WO2023098453A1 - 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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WO2023098453A1
WO2023098453A1 PCT/CN2022/131383 CN2022131383W WO2023098453A1 WO 2023098453 A1 WO2023098453 A1 WO 2023098453A1 CN 2022131383 W CN2022131383 W CN 2022131383W WO 2023098453 A1 WO2023098453 A1 WO 2023098453A1
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mutation
nitrile hydratase
fragment
mutant
mutated
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于慧敏
李付龙
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清华大学
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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Definitions

  • the application belongs to the fields of enzyme engineering and genetic engineering, and relates to a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant and applications thereof.
  • Nitrile hydratase is a class of metalloenzymes that can catalyze the hydration reaction of nitriles into amides.
  • the use of nitrile hydratase or microorganisms containing nitrile hydratase to catalyze the hydration of acrylonitrile and nicotinamide to produce acrylamide and nicotinamide is one of the most successful cases of industrial biotechnology replacing chemical methods in the production of bulk chemicals.
  • the biocatalytic method has the advantages of mild reaction conditions, low energy consumption, simple operation, high conversion rate, high product concentration and high product purity, and has become the mainstream method for the industrial production of acrylamide.
  • Nitrile hydratase is usually composed of ⁇ and ⁇ subunits (or called a and b subunits), and is found in various microorganisms such as Rhodococcus, Nocardia, Brevibacterium, Mycobacterium and Pseudomonas. distributed in both. Among them, Rhodococcus rhodochrous J1 (Nagasawa T, et al. Applied Microbiology and Biotechnology, 1993, 40 (2-3): 189-195), Rhodococcus rhodochrous R. ruber TH (Ma Y, et al. Bioresource Technology, 2010, 101 (1): 285-291) and its genetically engineered bacteria have been successfully applied to the industrial production of acrylamide.
  • the catalytic activity of the modified nitrile hydratase mutant still needs to be improved. It is of great significance to rationally design and modify the catalytic activity of nitrile hydratase for the industrial production of amides.
  • the purpose of this application is to provide a nitrile hydratase mutant on the basis of related technologies.
  • the nitrile hydratase mutant of the present application has better thermal stability and tolerance, and also has better activity.
  • the present application also provides the use of the nitrile hydratase mutant, specifically the use of the nitrile hydratase mutant to catalyze the synthesis of various amide compounds with important application value, including acrylamide and nicotinamide.
  • the application idea of this application is as follows: Starting from the wild-type nitrile hydratase, the applicant constructed a mutant library of nitrile hydratase through the rational design of the enzyme molecule, and obtained a series of mutants with improved catalytic activity. significantly improved; on this basis, the applicant introduced beneficial mutation sites into the improved nitrile hydratase improved by stability and tolerance, unexpectedly obtained the nitrile hydratase combination mutant described in the application, which can On the basis of the improved nitrile hydratase, the synergistic regulation effect is further exerted, and the activity is significantly improved while having better thermal stability and tolerance.
  • the application provides a nitrile hydratase mutant or a fragment thereof.
  • the mutant or a fragment thereof comprises the following mutations:
  • Mutation I a hydrophobic amino acid mutation on the domain between the secondary domain ⁇ -helix-1 and ⁇ -helix-2 located at the beginning of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation II distance from the active catalytic residue - C(S/T)LCSC(T/Y) Polar amino acid mutations on a range of random coil domains.
  • nitrile hydratase mutant or fragment thereof described in the present application wherein the wild-type nitrile hydratase is derived from Rhodococcus roseus, Rhodococcus pyridineophilus, Rhodococcus rubrum or Nocardia.
  • the wild-type nitrile hydratase has a sequence selected from:
  • Rhodococcus ruber TH The nitrile hydratase of wild-type Rhodococcus ruber (Rhodococcus ruber TH), the amino acid sequence of its ⁇ subunit is shown in SEQ ID NO: 1, the amino acid sequence of ⁇ subunit is shown in SEQ ID NO: 2, and the expression related structure See the Chinese patent application No. ZL200910076710.1 for details of the gene and regulatory gene sequence, the entire content of which is incorporated herein by reference. Rhodococcus ruber is also sometimes translated as Rhodococcus red.
  • Rhodococcus rhodochrous J1 Rhodococcus rhodochrous J1
  • Rhodococcus rhodochrous J1 Rhodococcus rhodochrous J1
  • the UPI number of its ⁇ subunit in the UniProt Archive database is P21220
  • the ⁇ subunit is P21219, the entire contents of which are incorporated herein by reference.
  • Rhodococcus rhodochrous M8 Rhodococcus rhodochrous M8
  • GenBank accession number of its ⁇ subunit is AAT79339.1
  • its ⁇ subunit is AAT79340.1, the entire content of which is incorporated herein by reference.
  • the UPI number of the nitrile hydratase from Rhodococcus pyridinivorans in the UniProt Archive database is Q2UZQ6, and the ⁇ subunit is Q2UZQ5, the entire content of which is incorporated herein by reference.
  • nitrile hydratase derived from Rhodococcus sp. the UPI number of its ⁇ subunit in the UniProt Archive database is Q59785, and the ⁇ subunit is Q59786, the entire contents of which are incorporated herein by reference.
  • the domain comprises fragment 1: -G(M/I)SW; optionally, the mutation is methylsulfide therein amino acid, isoleucine or tryptophan mutation; more alternatively, said methionine mutation is cysteine, aspartic acid, serine, alanine, valine, or
  • the isoleucine is mutated to cysteine, aspartic acid, serine, alanine, valine; and/or
  • the tryptophan is mutated to alanine, arginine, glutamic acid, aspartic acid, serine, threonine, asparagine, cysteine, valine, glutamine, phenylalanine acid, tyrosine, proline, glycine, leucine, isoleucine, methionine, lysine, or histidine.
  • the domain includes at least one of the following fragments:
  • Fragment 2 -(S/T)(S/T)(S/A)(E/D)(I/L/V/M/T)-;
  • Fragment 3 -G(Y/F)(A/S/T)(G/S)(E/R)(Q/H)(A/G)(H/E)-;
  • Fragment 4 -H(D/G)TGGMTGY-;
  • Fragment 5 -K(N/S)MNPL(G/E)HTR-.
  • the mutation is selected from one or more of the following:
  • one or more threonines are mutated to serine and/or one or more serines are mutated to glycine and/or aspartic acid or glutamic acid are mutated to threonine.
  • tyrosine is mutated to threonine or serine and/or glutamine is mutated to asparagine and/or threonine is mutated to serine and/or glutamic acid is mutated to aspartic acid;
  • aspartic acid is mutated to cysteine or valine and/or threonine is mutated to alanine and/or tyrosine is mutated to threonine or serine;
  • one or more asparagine is mutated to alanine and/or threonine is mutated to serine and/or arginine is mutated to valine and/or lysine is mutated to cysteine;
  • the mutant or its fragment described in the present application wherein, compared with the wild-type nitrile hydratase, the mutant or its fragment further comprises mutation III and/or mutation IV:
  • Mutation III Introduced salt bridge linkage in the domain between ⁇ -helix-7 and ⁇ -helix-1 of the secondary domain located at the beginning of the wild-type nitrile hydratase ⁇ subunit;
  • the structural domain that introduces the salt bridge includes fragment 6: -SFSLG-; more alternatively, the mutation is that serine is mutated to lysine and leucine is mutated to glutamic acid ;
  • the structural domain introducing a disulfide bond includes fragment 7: -GNGKD-, fragment 8: -VADP-; more alternatively, the mutation is that the aspartic acid and proline are mutated into cysteine;
  • Mutation IV a polar amino acid mutation located in the secondary structure ⁇ -helix-2 domain of the wild-type nitrile hydratase ⁇ subunit; optionally, it includes fragment 9: -RNKIG; more optionally, said mutation IV In order to mutate the paragine into serine.
  • the second aspect of the present application provides an isolated nucleic acid molecule, said nucleic acid molecule comprising a nucleotide sequence encoding said nitrile hydratase mutant or a fragment thereof;
  • the nucleotide sequence is obtained by base substitution based on the sequence of wild-type nitrile hydratase.
  • the third aspect of the present application provides an expression vector comprising the isolated nucleic acid molecule described in the second aspect of the present application.
  • the expression vector selected in the present application 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, phage or viral vectors in the art, etc., can The selected vectors were pET-28a and pNV18.1.
  • the nucleotide sequence of wild-type nitrile hydratase is inserted into pET-28a or pNV18.1 through molecular biological operations such as enzyme digestion and ligation, and recombinant expression plasmids are constructed, which are named as pET28a-Nh, pNV18.1-Nh; the coding gene of the nitrile hydratase mutant described in this application was constructed into a recombinant expression plasmid, named pET28a-Nh mutant and pNV18.1-Nh mutant respectively.
  • the fourth aspect of the present application provides a host cell comprising the isolated nucleic acid molecule or expression vector described in the second and third aspects of the present application.
  • the host cell is selected from Escherichia coli, Rhodococcus, Nocardia, Corynebacterium propionici, Bacillus subtilis, Corynebacterium glutamicum. This application can choose Rhodococcus ruber and Escherichia coli.
  • the isolated nucleic acid molecule described in the second aspect of the present application is directly inserted into the chromosome of the host bacterium, or the expression vector is introduced into the host bacterium by calcium chloride method or electroporation transformation method.
  • the fifth aspect of the present application provides a catalyst comprising the nitrile hydratase mutant or fragment thereof described in the first aspect of the present application and a preparation method thereof.
  • the catalyst includes three forms of whole cell catalyst, free protein catalyst and immobilized enzyme catalyst.
  • the whole cell catalyst refers to the whole cell obtained after the enrichment culture of the host cell constructed in the fourth aspect of the application and the induced expression of the target protein;
  • the free protein catalyst is the whole cell that is broken by ultrasonication or high-pressure homogenization and centrifuged
  • the crude enzyme solution obtained later also includes pure enzyme 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 sixth aspect of the present application provides the application of the nitrile hydratase mutant described in the first aspect of the present application in catalyzing the hydration reaction of nitrile compounds to prepare amides.
  • the nitrile compounds include acrylonitrile, nicotinonitrile, 2 - cyanopyrazine, cinnamonitrile, phenylacetonitrile, p-hydroxyphenylacetonitrile, etc.
  • said amides include acrylamide, nicotinamide, pyrazinamide, cinnamic amide, phenylacetamide and p-hydroxyphenylacetamide.
  • the applicant's beneficial mutation site significantly improves the activity of nitrile hydratase; the beneficial mutation site is further introduced into the improved nitrile hydratase with improved stability and tolerance, and the nitrile hydratase described in this application is unexpectedly obtained
  • the combined mutant can exert a synergistic regulatory effect on the basis of the improved nitrile hydratase, and further significantly improves the activity while having better thermostability and tolerance.
  • Fig. 1 shows the three-dimensional structure model that is derived from Rhodococcus rubra wild-type nitrile hydratase of the present application, wherein Fig. 1A is the three-dimensional crystal structure of wild-type nitrile hydratase and its catalytic active center and substrate channel; Fig. 1B is the bottom Molecular binding mode of the compound to wild-type nitrile hydratase.
  • Figure 2 shows the secondary domain characteristics of the wild-type nitrile hydratase derived from Rhodococcus rubrum of the present application, wherein Figure 2A is the secondary domain characteristics of the wild-type nitrile hydratase ⁇ subunit, and Figure 2B is the wild-type Secondary domain characterization of the nitrile hydratase beta subunit.
  • Figure 3 shows the secondary domain characteristics of wild-type nitrile hydratase alpha subunits from different sources of the present application.
  • Figure 4 shows the secondary domain characteristics of wild-type nitrile hydratase ⁇ subunits from different sources of the present application.
  • (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 has been isolated from a naturally occurring source.
  • a wild-type gene is the most frequently observed gene in a population, and is therefore arbitrarily designed to be the "normal” or "wild-type” form of the gene.
  • modified refers to a modification (for example, substitution, truncation or insertion), post-translational modification and/or Genes or gene products with functional properties (eg, altered properties).
  • Naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art.
  • a codon for methionine can be replaced by a codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutant monomer, and an arginine (R) to replace methionine (M).
  • CCT codon for arginine
  • R arginine
  • Nitrile hydratase is a metal-dependent enzyme whose active center contains non-heme iron atoms or cobalt atoms, which can be divided into Fe-type and Co-type.
  • nitrile hydratases all have two subunits of ⁇ and ⁇ (or called a and b subunits) in the form of heteromultimers, and the amino acid residues in the active center are relatively conservative (-C(S/ T) LCSC (T/Y)-).
  • Co-type nitrile hydratase was selected as the research object.
  • the applicant selected a variety of wild-type nitrile hydratases derived from Rhodococcus rhodochrous, Rhodococcus pyridineophilus, Rhodococcus rubrum or Nocardia.
  • nitrile hydratase On the basis of the related wild-type nitrile hydratase, through structural analysis and rational design, the applicant obtained multiple beneficial mutants. And further found that these beneficial mutations are concentrated in the specific secondary domain of wild-type nitrile hydratase.
  • the applicant first analyzed the crystal structure of nitrile hydratase derived from Rhodococcus rubrum (Fig. 1A), determined the amino acid residues of its active center, including (-CTLCSCY), and analyzed the secondary structure domain through structural analysis.
  • molecular docking is used to simulate the binding state of the substrate and the enzyme molecule, and the binding process of the substrate and the enzyme molecule is studied through molecular dynamics simulation, the interaction force between the substrate and the enzyme molecule is analyzed, and the influence is determined. Amino acid sites and their spatial structure positions that may affect the catalytic activity of enzymes. Subsequently, based on the remodeling of the substrate binding pocket, the pocket volume was increased, mutants were designed, and activity assays were performed to screen out multiple mutants with improved catalytic performance. The results showed that the obtained beneficial mutation sites were all concentrated in the wild-type nitrile hydration in specific secondary domains of the enzyme.
  • nitrile hydratases from Rhodococcus rhodococcus as a template, the structural models of nitrile hydratase from different sources were obtained through homology modeling. Through structural comparison, it was found that the specific secondary domains of these nitrile hydratases were highly conserved Therefore, this application also investigated the regulatory effect of the obtained beneficial mutation site on nitrile hydratase from other sources.
  • nitrile hydratases include:
  • nitrile hydratase derived from Rhodococcus ruber TH the amino acid sequence of its ⁇ subunit is shown in SEQ ID NO: 1
  • amino acid sequence of ⁇ subunit is shown in SEQ ID NO: 2
  • expression related structure See the Chinese patent application No. ZL200910076710.1 for details of the gene and regulatory gene sequence, the entire content of which is incorporated herein by reference.
  • Rhodococcus rhodochrous J1 Rhodococcus rhodochrous J1
  • Rhodococcus rhodochrous J1 Rhodococcus rhodochrous J1
  • the UPI number of its ⁇ subunit in the UniProt Archive database is P21220
  • the ⁇ subunit is P21219, the entire contents of which are incorporated herein by reference.
  • Rhodococcus rhodochrous M8 Rhodococcus rhodochrous M8
  • GenBank accession number of its ⁇ subunit is AAT79339.1
  • its ⁇ subunit is AAT79340.1, the entire content of which is incorporated herein by reference.
  • the UPI number of the nitrile hydratase from Rhodococcus pyridinivorans in the UniProt Archive database is Q2UZQ6, and the ⁇ subunit is Q2UZQ5, the entire content of which is incorporated herein by reference.
  • nitrile hydratase derived from Rhodococcus sp. the UPI number of its ⁇ subunit in the UniProt Archive database is Q59785, and the ⁇ subunit is Q59786, the entire contents of which are incorporated herein by reference.
  • the nitrile hydratase mutant or fragment thereof of the present application comprises the following mutations:
  • Mutation I a hydrophobic amino acid mutation on the domain between the secondary domain ⁇ -helix-1 and ⁇ -helix-2 located at the beginning of the wild-type nitrile hydratase ⁇ subunit;
  • Mutation II distance from the active center residue -C(S/T)LCSC(T/Y) Polar amino acid mutations on a range of random coil domains.
  • the domain comprises fragment 1: -G(M/I)SW; optionally, the mutation is methylsulfide therein amino acid, isoleucine or tryptophan mutation; more alternatively, said methionine mutation is cysteine, aspartic acid, serine, alanine, valine, or
  • the isoleucine is mutated to cysteine, aspartic acid, serine, alanine, valine; and/or
  • the tryptophan is mutated to alanine, arginine, glutamic acid, aspartic acid, serine, threonine, asparagine, cysteine, valine, glutamine, phenylalanine acid, tyrosine, proline, glycine, leucine, isoleucine, methionine, lysine, or histidine.
  • the domain includes at least one of the following fragments:
  • Fragment 2 -(S/T)(S/T)(S/A)(E/D)(I/L/V/M/T)-;
  • Fragment 3 -G(Y/F)(A/S/T)(G/S)(E/R)(Q/H)(A/G)(H/E)-;
  • Fragment 4 -H(D/G)TGGMTGY-;
  • Fragment 5 -K(N/S)MNPL(G/E)HTR-.
  • the mutation is selected from one or more of the following:
  • one or more threonines are mutated to serine and/or one or more serines are mutated to glycine and/or aspartic acid or glutamic acid are mutated to threonine.
  • tyrosine is mutated to threonine or serine and/or glutamine is mutated to asparagine and/or threonine is mutated to serine and/or glutamic acid is mutated to aspartic acid;
  • aspartic acid is mutated to cysteine or valine and/or threonine is mutated to alanine and/or tyrosine is mutated to threonine or serine;
  • one or more asparagine is mutated to alanine and/or threonine is mutated to serine and/or arginine is mutated to valine and/or lysine is mutated to cysteine;
  • the mutant or its fragment described in the present application wherein, compared with the wild-type nitrile hydratase, the mutant or its fragment further comprises mutation III and/or mutation IV:
  • Mutation III Introduced salt bridge linkage in the domain between ⁇ -helix-7 and ⁇ -helix-1 of the secondary domain located at the beginning of the ⁇ -subunit of wild-type nitrile hydratase;
  • the domain between the subunit secondary structure ⁇ -helix-10 and ⁇ -helix-4 introduces a disulfide bond;
  • the structural domain introducing the salt bridge includes fragment 6: -SFSLG-; more alternatively, the mutation is to mutate serine into lysine and/or leucine into gluten amino acid;
  • the structural domain introducing a disulfide bond includes fragment 7: -GNGKD-, fragment 8: -VADP-; more alternatively, the mutation is that the aspartic acid and proline are mutated into cysteine;
  • Mutation IV a polar amino acid mutation located in the secondary structure ⁇ -helix-2 domain of the wild-type nitrile hydratase ⁇ subunit; optionally, it includes fragment 9: -RNKIG; more optionally, said mutation IV In order to mutate the paragine into serine.
  • amino acid sequence of nitrile hydratase of the present application there are one or more deletions, substitutions or additions, specifically 1 to 20, optional 1 to 10, more optional 1 to 5, and further optional 1 to 2
  • a protein having an amino acid sequence composed of amino acids and having nitrile hydratase activity is also included in the nitrile hydratase of the present application.
  • Nitrile hydratase activity refers to the activity of catalyzing the hydration of nitrile compounds to produce amides.
  • the substrate nitrile compound
  • the substrate is reacted with nitrile hydratase under certain conditions, and the consumption of substrate and the increase of product are measured in unit time to calculate the activity of nitrile hydratase.
  • any nitrile compound can be used as long as the nitrile hydratase reacts, and acrylonitrile and nicotinonitrile can be selected.
  • the reaction conditions are general conditions for the hydration reaction, as long as the nitrile hydratase has catalytic activity, 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 with 1 to 10 carbon atoms, an optionally substituted cycloalkyl group or an aromatic group with 3 to 18 carbon atoms. group, or an optionally substituted saturated or unsaturated heterocyclic group.
  • the R group is an optionally substituted linear or branched alkyl or alkenyl group with 1 to 10 carbon atoms, an optionally substituted cycloalkyl group or an aromatic group with 3 to 18 carbon atoms. group, or an optionally substituted saturated or unsaturated heterocyclic group.
  • the nucleotide sequence encoding the nitrile hydratase mutant can be constructed into different types of recombinant vectors, and can also be directly integrated into the chromosome of the host bacterium.
  • the vector used include plasmid DNA, phage DNA, retrotransposon DNA, artificial chromosome DNA, and the like. Taking Escherichia coli and Rhodococcus as examples, PET series vectors and PNV series vectors can be selected.
  • the host that the transformant of the present application can use is not particularly limited as long as it can express the target nitrile hydratase after introducing the above-mentioned recombinant vector or the nucleotide sequence encoding the nitrile hydratase mutant.
  • bacteria such as Rhodococcus and Escherichia coli can be used, Yeast, animal cells, insect cells, plant cells, etc.
  • the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method of introducing DNA into bacteria.
  • a method using calcium ions, an electroporation method, etc. are mentioned.
  • the method for incorporating the nucleotide sequence encoding the nitrile hydratase mutant into bacteria is not particularly limited as long as it is a method of introducing DNA into bacteria.
  • use of homologous recombination, gene editing methods, etc. can be mentioned.
  • the applicant has designed the following mutant, wherein the amino acid sequence of the ⁇ subunit of the wild-type nitrile hydratase derived from Rhodococcus rhodococcus is as SEQ ID NO:1 Shown, the aminoacid sequence of ⁇ subunit is shown in SEQ ID NO:2, and described mutant sees table 1 for details:
  • Mutant 1 On the basis of wild-type nitrile hydratase, it has M ⁇ 45C(1) in fragment 1, indicating that the methionine in fragment 1 is mutated to cysteine.
  • the inventor designs BamH I and EcoR I enzyme cutting sites at both ends of the nucleotide sequence encoding nitrile hydratase, takes the plasmid pET-28a as a carrier, and uses endonuclease BamH I and EcoR I to the plasmid and in this embodiment
  • the nucleotide sequence of nitrile hydratase is double digested, and the gene fragment after digestion is recovered by nucleic acid electrophoresis (1.0% agarose) and the kit, and then the target gene fragment after digestion is connected to the plasmid vector after digestion middle.
  • 20 ⁇ L ligation system includes:
  • This application adopts the method of whole plasmid PCR to construct the recombinant plasmid containing the mutant gene of nitrile hydratase, first design the upstream and downstream primers containing the mutation site, use the plasmid pET28a-NH as template, and use PrimeSTAR HS DNA Polymerase (Takara company) to carry out the whole plasmid Amplify.
  • a recombinant plasmid containing a mutation in the gene sequence encoding nitrile hydratase was amplified by PCR.
  • the 20 ⁇ L PCR reaction system includes:
  • the forward primer is a specific primer used in the construction process of different mutants.
  • the reverse primers are specific primers used during the construction of different mutants. This section is limited to space and will not be described one by one, but those skilled in the art should know that designing primers based on known sequences and obtaining target products is a 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 E.coli top 10 competent cells by the heat shock method, and spread on LB plates containing kanamycin (50 ⁇ g/mL), placed at 37 Cultivate upside down in an incubator for about 12 hours. Single clones were picked for sequencing verification. After the sequencing was correct, 20% (V/V) glycerol was used for bacteria preservation and stored in a refrigerator at -70°C.
  • Nitrile hydratase combined mutants are obtained through multiple rounds of site-directed mutagenesis. After obtaining a single point mutant, use the recombinant plasmid containing the mutation site as a template to design upstream and downstream primers for the mutation site and perform full-plasmid PCR Amplify, and amplify the recombinant plasmid containing the gene sequence of the combined mutant encoding nitrile hydratase by PCR.
  • the 20 ⁇ L PCR reaction system includes:
  • the forward primers can use specific primers constructed for different mutants, which will not be described one by one due to space limitations, but those skilled in the art should know that designing primers on the basis of known sequences and obtaining target products is a skill in the art. Conventional technical means for technicians.
  • PCR stock solution was digested with Dpn I enzyme to remove the template sequence, it was transformed into E.coli top 10 competent cells by the heat shock method, and spread on LB plates containing kanamycin (50 ⁇ g/mL), placed at 37 Cultivate upside down in an incubator for about 12 hours. Single clones were picked for sequencing verification. After the sequencing was correct, 20% (V/V) glycerol was used for bacteria preservation and stored in a -70°C refrigerator.
  • the recombinant expression vectors prepared in Examples 3 and 4 were transformed into competent cells E.coli BL21 (DE3) by the heat shock method, coated with LB plates containing kanamycin (50 ⁇ g/mL), cultured at 37°C After overnight, pick a single colony and transfer it to LB liquid culture containing 50 ⁇ g/mL kanamycin, incubate at 37°C for 12 hours, send the sample for sequencing, and store the correct clone in a -70°C refrigerator to obtain Escherichia coli Genetically engineered bacteria as the host.
  • Inoculate the genetically engineered bacterium comprising the coding sequence of nitrile hydratase and its mutants into the LB liquid medium containing kanamycin resistance (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent (deionized water, pH 7.0), in a test tube containing LB medium (4mL, containing a final concentration of 50 ⁇ g/mL kanamycin), placed in a shaker at 37°C, and cultured at 200rpm for 10-12h, Obtain seed liquid.
  • Embodiment 6 Rhodococcus system construction of nitrile hydratase mutant and preparation of catalyst
  • the universal primers for nitrile hydratase were designed, which contained the homologous fragment of the suicide plasmid pYsacB and the nitrile hydratase gene fragment of the optimized version in the sequence of the universal primers, and were amplified by PCR.
  • the nitrile hydratase gene sequence containing the mutation site was added.
  • the optimized version of the suicide plasmid pYsacB1 is based on the suicide plasmid pYsacB by adding homology arm sequences of about 1000 bp at the upstream and downstream ends of the nitrile hydratase gene.
  • the obtained nitrile hydratase mutant gene was ligated with the digested suicide plasmid pYsacB,
  • 10 ⁇ L of ligation system includes:
  • a final concentration of 0.08mM Co 2+ should be added to the fermentation medium to induce expression for 48 hours. Afterwards, the cells were collected by centrifugation to obtain the nitrile hydratase Rhodococcus cell catalyst.
  • Embodiment 7 nitrile hydratase activity assay method
  • gas phase The production amount of the product is detected by gas phase, and the detection conditions are as follows: gas chromatography analysis conditions are as follows: American Thermo Fisher Trace 1300 gas chromatograph; Abel Bonded AB-I NOWAX chromatographic column (inner diameter 0.25mm, length 30m, film thickness 0.25 ⁇ m); FID detector. Column temperature, inlet temperature, and detector temperature were 19°C, 26°C, and 26°C, respectively; the carrier gas was nitrogen, in constant pressure mode, and the partial pressure was 10 8 kPa; the injection volume was 1 ⁇ L, split injection, and the split ratio was 50 :1.
  • gas chromatography analysis conditions are as follows: American Thermo Fisher Trace 1300 gas chromatograph; Abel Bonded AB-I NOWAX chromatographic column (inner diameter 0.25mm, length 30m, film thickness 0.25 ⁇ m); FID detector. Column temperature, inlet temperature, and detector temperature were 19°C, 26°C, and 26°C, respectively; the carrier gas was
  • Described enzyme activity (U) is defined as: under above-mentioned reaction condition, the enzyme amount needed for catalyzing 1 ⁇ mol substrate per minute is an enzyme activity unit, expressed with U.
  • Embodiment 8 nitrile hydratase thermal stability and tolerance evaluation
  • the method in example 7 measures the residual activity, and draws the inactivation curve, and the half-life of the catalyst is obtained by linear fitting, and the higher the half-life value, the better the stability, so as to measure the thermal stability of nitrile hydratase.
  • mutants constructed in Examples 3 and 4 the activity was determined according to the method of Example 7, and the results are shown in Table 2.
  • NH nitrile hydratase
  • mutants 1-6 The catalytic activity of 8-12, 15-19, 21-27, 29-31, 33-36, 38-40 and mutant 61 increased by 1.0-2.0 times; mutants 7, 13, 14, 20, 28, The catalytic activities of 32, 37, 41, 42-44, 49, 59, 61, 64 and mutant 66 were increased by 2.0-4.0 times; the catalytic activities of mutants 45-48, 52-58, 63 and mutant 69
  • the improvement times were 3.0-6.0 times; the catalytic activity of mutants 50, 51, 60, 65, 67, 68, 70-73, 75, 79-82 was 6.0-8.0 times, and that of mutants 74 and 76-78
  • the catalytic activity is increased by 8.0-10.0 times.
  • Embodiment 10 Nitrile hydratase and its mutant stability and tolerance assay results
  • Example 9 The mutants with salt bridges or disulfide bonds introduced in Example 9 were tested for stability and tolerance according to the method of Example 8.
  • the stability could be increased to 2.2 times that of the wild type, and the tolerance increased to 1.6 times that of the wild type; when disulfide bonds were introduced, the stability could be increased to 2.8 times that of the wild type, and the tolerance was increased to 2.0 times that of the wild type; when salt bridges and disulfide bonds were introduced at the same time, the stability could be improved Increased to 4.2 times of the wild type, tolerance increased to 5.0 times of the wild type.
  • the beneficial mutants obtained After introducing the designed mutation sites into nitrile hydratase, the beneficial mutants obtained not only have improved activity, but also improved thermal stability and tolerance, indicating that on the basis of salt bridge and disulfide bond modification Combination mutations exert a synergistic regulatory effect, and while having higher activity, their thermal stability and tolerance have also been improved.
  • the beneficial mutation sites obtained as in Example 1 are partially located around the active pocket, and these amino acids are functionally closely related to the catalytic function of nitrile hydratase, and in terms of structural positions, nitrile hydratases from different sources are spatially located in similar positions These amino acids are conserved, as shown in Figure 3 and Figure 4.
  • the present application investigated the regulatory effect of beneficial mutation sites as described in Example 1 on nitrile hydratase from other sources (Table 3). Firstly, the amino acid residues at similar positions in other nitrile hydratases were determined by multiple sequence alignment, and mutations and performance measurements were performed with reference to the mutants designed in Examples 1-10.
  • Nitrile hydratase from Rhodococcus rhodochrous J1 (Rhodococcus rhodochrous J1)
  • the UPI number of its ⁇ subunit in the UniProt Archive database is P21220
  • the ⁇ subunit is P21219.
  • Nitrile hydratase derived from Rhodococcus rhodochrous M8 (Rhodococcus rhodochrous M8)
  • the GenBank accession number of its ⁇ subunit is AAT79339.1
  • its ⁇ subunit is AAT79340.1.
  • Nitrile hydratase from Rhodococcus pyridinivorans RP Rhodococcus pyridinivorans
  • the UPI number of its ⁇ subunit in the UniProt Archive database is Q2UZQ6
  • the ⁇ subunit is Q2UZQ5.
  • Nitrile hydratase derived from Rhodococcus sp. (Rhodococcus sp.), the UPI number of its ⁇ subunit in the UniProt Archive database is Q59785, and the ⁇ subunit is Q59786.
  • Nocardia sp. JBRs Nocardia sp. JBRs

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Abstract

提供了一种腈水合酶突变体或其片段及其应用。与野生型腈水合酶相比，该腈水合酶突变体或其片段包含以下突变：突变I，位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-1和ɑ螺旋-2之间的结构域的疏水性氨基酸突变；突变II，距离活性中心残基-C(S/T)LCSC(T/Y)8A范围内的无规则卷曲结构域上的极性氨基酸突变。

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2021年11月30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202111448620.8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
本申请属于酶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
腈水合酶是一类可以催化腈类物质水合反应转化为酰胺类化合物的金属酶。利用腈水合酶或含腈水合酶的微生物，催化丙烯腈、烟腈水合生产丙烯酰胺和烟酰胺，是工业生物技术替代化学法生产大宗化学品最成功的案例之一。相比于化学法，生物催化法具有反应条件温和、能耗低、操作简单、转化率高、产物浓度高和产物纯度高等优点，目前已成为丙烯酰胺工业生产的主流方法。
腈水合酶通常由α和β两个亚基组成(或被称之a和b亚基)，在红球菌属、诺卡氏菌、短杆菌、分枝杆菌和假单胞菌等多种微生物中均有分布。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(Ma Y,et al.Bioresource Technology,2010,101(1):285-291)及其基因工程菌已被成功应用于丙烯酰胺的工业化生产。
丙烯腈水合反应过程强烈放热，且后期产物浓度较高，容易导致腈水合酶的快速失活，因此构建高活性、高稳定性的生物催化剂是丙烯酰胺工业生产的重要需求。目前报道的野生型腈水合酶多数都存在催化稳定性较差、催化活性不足等问题。很多研究通过构建盐桥、二硫键、Linker或者融合亚基等策略提高了腈水合酶的稳定性(Jiao S,et al.Applied Microbiology and Biotechnology,2020,104(3):1001-1012.)，但同时提高腈水合酶的活性和稳定性的报道较为少见。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。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。
清华大学首次报道了红色红球菌来源的腈水合酶，鉴定了其基因序列和氨基酸序列(专利号：CN200910076710.1)；基于分子模拟结果，对腈水合酶β亚基的141位丝氨酸、143位丝氨酸、144位亮氨酸进行置换，增加盐桥数量，显著提升了腈水合酶的耐热性、丙烯酰胺耐受性和超声耐受性(专利号：CN201110415465.X)；通过在α亚基和β亚基之间引入二硫键，进一步提升了腈水合酶的热稳定性和丙烯酰胺耐受性(专利号：CN201710456875.6)。
然而，上述改造的腈水合酶突变体，催化活性仍需提高。针对腈水合酶催化活性的提高进行理性设计改造对于酰胺类化合物的工业生产具有重要意义。
发明内容
因此，本申请的目的是在相关技术的基础上，提供一种来腈水合酶突变体。与相关技术相比，本申请的腈水合酶突变体在具有更好的热稳定性和耐受性的同时，还具有更好的活性。本申请还提供了所述腈水合酶突变体的用途，具体地所述腈水合酶突变体催化合成多种具有重要应用价值的酰胺类化合物，包括丙烯酰胺和烟酰胺的用途。
本申请的申请构思如下：申请人从野生型腈水合酶出发，通过酶分子的理性设计，构建了腈水合酶突变体库，获得了一系列催化活性提升的突变株，相比母本活性得到显著提升；在此基础上，申请人将有益突变位点引入到稳定性和耐受性提高的改良型腈水合酶中，意外的获得了本申请所述的腈水合酶组合突变体，其能在改良型腈水合酶的基础上，进一步发挥协同调控效应，在具有更好的热稳定性和耐受性的同时，显著地提高了活力。
本申请提供了如下的实施例：
一方面，本申请提供了一种腈水合酶突变体或其片段，与野生型腈水合酶相比，所述突变体或其片段包含以下的突变：
突变I：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-1和α螺旋-2之间的结构域上的疏水性氨基酸突变；
突变II：距离活性催化残基-C(S/T)LCSC(T/Y)
Figure PCTCN2022131383-appb-000001
范围内的无规则卷曲结构域上的极性氨基酸突变。
根据本申请所述的腈水合酶突变体或其片段，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、红色红球菌或诺卡氏菌。
具体地，所述野生型腈水合酶具有选自以下的序列：
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，表达相关的结构基 因和调控基因序列详见专利号ZL200910076710.1的中国申请专利，通过引用将其全部内容并入本文作为参考。红色红球菌Rhodococcus ruber有时也译作赤红球菌。
Figure PCTCN2022131383-appb-000002
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
根据本申请所述的腈水合酶突变体或其片段，其中，突变I中，所述结构域包含片段1：-G(M/I)SW；可选地，所述突变为其中的甲硫氨酸、异亮氨酸或色氨酸突变；更可选地，所述甲硫氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸，或
所述异亮氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸；和/或
所述色氨酸突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、苏氨酸、天冬酰胺、半胱氨酸、缬氨酸、谷氨酰胺、苯丙氨酸、酪氨酸、脯氨酸、甘氨酸、亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸或组氨酸。
根据本申请所述的腈水合酶突变体或其片段，其中，突变II中，所述结构域包括选自以下的片段中的至少一个：
片段2：-(S/T)(S/T)(S/A)(E/D)(I/L/V/M/T)-；
片段3：-G(Y/F)(A/S/T)(G/S)(E/R)(Q/H)(A/G)(H/E)-；
片段4：-H(D/G)TGGMTGY-；
片段5：-K(N/S)MNPL(G/E)HTR-。
任选地，所述突变选自以下的一种或多种：
片段2中，一个或多个苏氨酸突变为丝氨酸和/或一个或多个丝氨酸突变为甘氨酸和/或天冬氨酸或谷氨酸突变为苏氨酸。
片段3中，酪氨酸突变为苏氨酸或丝氨酸和/或谷氨酰胺突变为天冬酰胺和/或苏氨酸突变为丝氨酸和/或谷氨酸突变为天冬氨酸；
片段4中，天冬氨酸突变为半胱氨酸或缬氨酸和/或苏氨酸突变丙氨酸和/或酪氨酸突变为苏氨酸或丝氨酸；
片段5中，一个或多个天冬酰胺突变为丙氨酸和/或苏氨酸突变为丝氨酸和/或精氨酸突变为缬氨酸和/或赖氨酸突变为半胱氨酸；
根据本申请所述的腈水合酶突变体或其片段，其中，与野生型腈水合酶相比，所述突变体或其片段进一步包含突变III和/或突变IV：
突变III：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-7和β螺旋-1之间的结构域中的引入盐桥键；同时在位于野生型腈水合酶β亚基的二级结构α螺旋-10和β螺旋-4之间的结构域引入二硫键；
可选地，突变III中，引入盐桥键的结构域包括片段6：-SFSLG-；更可选地，所述突变为将其中的丝氨酸突变为赖氨酸和亮氨酸突变为谷氨酸；
和/或，
所述突变III中，引入二硫键的结构域包括片段7：-GNGKD-，片段8：-VADP-；更可选地，所述突变为将其中的天冬氨酸和脯氨酸突变为半胱氨酸；
和/或
突变IV：位于野生型腈水合酶β亚基的二级结构β螺旋-2结构域中的极性氨基酸突变；可选地，其包括片段9：-RNKIG；更可选地，所述突变IV为将其中的冬酰胺突变为丝氨酸。
本申请的第二方面提供了一种分离的核酸分子，所述核酸分子包含编码所述腈水合酶突变体或其片段的核苷酸序列；
可选地，所述核苷酸序列为在野生型腈水合酶的序列基础上，通过碱基置换获得。
本申请的第三方面提供了一种表达载体，其包含本申请第二方面中所述的分离的核酸分子。本申请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，可选载体为pET-28a和pNV18.1。
在一个具体的实施方案中，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中，构建得到重组表达质粒，分别命名为pET28a-Nh，pNV18.1-Nh；将本申请所述的腈水合酶突变体的编码基因，构建到重组表达质粒，分别命名为pET28a-Nh mutant，pNV18.1-Nh mutant。
本申请的第四方面提供了一种宿主细胞，其包含本申请第二和三方面中所述的分离的核酸分子或表达载体。所述宿主细胞选自大肠杆菌、红球菌、诺卡氏菌、丙酸棒杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌。本申请可选红色红球菌和大肠杆菌。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所述表达载体导入宿主菌。
本申请的第五方面提供包含本申请第一方面所述的腈水合酶突变体或其片段的催化剂及其制备方法。所述催化剂包括全细胞催化剂、游离蛋白催化剂及固定化酶催化剂三种形式。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所获的全细胞；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。
本申请的第六方面提供本申请第一方面中所述的腈水合酶突变体在催化腈类化合物发生水合反应制备酰胺类化合物中的应用，所述腈类化合物包括丙烯腈、烟腈、2-氰基吡嗪、肉桂腈、苯乙腈、对羟基苯乙腈等，所述酰胺类物质包括丙烯酰胺、烟酰胺、吡嗪酰胺、肉桂酰胺、苯乙酰胺和对羟基苯乙酰胺。
与相关技术相比，本申请的技术方案具有以下优势：
申请人的有益突变位点显著提高了腈水合酶活性；将有益突变位点进一步引入到稳定性和耐受性提高的改良型腈水合酶中，意外的获得了本申请所述的腈水合酶组合突变体，其能在改良型腈水合酶的基础上，发挥协同调控效应，在具有更好的热稳定性和耐受性的同时，进一步显著地提高了活力。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将结合附图详细说明本申请的技术方案：
图1示出为本申请的来源于红色红球菌野生型腈水合酶的三维结构模型，其中图1A为野生型腈水合酶的三维晶体结构及其催化活性中心和底物通道；图1B为底物与野生型腈水合酶的分子结合模式。
图2示出为本申请的来源于红色红球菌的野生型腈水合酶的二级结构域特征，其中图2A为野生型腈水合酶α亚基的二级结构域特征，图2B为野生型腈水合酶β亚基的二级结构域特征。
图3示出为本申请不同来源的野生型腈水合酶α亚基的二级结构域特征。
图4示出为本申请不同来源的野生型腈水合酶β亚基的二级结构域特征。
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。本申请所列举的具体实施例只作为本申请的范例，本申请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。为了更好地说明本申请，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，本申请同样可以实施。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，并且本申请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。
定义
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Qβ45R意指从β亚基起始N端处第45位的Q被R置换；或Qα42R意指从α亚基起始N端处第42位的Q被R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“或”。举例来说，Q87R/K意指Q87R或Q87K。
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/或功能特性质(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，申请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
在相关的野生型腈水合酶的的基础上，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二级结构域中。申请人首先解析了来源于红色红球菌的腈水合酶晶体结构(图1A)，通过结构分析确定其活性中心的氨基酸残基，包括(-CTLCSCY)，并对二级结构域进行了分析。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活性的氨基酸位点及其空间结构位置。随后基于底物结合口袋的重塑，增大口袋体积，设计突变体，并进行活力测定，筛选出多个催化性能提升的突变体，结果表明所获得有益突变位点都集中于野生型腈水合酶的特定的二级结构域中。
随后以来源于红色红球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特定二级结构域具有很高的保守性，因此本申请同样考察了所获的的有益突变位点在其他来源的腈水合酶的调控作用。这些腈水合酶包括：
来源于红色红球菌(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，表达相关的结构基因和调控基因序列详见专利号ZL200910076710.1的中国申请专利，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
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，通过引用将其全部内容并入本文作为参考。
进一步地，申请人还发现，在上述突变的基础上，如果进一步引入盐桥改造、二硫键，通过组合突变获得了催化性能更为优良的突变体。
与野生型腈水合酶相比，本申请的腈水合酶突变体或其片段包含以下的突变：
突变I：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-1和α螺旋-2之间的结构域上的疏水性氨基酸突变；
突变II：距离活性中心残基-C(S/T)LCSC(T/Y)
Figure PCTCN2022131383-appb-000003
范围内的无规则卷曲结构域上的极性氨基酸突变。
根据本申请所述的腈水合酶突变体或其片段，其中，突变I中，所述结构域包含片段1：-G(M/I)SW；可选地，所述突变为其中的甲硫氨酸、异亮氨酸或色氨酸突变；更可选地，所述甲硫氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸，或
所述异亮氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸；和/或
所述色氨酸突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、苏氨酸、天冬酰胺、半胱氨酸、缬氨酸、谷氨酰胺、苯丙氨酸、酪氨酸、脯氨酸、甘氨酸、亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸或组氨酸。
根据本申请所述的腈水合酶突变体或其片段，其中，突变II中，所述结构域包括选自以下的片段中的至少一个：
片段2：-(S/T)(S/T)(S/A)(E/D)(I/L/V/M/T)-；
片段3：-G(Y/F)(A/S/T)(G/S)(E/R)(Q/H)(A/G)(H/E)-；
片段4：-H(D/G)TGGMTGY-；
片段5：-K(N/S)MNPL(G/E)HTR-。
任选地，所述突变选自以下的一种或多种：
片段2中，一个或多个苏氨酸突变为丝氨酸和/或一个或多个丝氨酸突变为甘氨酸和/或天冬氨酸或谷氨酸突变为苏氨酸。
片段3中，酪氨酸突变为苏氨酸或丝氨酸和/或谷氨酰胺突变为天冬酰胺和/或苏氨酸突变为丝氨酸和/或谷氨酸突变为天冬氨酸；
片段4中，天冬氨酸突变为半胱氨酸或缬氨酸和/或苏氨酸突变丙氨酸和/或酪氨酸突变为苏氨酸或丝氨酸；
片段5中，一个或多个天冬酰胺突变为丙氨酸和/或苏氨酸突变为丝氨酸和/或精氨酸突变为缬氨酸和/或赖氨酸突变为半胱氨酸；
根据本申请所述的腈水合酶突变体或其片段，其中，与野生型腈水合酶相比，所述突变体或其片段进一步包含突变III和/或突变IV：
突变III：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-7和β螺旋-1之间的结构域中的引入盐桥键；同时在位于野生型腈水合酶b亚基的二级结构α螺旋-10和β螺旋-4之间的结构域引入二硫键；
可选地，突变III中，引入盐桥键的结构域包括片段6：-SFSLG-；更可选地，所述突变为将其中的丝氨酸突变为赖氨酸和/或亮氨酸突变为谷氨酸；
和/或，
所述突变III中，引入二硫键的结构域包括片段7：-GNGKD-，片段8：-VADP-；更可选地，所述突变为将其中的天冬氨酸和脯氨酸突变为半胱氨酸；
和/或
突变IV：位于野生型腈水合酶β亚基的二级结构β螺旋-2结构域中的极性氨基酸突变；可选地，其包括片段9：-RNKIG；更可选地，所述突变IV为将其中的冬酰胺突变为丝氨酸。
本申请的腈水合酶氨基酸序列中缺失、置换或附加有1个或多个、具体而言1～20个、可选1～10个、更可选1～5个、进一步可选1～2个的氨基酸而成的氨基酸序列且具有腈水合酶活性的蛋白质也包括在本申请的腈水合酶中。
腈水合酶的催化活性的测定
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-CONH 2  (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。
3.腈水合酶突变体的重组载体、转化体
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。
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的基础上，申请人设计了如下突变体，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
Figure PCTCN2022131383-appb-000004
Figure PCTCN2022131383-appb-000005
Figure PCTCN2022131383-appb-000006
突变体1：在野生型腈水合酶的基础上，具有片段1中Mβ45C(1)，表示片段1中的甲硫氨酸突变为半胱氨酸。
实施例2腈水合酶重组表达载体的构建
发明人将编码腈水合酶的核苷酸序列两端设计BamH I和EcoR I酶切位点，以质粒pET-28a为载体，使用内切酶BamH I和EcoR I对该质粒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连接到酶切后的质粒载体中。
20μL连接体系包括：
2μL 10×T4 DNA连接酶Buffer(Takara公司)；
5μL目的基因片段；
5μL质粒片段；
2μL T4 DNA连接酶；
8μL ddH 2O；
在16℃连接过夜，转化至DH5α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，并以此作为母本进行后续改造。
实施例3腈水合酶突变体的构建
本申请采用全质粒PCR的方法构建含有腈水合酶突变基因的重组质粒，首先设计含有突变位点的上下游引物，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100ng/μL)；
10μL 2×PrimeSTAR HS DNA聚合酶；
1.5μL正向引物(10μM)；
1.5μL反向引物(10μM)；
6μL ddH 2O。
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反应的条件如下：
(1)98℃预变性1min；
(2)98℃变性30s；
(3)引物的Tm-5℃退火10s；
(4)72℃延伸7min；
上述步骤(2)-(4)共进行30个循环，最后72℃延伸10min。
PCR原液经Dpn I酶消化去除模板序列后，利用热激法转化到E.coli top 10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。
20μL的PCR反应体系包括：
1μL母本/含有单个突变位点质粒模板(约100ng/μL)；
10μL 2×PrimeSTAR HS DNA Polymerase；
1.5μL正向引物(10μM)；
1.5μL反向引物(10μM)；
6μL ddH 2O。
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反应的条件如下：
(1)98℃预变性1min；
(2)98℃变性30s；
(3)(引物的Tm-5)℃退火10s；
(4)72℃延伸7min；
上述步骤(2)-(4)共进行30个循环，最后72℃延伸10min。
PCR原液经Dpn I酶消化去除模板序列后，利用热激法转化到E.coli top 10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℃冰箱内保存。
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中制备的重组表达载体利用热激法转化至感受态细胞E.coli BL21 (DE3)中，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OD 600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 Co 2+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。
同源重组双交换实验流程
1)以甲基化酶缺陷型大肠杆菌E.coli Trans110为宿主，构建并提取自杀质粒pS18mobsacBopt-amiE，转化R.ruber TH，复苏培养后涂布于含50μg/mL壮观霉素的平板培养基上，28℃培养3天长出菌落。
2)挑取单菌落，菌落PCR验证(一个引物在基因组上，位于上游同源臂的上游；一个引物在质粒上，位于下游同源臂的下游)，确保自杀质粒整合到正确位置。
3)单交换成功的菌落，接种到不含抗生素的种子培养基中，28℃、200rpm培养1天，稀释10～100倍，取200μL涂布于含100g/L蔗糖的平板上，28℃培养2～3天长出菌落。
4)挑取单菌落，进行菌落PCR(引物在基因组上，一个位于上游同源臂的上方，一个位于下游同源臂的下方)，验证是否发生基因敲除或者回复突变。
PCR反应的条件如下：
(1)98℃预变性1min；
(2)98℃变性30s；
(3)(引物的Tm-5)℃退火10s；
(4)72℃延伸2min；
上述步骤(2)-(4)共进行30个循环，最后72℃延伸10min。
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，
10μL的连接体系包括：
2μL目标基因；
3μL自杀质粒载体；
5μL连接试剂；
50℃下连接30min，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R.ruber TH9的感受态细胞内，复苏培养后涂布于含50μg/mL壮观霉素的平板培养基上，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。
PCR反应的条件如下：
(1)98℃预变性1min；
(2)98℃变性30s；
(3)(引物的Tm-5)℃退火10s；
(4)72℃延伸3min；
上述步骤(2)-(4)共进行30个循环，最后72℃延伸10min。
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OD 600达到30左右。此时按初始OD 600＝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 Co 2+，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10mM PBS(pH＝7.0)补足至4.5mL，在28℃水浴锅中放置10分钟使温度稳定。加入200μL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10 8kPa；进样体积为1μL，分流进样，分流比为50:1。
所述腈水合酶的活力计算方式如下：
Figure PCTCN2022131383-appb-000007
K为内标常数，其数值为0.6001；C ac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为丙烯酰胺的分子量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。
实施例8腈水合酶热稳定性和耐受性评价
将实施例5和6中获得的腈水合酶催化剂，用10mM PBS缓冲液进行重悬(pH＝7.0，10g/L)，在50℃恒温水浴中静止放置，每间隔15分钟取出样品，按照实施例7中的方法测定残余活力，并绘制出失活曲线，根据线性拟合出催化剂的半衰期，半衰期值越高说明稳定性越好，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10mM PBS缓冲液进行重悬(pH＝7.0，10g/L)，各取10mL重悬的催化剂分别放置于50mL三角瓶中，添加丙烯酰胺至终浓度为40％(v/v)，在30℃恒温水浴中静止放置1h后测定残余活力，并根据初始活力，计算活力相对值，当活力相对值越高说明耐受性越好，以此衡量腈水合酶的产物耐受性。
实施例9腈水合酶突变体的活力测定结果
如实施例3和4中所构建的突变体，按照实施例7的方法进行活力测定，结果见表2，在腈水合酶(NH)中引入所设计突变位点后，突变体1-6、8-12、15-19、21-27、29-31、33-36、38-40和突变体61的催化活力提高倍数为1.0-2.0倍；突变体7、13、14、20、28、32、37、41、42-44、49、59、61、64和突变体66的催化活力提高倍数为2.0-4.0倍；突变体45-48、52-58、63和突变体69的催化活力提高倍数为3.0-6.0倍；突变体50、51、60、65、67、68、70-73、75、79-82的催化活力提高倍数为6.0-8.0倍，突变体74、76-78的催化活力提高倍数为8.0-10.0倍。通过结构对比和底物结合口袋分析，有益突变体的口袋体积增大和极性环境的改变，有利于底物与酶的结合，是其催化活力提升的主要原因。
表2
编号 活力 编号 活力
野生型 100％ 突变体42 ++
突变体1 + 突变体43 ++
突变体2 + 突变体44 ++
突变体3 + 突变体45 +++
突变体4 + 突变体46 +++
突变体5 + 突变体47 +++
突变体6 + 突变体48 +++
突变体7 ++ 突变体49 ++
突变体8 + 突变体50 ++++
突变体9 + 突变体51 ++++
突变体10 + 突变体52 +++
突变体11 + 突变体53 +++
突变体12 + 突变体54 +++
突变体13 ++ 突变体55 +++
突变体14 ++ 突变体56 +++
突变体15 + 突变体57 +++
突变体16 + 突变体58 +++
突变体17 + 突变体59 ++
突变体18 + 突变体60 ++++
突变体19 + 突变体61 ++
突变体20 ++ 突变体62 +
突变体21 + 突变体63 +++
突变体22 + 突变体64 ++
突变体23 + 突变体65 ++++
突变体24 + 突变体66 ++
突变体25 + 突变体67 ++++
突变体26 + 突变体68 ++++
突变体27 + 突变体69 +++
突变体28 ++ 突变体70 ++++
突变体29 + 突变体71 ++++
突变体30 + 突变体72 ++++
突变体31 + 突变体73 ++++
突变体32 ++ 突变体74 +++++
突变体33 + 突变体75 ++++
突变体34 + 突变体76 +++++
突变体35 + 突变体77 +++++
突变体36 + 突变体78 +++++
突变体37 ++ 突变体79 ++++
突变体38 + 突变体80 ++++
突变体39 + 突变体81 ++++
突变体40 + 突变体82 ++++
突变体41 ++    
+:提高1.0-2.0倍，++：提高2.0-4.0倍；+++：提高3.0-6.0倍；
++++：提高6.0-8.0倍，+++++：提高8.0-10.0倍。
实施例10腈水合酶及其突变体稳定性和耐受性测定结果
将实施例9中引入盐桥或二硫键的突变体，按照实施例8的方法进行稳定性和耐受性测定，引入盐桥时稳定性均能提高至野生型的2.2倍，耐受性提高至野生型的1.6倍；引入二硫键时稳定性均能提高至野生型的2.8倍，耐受性提高至野生型的2.0倍；同时引入盐桥和二硫键时，稳定性均能提高至野生型的4.2倍，耐受性提高至野生型的5.0倍。
腈水合酶中引入所设计的突变位点后，所获得的有益突变体不仅活力有所 提升，其热稳定性和耐受性同样得到了提升，说明在盐桥和二硫键改造的基础上进行组合突变发挥了协同调控效应，在具有更高活力的同时，其热稳定性和耐受性也得到了提升。
实施例11关键氨基酸位点其他腈水合酶的调控作用
如实施例1所获得的有益突变位点部分位于活性口袋周围，这些氨基酸在功能上与腈水合酶的催化功能紧密相关，而在结构位置上，不同来源的腈水合酶中空间上位于相似位置的这些氨基酸具有保守性，如图3和图4所示。本申请考察了如实施例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3)。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1-10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催化活性和稳定性都同时得到了提高，说明这些突变位点对于不同来源的腈水合酶活力均具有协同调控效应。
Figure PCTCN2022131383-appb-000008
Figure PCTCN2022131383-appb-000009
Figure PCTCN2022131383-appb-000010
Figure PCTCN2022131383-appb-000011
Figure PCTCN2022131383-appb-000012
Figure PCTCN2022131383-appb-000013
Figure PCTCN2022131383-appb-000014
Figure PCTCN2022131383-appb-000015

Claims (11)

  1. 一种腈水合酶突变体或其片段，其中，与野生型腈水合酶相比，所述突变体或其片段包含以下至少一种突变：
    突变I：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-1和α螺旋-2之间的结构域上的疏水性氨基酸突变；
    突变II：距离活性中心残基
    Figure PCTCN2022131383-appb-100001
    范围内的无规则卷曲结构域上的极性氨基酸突变。
  2. 根据权利要求1所述的腈水合酶突变体或其片段，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、红色红球菌或诺卡氏菌。
  3. 根据权利要求1或2所述的腈水合酶突变体或其片段，其中，突变I中，所述结构域包含片段1：-G(M/I)SW-；
    可选地，所述突变为其中的甲硫氨酸、异亮氨酸或色氨酸突变；更可选地，所述甲硫氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸，或
    所述异亮氨酸突变为半胱氨酸、天冬氨酸、丝氨酸、丙氨酸、缬氨酸；和/或
    所述色氨酸突变为丙氨酸、精氨酸、谷氨酸、天冬氨酸、丝氨酸、苏氨酸、天冬酰胺、半胱氨酸、缬氨酸、谷氨酰胺、苯丙氨酸、酪氨酸、脯氨酸、甘氨酸、亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸或组氨酸。
  4. 根据权利要求1至3中任一项所述的腈水合酶突变体或其片段，其中，突变II中，所述结构域包括选自以下的片段中的至少一个：
    片段2：-(S/T)(S/T)(S/A)(E/D)(I/L/V/M/T)-；
    片段3：-G(Y/F)(A/S/T)(G/S)(E/R)(Q/H)(A/G)(H/E)-；
    片段4：-H(D/G)TGGMTGY-；
    片段5：-K(N/S)MNPL(G/E)HTR-；
    任选地，所述突变选自以下的一种或多种：
    片段2中，一个或多个苏氨酸突变为丝氨酸；和/或
    一个或多个丝氨酸突变为甘氨酸；和/或
    天冬氨酸或谷氨酸突变为苏氨酸；
    片段3中，酪氨酸突变为苏氨酸或丝氨酸；和/或
    谷氨酰胺突变为天冬酰胺；和/或
    苏氨酸突变为丝氨酸；和/或
    谷氨酸突变为天冬氨酸；
    片段4中，天冬氨酸突变为半胱氨酸或缬氨酸；和/或
    苏氨酸突变丙氨酸；和/或
    酪氨酸突变为苏氨酸或丝氨酸；
    片段5中，一个或多个天冬酰胺突变为丙氨酸；和/或
    苏氨酸突变为丝氨酸；和/或
    精氨酸突变为缬氨酸；和/或
    赖氨酸突变为半胱氨酸。
  5. 根据权利要求1至4中任一项所述的腈水合酶突变体或其片段，其中，与野生型腈水合酶相比，所述突变体或其片段进一步包含突变III和/或突变IV：
    突变III：位于野生型腈水合酶β亚基的起始端的二级结构域α螺旋-7和β螺旋-1之间的结构域中的引入盐桥键；同时在位于野生型腈水合酶β亚基的二级结构α螺旋-10和β螺旋-4之间的结构域引入二硫键；
    可选地，突变III中，引入盐桥键的结构域包括片段6：-SFSLG-；更可选地，所述突变为将其中的丝氨酸突变为赖氨酸和/或亮氨酸突变为谷氨酸；
    和/或，
    所述突变III中，引入二硫键的结构域包括片段7：-GNGKD-，片段8：-VADP-；更可选地，所述突变为将其中的天冬氨酸和脯氨酸突变为半胱氨酸；
    和/或
    突变IV：位于野生型腈水合酶β亚基的二级结构α螺旋-2结构域中的极性氨基酸突变；可选地，其包括片段9：-RNKIG；更可选地，所述突变IV为将其中的天冬酰胺突变为丝氨酸。
  6. 一种分离的核酸分子，所述核酸分子包含编码如权利要求1至5中任一项所述的腈水合酶突变体或其片段的核苷酸序列。
  7. 一种表达载体，其包含如权利要求6所述的分离的核酸分子；
    可选地，所述表达载体为质粒载体，可选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。
  8. 一种宿主细胞，其包含如权利要求6所述的分离的核酸分子或如权利要求7所述的表达载体；
    可选地，所述宿主细胞选自大肠杆菌、红球菌、诺卡氏菌、丙酸棒杆菌、枯草芽孢杆菌或谷氨酸棒状杆菌；
    更可选地，所述宿主细胞为红色红球菌或/和大肠杆菌E.coli BL21(DE3)。
  9. 一种催化剂，其包含如权利要求1至5中任一项所述的腈水合酶突变体或其片段；
    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。
  10. 根据权利要求9所述的催化剂在制备酰胺类化合物中的应用。
  11. 一种酰胺类化合物的制备方法，所述方法包括使用如权利要求9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、2-氰基吡嗪、肉桂腈、苯乙腈或对羟基苯乙腈；
    所述酰胺类物质选自丙烯酰胺、烟酰胺、吡嗪酰胺、肉桂酰胺、苯乙酰胺或对羟基苯乙酰胺。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116790573A (zh) * 2023-08-21 2023-09-22 清华大学 腈水合酶突变体及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317507A (zh) * 2021-11-30 2022-04-12 清华大学 腈水合酶突变体及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102216455A (zh) * 2008-11-14 2011-10-12 三井化学株式会社 腈水合酶突变体
CN102517271A (zh) * 2011-12-13 2012-06-27 清华大学 一种突变腈水合酶
CN102604921A (zh) * 2004-05-26 2012-07-25 三菱丽阳株式会社 改良型腈水合酶
CN103215242A (zh) * 2002-12-19 2013-07-24 三井化学株式会社 新型腈水合酶
CN103667228A (zh) * 2013-12-14 2014-03-26 江南大学 一种催化活力及热稳定性提高的真菌腈水解酶突变体及其构建方法
CN107177581A (zh) * 2017-06-16 2017-09-19 清华大学 一种改造腈水合酶及其应用
CN110139930A (zh) * 2016-12-28 2019-08-16 三井化学株式会社 突变型腈水合酶、编码该突变型腈水合酶的核酸、含有该核酸的表达载体及转化体、该突变型腈水合酶的制造方法、及酰胺化合物的制造方法
CN112501151A (zh) * 2020-12-09 2021-03-16 江南大学 一种腈水合酶突变体及其应用
CN113462677A (zh) * 2021-07-29 2021-10-01 浙江大学杭州国际科创中心 一种α亚基突变的腈水合酶突变体及其应用
CN114317507A (zh) * 2021-11-30 2022-04-12 清华大学 腈水合酶突变体及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621600B (zh) * 2021-09-17 2023-06-27 无锡新晨宇生物工程有限公司 一种高活性腈水合酶突变体及其应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103215242A (zh) * 2002-12-19 2013-07-24 三井化学株式会社 新型腈水合酶
CN102604921A (zh) * 2004-05-26 2012-07-25 三菱丽阳株式会社 改良型腈水合酶
CN102216455A (zh) * 2008-11-14 2011-10-12 三井化学株式会社 腈水合酶突变体
CN102517271A (zh) * 2011-12-13 2012-06-27 清华大学 一种突变腈水合酶
CN103667228A (zh) * 2013-12-14 2014-03-26 江南大学 一种催化活力及热稳定性提高的真菌腈水解酶突变体及其构建方法
CN110139930A (zh) * 2016-12-28 2019-08-16 三井化学株式会社 突变型腈水合酶、编码该突变型腈水合酶的核酸、含有该核酸的表达载体及转化体、该突变型腈水合酶的制造方法、及酰胺化合物的制造方法
CN107177581A (zh) * 2017-06-16 2017-09-19 清华大学 一种改造腈水合酶及其应用
CN112501151A (zh) * 2020-12-09 2021-03-16 江南大学 一种腈水合酶突变体及其应用
CN113462677A (zh) * 2021-07-29 2021-10-01 浙江大学杭州国际科创中心 一种α亚基突变的腈水合酶突变体及其应用
CN114317507A (zh) * 2021-11-30 2022-04-12 清华大学 腈水合酶突变体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIROMI TAKARADA, YOSHIAKI KAWANO, KOUICHI HASHIMOTO, HIROSHI NAKAYAMA, SHUNSAKU UEDA, MASAFUMI YOHDA, NOBUO KAMIYA, NAOSHI DOHMAE,: "Mutational Study on αGln90 of Fe-Type Nitrile Hydratase from Rhodococcus sp. N771", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, vol. 70, no. 4, 1 April 2006 (2006-04-01), pages 881 - 889, XP055101435, ISSN: 09168451, DOI: 10.1271/bbb.70.881 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116790573A (zh) * 2023-08-21 2023-09-22 清华大学 腈水合酶突变体及其应用
CN116790573B (zh) * 2023-08-21 2023-11-21 清华大学 腈水合酶突变体及其应用

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