not described in detail, 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.
  • 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 ⁇ g/mL) and placed in an incubator at 37°C for inversion culture for about 12 hours. Single clones were selected for sequencing verification. After the sequencing was correct, 20% (V/V) glycerol was used to preserve the bacteria and stored in a refrigerator at -70°C.
  • the nitrile hydratase combination mutants are obtained by multiple rounds of site-directed mutagenesis. After obtaining a single point mutant, a recombinant plasmid containing the mutation site is used as a template, upstream and downstream primers of the mutation site are designed, and whole-plasmid PCR amplification is performed. A recombinant plasmid containing a gene sequence encoding the nitrile hydratase combination mutants is amplified by PCR.
  • 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.
  • 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 ⁇ g/mL) and placed in an incubator at 37°C for about 12 hours. Single clones were picked for sequencing verification.
  • the genetically engineered Escherichia coli prepared in Examples 3 and 4 were coated on LB plates containing kanamycin (50 ⁇ g/mL) and cultured overnight at 37°C. Single colonies were picked and transferred to LB liquid culture containing 50 ⁇ g/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 Escherichia coli as the host.
  • 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 is based on the suicide plasmid pYsacB, with the addition of homologous arm sequences of about 1000 bp at both ends of the nitrile hydratase gene.
  • the obtained nitrile hydratase mutant gene was connected to the suicide plasmid pYsacB after enzyme digestion using the Gibson Assembly seamless cloning kit.
  • the 20 ⁇ L ligation system includes:
  • a final concentration of 0.08 mM Co 2+ was added to the fermentation medium. After induced expression for 48 hours, the cells were collected by centrifugation to obtain the Rhodococcus bacteria cell catalyst of nitrile hydratase.
  • the method for determining the activity of nitrile hydratase on the substrate acrylonitrile is as follows: Take 50-100 ⁇ L 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 ⁇ L of acrylonitrile, mix well and react for 5 minutes, and add 200 ⁇ L of 3 mol/L hydrochloric acid to terminate the reaction.
  • the amount of product generated was detected by gas phase, and the detection conditions were as follows: Thermo Fisher Trace 1300 gas chromatograph; Abel Bonded AB-I NOWAX column (inner diameter 0.25mm, length 30m, film thickness 0.25 ⁇ m); FID detector.
  • the 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;
  • the injection volume was 1 ⁇ L, split injection, and the split ratio was 30:1.
  • the activity of the nitrile hydratase is calculated as follows:
  • the enzyme activity (U) is defined as: under the above reaction conditions, the amount of enzyme required to catalyze 1 ⁇ mol of substrate per minute is one enzyme activity unit, represented by U.
  • mutant M133 After testing, the substrate consumption rate of mutant M133 is shown in FIG2 . Compared with the wild-type nitrile hydratase, it still has good catalytic ability and can catalyze the substrate to react.
  • nitrile hydratase catalysts obtained in Examples 5 and 6 were resuspended in deionized water (10 g/L), placed in a constant temperature water bath at 55° C., and samples were taken out at intervals of 2 h.
  • the residual activity was determined according to the method in Example 7, and the inactivation curve of the catalyst was plotted.
  • the enzyme inactivation half-life was obtained by linear fitting. The higher the half-life, the better the stability, which was used to measure the thermal stability of nitrile hydratase.
  • the nitrile hydratase catalysts obtained in Examples 5 and 6 were resuspended in deionized water (10 g/L), and an appropriate amount of the catalyst was placed in a 10-mL centrifuge tube, and acrylamide was added to a final concentration of 50% (v/v). After standing still in a constant temperature water bath at 28° C. for 2 h, the residual activity of the catalyst was measured, and the inactivation curve of the catalyst was drawn. The enzyme inactivation half-life was linearly fitted. The higher the half-life, the better the stability, which was used to measure the product tolerance of the nitrile hydratase.
  • mutant M133 After testing, the residual activity of mutant M133 is shown in FIG3 , which still has better activity than the wild-type nitrile hydratase.
  • the mutants constructed in Examples 3 and 4 were subjected to stability and tolerance tests according to the method of Example 8. The results showed that the thermal stability and tolerance of the obtained nitrile hydratase mutants were improved to varying degrees.
  • the product tolerance of mutants M1-M35 increased by 5.1-6.0 times, and the thermal stability of mutants M1-M35 increased by 5.1-6.0 times;
  • the product tolerance of mutants M36-M119, M121-M125, M127-M130, M133 and M134 increased by 6.1-8.0 times;
  • the thermal stability of mutants M36-M119, M121-M125, M127-M130, M133 and M134 increased by 6.1-8.0 times;
  • the product tolerance of mutants M120, M126, M131, M132, M135-M139 increased by 8.1-12.0 times.
  • Example 1 This application studies the regulatory effect of the beneficial mutation sites described in Example 1 on nitrile hydratases from other sources (Table 1). First, the amino acid residues located at similar positions in other nitrile hydratases were determined by multiple sequence alignment, and the mutants designed in Examples 1-9 were used for mutation and performance determination.
  • the nitrile hydratase derived from Rhodococcus rhodochrous J1 has a UPI number of P21220 for its ⁇ subunit in the UniProt Archive database, and P21219 for its ⁇ subunit.
  • the nitrile hydratase derived from Rhodococcus rhodochrous M8 has a GenBank accession number of AAT79339.1 for its ⁇ subunit and AAT79340.1 for its ⁇ subunit.
  • the nitrile hydratase from Rhodococcus pyridinivorans RP has a UPI number of Q2UZQ6 for its ⁇ subunit and Q2UZQ5 for its ⁇ subunit in the UniProt Archive database.
  • the nitrile hydratase from Rhodococcus sp. has a UPI number of Q59785 for its ⁇ subunit and Q59786 for its ⁇ subunit in the UniProt Archive database.
  • the obtained mutant nitrile hydratase has enzyme activity, and the thermal stability and product tolerance are improved, indicating that these mutation sites have a beneficial regulatory effect on the thermal stability and product tolerance of nitrile hydratase from different sources.
  • the results are shown in Table 3.
  • ⁇ 110R/ ⁇ 152D/ ⁇ 17D indicates the simultaneous existence of amino acid sites 110, 152, and 17 in the ⁇ subunit. Other descriptions in this way also indicate the simultaneous existence.

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Abstract

一种腈水合酶突变体及其应用，腈水合酶突变体为野生型腈水合酶的柔性环结构域上的氨基酸残基发生突变，包含突变1-3中的任意的一项或其组合：突变1：位于野生型腈水合酶β亚基的二级结构域S1和S2之间的柔性环上的极性氨基酸突变；突变2：位于野生型腈水合酶β亚基的起始端的二级结构域H6和H7之间的柔性环上的极性氨基酸突变；突变3：位于野生型腈水合酶β亚基的起始柔性环L1上的疏水性氨基酸突变。腈水合酶突变体能在野生型腈水合酶的基础上，包含上述任意一项或其组合的突变，其单独或协同发挥调控效应，具有一定酶活力，具有良好的热稳定性和耐受性。

Description

腈水合酶突变体及其应用
相关申请的交叉引用
本申请要求享有于2023年8月21日提交的名称为“腈水合酶突变体及其应用”的中国专利申请202311054507.0的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本发明属于蛋白质工程和基因工程领域，涉及一种腈水合酶突变体，包含该突变体的基因工程菌及其应用。
背景技术
酰胺类化合物是重要的有机合成中间体和化学品，被广泛应用于医药、农药、饲料、食品、环保、采油、材料等领域。从腈类化合物出发通过水合反应可获得相应的酰胺化合物，也是工业上制备此类化合物的重要方法之一。腈水合酶(Nitrile hydratase,简称NHase,EC 4.2.1.84)是一类能够将腈化合物通过水合作用转化生成相应酰胺类物质的多亚基酶。利用腈水合酶催化剂，催化丙烯腈、烟腈水合生产丙烯酰胺和烟酰胺，是生物技术替代化学法工业化生产大宗化学品最成功的案例之一。
腈水合酶通常由α和β两个亚基组成(或被称之α和β亚基)，在红球菌属、诺卡氏菌、短杆菌和假单胞菌等多种微生物中均有分布。早在1980年，日本学者Asano首次在玫瑰色红球菌R.rhodochrous J1中发现此类酶并将其定义为腈水合酶(Asano Y,et al.Agricultural and Biological Chemistry,1982,46(5):1165-1174.)。利用腈水合酶催化丙烯腈生产丙烯酰胺已有数十年的研究历程，所使用的生产菌株也已经历了三代，其中玫瑰色红球菌Rhodococcus rhodochrous J1(Nagasawa T,et al.Applied Microbiology and Biotechnology,1993,40(2-3):189-195)、红色红球菌R.ruber TH及其基因工程菌被广泛地用于工业化生产丙烯酰胺(Ma Y,et al.Bioresource Technology,2010,101(1):285-291)。
在催化丙烯腈的水合过程中，腈水合酶催化剂往往面临着水合反应的放热效应引起温度骤升问题，导致酶的失活以及细胞的破裂，造成经济效益下降。此外高产物浓度下对细胞的毒害作用较大，会加催化剂的失活。因此构建高稳定性、高产物耐受性的新一代催化剂是酰胺化合物工业生产的重要需求。野生型腈水合酶多数都存在稳定性、产物耐受性较差、催化活性不足等问题。为了进一步提高腈水合酶的生产性能，研究者从在酶分子层面上通过盐桥和二硫键的设计及分子伴侣的引入等多个方面进行了深入的研究，并提高了腈水合酶的稳定性(Liu Y,et al.Applied Biochemistry and Biotechnology,2016,180(1):3-9；Ma Y,et al.Journal of Industrial Microbiology &  Biotechnology,2012,39(10):1421-1430；Tian Y,et al.Journal of Microbiology and Biotechnology,2016,26(2):337-346；Jiao S,et al.Applied Microbiology and Biotechnology,2020,104(3):1001-1012.)。
随着生物信息学技术和蛋白质晶体学科的飞速发展，计算机模拟和蛋白质晶体结构被越来越多地应用于指导蛋白质工程研究中，从而发展出半理性设计、理性设计等多种蛋白质工程手段。尽管一些腈水合酶的晶体结构已经被解析，但由于腈水合酶催化机理较为复杂，至今仍无定论，增加了理性设计和半理性设计的难度，因此关于腈水合酶改造进展较为缓慢，而为了满足工业需求，如何快速高效地获取催化性能提升的新型工业催化剂成为研究的热点和难点。这主要是由于腈水合酶催化机理较为复杂，增加了理性设计和半理性设计的难度，因此关于腈水合酶改造进展较为缓慢。
发明内容
因此，本申请的目的是在现有技术的基础上，提供一种来腈水合酶突变体。本申请腈水合酶突变体能在野生型腈水合酶的基础上，包含上述突变，其单独或发挥协同调控效应，具有一定酶活力，且具有更好的热稳定性和耐受性。
第一方面，本申请实施例提供了一种腈水合酶突变体，基于野生型腈水合酶，所述腈水合酶突变体为野生型腈水合酶的柔性环结构域上的氨基酸残基发生突变，包含突变1-3中的任意的一项或其组合：
突变1：位于野生型腈水合酶β亚基的二级结构域S1和S2之间的柔性环上的极性氨基酸突变；
突变2：位于野生型腈水合酶β亚基的起始端的二级结构域H6和H7之间的柔性环上的极性氨基酸突变；
突变3：位于野生型腈水合酶β亚基的起始柔性环L1上的疏水性氨基酸突变。
根据本申请一个方面的实施例，野生型腈水合酶来源于玫瑰色红球菌、嗜吡啶红球菌、红色红球菌、诺卡氏菌中的任意一种。
根据本申请一个方面的实施例，腈水合酶突变体满足下列条件中的至少一项：
1)突变1中，β亚基的152位氨基酸位点发生突变；
2)突变2中，β亚基的110位氨基酸位点发生突变；
3)突变3中，β亚基的17位氨基酸位点发生突变。
根据本申请一个方面的实施例，152位氨基酸位点的氨基酸残基突变为半胱氨酸、苏氨酸，谷氨酸、天冬氨酸、缬氨酸、丝氨酸、甘氨酸、谷氨酰胺中的任意一种。
根据本申请一个方面的实施例，110位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、丝氨酸、苏氨酸、缬氨酸、苯丙氨酸、亮氨酸、异亮氨酸、赖氨酸、组氨酸中的任意一种。
根据本申请一个方面的实施例，17位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、半胱氨酸、赖氨酸、组氨酸中的任意一种。
根据本申请一个方面的实施例，腈水合酶突变体包括突变1和突变3的任意组合。
根据本申请一个方面的实施例，腈水合酶突变体包括突变1和突变2的任意组合
根据本申请一个方面的实施例，腈水合酶突变体包括突变2和突变3的任意组合。
根据本申请一个方面的实施例，腈水合酶突变体包括突变1、突变2和突变3。
根据本申请一个方面的实施例，腈水合酶突变体包含β亚基167位氨基酸位点的氨基酸残基突变为丝氨酸，α亚基174位的氨基酸位点的氨基酸残基突变为亮氨酸，α亚基178位的氨基酸位点的氨基酸残基突变为谷氨酸中的至少一种。
根据本申请一个方面的实施例，腈水合酶突变体包含：结构域突变，其包括：β亚基141位氨基酸位点的氨基酸残基突变为赖氨酸，143位氨基酸位点的氨基酸残基突变为赖氨酸，144位氨基酸位点的氨基酸残基突变为谷氨酸，215位氨基酸位点的氨基酸残基突变为半胱氨酸，α亚基133位氨基酸位点的氨基酸残基突变为半胱氨酸。
根据本申请一个方面的实施例，腈水合酶突变体包括17A/17R/17E/17D/17S/17C/17K/17H、110A/110R/110S/110T/110V/110F/110L/110I/110K、152C/152A/152T/152E/152D/152V/152S/152Q/152G中的至少一项。
根据本申请一个方面的实施例，β17S/β110R/β152C、β17R/β110R/β152D、β17S/β110K/152C、β17S/β110R/β152C、β17S/β110R/β152C、β110K/β152T、β110K/β152D、β110R/β152T、β110K/β17E、β110K/β17C、β110K/β17D、β152S/β17H、β152S/β17E、β110K/β152S/β17E、β17A/β110K/β152G/α174L/α178E、β17E/β110R/β152S/α174L/α178E、β17S/β110V/β152S/βS141K/β143K/β144E、β17D/β110K/β152V/α174L/α178Eβ110R/β152S/β17D、β17E/β110K/β152S/β167S/α174L/α178E、β17E/β110K/β152S/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E、β17R/β110S/β152Q/β167S/α174L/α178E/β215C/α133C//βS141K/β143K/β144E、β17D//β110F/β152C/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E中的任意一项。
第二方面，本申请实施例提供了一种分离的核酸分子，核酸分子包含编码如第一方面的腈水合酶突变体的核苷酸序列。
第三方面，本申请实施例提供了一种表达载体，其包含第二方面的核酸分子。
根据本申请一个方面的实施例，表达载体为质粒载体。
根据本申请一个方面的实施例，表达载体为pET系列、穿梭载体、噬菌体或病毒载体。
第三方面，本申请实施例提供了一种宿主细胞，其包含第二方面的核酸分子或第三方面的表达载体。
根据本申请一个方面的实施例，宿主细胞包括大肠杆菌、红球菌、诺卡氏菌、枯草芽孢杆菌或谷氨酸棒状杆菌中的任意一种。
根据本申请一个方面的实施例，宿主细胞为红色红球菌或/和大肠杆菌E.coli BL21(DE3)。
第四方面，本申请提供了一种催化剂，其包含第一方面的腈水合酶突变体。
根据本申请一个方面的实施例，所述催化剂为全细胞催化剂、游离蛋白催化剂或固定化酶催化剂。
第五方面，本申请实施例提供了一种酰胺类化合物的制备方法，方法包括使用第一方面或第四方面的催化剂催化腈类化合物发生水合反应，得到酰胺类化合物。
根据本申请一个方面的实施例，腈类化合物选自丙烯腈、烟腈、肉桂腈、苯乙腈或对羟基苯乙腈。
根据本申请一个方面的实施例，酰胺类物质选自丙烯酰胺、烟酰胺、肉桂酰胺、苯乙酰胺或对羟基苯乙酰胺。
本申请的技术方案至少具有以下优势：
本申请实施例的腈水合酶突变体具有上述突变位点，显著提高了腈水合酶的热稳定性、产物耐受性；将上述突变位点进一步引入到不同来源的腈水合酶中，意外地获得了本申请的腈水合酶突变位点在不同来源的腈水合酶中同样发挥调控效应，可进一步提升腈水合酶的热稳定性和产物耐受性，且具有一定的活力。
本申请实施例能够提供具有热稳定性、产物耐受性，或者催化活性的腈水合酶突变体。腈水合酶突变体在酰胺化合物的制造上是非常有用的。
本申请实施例能够进一步提供编码上述突变型腈水合酶的核酸分子、包含编码上述突变型腈水合酶的核酸分子的表达载体、包含该表达载体的转化体。本申请实施例能够进一步提供酰胺类化合物的制备方法，该方法利用了从该转化体的培养物中提取的腈水合酶及其制造、以及使用了前述腈水合酶或者使用了该培养物或该培养物的处理物。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将结合附图详细说明本申请的技术方案：
图1示出了基于红色红球菌的腈水合酶空间构象的包含本申请实施例的突变1-3位点的空间位置示意图。
图2示出了野生型腈水合酶与本申请实施例的突变体M133腈水合酶的底物消耗效果图。
图3示出了野生型腈水合酶与本申请实施例的突变体M133腈水合酶的残余活力效果图。
具体实施方式
下面将详细描述本申请的各个方面的特征和示例性实施例。本发明所列举的具体实施例只作为本申请的范例，本发明并不限制于下文所描述的具体实施例。
对于本领域技术人员而言，任何对下文所述的实施例进行的等同修改和替代也都在本申请的范畴之中。因此，在不脱离本申请的精神和范围下所作的均等变换和修改，都应涵盖在本申请的范围内。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所有试剂或仪器未注明生产厂商者，均为可以通过市购的常规产品。为了更好地说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实施例中，对于本领域技术人员熟知的方法、手段、器材和步骤未作详细描述，以便凸显本申请的主旨。
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。如无特殊说明，本说明书中所使用的单位均为国际标准单位，并且本发明中出现的数值和数值范围，应当理解为包含了工业生产中所不可避免的系统性误差。
在本文的所有讨论中，使用针对氨基酸的标准单字母代码。也使用标准的取代记法，即β110A意指从β亚基起始N端处第110位的氨基酸残基被丙氨酸(A)置换；或α174L意指从α亚基起始N端处第174位的氨基酸残基被亮氨酸(L)置换。
对于特定位置处的不同氨基酸通过/符号分隔的本文段落中，“/”符号意指“和”或者“或”。举例来说β17S/β110R/β152S/βS141K/β143K/β144E/β215C/α133C可以表示上述突变均存在于腈水合酶中。
对于特定位置处的不同氨基酸通过/符号分隔的本文段落中，并用括号包括，其中/符号意指“或”，()符号表明同一位置处的氨基酸。举例来说，(Y/F)(A/S/T)意指两个氨基酸残基可以为YA、YS、YT、FA、FS或FT。
在不同位置处通过/符号分隔的本文段落中，“/”符号意指“和”以使得Y51/N55是Y51和N55。
术语“野生型”是指与天然存在的来源分离的基因或基因产物。野生型基因是群体中最常观察到的基因，并且因此被任意设计为基因的“正常”或“野生型”形式。相反，术语“经修饰的”，“突变体”或“变体”是指与野生型基因或基因产物相比显示序列的修饰(例如，取代、截短或插入)，翻译后修饰和/或功能特性(例如，改变的特性)的基因或基因产物。注意，天然存在的突变体可以被分离；通过与野生型基因或基因产物相比其具有改变的特性这一事实来鉴定这些突变体。用于引入或取代天然存在的氨基酸的方法在本领域是众所周知的。举例来说，可通过在编码突变单体的多核苷酸中的相关位置处用精氨酸的密码子(CGT)置换甲硫氨酸的密码子(ATG)，而用精氨酸(R)来取代甲硫氨酸(M)。用于引入或取代非天然存在的氨基酸的方法在本领域也是众所周知的。
野生型腈水合酶
腈水合酶(nitrile hydratase，NHase)是一种金属依赖性酶，活性中心含有非血红素铁原子或钴原子，可分为Fe-型和Co-型两种。目前已知的腈水合酶均有α和β两个亚基(或被称之a和b亚基)以异源多聚体形式存在，活性中心的氨基酸残基较为保守(-C(S/T)LCSC(T/Y)-)。本研究选择Co-型腈水合酶为研究对象。具体地，发明人选取了来源于玫瑰色红球菌、嗜吡啶红球菌、红色红球菌或诺卡氏菌的多种野生型腈水合酶。
腈水合酶突变体
本申请的技术构思如下：发明人从野生型腈水合酶出发，解析了该酶的晶体结构信息，通过结构分析、分子动力学模拟和酶分子序列比对，锁定了调控关键氨基酸残基，通过定向设计，构建了腈水合酶突变体库，获得了一系列催化性能提升的突变株，相比母本稳定性和产物耐受性得到显著提升；在此基础上，发明人将有益突变位点引入到不同来源的腈水合酶中，意外地获得了本申请腈水合酶突变位点在不同来源的腈水合酶中同样发挥调控效应，可进一步提升腈水合酶的热稳定性和产物耐受性。
以腈水合酶分子为研究对象，通过结构-功能关系研究，分析底物与酶分子间相互作用，并对酶分子进行重构，从分子水平上提升其稳定性和产物耐受性。
本申请的目的是通过以下技术方案实现的：
第一方面，本申请实施例提供了一种腈水合酶突变体，基于野生型腈水合酶，所述腈水合酶突变体为野生型腈水合酶的柔性环结构域上的氨基酸残基发生突变，包含突变1-3中的任意的一项或其组合：
突变1：位于野生型腈水合酶β亚基的二级结构域S1和S2之间的柔性环上的极性氨基酸突变；
突变2：位于野生型腈水合酶β亚基的起始端的二级结构域H6和H7之间的柔性环上的极性氨基酸突变；
突变3：位于野生型腈水合酶β亚基的起始柔性环L1上的疏水性氨基酸突变。
根据本申请实施例，二级结构域S1和S2，二级结构域H6和H7，起始柔性环L1中的位置可以如图1所示。
在现有的野生型腈水合酶的基础上，通过结构分析和理性设计，申请人获得了多个有益突变体。并进一步发现这些有益的突变都集中于野生型腈水合酶的特定的一级结构(氨基酸种类的突变)和/或二级结构域中。
进一步发现这些有益的突变位点在空间结构中的分布。发明人在结构基础上利用分子对接模拟底物与酶分子的结合状态，并通过分子动力学模拟，研究了底物与酶分子的结合过程，分析底物与酶分子间的相互作用力，确定了影响可能影响酶催化性能的氨基酸位点，并结合多序列比对分析锁定候选氨基酸残基，并依据结构位置和进化保守性分析进行定向设计，构建突变体库，并通过催化性能评价，筛选出多个稳定性和产物耐受性提升的突变体。
以来源于红色红球菌TH、玫瑰色红球菌J1、玫瑰色红球菌M8、嗜吡啶红球菌、诺卡氏菌的腈水合酶为模板，通过同源建模获得不同来源的腈水合酶的结构模型，通过结构比对发现这些腈水合酶中一级结构和特定二级结构域具有很高的保守性，因此本申请考察了所获的有益突变位点在其他来源的腈水合酶的调控作用。
作为示例，基于，红色红球菌(Rhodococcus ruber TH)的腈水合酶，其β亚基的氨基酸序列如SEQ ID NO:1所示，α亚基的氨基酸序列如SEQ ID NO:2所示；本申请的腈水合酶突变体在上述的野生型腈水合酶的氨基酸序列的基础上，包含上述任意一项或其任意组合的突变。
在一些可选的实施方式中，野生型腈水合酶来源于玫瑰色红球菌、嗜吡啶红球菌、红色红球菌、诺卡氏菌中的任意一种。
根据本申请实施例，野生型腈水合酶可以来自野生型腈水合酶来源于玫瑰色红球菌、嗜吡啶红球菌、红色红球菌或诺卡氏菌中的任意一项。
具体地，野生型腈水合酶具有选自以下的序列：
1)野生型红色红球菌(Rhodococcus ruber TH)的腈水合酶，其β亚基的氨基酸序列如SEQ ID NO:1所示，α亚基的氨基酸序列如SEQ ID NO:2所示，表达相关氨基酸序列的结构基因和调控基因序列详见专利号ZL200910076710.1的中国发明专利，将其全部内容通过引用并入本文。红色红球菌Rhodococcus ruber有时也译作赤红球菌。

2)来源于玫瑰色红球菌J1(Rhodococcus rhodochrous J1)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为P21220，α亚基为P21219，其全部内容通过引用并入本文。
3)来源于玫瑰色红球菌M8(Rhodococcus rhodochrous M8)的腈水合酶，其β亚基的GenBank登录号为AAT79339.1，α亚基为AAT79340.1，其全部内容通过引用并入本文。
4)来源于嗜吡啶红球菌(Rhodococcus pyridinivorans)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q2UZQ6，α亚基为Q2UZQ5，其全部内容通过引用并入本文。
5)来源于红球菌属(Rhodococcus sp.)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q59785，α亚基为Q59786，其全部内容通过引用并入本文。
6)来源于诺卡氏菌(Nocardia sp.JBRs)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q8GE66，α亚基为Q8GE67，其全部内容通过引用并入本文。
在一些可选的实施方式中，腈水合酶突变体满足下列条件中的至少一项：
1)突变1中，β亚基的152位氨基酸位点发生突变；
2)突变2中，β亚基的110位氨基酸位点发生突变；
3)突变3中，β亚基的17位氨基酸位点发生突变。
根据本申请实施例，β亚基的152位氨基酸位点发生突变可以理解为：β亚基的自起始N端处第152位氨基酸位点发生突变。β亚基的110位氨基酸位点发生突变可以理解为：β亚基的自起始N端处第110位氨基酸位点发生突变。β亚基的17位氨基酸位点发生突变可以理解为：β亚基自起始N端处第17位氨基酸位点发生突变。上述突变可以任意地单独存在，或者任意的组合。
在一些可选的实施方式中，152位氨基酸位点的氨基酸残基突变为半胱氨酸、苏氨酸，谷氨酸、天冬氨酸、缬氨酸、丝氨酸、甘氨酸、谷氨酰胺中的任意一种。
根据本申请实施例，可以理解为β亚基的152位氨基酸位点的氨基酸残基突变为152C、152T、152E、152D、152V、152S、152Q、152G中任意一种。
在一些可选的实施方式中，110位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、丝氨酸、苏氨酸、缬氨酸、苯丙氨酸、亮氨酸、异亮氨酸、赖氨酸、组氨酸中的任意一种。
根据本申请实施例，可以理解为110位氨基酸位点的氨基酸残基突变为110A、110R、110S、110T、110V、110F、110L、110I、110K中任意一种。
在一些可选的实施方式中，17位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、半胱氨酸、赖氨酸、组氨酸中的任意一种。
根据本申请实施例，可以理解为β亚基自起始N端处第17位氨基酸位点的氨基酸残基突变为17A、17R、17E、17D、17S、17C、17K、17H中任意一种。
在一些可选的实施方式中，腈水合酶突变体包括突变1和突变2的任意组合。示例性地，突变体M26-M30。
在一些可选的实施方式中，腈水合酶突变体包括突变1和突变3的任意组合。示例性地，突变体M48-M54。
在一些可选的实施方式中，腈水合酶突变体包括突变2和突变3的任意组合。示例性地，突变体M39-M47。
在一些可选的实施方式中，腈水合酶突变体包括突变1、突变2和突变3。示例性地，突变体M56-M69。
根据本申请一个方面的实施例，腈水合酶突变体包含β亚基167位氨基酸位点的氨基酸残基突变为丝氨酸，α亚基174位的氨基酸位点的氨基酸残基突变为亮氨酸，α亚基178位的氨基酸位点的氨基酸残基突变为谷氨酸中的至少一种。
可以理解为：β亚基自起始N端处第167位氨基酸为β167S；α亚基自起始N端处第174位氨基酸为α174L；α亚基的自起始N端处第178位氨基酸为α178E。上述的突变可以单独存在后，与突变1-突变3中任意组合的突变进行组合。上述的突变可以任意地组合存在后，与突变1-突变3中任意组合的突变进行组合。
在上述突变体的基础上，腈水合酶突变体进一步包含β亚基167位天冬酰胺突变为丝氨酸，α亚基174位的甘氨酸突变为亮氨酸，α亚基178位的天冬氨酸突变为谷氨酸中的任意一种或其组合。本实施例的上述的突变可以任意的组合存在后，与上述的任意实施例且包含突变1-突变3中任意组合的突变进行组合。
在一些可选的实施方式中，腈水合酶突变体包含：结构域突变，其包括：β亚基141位氨基酸位点的氨基酸残基突变为赖氨酸，143位氨基酸位点的氨基酸残基突变为赖氨酸，144位氨基酸位点的氨基酸残基突变为谷氨酸，215位氨基酸位点的氨基酸残基突变为半胱氨酸，α亚基133位氨基酸位点的氨基酸残基突变为半胱氨酸。
可以理解为：β亚基自起始N端处第141位氨基酸为β141K；β亚基自起始N端处第143位氨基酸为β143K；β亚基自起始N端处第144位氨基酸为β144E；β亚基 自起始N端处第215位氨基酸为β215C；α亚基自起始N端处第133位氨基酸为α133C。上述突变同时存在于该结构域突变中。可以与其他实施例的突变任意的组合。
在上述突变体或野生型的基础上，进一步引入盐桥改造、二硫键，所述腈水合酶突变体包含：β亚基141丝氨酸突变为赖氨酸、143位丝氨酸突变为赖氨酸和144位亮氨酸突变为谷氨酸的组合，215位天冬氨酸突变为半胱氨酸、α亚基133位脯氨酸突变为半胱氨酸的组合，并与上述实施例的任意突变体任意的组合且共同存在。
在一些可选的实施方式中，腈水合酶突变体包括17A/17R/17E/17D/17S/17C/17K/17H、110A/110R/110S/110T/110V/110F/110L/110I/110K、152C/152A/152T/152E/152D/152V/152S/152Q/152G中的至少一项。
可以理解为：β亚基自起始N端处第17位氨基酸可以为17A、17R、17E、17D、17S、17C、17K、17H中任意一种，β亚基自起始N端处第110位氨基酸为110A、110R、110S、110T、110V、110F、110L、110I、110K中任意一种。β亚基自起始N端处第152位氨基酸为152C、152T、152E、152D、152V、152S、152Q、152G中任意一种，本申请实施例的腈水合酶突变体包含上述三种突变中的任意一种，或任意一种的随机组合。
在一些可选的实施方式中，β17S/β110R/β152C、β17R/β110R/β152D、β17S/β110K/152C、β17S/β110R/β152C、β17S/β110R/β152C、β110K/β152T、β110K/β152D、β110R/β152T、β110K/β17E、β110K/β17C、β110K/β17D、β152S/β17H、β152S/β17E、β110K/β152S/β17E、β17A/β110K/β152G/α174L/α178E、β17E/β110R/β152S/α174L/α178E、β17S/β110V/β152S/βS141K/β143K/β144E、β17D/β110K/β152V/α174L/α178Eβ110R/β152S/β17D、β17E/β110K/β152S/β167S/α174L/α178E、β17E/β110K/β152S/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E、β17R/β110S/β152Q/β167S/α174L/α178E/β215C/α133C//βS141K/β143K/β144E、β17D//β110F/β152C/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E中的任意一项。
可以理解为：与野生型腈水合酶相比，或与相关技术中的突变型的腈水合酶相比，腈水合酶的位点包括其中三个位点的突变，突变的具体形式包括：17S/110R/152C、17R/110R/152D、17S/110K/152C、17S/110R/152C、17S/110R/152C等。例如，17S/110R/152C可以理解为：β亚基自起始N端处17位氨基酸为丝氨酸，β亚基自起始N端处第110位氨基酸为精氨酸；β亚基自起始N端处第152位氨基酸为半胱氨酸。
腈水合酶的催化活性
腈水合酶活性是指催化腈化合物水合产生酰胺类化合物的活性。将底物(腈类化合物)与腈水合酶在一定条件下进行反应，在单位时间内测定底物的消耗量和产物的增加量计算腈水合酶的活性。作为底物，只要腈水合酶发生反应，则不论怎样的腈化合物都可以使用，可选为丙烯腈和烟腈。反应条件是水合反应的通用条件，只要能够保证腈水合酶具有催化活性即可，底物的消耗量和产物的增加量可通过HPLC和GC进行检测和定量分析。
腈类化合物的通式如(1)所示：
R-CN  (1)
此处，R基团为任选被取代的碳原子数1～10的直链状或者支链状的烷基或烯基、任选被取代的碳原子数3～18的环烷基或芳基、或者任选被取代的饱和或不饱和杂环基。
酰胺类化合物的通式如(2)所示：
R-CONH2  (2)
此处，R基团为任选被取代的碳原子数1～10的直链状或者支链状的烷基或烯基、任选被取代的碳原子数3～18的环烷基或芳基、或者任选被取代的饱和或不饱和杂环基。
经研究，本申请记载的野生型腈水合酶和突变型腈水合酶分别具有催化活性。
第二方面，本申请提供了一种分离的核酸分子，核酸分子包含编码如第一方面的腈水合酶突变体的核苷酸序列。
根据本申请实施例，核酸分子包含编码所述腈水合酶突变体的核苷酸序列。核苷酸序列可以为在野生型腈水合酶的序列基础上，通过碱基突变获得。
腈水合酶突变体的重组载体、转化体
第三方面，本申请提供了一种表达载体，其包含第二方面的核酸分子。
根据本申请实施例，选择的表达载体可以在原核或真核细胞的各种宿主中稳定存在并可自主复制，如本领域中的常规质粒(pET系列)、穿梭载体pNV18.1、噬菌体或病毒载体等。
在一些可选的实施方式中，表达载体为质粒载体。
在一些可选的实施方式中，表达载体为pET系列、穿梭载体、噬菌体或病毒载体；可选地，所述表达载体为pET-28a或pNV18.1。
根据本申请实施例，通过酶切、连接等分子生物学操作，将野生型腈水合酶的核苷酸序列插入到pET-28a或pNV18.1等表达载体中，构建得到重组表达质粒，分别命名为pET28a-Nh，pNV18.1-Nh。根据本申请实施例，可以将本申请实施例的腈水合酶突变体的编码基因，构建到重组表达质粒。
第三方面，本申请提供了一种宿主细胞，其包含第二方面的核酸分子或第三方面的表达载体。
根据本申请实施例，可以将本申请第二方面所述的分离的核酸分子直接插入宿主菌的染色体，或采用氯化钙法或电穿孔转化法将第三方面的表达载体导入宿主菌。
在一些可选的实施方式中，宿主细胞包括大肠杆菌、红球菌、诺卡氏菌、枯草芽孢杆菌或谷氨酸棒状杆菌中的任意一种。
在一些可选的实施方式中，宿主细胞为红色红球菌或/和大肠杆菌E.coli BL21(DE3)。
第四方面，本申请提供了一种催化剂，其包含第一方面的腈水合酶突变体。
根据宿主细胞的不同，编码腈水合酶突变体的核苷酸序列可以构建到不同类型的重组载体，也可以直接整合到宿主菌的染色体中。作为使用的载体，可列举出质粒DNA、噬菌体DNA、反转录转座子DNA、人工染色体DNA等。以大肠杆菌和红色红球菌为例，可选为PET系列载体和PNV系列载体。
本申请的转化体能使用的宿主只要能够在导入上述重组载体或编码腈水合酶突变体的核苷酸序列后表达目标腈水合酶，就没有特别限定，例如可以使用红球菌、大肠杆菌等细菌、酵母、动物细胞、昆虫细胞、植物细胞等。
作为向细菌导入重组载体的方法，只要是向细菌中导入DNA的方法，就没有特别的限定。例如可列举出使用钙离子的方法、电穿孔法等。
作为向细菌中整合编码腈水合酶突变体的核苷酸序列的方法，只要是向细菌中导入DNA的方法，就没有特别的限定。例如可列举出使用同源重组、基因编辑法等。
催化剂以及酰胺类化合物的制备方法
在一些可选的实施方式中，催化剂为全细胞催化剂、游离蛋白催化剂或固定化酶催化剂。
根据本申请实施例，全细胞催化剂是指经本申请第四方面构建的宿主细胞经富集培养和目标蛋白的诱导表达后获得的全细胞。游离蛋白催化剂是将全细胞经超声破碎或高压匀浆破碎并通过离心后获得的粗酶液，亦包括通过蛋白纯化手段获得的纯酶。固定化酶催化剂即为选择不同的固定化载体，将游离蛋白催化剂进行固定化操作，从而获得不同形式的固定化腈水合酶突变体。
第五方面，本申请提供了一种酰胺类化合物的制备方法，方法包括使用第一方面或第四方面的催化剂催化腈类化合物发生水合反应，得到酰胺类化合物；
在一些可选的实施方式中，腈类化合物选自丙烯腈、烟腈、肉桂腈、苯乙腈或对羟基苯乙腈。
在一些可选的实施方式中，酰胺类物质选自丙烯酰胺、烟酰胺、肉桂酰胺、苯乙酰胺或对羟基苯乙酰胺。
下述实施例更具体地描述了本申请公开的内容，这些实施例仅仅用于阐述性说明，因为在本申请公开内容的范围内进行各种修改和变化对本领域技术人员来说是明显的。实施例中使用的所有试剂都可商购获得或是按照常规方法进行合成获得，并且可直接使用而无需进一步处理，以及实施例中使用的仪器均可商购获得。
实施例
实施例1腈水合酶突变体的序列
在红色红球菌来源的野生型腈水合酶(WT)的基础上，设计了如下突变体，其中，所述红色红球菌来源的野生型腈水合酶的β亚基的氨基酸序列如SEQ ID NO:1所示，α亚基的氨基酸序列如SEQ ID NO:2所示，所述突变体详见表1：
表1


实施例2腈水合酶重组表达载体的构建
在大肠杆菌细胞内，以质粒pET-28a为表达载体，发明人将编码腈水合酶的核苷酸序列两端设计Nco I和Xho I的酶切位点，使用限制性内切酶Nco I和Xho I对质粒载体和本实施例中腈水合酶的核苷酸序列进行双酶切，利用核酸电泳(1.0％琼脂糖)和试剂盒回收酶切后的基因片段，然后将酶切后的目的基因片段酶切后的质粒载体进行连接。
10μL连接体系包括：
1μL 10×T4 DNA连接酶Buffer(Takara公司)；
2.5μL目的基因片段；
2.5μL质粒片段；
1μL T4 DNA连接酶；
3μL ddH2O；
在16℃下连接过夜，转化至BL21(DE3)感受态细胞内，挑取单克隆子测序验证，提取测序正确的重组质粒，获得包含腈水合酶编码基因的重组表达载体。
实施例3腈水合酶突变体的构建
本申请采用全质粒扩增法构建含有腈水合酶突变基因的重组质粒。将所需突变体位点设计上下游引物，以质粒pET28a-NH为模板，使用PrimeSTAR HS DNA Polymerase(Takara公司)进行全质粒扩增。通过PCR扩增出含有编码腈水合酶基因序列发生突变的重组质粒。
20μL的PCR反应体系包括：
1μL pET28a-NH质粒模板(约220ng/μL)；
10μL 2×PrimeSTAR HS DNA聚合酶；
1.0μL正向引物(10μM)；
1.0μL反向引物(10μM)；
7μL ddH2O。
所述正向引物为针对不同突变体构建过程中所使用的特定引物。所述反向引物为针对不同突变体构建过程中所使用的特定引物。该处限于篇幅不一一赘述，但是本领域技术人员应该知晓，在已知序列的基础上设计引物并获得目的产物是本领域技术人员的常规技术手段。
PCR原液经Dpn I酶消化去除模板序列后，利用热激法转化到BL21(DE3)感受态细胞中，并涂布于含有卡那霉素(50μg/mL)的LB平板中，置于37℃培养箱中倒置培养约12h。挑取单克隆子进行测序验证，测序正确后用20％(V/V)的甘油进行保菌，置于-70进冰箱内保存。
实施例4腈水合酶组合突变体的构建
腈水合酶组合突变体是通过多轮定点突变的方式获得的，在获取单个点突变体后，以含有该突变位点的重组质粒为模板，设计突变位点的上下游引物，进行全质粒PCR扩增，通过PCR扩增出含有编码腈水合酶组合突变体基因序列的重组质粒。
所述正向引物可以使用针对不同突变体构建过的特定引物，该处限于篇幅不一一赘述，但是本领域技术人员应该知晓，在已知序列的基础上设计引物并获得目的产物是本领域技术人员的常规技术手段。
PCR原液经Dpn I酶消化去除模板序列后，利用热激法转化到BL21(DE3)感受态细胞中，并涂布于含有卡那霉素(50μg/mL)的LB平板中，置于37℃培养箱中倒置培养约12h。挑取单克隆子进行测序验证。
实施例5腈水合酶及其突变体的大肠杆菌基因工程菌构建及催化剂的制备
将实施例3和4中制备的大肠杆菌基因工程菌，涂布含卡那霉素(50μg/mL)的LB平板，37℃，培养过夜后，挑取单菌落转接含50μg/mL卡那霉素的LB液体培养中，37℃培养12h，送样进行测序，将正确的克隆子保存到-70℃冰箱，以此获得大肠杆菌为宿主的基因工程菌。
将包含腈水合酶及其突变体的编码序列的基因工程菌接种到含有卡那霉素抗性的LB液体培养基(蛋白胨10g/L，酵母膏5g/L，氯化钠10g/L，溶剂为去离子水，pH＝7.0)中，在含有LB培养基的试管(4mL，含终浓度50μg/mL卡那霉素)中，置于37℃摇床内，200rpm转速下培养10～12h，得到种子液。
在超净工作台中将试管内的种子液转接到含有LB培养基的摇瓶(100mL，含终浓度50μg/mL卡那霉素)中。将含有种子液的LB培养基置于37℃摇床内，200rpm转速下培 养2～3h。待培养液的OD600值达到0.6～0.8时，添加终浓度为0.1～0.8mM的IPTG和0.1-0.8mM Co2+离子进行诱导表达，诱导温度为16～37℃。可选的IPTG浓度为0.2mM，可选钴离子浓度为0.2mM，可选的诱导温度为16℃。在可选条件下诱导24h后，离心收集细胞，获得腈水合酶的大肠杆菌细胞催化剂，置于-70℃冰箱内保存。
称取10g冻存湿细胞，加入到100mL的缓冲液A(25mM Tris-HCl，pH 8.0；300mM NaCl，10mM咪唑，375μL/L巯基乙醇)中，终浓度为10g/L，放置在室温融解，过滤去除未完全溶解的块状菌体。然后使用高压匀浆机进行破碎，压力控制在700-800bar，破碎过程中利用低温循环设备进行降温，循环破碎2-3次即可。将收集到的破碎液离心，收集上清液，获得游离的腈水合酶催化剂。
称取适量固定化氨基树脂材料，用磷酸钾缓冲液(100mM,pH 7.0)平衡，将处理好的树载体加入到缓冲液中，载体与溶液的比例为1/5(w/v)，然后加入戊二醛溶液(50％V/V)至终浓度为2％。在振荡器中(16℃,200rpm)活化2-3h后，使用去离子水清洗活化后的载体，去除残余的戊二醛。称取适量活化后的固定化载体置于缓冲液中，加入酶溶液，载体和酶溶液的比例仍为1/5(w/v)。将其置于恒温摇床或振荡器中(16℃,200rpm)进行固定化，时间为8h。然后用缓冲液清洗固定化酶以除去残余的酶液，然后将其置于4℃冰箱内保存备用。
实施例6腈水合酶突变体的红球菌体系构建及催化剂的制备
分别以实施例3和4中制备的重组表达载体为模板，设计腈水合酶的通用引物，该通用引物序列中包含优化版的自杀质粒pYsacB的同源片段和腈水合酶基因片段，通过PCR扩增出含有突变位点的腈水合酶基因序列。该优化版的自杀质粒pYsacB1是在自杀质粒pYsacB的基础上添加了腈水合酶基因上下游两端的各1000bp左右的同源臂序列。通过Gibson Assembly无缝克隆试剂盒，将获得的腈水合酶突变基因与酶切后的自杀质粒pYsacB进行连接，
20μL的连接体系包括：
4μL目标基因；
6μL自杀质粒载体；
10μL连接试剂；
55℃下连接50min，将连接液转换至Top 10感受态细胞内，挑起单克隆测序验证后，提取重组自杀质粒，将构建成功的重组自杀质粒利用电转化法，转化至红球菌的感受态细胞内，复苏培养后涂布于含抗生素的平板培养基上，20-37℃下培养；然后挑取单菌落，菌落PCR验证，验证自杀质粒整合到红球菌基因组中的正确位置；将单交换成功的菌落，接种到不含抗生素的种子培养基中，20-37℃下培养12h，稀释100倍，取200μL涂布于含100g/L蔗糖的平板上，28℃培养，待长出单菌落后，进行菌落 PCR，验证腈水合酶基因序列整合到基因组上，并送测序公司，将测序正确的菌株置于-70℃冰箱保存备用。
将上述构建的包含腈水合酶突变体的编码序列的红球菌接种到种子培养基中，培养至菌体OD600达到30左右。此时按初始OD600＝3.0的接种量将种子培养基接入50mL发酵培养基中，对于腈水合酶的诱导表达，需向发酵培养基中加入终浓度为0.08mM Co2+，诱导表达48h后，离心收集细胞，获得腈水合酶红球菌细胞催化剂。
称取10g冻存湿细胞，加入到100mL的缓冲液A(25mM Tris-HCl，pH 8.0；300mM NaCl，10mM咪唑，375μL/L巯基乙醇)中，终浓度为10g/L，放置在室温融解，过滤去除未完全溶解的块状菌体。然后使用高压匀浆机进行破碎，压力控制在1200-1500bar，破碎过程中利用低温循环设备进行降温，循环破碎2-3次即可。将收集到的破碎液离心，收集上清液，获得游离的腈水合酶催化剂。
称取适量固定化载体，用磷酸钾缓冲液(100mM,pH 7.0)平衡，将处理好的树载体加入到缓冲液中，载体与溶液的比例为1/5(w/v)，然后加入戊二醛溶液(50％)至终浓度为2％V/V。在振荡器中(16℃,200rpm)活化2-3h后，使用去离子水清洗活化后的载体，去除残余的戊二醛。称取适量活化后的固定化载体置于缓冲液中，加入酶溶液，载体和酶溶液的比例仍为1/5(w/v)。将其置于恒温摇床或振荡器中(16℃,200rpm)进行固定化，时间为8h。然后用缓冲液清洗固定化酶以除去残余的酶液，然后将其置于4℃冰箱内保存备用。
实施例7腈水合酶活力测定方法
腈水合酶对底物丙烯腈活性测定方法如下：取50～100μL催化剂(细胞、游离酶、固定化酶)加入离心管中，加入纯水补足至4.5mL，在28℃水浴锅中放置10分钟使温度稳定。加入100μL丙烯腈，混匀后反应5分钟，加入200μL的3mol/L盐酸终止反应。取1mL反应液13000×g离心2分钟后，取500μL上清液与500μL的内标溶液(40g/L乙酰胺)混匀后，进行气相色谱分析。测定丙烯酰胺与乙酰胺的面积比，利用内标法测定产物丙烯酰胺的浓度，计算活力。
通过气相检测产物的生成量，检测条件为：气相色谱分析条件如下：美国赛默飞Trace 1300气相色谱仪；Abel Bonded AB-I NOWAX色谱柱(内径0.25mm，长度30m，膜厚0.25μm)；FID检测器。柱温、进样口温度、检测器温度分别19℃、26℃、26℃；载气为氮气，恒压模式，分压108kPa；进样体积为1μL，分流进样，分流比为30:1。
所述腈水合酶的活力计算方式如下：
K为内标常数，其数值为0.6001；Cac为乙酰胺浓度40g/L；V总为总体积，其数值为5mL；V为实际加入的催化剂体积；t为反应时间；MW为丙烯酰胺，分子量71；U为总酶活，单位为μmol丙烯酰胺/(min·mL菌液)。
所述酶活力(U)定义为：在上述反应条件下，每分钟催化1μmol底物所需要的酶量为一个酶活单位，用U表示。
经检测，突变体M133的底物消耗率如图2所示，相比野生型的腈水合酶，仍具有良好的催化能力，能催化底物进行反应。
实施例8腈水合酶热稳定性和产物耐受性评价
将实施例5和6中获得的腈水合酶催化剂，用去离子水进行重悬(10g/L)，在55℃恒温水浴中静止放置，间隔2h取出样品，按照实施例7中的方法测定残余活力，并绘制催化剂的失活曲线，通过线性拟合的酶失活半衰期，半衰期越高，表明稳定性越好，以此衡量腈水合酶的热稳定性。
同样，将实施例5和6中获得的腈水合酶催化剂，用去离子水进行重悬(10g/L)，各取适量催化剂放置于10-mL的离心管中，添加丙烯酰胺至终浓度为50％(v/v)，在28℃恒温水浴中静止放置2h后测定催化剂的残余活力，绘制催化剂的失活曲线，通过线性拟合的酶失活半衰期，半衰期越高，表明稳定性越好，以此衡量腈水合酶的产物耐受性。
经检测，突变体M133的残余活力如图3所示，相比野生型的腈水合酶，仍具有较佳的活力。
实施例9腈水合酶及其突变体稳定性和耐受性测定结果
如实施例3和4中所构建的突变体，按照实施例8的方法进行稳定性和耐受性测定，结果表明获得腈水合酶突变体其热稳定性和耐受性的均有不同提升。突变体M1-M35的产物耐受性提高倍数在5.1-6.0倍，突变体M1-M35的热稳定性的提高倍数在5.1-6.0倍；突变体M36-M119、M121-M125、M127-M130、M133和M134的产物耐受性提高倍数在6.1-8.0倍；突变体M36-M119、M121-M125、M127-M130、M133和M134的热稳定性的提高倍数在6.1-8.0倍；突变体M120、M126、M131、M132、M135-M139的产物耐受性提高倍数在8.1-12.0倍。
表2

表2中，+:提高5.1-6.0倍，++：提高6.1-8.0倍；+++：提高8.1-12.0倍。
实施例10关键氨基酸位点对于不同来源的腈水合酶的调控作用
本申请研究了实施例1所述的有益突变位点在其他来源的腈水合酶的调控作用(表1)。首先通过多序列比对确定了其他腈水合酶中的位于相似位置的氨基酸残基，并参照实施例1-9中设计的突变体进行突变和性能测定。来源于玫瑰色红球菌J1(Rhodococcus rhodochrous J1)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为P21220，α亚基为P21219。来源于玫瑰色红球菌M8(Rhodococcus rhodochrous M8)的腈水合酶，其β亚基的GenBank登录号为AAT79339.1，α亚基为AAT79340.1。
来源于嗜吡啶红球菌RP(Rhodococcus pyridinivorans)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q2UZQ6，α亚基为Q2UZQ5。
来源于红球菌属R.sp(Rhodococcus sp.)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q59785，α亚基为Q59786。
来源于诺卡氏菌JBR(Nocardia sp.JBRs)的腈水合酶，其β亚基在UniProt Archive数据库中的UPI编号为Q8GE66，α亚基为Q8GE67。
获得的突变腈水合酶的具有酶活性，且热稳定性和产物耐受性均得到了提高，说明这些突变位点对于不同来源的腈水合酶的热稳定性和产物耐受性均具有有益调控效应。结果如表3所示。
如β110R/β152D/β17D表示β亚基中110氨基酸位点、152氨基酸位点、17氨基酸位点同时存在的情况，其他以此的描述方式也表示同时存在的情况。
表3中，++：提高6.1-8.0倍；+++：提高8.1-12.0倍。

Claims (13)

  1. 一种腈水合酶突变体，其中，基于野生型腈水合酶，所述突变体或其片段为所述野生型腈水合酶的柔性环结构域上的氨基酸残基发生突变，包含突变1-3中的任意的一项或其组合：
    突变1：位于野生型腈水合酶β亚基的二级结构域S1和S2之间的柔性环上的极性氨基酸突变；
    突变2：位于野生型腈水合酶β亚基的起始端的二级结构域H6和H7之间的柔性环上的极性氨基酸突变；
    突变3：位于野生型腈水合酶β亚基的起始柔性环L1上的疏水性氨基酸突变。
  2. 根据权利要求1所述的腈水合酶突变体，其中，所述野生型腈水合酶来源于玫瑰色红球菌、嗜吡啶红球菌、红色红球菌、诺卡氏菌中的任意一种。
  3. 根据权利要求1或2所述的腈水合酶突变体，其中，所述腈水合酶突变体满足下列条件中的至少一项：
    1)突变1中，β亚基的152位氨基酸位点发生突变，可选地，所述152位氨基酸位点的氨基酸残基突变为半胱氨酸、苏氨酸，谷氨酸、天冬氨酸、缬氨酸、丝氨酸、甘氨酸、谷氨酰胺中的任意一种；
    2)突变2中，β亚基的110位氨基酸位点发生突变，可选地，所述110位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、丝氨酸、苏氨酸、缬氨酸、苯丙氨酸、亮氨酸、异亮氨酸、赖氨酸、组氨酸中的任意一种；
    3)突变3中，β亚基的17位氨基酸位点发生突变；
    可选地，所述17位氨基酸位点的氨基酸残基突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、半胱氨酸、赖氨酸、组氨酸中的任意一种。
  4. 根据权利要求1-3任一项所述的腈水合酶突变体，其中，所述腈水合酶突变体包括突变1和突变3的任意组合；和/或，
    所述腈水合酶突变体包括突变1和突变2的任意组合；和/或，
    所述腈水合酶突变体包括突变1、突变2和突变3的组合。
  5. 根据权利要求1-4中任一项所述的腈水合酶突变体，其中，所述腈水合酶突变体包括突变2和突变3的任意组合。
  6. 根据权利要求1-5中任一项所述的腈水合酶突变体，其中，所述突变体或其片段包含β亚基167位氨基酸位点的氨基酸残基突变为丝氨酸，α亚基174位的氨基酸位点的氨基酸残基突变为亮氨酸，α亚基178位的氨基酸位点的氨基酸残基突变为谷氨酸中的至少一种。
  7. 根据权利要求1-6中任一项所述的腈水合酶突变体，其中，所述突变体或其片段包含：结构域突变，其包括：β亚基141位氨基酸位点的氨基酸残基突变为赖氨酸，143位氨基酸位点的氨基酸残基突变为赖氨酸，144位氨基酸位点的氨基酸残基突变为谷氨酸，215位氨基酸位点的氨基酸残基突变为半胱氨酸，α亚基133位氨基酸位点的氨基酸残基突变为半胱氨酸。
  8. 根据权利要求1-7所述的腈水合酶突变体，其中，所述突变体或其片段包括17A/17R/17E/17D/17S/17C/17K/17H、110A/110R/110S/110T/110V/110F/110L/110I/110K、152C/152A/152T/152E/152D/152V/152S/152Q/152G中的至少一项；
    可选地，β17S/β110R/β152C、β17R/β110R/β152D、β17S/β110K/152C、β17S/β110R/β152C、β17S/β110R/β152C、β110K/β152T、β110K/β152D、β110R/β152T、β110K/β17E、β110K/β17C、β110K/β17D、β152S/β17H、β152S/β17E、β110K/β152S/β17E、β17A/β110K/β152G/α174L/α178E、β17E/β110R/β152S/α174L/α178E、β17S/β110V/β152S/βS141K/β143K/β144E、β17D/β110K/β152V/α174L/α178Eβ110R/β152S/β17D、β17E/β110K/β152S/β167S/α174L/α178E、β17E/β110K/β152S/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E、β17R/β110S/β152Q/β167S/α174L/α178E/β215C/α133C//βS141K/β143K/β144E、β17D//β110F/β152C/β167S/α174L/α178E/β215C/α133C/βS141K/β143K/β144E中的任意一项。
  9. 一种分离的核酸分子，其中，所述核酸分子包含编码权利要求1至8中任一项所述的腈水合酶突变体的核苷酸序列。
  10. 一种表达载体，其中，包含权利要求9所述的核酸分子；
    可选地，所述表达载体为质粒载体，可选为pET系列、穿梭载体、噬菌体或病毒载体；
    可选地，所述表达载体为pET-28a或pNV18.1。
  11. 一种宿主细胞，其中，包含权利要求9所述的核酸分子或权利要10所述的表达载体；
    可选地，所述宿主细胞包括大肠杆菌、红球菌、诺卡氏菌、枯草芽孢杆菌或谷氨酸棒状杆菌中的任意一种；
    可选地，所述宿主细胞为红色红球菌或/和大肠杆菌E.coli BL21。
  12. 一种催化剂，其中，包含权利要求1至8中任一项所述的腈水合酶突变体；
    可选地，所述催化剂为全细胞催化剂、游离蛋白催化剂或固定化酶催化剂。
  13. 一种酰胺类化合物的制备方法，其中，所述方法包括使用权利要求1至8中任一项所述的腈水合酶突变体或权利要求12所述的催化剂催化腈类化合物发生水合反应，得到酰胺类化合物；
    可选地，所述腈类化合物选自丙烯腈、烟腈、肉桂腈、苯乙腈或对羟基苯乙腈；
    可选地，所述酰胺类物质选自丙烯酰胺、烟酰胺、肉桂酰胺、苯乙酰胺或对羟基苯乙酰胺。
PCT/CN2023/115259 2023-08-21 2023-08-28 腈水合酶突变体及其应用 Pending WO2025039280A1 (zh)

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