Cyanohydrinase variants with improved specific activity and thermal stability and uses thereof

Abstract

The present application provides a nitrile hydratase variant with improved specific activity and thermal stability, which is obtained by point mutation of nitrile hydratase from Klebsiella oxytoca. The present application also relates to the use of the enzyme in the preparation of dextrometorphan by biocatalytic technology. The use of the nitrile hydratase variant of the present application in the catalytic production of dextrometorphan has high substrate conversion rate, product e.e. value > 99%, and low enzyme input, which effectively reduces the cost of dextrometorphan production by biocatalytic synthesis. Meanwhile, the thermal stability of the enzyme is greatly improved, which is beneficial to storage and use.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a nitrile hydratase variant with improved specific activity and thermal stability and its application in the catalytic synthesis of amide ketoprofen. Background Technology

[0002] Ketoprofen is a 2-arylpropionic acid class of nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs all have a chiral center, and only the dextrorotatory isomer has anti-inflammatory, antirheumatic, and analgesic effects. The levorotatory isomer has almost no pharmacological activity and has toxic side effects. {He Fengci et al. Research progress on ketoprofen and its chiral enantiomers [J]. China Pharmacy, 2004, 15(4): 244-246; Mei Zhinan et al. Pharmacokinetic and pharmacodynamic properties of dextrorotatory ketoprofen [J]. Chinese Journal of New Drugs, 1998, 7(005): 339-341}.

[0003] Currently, the main synthetic methods for commercially available (S)-(+)-ketoprofen include traditional chemical synthesis, chiral resolution, and bioenzymatic synthesis. Chemical synthesis mainly involves asymmetric synthesis, which can be achieved through a 6-step reaction. Allyl alcohol is prepared into (2S, 3S) epoxides via sharpless epoxidation. Then, a chiral shift reagent is added, and after enantioselective hydrogenolysis, diols are generated. Under the catalysis of RuO4 / NaIO4, dextrorotatory ketoprofen is finally obtained, with an ee value of 98%. This method has relatively ideal yield and enantioselectivity, but it requires the use of traditional chemical catalysts to promote rapid reaction. Most of these catalysts are highly toxic, flammable, and cause environmental pollution, and may also introduce harmful byproducts.

[0004] Another chiral resolution method, patent CN101928214A, uses domestically inexpensive and readily available (-)-glucocynylamine as a resolving agent to resolve (RS)-ketoprofen. After mixing in a certain proportion, the mixture is heated to reflux and then cooled to crystallize, achieving a ketoprofen utilization rate of 70%. However, this method is time-consuming and complex. Currently, there are also methods using polymer imprinting and chromatography to resolve enantiomers of ketoprofen {Jiang Jigang et al. Resolution of racemic ketoprofen using polymer imprinting [J]. Analytical Laboratory, 2004, 23(003):56-58, patent CN102516068B}. These methods lack cost advantages and are difficult to market directly. There are also studies using microbial enzymatic methods for ketoprofen resolution. Ma Yan et al. used microbial strains obtained by ultraviolet mutagenesis to ferment under certain conditions, and used cell-mediated resolution (RS)-ketoprofen. At the same time, they also used Novozym lipase 435 to catalyze the esterification resolution of ketoprofen {Ma Yan et al. Study on esterification resolution of ketoprofen catalyzed by Novozym 435 [J]. Journal of Nanjing University of Technology (Natural Science Edition), 2015, 37(6):54-60,68.}. Patent CN103194467A expressed a thermophilic esterase from archaea in Escherichia coli, and used cell-mediated catalysis of ketoprofen ethyl ester reaction to generate dextro-ketoprofen. The biggest drawback of this method is the low substrate concentration, long reaction time, and product ee value of only 90%. The disadvantage of using esterase is that the resolution rate and product ee value are not ideal, and the problem of low substrate concentration is also difficult to solve.

[0005] Bioenzymatic synthesis can solve the problems of low substrate concentration and low product ee value to a certain extent. There are also reports on the preparation of optically pure ketoprofen using a dual-enzyme system of nitrile hydratase and amidase. Norman Layh et al. screened the strain Rhodococcus sp. C3II, which can convert ketoprofen nitrile into (S)-(+)-amide ketoprofen with an ee value of 99%, but the conversion rate is only 12% {Layh N, Knackmuss HJ, Stolz A. Enantioselective hydrolysis of ketoprofen amide by Rhodococcus sp. C3II and Rhodococcus erythropolis MP 50[J]. Biotechnology Letters, 1995, 17(2): 187-192.}.

[0006] R. Bauer et al. used a nitrile hydratase purified from Agrobacterium tumefaciens to catalyze ketoprofen nitrile (cyano ketoprofen). The yield of (S)-(+)-amide ketoprofen was 30%, and the optical purity was greater than 90%. When the conversion rate was 50%, the optical purity decreased to 80%. When the conversion rate reached 100%, racemic amide ketoprofen was generated. {Bauer R, Knackmuss HJ, Stolz A. Enantioselective hydration of 2-arylpropionitriles by a nitrile hydratase from Agrobacterium tumefaciens strain d3[J]. Applied Microbiology and Biotechnology, 1998, 49(1): 89-95.}

[0007] The conversion rate or optical purity of the methods reported above still cannot meet the production requirements, and it is difficult to achieve both optical purity and conversion rate.

[0008] Nitrile hydratases are multi-subunit metalloenzymes, consisting of a polymeric structure composed of α and β subunits. Based on the different metal ions contained in the center of the nitrile hydratase, they can be divided into iron-type and cobalt-type nitrile hydratases. Furthermore, the correct expression of nitrile hydratases requires the participation of accessory proteins. Nitrile hydratases derived from *Klebsiella oxytoca* are cobalt-type nitrile hydratases, capable of catalyzing the hydrolysis of various nitrile compounds. Vojtěch Vejvoda et al. reported that the nitrile hydratase of Klebsiella oxytoca can hydrolyze dibenzonitrile, 3-toluenenitrile, 3-chlorobenzonitrile, 4-chlorobenzonitrile, 3-hydroxybenzonitrile, 4-aminobenzonitrile, propionitrile, butyronitrile, valerate, and 2-methyl-3-butyronitrile [Vojtěch Vejvoda, Ludmila Martínková, Alicja B.Veselá, Kaplan, O., Lutz-Wahl, S., & Fischer, L., et al. (2011). Biotransformation of nitriles to hydroxamic acids via a nitrile hydratase–amidase cascade reaction. Journal of Molecular Catalysis B Enzymatic, 71(1-2), 51-55.]. EP1842907A1 discloses that the nitrile hydratase of Klebsiella oxytoca can hydrolyze 2-phenylpropionitrile, mandeonitrile, benzyl nitrile, phenylacetonitrile, 2-phenylbutyronitrile, 2-aminophenylacetonitrile, butyronitrile, and methacrylonitrile, and has good S-chiral selectivity.Fa-Mou Guo et al. reported that the nitrile hydratase of Klebsiella oxytoca can hydrolyze propionitrile, butyric acid nitrile, isobutyric acid nitrile, acrylonitrile, methacrylonitrile, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, benzyl nitrile, phenylacetonitrile, α-methylbenzonitrile, mandelic acid nitrile, 2,2-dimethylcyclopropionitrile, and 2-(4-chlorophenyl)3-methylbutyric acid nitrile, while showing good S-chirality selectivity for α-methylbenzonitrile, mandelic acid nitrile, 2,2-dimethylcyclopropionitrile, and 2-(4-chlorophenyl)3-methylbutyric acid nitrile [Guo, FM, Wu, JP, Yang, LR, & Xu, G.. (2015). Overexpression of a nitrile hydratase from klebsiella oxytoca kctc1686in escherichia coli and its biochemical characterization. Biotechnology and bioprocess engine. e.ring, 20(6), 995-1004.].

[0009] This study is the first to discover that nitrile hydratase NH3 derived from Klebsiella oxytoca can catalyze the hydrolysis of cyanoketoneprofen with an ee value exceeding 98%. It shows great potential for application in the production of (S)-(+)-amide-ketoprofen. However, nitrile hydratase NH3 exhibits low enzyme activity and stability during the catalytic conversion of cyanoketoneprofen to (S)-(+)-amide-ketoprofen, resulting in a large enzyme dosage and high production costs. Summary of the Invention

[0010] This invention aims to at least partially solve one of the technical problems in the related art. To this end, the inventors of this application, based on nitrile hydratases derived from Klebsiella oxytoca, screened several nitrile hydratase variants with improved specific activity and thermostability through rational design and directed evolution. These variants have a specific activity of 110.1% to 526.2% of the parent nitrile hydratase, and also exhibit significantly improved stability when stored at room temperature.

[0011] In a first aspect of the invention, the invention proposes a nitrile hydratase variant with improved specific activity and thermal stability.

[0012] According to an embodiment of the present invention, the nitrile hydratase variant comprises an α subunit and a β subunit, the α subunit amino acid sequence being as shown in SEQ ID NO:1 or containing mutations selected from one or more positions corresponding to SEQ ID NO:1: position 8, position 36, position 127, position 149, position 172, position 199, and the β subunit amino acid sequence being as shown in SEQ ID NO:2 or containing mutations selected from one or more positions corresponding to SEQ ID NO:2: position 35, position 37, position 41, position 78, position 101, position 122, position 127, position 170, position 181, position 204, position 208, and at least one of the sequences of the α subunit and the β subunit is obtained by mutation;

[0013] The amino acid positions of the α and β subunits of the nitrile hydratase variant are numbered relative to the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0014] According to an embodiment of the present invention, the α subunit amino acid sequence of the nitrile hydratase variant contains mutations selected from one or more positions corresponding to SEQ ID NO:1: amino acid 8 is mutated to arginine, amino acid 36 is mutated to cysteine, amino acid 149 is mutated to proline, amino acid 172 is mutated to proline, and amino acid 199 is mutated to proline; the β subunit amino acid sequence of the nitrile hydratase variant contains mutations selected from one or more positions corresponding to SEQ ID NO:1. Mutations at one or more positions of NO:2: Amino acid at position 35 is mutated to cysteine; amino acid at position 37 is mutated to alanine, cysteine, glycine, leucine, isoleucine, methionine, serine, or tryptophan; amino acid at position 41 is mutated to alanine; amino acid at position 78 is mutated to arginine; amino acid at position 101 is mutated to arginine; amino acid at position 122 is mutated to cysteine; amino acid at position 127 is mutated to alanine; amino acid at position 170 is mutated to cysteine; amino acid at position 181 is mutated to cysteine; amino acid at position 204 is mutated to arginine; amino acid at position 208 is mutated to leucine.

[0015] According to embodiments of the present invention, the nitrile hydratase variant comprises a combination of at least two of the following mutation sites:

[0016] The α subunit is located at positions 8, 36, 149, and 199; the β subunit is located at positions 35, 78, 101, 122, 127, 170, 181, and 208.

[0017] According to embodiments of the present invention, the mutation of the nitrile hydratase variant is selected from one of the following groups:

[0018] αL36C,βA35C;

[0019] βA122C,βV170C;

[0020] βV170C, βE181C;

[0021] αL36C, βA35C, βA122C, βV170C;

[0022] αL36C, βA35C, βV170C, βE181C;

[0023] Any one of αT8R / αA149P / αA172P / αN199P / βT78R / βA101R / βD127A / βT204R / βR208L / βF41A;

[0024] Any one of βF37A / βF37C / βF37G / βF37I / βF37L / βF37M / βF37S / βF37W;

[0025] αT8R, αN199P;

[0026] αT8R,βD127A;

[0027] αA172P,βT78R;

[0028] αA149P,βA101R;

[0029] βT78R,βA101R;

[0030] βR208L,βA101R.

[0031] According to embodiments of the present invention, the α-subunit amino acid sequence of the nitrile hydratase variant is selected from the sequences shown in SEQ ID NO:1, 5, 11, 12, 13, 14, 29, and the β-subunit amino acid sequence is selected from the sequences shown in SEQ ID NO:2, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31.

[0032] According to embodiments of the present invention, the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 are derived from Klebsiella oxytoca.

[0033] In a second aspect, the present invention provides a nucleotide encoding the above-described nitrile hydratase variant.

[0034] The present invention also provides an expression vector comprising nucleotides encoding nitrile hydratase variants and accessory proteins, and a transformed host cell.

[0035] According to embodiments of the present invention, the accessory protein is selected from accessory proteins of nitrile hydratases from any source, preferably accessory proteins with an amino acid sequence as shown in SEQ ID NO:32.

[0036] According to an embodiment of the present invention, the expression vector is the pET28a vector;

[0037] Optionally, the host cells include Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae.

[0038] In a third aspect, the present invention provides a method for preparing dextromethorphan ketoprofen using a nitrile hydratase variant. According to embodiments of the invention, the method comprises subjecting cyanoketoneprofen to an amidation reaction catalyzed by the nitrile hydratase variant described herein to obtain dextromethorphan ketoprofen.

[0039] According to an embodiment of the present invention, the method for preparing dextromethorphan ketoprofen described above can be represented as follows:

[0040]

[0041] The inventors discovered that nitrile hydratase NH3 variants with certain amino acid site mutations on the α or β subunits exhibit varying degrees of improvement in specific activity and thermal stability compared to the parent nitrile hydratase NH3. Using nitrile hydratase variants to prepare dextromethorphan can significantly reduce the amount of enzyme added in the catalytic reaction, effectively lowering production costs. At the same time, the enzyme's storage stability is also greatly improved.

[0042] According to embodiments of the present invention, the above-described method for preparing dextromethorphan may further include at least one of the following additional technical features:

[0043] According to embodiments of the present invention, the α-subunit amino acid sequence of the nitrile hydratase variant is selected from the sequences shown in SEQ ID NO:1, 5, 11, 12, 13, 14, 29, and the β-subunit amino acid sequence is selected from the sequences shown in SEQ ID NO:2, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31.

[0044] According to embodiments of the present invention, the nitrile hydratase variant is provided in the form of wet cell culture of nitrile hydratase fermentation cells, spray-dried enzyme powder, or freeze-dried enzyme powder.

[0045] According to a specific embodiment of the present invention, the nitrile hydratase variant fermentation cells are obtained by: activating Escherichia coli cells transformed with a plasmid carrying a nucleic acid sequence expressing a nitrile hydratase variant and a helper protein; and then performing basal fermentation culture on the activated bacterial culture to obtain OD. 600 The basic fermentation culture medium is 30-35 μL; the basic fermentation culture medium is then subjected to induction culture, which is carried out in the presence of IPTG and CoCl2, and the induction culture time is 4-24 h.

[0046] According to an embodiment of the present invention, the plasmid is an expression plasmid containing a nitrile hydratase variant nucleic acid sequence obtained by using a nucleotide sequence containing nitrile hydratase NH3 as a template and introducing one or more amino acid mutations or substitutions into the nitrile hydratase NH3 sequence through site-directed mutagenesis.

[0047] According to an embodiment of the present invention, the accessory protein is an accessory protein of nitrile hydratase NH3.

[0048] According to an embodiment of the present invention, the mass ratio of the wet cell, spray-dried enzyme powder, or freeze-dried enzyme powder of the nitrile hydratase variant to cyanoprofen is (0.15-4):1, such as 0.2:1, 0.5:1, or 1:1.

[0049] According to an embodiment of the present invention, the wet cell is obtained by centrifuging and washing the fermentation broth of a nitrile hydratase variant.

[0050] According to an embodiment of the present invention, the spray-dried enzyme powder is obtained by spray drying a nitrile hydratase variant fermentation broth.

[0051] According to an embodiment of the present invention, the freeze-dried enzyme powder is obtained by centrifuging, washing, pre-cooling at ultra-low temperature, and freeze-drying the fermentation broth of a nitrile hydratase variant.

[0052] According to an embodiment of the present invention, the nitrile hydratase variant is dispersed in an organic solvent to carry out the catalytic reaction;

[0053] Optionally, the organic solvent is selected from toluene, ethyl acetate, and isopropyl acetate, preferably ethyl acetate;

[0054] Optionally, the concentration of the organic solvent is 5% to 15%, preferably 10%.

[0055] According to an embodiment of the present invention, the cyanoketone profen is dissolved in a buffer solution to carry out the catalytic reaction;

[0056] Optionally, the buffer solution is selected from sodium phosphate, potassium phosphate, and Tris-HCl, preferably sodium phosphate buffer solution;

[0057] Optionally, the concentration of the buffer solution is 0.03–0.5 M, and the pH is 7.0–8.0.

[0058] According to an embodiment of the present invention, the catalytic reaction temperature is 25°C to 35°C, and the reaction time is 6h to 24h.

[0059] definition

[0060] In this invention, the nitrile hydratase derived from Klebsiella oxytoca is named NH3, and as used herein, the terms "nitrile hydratase parent" and "nitrile hydratase NH3 parent" refer to the nitrile hydratase derived from Klebsiella oxytoca.

[0061] As used in this article, the term "nitrile hydratase" refers to a class of enzymes that can catalyze the conversion of nitrile substances into corresponding amide substances.

[0062] As used herein, the term "nitrile hydratase variant" means a nitrile hydratase whose amino acid sequence has been modified, wherein one or more amino acid residues of the nitrile hydratase have been replaced by other amino acid residues, and / or one or more amino acid residues have been deleted from the nitrile hydratase, and / or one or more amino acid residues have been added to and / or inserted into the nitrile hydratase.

[0063] As used in this article, the term "IPTG" refers to isopropyl-β-D-thiogalactopyranoside, a commonly used molecular biology reagent that acts as an inducer of β-galactosidase and β-galactosylpermease.

[0064] In this paper, the term "ee" refers to enantiomeric excess, which means that one enantiomer is in excess of another. The ee value is used to describe the optical purity of a substance.

[0065] In the naming of mutation points in this paper, the first letter represents the α subunit and β represents the β subunit; the second letter represents the parent amino acid of nitrile hydratase NH3; the numbers from the third to the second to last represent the position corresponding to the parent sequence number; the last letter represents the amino acid of the mutant; for example, the mutation point "αL36C" in the paper means that the mutation point is obtained by mutating leucine at position 36 of the α subunit amino acid sequence number of nitrile hydratase NH3 to cysteine. Attached Figure Description

[0066] Figure 1 The pET28a-NH3 plasmid pattern is shown;

[0067] Figure 2The image shows an SDS-PAGE electrophoresis diagram of the nitrile hydratase NH3 protein. Detailed Implementation

[0068] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0069] This invention provides a method for preparing fermentation cells containing a nitrile hydratase variant, and utilizes the fermentation cells for enzyme-catalyzed reactions. The pathway for synthesizing dextromethorphan using the nitrile hydratase variant is as follows:

[0070]

[0071] The embodiments of the present invention will now be described in more detail.

[0072] Example 1: Construction of the parental *Escherichia coli* strain expressing nitrile hydratase NH3

[0073] Based on the amino acid sequences of the α-subunit (SEQ ID NO:1), β-subunit (SEQ ID NO:2), and accessory protein (SEQ ID NO:32) of the nitrile hydratase NH3 parent, nucleotide sequences were optimized according to the *E. coli* codons to obtain the following results: α-subunit nucleotide sequence as shown in SEQ ID NO:3, β-subunit nucleotide sequence as shown in SEQ ID NO:4, and accessory protein nucleotide sequence as shown in SEQ ID NO:33. An RBS site was added before each of the above three nucleotide sequences. According to an embodiment of the present invention, the selected RBS sequence was AAAAGAGGAGAAA. These sequences were then spliced ​​together to obtain a tandem gene (in this embodiment, the sequence was RBS-α-subunit-RBS-β-subunit-RBS-accessory protein), which was submitted to General Biosystems (Anhui) Co., Ltd. for synthesis.

[0074] Then, using gene recombination technology, the synthetic tandem gene was cloned between the XbaI and HindIII sites of the pET28a vector to obtain the pET28a-NH3 plasmid, as shown in the plasmid map. Figure 1As shown. The pET28a-NH3 plasmid was transformed into BL21(DE3) competent cells using the heat shock method. The transformation medium was plated on LB+Kan plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin, 15 g / L agar powder) and cultured overnight at 37°C. The transformed strain was named BL21-NH3 after being verified by PCR and sequencing. After amplification by LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin, 15 g / L agar powder) at 37°C, sterile glycerol was added to a final concentration of 15–20% and the cells were stored at -80°C.

[0075] Example 2: Induced expression of the NH3 parent of nitrile hydratase

[0076] Single colonies of the BL21-NH3 strain, frozen at -80℃, were isolated by streaking on LB+Kan plates. Single colonies from the transformation plates were picked and transferred to 5 mL of liquid LB+Kan medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin) and incubated overnight at 37℃ and 250 g / min for approximately 12 h.

[0077] Take 2 mL of the overnight activated bacterial culture and transfer it to 200 mL of fresh LB+Kan liquid. Incubate at 37℃ and 250 g / min until the OD reaches 0.5-0.8.

[0078] Take the cultured bacterial solution, add IPTG to a final concentration of 1 mM and CoCl2 to a final concentration of 1 mM, and incubate at 30℃ and 250 g / min for 5 h.

[0079] After induction, all bacterial culture was collected and centrifuged at 4000 g / min and 4℃ to collect bacterial cells. An appropriate amount of bacterial cells was subjected to ultrasonic disruption, followed by centrifugation at 10000 g / min and 4℃ for 10 min. The supernatant was then analyzed by SDS-PAGE to detect protein expression. Figure 2 As shown.

[0080] Example 3: Construction and Induced Expression of Nitrile Hydrate Hydrate Enzyme NH3 Variant Escherichia coli

[0081] Using molecular biology techniques known in the art, pET28a-NH3 plasmid was used as a template. One or more amino acid mutations (substitutions) were introduced into the parental NH3 cell using the TAKARA site-directed mutagenesis kit (Code NO. R401) to obtain various NH3 variant expression plasmids. The NH3 variant expression plasmids were transformed into BL21(DE3) competent cells using the heat shock method. The transformation solution was plated on LB+Kan plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin, 15 g / L agar powder) and incubated overnight at 37°C. After the transformants were verified to be correct by PCR and sequencing, they were amplified by culturing in LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin, 15 g / L agar powder) at 37°C. After adding sterile glycerol to a final concentration of 15–20%, the mixture was stored at -80°C. The constructed nitrile hydratase NH3 variants are shown in Table 1.

[0082] The nitrile hydratase NH3 variant was induced to express in the same manner as in Example 2.

[0083] Example 4: Detection of enzyme activity, stability, and specific activity calculation of nitrile hydratase NH3 parent and NH3 variant.

[0084] Enzyme activity detection and specific activity calculation: Nitrile hydratase NH3 parental and NH3 variant bacterial cultures were prepared according to the methods described in Examples 2 and 3, respectively. The wet bacterial cells were collected by centrifugation at 4000g for 30 min at 4℃. The cells were then homogenized using a high-pressure homogenizer at 800-1000 bar at 4℃. The supernatant was collected by centrifugation at 4000g for 30 min at 4℃. The pH of the supernatant was adjusted to 4.0-4.5 with citric acid, filtered, and the conductivity was measured to be <10 mS / CM. The filtrate was subjected to ion exchange chromatography (UniGel 80-SP packing material) and hydrophobic chromatography (Monomix MC30-Hic Butyl packing material) to obtain nitrile hydratase NH3 parental and NH3 variant proteins with a purity of over 98%, respectively.

[0085] Purified nitrile hydratase NH3 parent and NH3 variant protein were added separately to 100 mL reaction flasks. 40 mL of 0.1 mol / L pH 7.5 phosphate buffer was added to each flask, and the mixture was stirred thoroughly and incubated at 30 °C for 10 min. 2.4 g of cyanoprofen was weighed into 10 mL EP tubes, and 4 mL of ethyl acetate was added. The tubes were vortexed for 10 min until fully dissolved, and then added dropwise to the reaction flasks over approximately 1 min. The reaction was incubated at 30 °C for 30 min. Extraction was performed using 20 mL of dichloromethane. 50 μL of the extract was collected, dried at 30–40 °C, and analyzed by HPLC. HPLC conditions were as follows: the dried sample was dissolved in acetonitrile, and a Welch Topsil 5u C18 100A 4.6 × 150 mm column was used. The column temperature was controlled at 25 °C. Elution was performed isocratically for 25 min with a mobile phase of 2% phosphate buffer-43% ACN-55% H2O. Detection was performed at 255 nm. Enzyme activity is defined as the amount of enzyme required to convert 1 micromolar of substrate within 1 minute at 30°C, which is one unit of activity (U). Following the manufacturer's instructions, the protein concentrations of the purified NH3 parent and NH3 variant were determined using the TAKARA Bradford Protein Assay Kit (Code NO. T9310A). Specific activity was then calculated using the formula (Specific Activity = Enzyme Activity / Enzyme Protein Concentration).

[0086] Stability testing: The purified NH3 parent and NH3 variant protein solutions were dialyzed to replace the solvent with 0.1M pH 7.5 sodium phosphate buffer, and the initial specific activity was measured. Then, the specific activity was measured after incubation at 25°C for 3 days. The remaining specific activity percentage was calculated using the formula (remaining specific activity percentage = specific activity after 3 days at 25°C / initial specific activity * 100%). A higher remaining specific activity percentage indicates better stability, or vice versa.

[0087] The relative specific activity and stability data of nitrile hydratase NH3 parent and NH3 variant are shown in Table 1. Compared with the NH3 parent, the NH3 variant showed varying degrees of improvement in specific activity and / or stability.

[0088] Table 1. Parental and variant numbers, mutation sites, specific activities, stability, and amino acid sequences of NH3. (SEQ ID NO)

[0089]

[0090]

[0091] *Mutation site explanation: The first letter represents the parent amino acid NH3; the second letter: α represents the α subunit, β represents the β subunit; the numbers from the third letter up to the penultimate letter represent the positions corresponding to the parent sequence number; the last letter represents the amino acid of the mutant. Multiple mutation sites are separated by commas.

[0092] Example 5: 50L tank fermentation of nitrile hydratase NH3 parent and NH3 variant

[0093] Both the parent strain of nitrile hydratase NH3 from Example 1 and the strain of nitrile hydratase NH3 variant from Example 3, frozen at -80℃, were activated overnight. Then, 1 mL of each strain was transferred to 1000 mL of fresh liquid LB+Kan medium and cultured at 37℃ and 250 g / min for approximately 10 h. Afterward, each strain was inoculated into a 50 L fermenter (fermentation medium: 121 g glucose monohydrate, 500 g yeast extract, 250 g yeast peptone, 100 g NaCl, 25 g Na2HPO4, 20 g MgSO4·7H2O, 100 g KH2PO4, 50 g (NH4)2SO4, 55 g citric acid monohydrate, 75 g NaOH, 25 mL GPE antifoaming agent. Sterilized at 118℃ for 30 min, resulting in a volume of approximately 25 L after sterilization). Fermentation was then carried out according to the following process.

[0094] Basic culture stage: control the culture temperature at 37.0℃, the aeration rate at 1:1 vvm, and adjust the dissolved oxygen level of the fermentation broth to be higher than 50% by stirring and aeration, and the tank pressure at 40-70 kPa.

[0095] Glucose supplementation phase: When the pH of the fermentation broth rebounded to 7.15, glucose solution was added, with an initial supplementation rate of 6.6 g / Lh, increasing by 0.7 g / Lh every 0.5 h, until the glucose supplementation rate reached 10.0 g / Lh. This rate was maintained until the induction phase. The culture temperature during the glucose supplementation phase was 34.0℃, with an aeration rate of 1:1.3 vvm. The dissolved oxygen level in the fermentation broth was maintained above 30% through stirring and aeration.

[0096] Induction stage: Fermentation broth OD 600 When the pH value is between 30 and 35, induction begins by injecting a single batch of 1 mM IPTG solution and a 1 mM CoCl2 solution. Before induction, lower the culture temperature to 30°C. After induction, increase the glucose replenishment rate to 3.3–3.6 g / L h, and maintain the dissolved oxygen level in the fermentation broth at 20%–50%. Maintain this rate until fermentation is complete, with induction lasting approximately 15 hours.

[0097] After fermentation, the fermentation broth was immediately converted into wet cells and enzyme powder and stored at 4°C.

[0098] Example 6: Preparation of wet cells and enzyme powder of nitrile hydratase NH3 parent and NH3 variant

[0099] Preparation of wet cells: 500 mL of fermentation broth from the nitrile hydratase NH3 parent and the nitrile hydratase NH3 variant obtained in Example 5 were centrifuged at 4000 g for 30 min at 4 °C to collect wet cells. The cells were washed with physiological saline and then centrifuged again to remove excess saline, yielding the nitrile hydratase wet cells. The wet cells were stored at 4 °C.

[0100] Preparation of spray-dried enzyme powder: 500 mL of fermentation broth from the nitrile hydratase NH3 parent and the nitrile hydratase NH3 variant obtained in Example 5 were spray-dried in a spray drying oven (with parameters set as inlet temperature 170℃, outlet temperature 80℃, and feed flow rate 700 mL / h) to obtain nitrile hydratase spray-dried enzyme powder. The spray-dried enzyme powder was stored at 4℃.

[0101] Preparation of lyophilized enzyme powder: Using 500 mL of fermentation broth from the nitrile hydratase NH3 parent and the nitrile hydratase NH3 variant obtained in Example 5, centrifuge at 4000 g for 30 min at 4℃ to retain the bacterial cells. Wash the bacterial cells with physiological saline, then centrifuge again as described above to remove excess saline. Add 5% sucrose solution as a preservative. Resuspend the bacterial cells and place them on a partition in a freezer chamber. Pre-cool in an ultra-low temperature freezer (-70℃) for 12 h. Then freeze-dry in a desiccant for 48 h to obtain lyophilized enzyme powder. Store the lyophilized enzyme powder at 4℃.

[0102] Example 7: Synthesis of D-Damidokeprofen from Cyanobenzocatalyzed by wet cell cyanoketoneprofen using nitrile hydratase NH3 parent and NH3 variant.

[0103] 2.0–20 g of wet cells of the nitrile hydratase NH3 parent and the nitrile hydratase NH3 variant from Example 6 were uniformly dispersed in 100 mL of 0.1 M pH 7.5 sodium phosphate buffer and incubated at 30 °C for 10 min. Then, 6 g of cyanoprofen substrate dissolved in 10 mL of ethyl acetate was added dropwise to the reaction flask, and the reaction was maintained at this temperature. After 24 h of reaction, 2 mL of the sample was taken, extracted with 4 mL of dichloromethane, and 50 μL of the extract was collected, dried, and the content of each component was determined by HPLC. The HPLC conditions were as follows:

[0104] (1) Sample preparation: The dried extract phase was dissolved in acetonitrile and prepared to a concentration of 1 mg / ml.

[0105] (2) Detection conditions: HPLC normal phase was used, with a column (Welch Topsil 5u C18 100A, 4.6×150mm), detection wavelength: 250nm, column temperature: 25℃, injection volume: 5ul, mobile phase: phosphate buffer (68.0g of potassium dihydrogen phosphate was dissolved in water and diluted to 1000ml, and the pH was adjusted to 3.5±0.1 with phosphoric acid)-acetonitrile-water (2:43:55), elution conditions: isocratic elution, run time: 25min.

[0106] (3) Calculation of ee value: ee = (molar content of dextrorotatory KPA - molar content of levorotatory KPA) / (molar content of dextrorotatory KPA + molar content of levorotatory KPA) * 100%.

[0107] (4) Conversion rate calculation: Conversion rate = KPA molar content / initial KPN molar content * 100%.

[0108] (5) Wherein, KPN is the substrate cyanoketoneprofen and KPA is the product amide ketoneprofen.

[0109] Finally, the required amount of wet bacterial cells to be added to achieve a 45% conversion rate for both the nitrile hydratase NH3 parent and each nitrile hydratase NH3 variant was determined, and the ee value at this point was measured. The results are shown in Table 2.

[0110] The results showed that, compared with the NH3 parent, the NH3 variant required a smaller amount of wet cell to achieve a 45% conversion rate in the same reaction time, while the ee value remained above 98%, which effectively reduced production costs.

[0111] Example 8: Synthesis of D-Amamidokeprofen from Cyanokeprofen Catalyzed by Spray-dried Powder of Nitrile Hydrate Enzyme NH3 Parent and NH3 Variant

[0112] 0.2–10 g of the nitrile hydratase NH3 parent and nitrile hydratase NH3 variant spray-dried powder from Example 6 were uniformly dispersed in 100 mL of 0.1 M pH 7.5 sodium phosphate buffer and incubated at 30 °C for 10 min. Then, 6 g of cyanoprofen substrate dissolved in 10 mL of ethyl acetate was added dropwise to the reaction flask, and the reaction was maintained at this temperature. After 24 h of reaction, 2 mL of sample was taken, extracted with 4 mL of dichloromethane, and 50 μL of the extract phase was collected and dried. The HPLC method of Example 7 was used for detection. Finally, the amount of wet bacterial cells required to achieve a 45% conversion rate for the nitrile hydratase NH3 parent and each nitrile hydratase NH3 variant was determined, and the ee value at this point was calculated according to the method of Example 7. The results are shown in Table 2.

[0113] The results showed that, compared with the parent NH3, the NH3 variant required a significantly lower amount of spray-dried powder to achieve a 45% conversion rate in the same reaction time, while still maintaining an ee value of over 98%, which effectively reduced production costs.

[0114] Example 9: Synthesis of D-aminoketoprofen from lyophilized powders of nitrile hydratase NH3 parent and NH3 variant catalyzed by cyanoketoneprofen.

[0115] 0.2–2 g of the lyophilized powder of the nitrile hydratase NH3 parent and the nitrile hydratase NH3 variant from Example 6 were uniformly dispersed in 100 mL of 0.1 M pH 7.5 sodium phosphate buffer and incubated at 30 °C for 10 min. Then, 6 g of cyanoprofen substrate dissolved in 10 mL of ethyl acetate was added dropwise to the reaction flask, and the reaction was maintained at this temperature. After 24 h of reaction, 2 mL of sample was taken, extracted with 4 mL of dichloromethane, and 50 μL of the extract was collected and dried. The HPLC method of Example 7 was used for detection. Finally, the amount of wet bacterial cells required to achieve a 45% conversion rate of the nitrile hydratase NH3 parent and each nitrile hydratase NH3 variant was determined, and the ee value at this point was calculated according to the method of Example 7. The results are shown in Table 2.

[0116] The results showed that, compared with the parent NH3, the amount of lyophilized powder used by the NH3 variant was reduced when the conversion rate reached 45% in the same reaction time, while the ee value remained above 98%, which can effectively reduce production costs.

[0117] Table 2. Dosage and corresponding ee values ​​of wet, spray-dried, and lyophilized cyanoketone-profen by NH3 parent and NH3 variants.

[0118]

[0119]

[0120] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0121] sequence list

[0122] >SEQ ID NO 1

[0123] MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH

[0124] >SEQ ID NO 2

[0125] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0126] >SEQ ID NO 3

[0127] ATGAGCCATGATCATGATCATACCGAACCGCCGACCGAAATTGCCCTGCGTGTTAAAGCCCTGGAAAGCCTGCTGACCGAAAAAGGCCTGGTTGATCCGGCCGCCCTGGATGAACTGGTTGATACCTATGAAAATCGCATTGGTCCGCGTAATGGTGCCCTGGTTGTTGCAAAAGCCTGGACCGATCCGGCATATAAACAGCGTCTGCTGACCAATGCAACCGAAGCAATTGCAGAACTGGGTTTTAGCGGTGTTCAGGGCGAAGATATGCTGGTGGTTGAAAATAGTCCGACCGTGCATAATATGACCGTGTGTACCCTGTGCAGTTGTTATCCGTGGCCGACCCTGGGCCTGCCGCCTGCATGGTATAAAAGCGCCCCGTATCGCAGTCGCGTTGTTATTGATCCGCGTGGCGTTCTGGCCGAATTTGGTGTGAGTGTTCCGGCAGATAAAGAAGTGCGCGTTTGGGATAGTAGCGCCGAACTGCGCTATCTGGTGCTGCCGGAACGTCCGGCAGGCACCGAAGGCTGGAGCGAAGAACAGCTGGTGGAACTGGTTACCCGTGATAGTATGATTGGCACCGGCTTTCCGAAAAATCCGGCAGATCTGCATTAA

[0128] >SEQ ID NO 4

[0129] ATGAACGGTGTGCATGATCTGGGTGGTATGCATGGTCTGGGTCCGATTGCCCCGCCGGCAGATGAACCGGTGTTTGCACATCAGTGGGAACGCCGCATTTTTGCCCTGTTTGTGCCGCTGTTTGGTGGTGGTCATTTTAATGTGGATCAGTTTCGCCATGCAATTGAACGTATGGATCCGGCCCATTATCTGCAGGGCACCTATTATGAACATTGGCTGCATGCCTTTGAAACCCTGCTGATTGAAGGTGGCGCAATTAGTCGTGCCGAACTGGATGCACGTATTAAGCAGATTGGTGGTGCACAGATTATGGCAGTGGTTACCCGCGATATGATTGAACCGATTGTTCGTACCGGTGCCAGTGCACGTGTGGCCGCAGATGTTGCCGCCCGTTTTAAAGTTGGCGATACCGTTCGTGCAAAAAATATTAATCCGACCACCCATACCCGCCTGCCGCGCTATGTTCGCGGCCGTGTTGGTACCATTGAAATTGATCATGGCGTTTTTGTGACCCCGGATACCGTGGCCCACGGTAAAGGTGAACATCCGCAGCATGTGTATTGCGTGCGCTTTGCCGCAGTGGAACTGTGGGGCAGCGATGTGAGTGGTACCGATAATGTTCGCATTGATCTGTGGGATGATTATCTGGAAAAAGCCTAA

[0130] >SEQ ID NO 5

[0131] MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAACDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH

[0132] >SEQ ID NO 6

[0133] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0134] >SEQ ID NO 7

[0135] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASCRVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0136] >SEQ ID NO 8

[0137] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGCHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0138] >SEQ ID NO 9

[0139] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASCRVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0140] >SEQ ID NO 10

[0141] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGCHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0142] >SEQ ID NO 11

[0143] MSHDHDHREPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH

[0144] >SEQ ID NO 12

[0145] MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPPDKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH

[0146] >SEQ ID NO 13

[0147] MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPPGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH

[0148] >SEQ ID NO 14

[0149] MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKPPADLH

[0150] >SEQ ID NO 15

[0151] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFERLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0152] >SEQ ID NO 16

[0153] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0154] >SEQ ID NO 17

[0155] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAAAVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0156] >SEQ ID NO 18

[0157] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGRDNVRIDLWDDYLEKA

[0158] >SEQ ID NO 19

[0159] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVLIDLWDDYLEKA

[0160] >SEQ ID NO 20

[0161] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALAVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0162] >SEQ ID NO 21

[0163] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALCVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0164] >SEQ ID NO 22

[0165] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALGVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0166] >SEQ ID NO 23

[0167] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALIVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0168] >SEQ ID NO 24

[0169] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALLVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0170] >SEQ ID NO 25

[0171] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALMVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0172] >SEQ ID NO 26

[0173] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALSVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0174] >SEQ ID NO 27

[0175] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALWVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0176] >SEQ ID NO 28

[0177] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLAGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0178] >SEQ ID NO 29

[0179] MSHDHDHREPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKPPADLH

[0180] >SEQ ID NO 30

[0181] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFERLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA

[0182] >SEQ ID NO 31

[0183] MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVLIDLWDDYLEKA

[0184] >SEQ ID NO 32

[0185] MAETIDYELVNLPRDDEGPVFEEPWQAQVFSLTVHLHKAGHFTWPQWVQTFSREIGRSPALPGESVNAAYYRQWAAALEHMVAKIGLAGLVDVAGRTEEWRQAYINTPHGHPVLLANAACPPAHDHHHHVPERSPVAISPACPV

[0186] >SEQ ID NO 33

[0187] ATGGCCGAAACCATTGATTATGAACTGGTGAATCTGCCGCGCGATGATGAAGGTCCGGTTTTTGAAGAACCGTGGCAGGCACAGGTTTTTAGCCTGACCGTGCATCTGCATAAAGCCGGCCATTTTACCTGGCCGCAGTGGGTTCAGACCTTTAGTCGTGAAATTGGCCGTAGTCCGGCACTGCCGGGTGAAAGCGTTAATGCCGCATATTATCGCC AGTGGGCAGCCGCCCTGGAACATATGGTTGCCAAAATTGGTCTGGCAGGCCTGGTTGATGTTGCAGGCCGCACCGAAGAATGGCGTCAGGCCTATATTAATACCCCGCATGGTCATCCGGTGCTGCTGGCAAATGCAGCATGTCCGCCGGCACATGATCATCATCATCATGTTCCGGAACGTAGTCCGGTGGCCATTAGTCCGGCCTGTCCGGTGTAA SEQUENCE LISTING <110> Yichang Dongyangguang Biochemical Pharmaceutical Co., Ltd. <120> Nitrile hydratase variants with improved specific activity and thermal stability and their applications <130> PP1175CN <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 204 <212> PRT <213> Klebsiella oxytoca <400> 1 Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His 195 200 <210> 2 <211> 219 <212> PRT <213> Klebsiella oxytoca <400> 2 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 3 <211> 615 <212> DNA <213> Artificial sequence <400> 3 atgagccatg atcatgatca taccgaaccg ccgaccgaaa ttgccctgcg tgttaaagcc 60 ctggaaagcc tgctgaccga aaaaggcctg gttgatccgg ccgccctgga tgaactggtt 120 gatacctatg aaaatcgcat tggtccgcgt aatggtgccc tggttgttgc aaaagcctgg 180 accgatccgg catataaaca gcgtctgctg accaatgcaa ccgaagcaat tgcagaactg 240 ggttttagcg gtgttcaggg cgaagatatg ctggtggttg aaaatagtcc gaccgtgcat 300. aatatgaccg tgtgtaccct gtgcagttgt tatccgtggc cgaccctggg cctgccgcct 360 gcatggtata aaagcgcccc gtatcgcagt cgcgttgtta ttgatccgcg tggcgttctg 420 gccgaatttg gtgtgagtgt tccggcagat aaagaagtgc gcgtttggga tagtagcgcc 480 gaactgcgct atctggtgct gccggacgt ccggcaggca ccgaaggctg gagcgaagaa cagctggtgg aactggttac ccgtgatagt atgattggca ccggctttcc gaaaaatccg gcagatctgc attack 615 <210> 4 <211> 660 <212> DNA <213> The snowstorm <400> 4 atgaacggtg tgcatgatct gggtggtatg catggtctgg gtccgattgc cccgccggca 120. gatgaaccgg tgtttgcaca tcagtgggaa cgccgcattt ttgccctgtt tgtgccgctg tttggtggtg gtcattttaa tgtggatcag tttcgccatg caattgaacg tatggatccg gcccattatc tgcagggcac ctattatgaa cattggctgc atgcctttga aaccctgctg 240 attgaaggtg gcgcaattag tcgtgccgaa ctggatgcac gtattaagca gattggtggt 300 gcacagatta tggcagtggt tacccgcgat atgattgaac cgattgttcg taccggtgcc 360 agtgcacgtg tggccgcaga tgttgccgcc cgttttaaag ttggcgatac cgttcgtgca 420 aaaaatatta atccgaccac ccatacccgc ctgccgcgct atgttcgcgg ccgtgttggt 480 accattgaaa ttgatcatgg cgtttttgtg accccggata ccgtggccca cggtaaaggt 540 gaacatccgc agcatgtgta ttgcgtgcgc tttgccgcag tggaactgtg gggcagcgat 600 gtgagtggta ccgataatgt tcgcattgat ctgtgggatg attatctgga aaaagcctaa 660 <210> 5 <211> 204 <212> PRT <213> Artificial sequence <400> 5 Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Cys Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His 195 200 <210> 6 <211> 219 <212> PRT <213> Artificial Sequence <400> 6 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 7 <211> 219 <212> PRT <213> Artificial Sequence <400> 7 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Cys Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 8 <211> 219 <212> PRT <213> Artificial Sequence <400> 8 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Cys His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 9 <211> 219 <212> PRT <213> Artificial sequence <400> 9 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Cys Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 10 <211> 219 <212> PRT <213> Artificial Sequence <400> 10 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Cys His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 11 <211> 204 <212> PRT <213> Artificial Sequence <400> 11 Met Ser His Asp His Asp His Arg Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His 195 200 <210> 12 <211> 204 <212> PRT <213> Artificial Sequence <400> 12 Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Pro Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His 195 200 <210> 13 <211> 204 <212> PRT <213> Artificial Sequence <400> 13 Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Pro Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His 195 200 <210> 14 <211> 204 <212> PRT <213> Artificial Sequence <400> 14 Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Pro Pro Ala Asp Leu His 195 200 <210> 15 <211> 219 <212> PRT <213> Artificial Sequence <400> 15 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Arg Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 16 <211> 219 <212> PRT <213> Artificial Sequence <400> 16 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 17 <211> 219 <212> PRT <213> Artificial Sequence <400> 17 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Ala Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 18 <211> 219 <212> PRT <213> Artificial Sequence <400> 18 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Arg Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 19 <211> 219 <212> PRT <213> Artificial Sequence <400> 19 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Leu 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 20 <211> 219 <212> PRT <213> Artificial Sequence <400> 20 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Ala Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 21 <211> 219 <212> PRT <213> Artificial Sequence <400> 21 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Cys Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 22 <211> 219 <212> PRT <213> Artificial Sequence <400> 22 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Gly Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 23 <211> 219 <212> PRT <213> Artificial Sequence <400> 23 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Ile Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 24 <211> 219 <212> PRT <213> Artificial Sequence <400> 24 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Leu Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 25 <211> 219 <212> PRT <213> Artificial Sequence <400> 25 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Met Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 26 <211> 219 <212> PRT <213> Artificial sequence <400> 26 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Ser Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 27 <211> 219 <212> PRT <213> Artificial Sequence <400> 27 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Trp Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 28 <211> 219 <212> PRT <213> Artificial Sequence <400> 28 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Ala Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 29 <211> 204 <212> PRT <213> Artificial Sequence <400> 29 Met Ser His Asp His Asp His Arg Glu Pro Pro Thr Glu Ile Ala Leu 1 5 10 15 Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp 20 25 30 Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly 35 40 45 Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala 50 55 60 Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu 65 70 75 80 Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser 85 90 95 Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro 100 105 110 Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr 115 120 125 Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly 130 135 140 Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala 145 150 155 160 Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly 165 170 175 Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile 180 185 190 Gly Thr Gly Phe Pro Lys Pro Pro Ala Asp Leu His 195 200 <210> 30 <211> 219 <212> PRT <213> Artificial Sequence <400> 30 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Arg Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 31 <211> 219 <212> PRT <213> Artificial Sequence <400> 31 Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile 1 5 10 15 Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg 20 25 30 Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val 35 40 45 Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu 50 55 60 Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu 65 70 75 80 Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys 85 90 95 Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile 100 105 110 Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val 115 120 125 Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn 130 135 140 Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly 145 150 155 160 Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala 165 170 175 His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala 180 185 190 Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Leu 195 200 205 Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala 210 215 <210> 32 <211> 144 <212> PRT <213> Artificial Sequence <400> 32 Met Ala Glu Thr Ile Asp Tyr Glu Leu Val Asn Leu Pro Arg Asp Asp 1 5 10 15 Glu Gly Pro Val Phe Glu Glu Pro Trp Gln Ala Gln Val Phe Ser Leu 20 25 30 Thr Val His Leu His Lys Ala Gly His Phe Thr Trp Pro Gln Trp Val 35 40 45 Gln Thr Phe Ser Arg Glu Ile Gly Arg Ser Pro Ala Leu Pro Gly Glu 50 55 60 Ser Val Asn Ala Ala Tyr Tyr Arg Gln Trp Ala Ala Ala Leu Glu His 65 70 75 80 Met Val Ala Lys Ile Gly Leu Ala Gly Leu Val Asp Val Ala Gly Arg 85 90 95 Thr Glu Glu Trp Arg Gln Ala Tyr Ile Asn Thr Pro His Gly His Pro 100 105 110 Val Leu Leu Ala Asn Ala Ala Cys Pro Pro Ala His Asp His His His 115 120 125 His Val Pro Glu Arg Ser Pro Val Ala Ile Ser Pro Ala Cys Pro Val 130 135 140 <210> 33 <211> 435 <212> DNA <213> Artificial sequence <400> 33 atggccgaaa ccattgatta tgaactggtg aatctgccgc gcgatgatga aggtccggtt 60 tttgaagaac cgtggcaggc acaggttttt agcctgaccg tgcatctgca taaagccggc 120 cattttacct ggccgcagtg ggttcagacc tttagtcgtg aaattggccg tagtccggca 180 ctgccgggtg aaagcgttaa tgccgcatat tatcgccagt gggcagccgc cctggaacat 240 atggttgcca aaattggtct ggcaggcctg gttgatgttg caggccgcac cgaagaatgg 300 cgtcaggcct atattaatac cccgcatggt catccggtgc tgctggcaaa tgcagcatgt 360 ccgccggcac atgatcatca tcatcatgtt ccggaacgta gtccggtggc cattagtccg 420 gcctgtccgg tgtaa 435

Claims

1. A nitrile hydratase variant with improved specific activity and thermostability, said nitrile hydratase variant comprising an α subunit and a β subunit, characterized in that, The α-subunit amino acid sequence and β-subunit amino acid sequence of the nitrile hydratase variant are selected from the following combinations: The α-subunit amino acid sequence is shown in SEQ ID NO: 12, and the β-subunit amino acid sequence is shown in SEQ ID NO: 16; The α-subunit amino acid sequence is shown in SEQ ID NO: 1, and the β-subunit amino acid sequence is shown in SEQ ID NO: 30; The α-subunit amino acid sequence is shown in SEQ ID NO: 1, and the β-subunit amino acid sequence is shown in SEQ ID NO:

31.

2. A nucleotide encoding the nitrile hydratase variant of claim 1.

3. An expression vector comprising the nucleotide of claim 2, the vector further comprising a nucleotide encoding an accessory protein selected from accessory proteins of nitrile hydratases of any origin.

4. The expression vector according to claim 3, wherein the amino acid sequence of the accessory protein is shown in SEQ ID NO:

32.

5. A host cell comprising the expression vector of claim 3 or 4.

6. The host cell as described in claim 5 includes Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae.

7. A method for preparing dextromethorphan using the nitrile hydratase variant of claim 1, characterized in that, Cyanoketoneprofen was subjected to an amidation reaction catalyzed by a nitrile hydratase variant to obtain dextromethorphan.

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