A cyanidase mutant and its application in the degradation of cyanide-containing compounds

Abstract

This application relates to the field of bioengineering technology, specifically to a cyanide dihydrate mutant. The amino acid sequence of the cyanide dihydrate mutant, compared to the wild-type cyanide dihydrate enzyme sequence, involves mutations at one or more amino acids at positions 34, 97, 225, 301, and 307. The mutated amino acid is selected from arginine, aspartic acid, asparagine, or lysine. Compared to the wild-type cyanide dihydrate mutant, the cyanide dihydrate mutant of this invention exhibits significantly improved cyanide dihydrate enzyme activity, as well as strong catalytic activity, alkali resistance, and high-temperature resistance. It retains high enzyme activity in alkaline solvent reaction systems and can degrade cyanide under high-temperature conditions, demonstrating excellent prospects for industrial application.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, specifically to a cyanide dihydrate mutant and its application in the degradation of cyanide-containing compounds. Background Technology

[0002] Cyanide is a class of highly toxic compounds containing a cyanide group (CN-), commonly including potassium cyanide, sodium cyanide, and hydrogen cyanide. Cyanide is widely used in industries such as mining, jewelry, nylon, plastics, and pharmaceuticals, resulting in the discharge of large amounts of cyanide-containing wastewater. The pollutants contained in cyanide-containing wastewater vary depending on its source. In practical treatment, achieving the simultaneous removal of these pollutants is key to improving treatment efficiency and reducing treatment steps, thus contributing to increased economic benefits. Currently, biological, physical, and chemical methods can be used to treat and remove cyanide. Chemical and physical methods, which treat cyanide through acidification or chemical oxidation, have high investment costs and produce toxic byproducts, undoubtedly increasing the workload from an environmental perspective. However, biological methods for treating cyanide-containing wastewater require no auxiliary agents, are cost-effective, and better align with the requirements of green environmental protection, energy conservation, and emission reduction for sustainable development.

[0003] Cyanide hydratase can convert HCN into formic acid and ammonia. The product formic acid has low toxicity, does not require cofactors, and has favorable degradation characteristics.

[0004] However, one of the biggest problems with enzymatic treatment is that cyanide-containing wastewater is usually maintained at a high pH value. At neutral pH, cyanide is easily volatilized, forming hydrogen cyanide gas. Therefore, cyanide-containing wastewater needs to be kept relatively alkaline to limit the formation of hydrogen cyanide. Most wild-type cyanide-reducing bacteria require a weakly alkaline environment for reaction, and are easily inactivated at higher pH conditions, exhibiting poor thermal stability. For example, the cyanidase DN25 disclosed in patent CN201610342894.1 shows a sharp decrease in enzyme activity at pH above 8 and reaction temperature above 30℃. Currently, protein engineering technology is being used to address this problem, studying the kinetics, thermal stability, and pH of cyanidase. For instance, the cyanidase mutant in patent CN202410788676.5, through molecular modification, effectively improves its thermal stability to cyanide. However, it has not significantly improved the problems of low pH tolerance and poor high-temperature resistance in the application of cyanide-containing wastewater degradation.

[0005] Therefore, it is urgent to solve the problems of low pH tolerance and poor high temperature tolerance of existing cyanide hydrates in order to better play a role in practical applications such as the degradation of cyanide-containing wastewater. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, and to solve the technical problems of low pH tolerance and poor heat resistance of cyanide dihydrate enzyme in the prior art, the purpose of this invention is to provide a cyanide dihydrate enzyme mutant and its application in the degradation of cyanide-containing compounds. Through protein engineering technology, a cyanide dihydrate enzyme mutant capable of efficiently catalyzing inorganic cyanides such as potassium cyanide is obtained. This invention constructs a cyanide dihydrate enzyme mutant capable of catalyzing cyanide-containing compounds, including the mutant protein-coding gene, a recombinant vector containing the gene, and recombinant genetically engineered bacteria transformed from the recombinant vector. This solves the problems of low pH tolerance and poor heat resistance of cyanide dihydrate enzyme in the degradation of cyanide-containing wastewater.

[0007] To achieve the above and other related objectives, the first aspect of this application provides a cyanidase mutant, wherein the amino acid sequence of the cyanidase mutant is mutated at one or more of the following positions: positions 34, 97, 225, 301, and 307, relative to the amino acid sequence of the wild-type cyanidase. The mutated amino acid is selected from arginine, aspartic acid, asparagine, or lysine. The amino acid sequence of the wild-type cyanidase is shown in SEQ ID NO.1.

[0008] In some embodiments of this application, the cyanidase mutant, compared with wild-type cyanidase, can improve the pH tolerance and heat tolerance of cyanidase.

[0009] In some embodiments of this application, the amino acid sequence of the cyanidase mutant is selected from one or more of the following:

[0010] 1) The glutamic acid at position 34 is mutated to lysine;

[0011] 2) The lysine at position 97 is mutated to arginine;

[0012] 3) The lysine residue at position 225 is mutated to asparagine;

[0013] 4) The serine at position 301 is mutated to aspartic acid;

[0014] 5) The histidine at position 307 is mutated to lysine.

[0015] In some embodiments of this application, the amino acid sequence of the cyanide dihydrate mutant is selected from any of the following:

[0016] 1) The lysine at position 97 is mutated to arginine, and the lysine at position 225 is mutated to asparagine;

[0017] 2) The glutamic acid at position 34 is mutated to lysine, and the lysine at position 97 is mutated to arginine;

[0018] 3) The lysine at position 97 is mutated to arginine, the lysine at position 225 is mutated to asparagine, and the serine at position 301 is mutated to aspartic acid.

[0019] 4) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, and histidine at position 307 is mutated to lysine.

[0020] 5) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, and serine at position 301 is mutated to aspartic acid.

[0021] 6) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine; serine at position 301 is mutated to aspartic acid; and histidine at position 307 is mutated to lysine.

[0022] In some embodiments of this application, the amino acid sequence of the cyanidase mutant is selected from any of the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Preferably, the amino acid sequence of the cyanidase mutant is shown in SEQ ID NO: 12.

[0023] A second aspect of this application provides an isolated polynucleotide encoding the aforementioned dicyandihydrase mutant.

[0024] A third aspect of this application provides a construct containing the aforementioned polynucleotides.

[0025] The fourth aspect of this application provides a host cell containing the aforementioned construct or whose genome integrates the aforementioned polynucleotides.

[0026] The fifth aspect of this application provides a method for producing cyanidase, comprising culturing the aforementioned host cells under conditions suitable for the expression of the hexokinase mutant to obtain the cyanidase mutant.

[0027] The sixth aspect of this application provides the use of the above-mentioned cyanidase mutant, polynucleotide, construct, or host cell in the degradation of cyanide-containing compounds.

[0028] In some embodiments of this application, the cyanide-containing compound is selected from inorganic cyanides.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] Cyanide dihydrate mutants were obtained through random and site-directed mutagenesis screening. Compared with wild-type cyanide dihydrate CynD, the cyanide dihydrate mutants of this invention exhibit significantly improved cyanide dihydrate enzyme activity, thereby shortening reaction time and reducing degradation costs. The cyanide dihydrate mutants of this invention show 4.89 to 9.06 times higher enzyme activity than the wild type at 45°C and pH 9.0, meaning that they can still retain high enzyme activity in alkaline solvent reaction systems and can also degrade cyanide under high temperature conditions, showing great promise for industrial application. Attached Figure Description

[0031] Figure 1 The graph shows a comparison of the enzyme activities of wild-type dicyandihydrate and mutants of K97R, K225N, S301D, K97R / K225N, and K97R / K225N / S301D to potassium cyanide substrate in the range of 30-40℃.

[0032] Figure 2 The graph shows a comparison of the enzyme activities of wild-type dicyandihydrate and E34K, K97R, H307K, E34K / K97R, and E34K / K97R / H307K mutants to potassium cyanide in the pH range of 7.5–9.5. Detailed Implementation

[0033] To make the inventive objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described below with reference to embodiments. It should be understood that the embodiments are only for explaining the invention and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this description.

[0034] Through extensive research and exploration, this application successfully discovered a mutant of cyanide dihydrate enzyme. Compared with the corresponding wild-type cyanide dihydrate enzyme, the mutant of this application has significant enhancements, and exhibits significant high-temperature resistance and pH alkali resistance. Based on this, the present invention was completed.

[0035] This application provides a cyanidase mutant, wherein the amino acid sequence of the cyanidase mutant is mutated at one or more of the following positions (34, 97, 225, 301, and 307) compared to the amino acid sequence of the wild-type cyanidase, and the mutated amino acid is selected from arginine, aspartic acid, asparagine, or lysine; the amino acid sequence of the wild-type cyanidase is shown in SEQ ID NO.1.

[0036] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL(SEQ ID NO.1)。

[0037] ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTGAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCAAAAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAAGAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGAGCCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG(SEQ ID NO.2).

[0038] In some embodiments of this application, the amino acid sequence of the cyanidase mutant is selected from one or more of the following:

[0039] 1) The glutamic acid at position 34 is mutated to lysine;

[0040] 2) The lysine at position 97 is mutated to arginine;

[0041] 3) The lysine residue at position 225 is mutated to asparagine;

[0042] 4) The serine at position 301 is mutated to aspartic acid;

[0043] 5) The histidine at position 307 is mutated to lysine;

[0044] In a specific embodiment, when the glutamic acid at position 34 is mutated to lysine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.3, specifically:

[0045] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0046] In a specific embodiment, when the lysine at position 97 is mutated to arginine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.4, specifically:

[0047] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0048] In a specific embodiment, when the lysine at position 225 is mutated to asparagine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.5, specifically:

[0049] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0050] In a specific embodiment, when the serine at position 301 is mutated to aspartic acid, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.6, specifically:

[0051] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0052] In a specific embodiment, when the histidine at position 307 is mutated to lysine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.7, specifically:

[0053] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0054] In some embodiments of this application, the amino acid sequence of the cyanide dihydrate mutant is selected from any of the following:

[0055] 1) The lysine at position 97 is mutated to arginine, and the lysine at position 225 is mutated to asparagine;

[0056] 2) The glutamic acid at position 34 is mutated to lysine, and the lysine at position 97 is mutated to arginine;

[0057] 3) The lysine at position 97 is mutated to arginine, the lysine at position 225 is mutated to asparagine, and the serine at position 301 is mutated to aspartic acid.

[0058] 4) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, and histidine at position 307 is mutated to lysine.

[0059] 5) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, and serine at position 301 is mutated to aspartic acid.

[0060] 6) Glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine; serine at position 301 is mutated to aspartic acid; and histidine at position 307 is mutated to lysine.

[0061] In a specific embodiment, when the lysine at position 97 is mutated to arginine and the lysine at position 225 is mutated to asparagine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.8, specifically:

[0062] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0063] In a specific embodiment, when glutamic acid at position 34 is mutated to lysine and lysine at position 97 is mutated to arginine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.9, specifically:

[0064] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0065] In a specific embodiment, when lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, and serine at position 301 is mutated to aspartic acid, the amino acid sequence of the cyanidase mutant is shown in SEQ ID NO. 10, specifically:

[0066] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0067] In a specific embodiment, when glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, and histidine at position 307 is mutated to lysine, the amino acid sequence of the cyanidase mutant is as shown in SEQ ID NO.11, specifically:

[0068] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0069] In a specific embodiment, when glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, and serine at position 301 is mutated to aspartic acid, the amino acid sequence of the cyanidase mutant is shown in SEQ ID NO.12, specifically:

[0070] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0071] In a specific embodiment, when glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, serine at position 301 is mutated to aspartic acid, and histidine at position 307 is mutated to lysine, the amino acid sequence of the cyanidase mutant is shown in SEQ ID NO.13, specifically:

[0072] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGL MCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0073] In some embodiments of this application, the amino acid sequence of the cyanidase mutant is selected from any of the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Preferably, the amino acid sequence of the cyanidase mutant is shown in SEQ ID NO: 12.

[0074] The cyanidase mutant provided in this application, compared with the wild-type cyanidase, can improve the enzyme activity, pH tolerance, and heat resistance of cyanidase.

[0075] This application also provides an isolated polynucleotide encoding the aforementioned dicyandihydrate mutant.

[0076] The polynucleotide can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded.

[0077] The full-length nucleotide sequence or fragments of the hydrolase mutant of this application can generally be obtained by PCR amplification, recombination, or artificial synthesis. One method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0078] Based on the already disclosed amino acid sequence of the cyanidase, due to the degeneracy of codons, those skilled in the art can obtain the nucleotide sequence of the p-linked polynucleotide while keeping its encoding amino acid sequence unchanged. This is a conventional technique in the art.

[0079] In one specific embodiment of this application, the polynucleotide is selected from any of the nucleotide sequences shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.17.

[0080] SEQ ID NO.14:ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGA TCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTGAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG。.

[0081] SEQ ID NO.15:ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAAGAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGAGCCCGACCGATGTGGTGAAGAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG。.

[0082] SEQ ID NO.16:ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG。.

[0083] SEQ ID NO.17:ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGA TCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGAAGAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG。.

[0084] This application also provides a construct that further includes additional expression regulatory elements configured to be operatively linked to an optional nucleotide sequence. The construct is typically obtained by inserting the isolated polynucleotide into a suitable vector, which can be selected by those skilled in the art. For example, the vector can be an expression vector or a cloning vector. Furthermore, the vector type can be a plasmid, bacteriophage, bacteriophage derivative, animal virus, or entrapment, etc. Preferably, it is pET-28a.

[0085] This application also provides a host cell containing the aforementioned construct or whose genome integrates exogenous polynucleotides. The construct is transformed, transduced, or transfected into the host cell using conventional methods in the art, such as chemical transformation using calcium chloride or high-voltage electroporation. Specifically, the host cell can be a prokaryotic or eukaryotic cell, preferably *Escherichia coli*, *Bacillus subtilis*, yeast (such as *Pichia pastoris*, *Saccharomyces cerevisiae*), or various animal and plant cells. More preferably, the host cell is a genetically engineered bacterium commonly used in the art, such as *Escherichia coli*, *Bacillus subtilis*, *Pichia pastoris*, or *Saccharomyces cerevisiae*. Preferably, the host cell is *Escherichia coli* BL21(DE3) or *Escherichia coli* Top 10. The *Escherichia coli* strain must be able to express the aforementioned cyanidase mutant, thereby providing the conditions for the existence of the cyanidase mutant. Suitable methods for constructing the aforementioned *Escherichia coli* strain should be known to those skilled in the art.

[0086] This application also provides a method for producing cyanidase, comprising the following steps: culturing the aforementioned host cells under the conditions for expressing the cyanidase mutant to obtain the cyanidase mutant. Suitable induction expression methods should be known to those skilled in the art. For example, the aforementioned host cells can be induced under suitable conditions to provide cyanidase. Another example is inoculating a single clone of *E. coli* in LB liquid medium and culturing at 37°C for 8–12 h; then, at an inoculation ratio of 2–5%, inoculating into 2xYT culture medium and continuing to culture at 37°C, while continuously monitoring cell growth. When the cells reach the ideal stage of logarithmic growth, IPTG is added for induction to promote cyanidase expression, with the induction time controlled at 3–12 h, specifically 3–6 h, 6–8 h, or 8–12 h, etc.

[0087] This application also provides the use of the aforementioned cyanide dihydrate mutant, polynucleotide, construct, or the aforementioned host cell in the degradation of cyanide-containing compounds or in the preparation of catalysts.

[0088] In some embodiments of this application, the cyanide-containing compound is selected from inorganic cyanides. Preferably, the inorganic cyanide is selected from potassium cyanide, sodium cyanide, hydrogen cyanide, or similar inorganic cyanides.

[0089] The cyanidase mutant provided by this invention can effectively improve the stability of cyanidase. Experimental data show that, in the presence of the cyanidase mutant of this application, compared with wild-type cyanidase, it has higher enzyme activity, heat resistance, and alkali resistance, thereby shortening the reaction time and reducing degradation costs. It can still retain high enzyme activity in alkaline solvent reaction systems and can also degrade cyanide under high-temperature conditions, showing great promise for industrial application.

[0090] The invention of this application will be further illustrated by the following embodiments, but this does not limit the scope of this application.

[0091] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0092] Example 1

[0093] Construction of a site-directed saturation mutant library for cyanidase CynD

[0094] The pET-28a-CynD plasmid was constructed based on the cyanidase CynD, and transformed into the host bacterium E. coli BL21(DE3) to construct the engineered bacterium E. coli BL21(DE3) / pET-28b-CynD. The amino acid sequence of the cyanidase CynD is: SEQ ID No.1: MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLG GLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL;

[0095] The nucleotide sequence is: SEQ ID No.2:ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGC AGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTGAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCAAAAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAAGAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGAGCCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG。.

[0096] Site-directed saturation mutagenesis of the cyanidase CynD gene sequence was performed using whole plasmid PCR amplification technology. The template used was the recombinant plasmid pET28a-CynD of cyanidase CynD.

[0097] The conventional whole plasmid PCR amplification technique was used. The PCR amplification reaction system was as follows: 0.5 μL plasmid template, 1 μL each of a pair of primers, 10 μL PrimeSTARHS (Premix), and ddH2O added to 20 μL. The primers were degenerate primers (containing the NNK codon) for the mutation sites synthesized by a gene synthesis company. The primer sequences for the six mutation sites are shown in Table 1.

[0098] Table 1 Primer sequences

[0099]

[0100]

[0101] The PCR amplification reaction system is as follows: (1) 98℃ pre-denaturation for 3 min; (2) 98℃ denaturation for 10 s; (3) 55℃ annealing for 15 s; (4) 72℃ extension for 5 min. Steps (2)-(4) are performed for a total of 20 cycles. Finally, the product is extended at 72℃ for 7 min and stored at 4℃.

[0102] 3 μL of the PCR amplification product was subjected to nucleic acid electrophoresis to detect the PCR bands. A single bright band with a position greater than 5000 markers was considered a successful PCR. Next, 2 μL of CutOne buffer and 1 μL of DpnI enzyme were added to the PCR product tube for rapid enzyme digestion to eliminate the template plasmid. The mixture was vortexed and placed in a 37°C water bath for 1 hour to construct the mutant expression plasmid. If there were two mutations, a second round of PCR ligation was performed using primers for the second mutation to construct a double-mutant expression plasmid. If there were three mutations, a third round of PCR ligation was performed using primers for the third mutation to construct a triple-mutant expression plasmid. The plasmid was then transformed into E. coli BL21(DE3) competent cells and evenly spread on LB agar plates containing 50 μg / mL kanamycin. The cells were incubated overnight at 37°C to construct a random mutant library using site-directed saturation mutagenesis.

[0103] The cyanidase mutant of this application can be generated and verified by introducing a mutation into the wild-type cyanidase CynD gene. Based on the disclosed amino acid sequence and coding gene, those skilled in the art can also construct an expression vector for the cyanidase CynD mutant by synthesizing the corresponding gene and transforming it into *E. coli* to express the corresponding mutant. The amino acid sequence of the cyanidase mutant is shown in Table 2.

[0104] Single clones were selected and cultured in deep-well plates. Each well of the primary plate contained 300 μL of LB medium (containing 50 μL g / mL kanamycin) and was incubated overnight at 37°C and 350 rpm. 50 μL of the primary seed culture was transferred to each well of a secondary plate containing 400 μL of LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C and 350 rpm for 3-4 hours. 40 μL of 1 mM IPTG (final concentration 0.1 mM) was added to each well for induction, and the plates were incubated at 25°C for another 16-18 hours. The deep-well plates were centrifuged at 3500 rpm and 4°C for 10 minutes, the supernatant was discarded, and 400 μL of lysis buffer (100 mM KPB solution, pH 7.5, containing 0.75 mg / L lysozyme) was added to each well. The plates were shaken to resuspend the cells and lysed at 37°C for 2 hours, followed by centrifugation at 3500 rpm and 4°C for 10 minutes.

[0105] Take 50 μL of the cell lysate from each well, centrifuge, and transfer the supernatant to a new deep-well plate. Add 450 μL of phosphate buffer (0.1 M, pH 7.5) containing 500 mg / L potassium cyanide. Incubate at 37°C and 350 rpm for 15 min. Stop the reaction by adding 50 μL of 5 M sodium hydroxide solution to each well and centrifuge at 3500 rpm and 4°C for 10 min. Take 25 μL of supernatant from each well and add it to a deep-well plate containing 475 μL of phosphate buffer (0.1 M, pH 7.5), dilute 10-fold, and analyze the ammonia nitrogen content using Nessler's reagent spectrophotometry. Add 10 μL of sodium tartrate and 10 μL of Nessler's reagent to the above sample, mix well, and incubate at 25°C for 10 min. Transfer 200 μL from each well to a microplate and analyze the OD value. 420 The absorbance was measured to screen mutants with significantly higher ammonia nitrogen production than the wild type, and the corresponding bacterial culture in the first-level deep well plate was stored at -80℃.

[0106] Table 2. Amino acid sequences of cyanidase mutants

[0107]

[0108]

[0109]

[0110] Example 2

[0111] Culture and expression of cyanidase CynD and its mutants

[0112] The cyanidase mutant glycerol bacterium, which showed a significantly higher ammonia nitrogen production than the wild type in Example 1, was streaked onto a plate and incubated overnight at 37°C. Single colonies were then picked and inoculated into 10 mL LB tubes containing kanamycin (50 μg / mL). The tubes were incubated at 37°C and 220 rpm for 12-16 h. Then, a 2% (v / v) inoculation was added to 150 mL Erlenmeyer flasks containing LB medium and incubated at 37°C and 220 rpm with shaking. The culture was continued until the absorbance reached 0. 600 When the cytosolic concentration reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM is added for induction at 25°C. After 18 hours of induction, the culture medium is centrifuged to obtain resting cells.

[0113] The obtained resting cells were suspended in potassium phosphate buffer (KPB, 100 mM, pH 7.5), sonicated in an ice-water bath, and the supernatant was collected by centrifugation. This supernatant was the crude enzyme solution of the recombinant enzyme, and its expression was verified. Based on the disclosed amino acid sequence and coding gene, those skilled in the art can also construct an expression vector for the CynD mutant of cyanidase by synthesizing the corresponding gene and transforming it into *E. coli* to express the corresponding mutant.

[0114] Example 3 – Construction and screening of cyanidase mutants with good heat tolerance

[0115] The crude enzyme solution of the recombinant enzyme obtained in Example 2 was used to construct and screen high-temperature resistant dicyandihydrate mutants.

[0116] Take 10 mL of phosphate buffer (0.1 M, pH 7.5) containing 500 mg / L potassium cyanide, add 0.1 mL of crude enzyme solution, and react the resulting mixture at 30 °C for 15 min in an apparatus with constant stirring and temperature. Accurately transfer 0.5 mL of the reaction solution and add 0.5 mL of 0.5 M sodium hydroxide to terminate the reaction with solvent. Dilute the terminated reaction solution by a certain factor and centrifuge to remove impurities. Detect the ammonia nitrogen content using Nessler's reagent spectrophotometry.

[0117] Nessler's reagent spectrophotometric method: Take 1 mL of the diluted sample, add 20 μL of sodium tartrate and 20 μL of Nessler's reagent, mix well, and let stand at 25°C for 10 min. Observe the sample at OD... 420 The absorbance was measured at the point.

[0118] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μM of product (ammonia nitrogen) per minute at 30°C and pH 7.5. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μM of product (ammonia nitrogen) per minute.

[0119] The enzyme activity of these cyanidase mutants was measured at 30-50℃ to determine their high-temperature tolerance. Mutants with higher enzyme activity than wild-type were selected for sequencing and rescreening to determine the mutation location. Compared with wild-type cyanidase, the enzyme activities of cyanidase mutants K97R, K225N, and S301D were significantly increased.

[0120] The amino acid mutations at the three sites were combined, and the plasmid construction method of Example 1 was used to construct multiple site mutant expression plasmids. These plasmids were then transformed into E. coli BL21(DE3) competent cells for culture and expression to obtain crude enzyme solutions of recombinant enzymes. The enzyme activity of these cyanidase mutants was measured at 30-50℃. Mutants with enzyme activity greater than wild-type were selected for sequencing and rescreening to determine the mutation location.

[0121] Enzyme activity was detected by examining single and combined mutations. Results are shown below. Figure 1 As shown, within a temperature range of 30-50℃, the enzyme activities of the cyanidase mutants K97R, K225N, S301D, K97R / K225N, and K97R / K225N / S301D were significantly improved compared to the wild-type cyanidase. In particular, the cyanidase mutant K97R / K225N / S301D retained an enzyme activity of 0.94 U / mg at a relatively high reaction temperature of 50℃, which is 12.5 times higher than that of the wild type, demonstrating its excellent enzyme activity and thermostability.

[0122] Example 4 – Construction and screening of cyanidase mutants with good alkali resistance

[0123] The crude enzyme solution of the recombinant enzyme obtained in Example 2 was used to construct and screen alkali-resistant cyanidase mutants. 10 mL of phosphate buffer (0.1 M, pH 7.5) containing 500 mg / L potassium cyanide was taken, and 0.1 mL of crude enzyme solution was added. The resulting mixture was reacted at 30 °C for 15 min in a device with constant stirring and temperature. 0.5 mL of the reaction solution was accurately transferred and 0.5 mL of 0.5 M sodium hydroxide was added to terminate the reaction with solvent. The terminated reaction solution was diluted by a certain factor and centrifuged to remove impurities. The ammonia nitrogen content was detected by Nessler's reagent spectrophotometry.

[0124] Nessler's reagent spectrophotometric method: Take 1 mL of the diluted sample, add 20 μL of sodium tartrate and 20 μL of Nessler's reagent, mix well, and let stand at 25°C for 10 min. Observe the sample at OD... 420 The absorbance was measured at the point.

[0125] The enzyme activities of these cyanidase mutants were measured at pH 7.5–9.5 to determine their alkali tolerance. Mutants with higher enzyme activity than the wild-type were sequenced and rescreened to determine the mutation location. Compared to the wild-type cyanidase, the enzyme activities of the cyanidase mutants E34K, K97R, and H307K were significantly increased. Amino acid mutations at the three sites were combined, and multiple mutant expression plasmids were constructed using the plasmid construction method described in Example 1. These plasmids were then transformed into E. coli BL21(DE3) competent cells for expression, yielding crude recombinant enzyme solutions. The enzyme activities of these cyanidase mutants were measured at pH 7.5–9.5, and mutants with higher enzyme activity than the wild-type were selected for sequencing and rescreening to determine the mutation location.

[0126] Enzyme activity was detected by examining single and combined mutations. Results are shown below. Figure 2 As shown, under catalytic reaction conditions with a reaction pH of 8.0-9.5, the enzyme activities of the cyanidase mutants E34K, K97R, H307K, E34K / K97R, and E34K / K97R / H307K were significantly increased compared to the wild-type cyanidase. The enzyme activity of the wild-type cyanidase showed a decreasing trend at reaction pH of 7.5-9.0. The optimal reaction pH for mutant H307K was 8.0. The optimal reaction pH for mutants E34K, K97R, E34K / K97R, and E34K / K97R / H307K was 8.5. When the reaction pH was 9.5, the wild-type cyanidase was inactivated, while the cyanidase mutant E34K / K97R / H307K still retained 0.62 U / mg enzyme activity, demonstrating its excellent alkali resistance.

[0127] Example 5

[0128] Construction and screening of cyanidase mutants with significantly improved heat and alkali resistance

[0129] Combining some amino acid mutations from Examples 3 and 4, and using the method for constructing mutant plasmids as described in Example 1, mutants at multiple sites were constructed and then transformed into E. coli BL21(DE3) competent cells for culture and expression to obtain crude enzyme solutions of recombinant enzymes. The aim was to integrate cyanidase mutants with both high thermostable and alkali-resistant properties. Given that the amino acid sequences and coding genes have been disclosed, those skilled in the art can also construct expression vectors for cyanidase CynD mutants by synthesizing the corresponding genes and transforming them into E. coli to express the corresponding mutants. Following the cyanidase activity assay method described in Example 3, the enzyme activity of these cyanidase mutants was measured at a reaction pH of 9.0 and a reaction temperature of 45°C to determine their alkali and thermostable resistance. Mutants with enzyme activities greater than the wild type were selected for sequencing and rescreening to determine the mutation location. The enzyme activity test results are shown in Table 3.

[0130] Table 3. Results of enzyme activity assay for cyanidin mutants.

[0131] Combined mutation sites Protein types Mutant number Enzyme activity (U / mg) WT wild type / 0.167 K97R / K225N / S301D mutant M-1 1.031 E34K / K97R / H307K mutant M-2 0.818 E34K / K97R / K225N / S301D mutant M-3 1.514 E34K / K97R / K225N / S301D / H307K mutant M-4 1.421

[0132] As shown in Table 2, under the catalytic conditions of pH 9.0 and reaction temperature 45℃, the enzyme activities of mutants K97R / K225N / S301D, E34K / K97R / H307K, E34K / K97R / K225N / S301D, and E34K / K97R / K225N / S301D / H307K were significantly improved compared with wild-type dicyandihydrate enzyme. In particular, the cyandihydrate enzyme mutant E34K / K97R / K225N / S301D, under the catalytic conditions of pH 9.0 and reaction temperature 45℃, still retained an enzyme activity of 1.421 U / mg, which was 8.5 times higher than that of wild-type enzyme, showing extremely excellent alkali resistance and high temperature resistance.

[0133] Example 6

[0134] 1. Wild-type cyanide dihydrate enzyme degrades cyanide-containing wastewater

[0135] The wild-type Escherichia coli expressing cyanidase was cultured and induced to produce enzyme according to the method described in Example 2. The cells were then broken to obtain a crude enzyme solution containing the wild-type cyanidase.

[0136] 100 mL of cyanide-containing wastewater with pH 8.0 or pH 9.0 (initial cyanide concentration of 430 mg / L) was added to a 250 mL three-necked flask. 1 mL of cell lysate containing the wild-type cyanide dihydrate protein was added. The reaction was carried out in a water bath at 35-45℃. Samples were taken at time intervals, and the residual cyanide concentration in the wastewater after degradation was determined by silver nitrate titration. This was to test the time required for the wild-type cyanide dihydrate to degrade the cyanide concentration in the wastewater to below 5 mg / L at different temperatures and pH. The results are shown in Table 4.

[0137] Table 4

[0138]

[0139] As shown in Table 4, the wastewater degradation rate decreases with increasing reaction temperature and pH. When the reaction pH is 8.0, within the reaction temperature range of 35-45℃, cyanide in the wastewater can be degraded by more than 99% with prolonged reaction time. When the reaction pH is 9.0, the degradation effect of cyanide in the wastewater is poor within the reaction temperature range of 35-45℃.

[0140] 2. Degradation of cyanide-containing wastewater by cyanide dihydrate synthase mutant

[0141] The Escherichia coli expression strain M-3, which is the cyanidase mutant strain E34K / K97R / K225N / S301D, from Example 5, was cultured and induced to produce enzyme in accordance with the method described in Example 2. The cells were then broken to obtain a crude enzyme solution containing the cyanidase mutant.

[0142] 100 mL of cyanide-containing wastewater (pH 9.0 or pH 9.5, initial cyanide concentration 430 mg / L) was added to a 250 mL three-necked flask. 1 mL of cell lysate containing the cyanide dihydrate mutant protein was added. The reaction was carried out in a water bath at 40-50 °C. Samples were taken at time intervals, and the residual cyanide concentration in the wastewater after degradation was determined by silver nitrate titration. This was to test the time required for the cyanide dihydrate mutant to degrade the cyanide concentration in the wastewater to below 5 mg / L at different temperatures and pH levels. The results are shown in Table 5.

[0143] The above silver nitrate titration method for detecting cyanide concentration refers to the national standard HJ-484-2009.

[0144] Table 5

[0145]

[0146]

[0147] Table 4 shows that as the reaction temperature and pH increase, the wastewater degradation rate decreases. When the reaction pH is 9.0, within the reaction temperature range of 40-50℃, cyanide in the wastewater can be degraded by more than 99% with the extension of reaction time. When the reaction pH is 9.5, within the reaction temperature range of 40-45℃, cyanide in the wastewater can also be degraded by more than 99% with the extension of reaction time. When the reaction pH is 9.5 and the reaction temperature is 50℃, the cyanide degradation rate in the wastewater is 92.27% after 4 hours of reaction.

[0148] In summary, this invention has improved cyanidase by employing random mutagenesis and site-directed saturation mutagenesis techniques, successfully screening out cyanidase mutants that, compared to wild-type cyanidase, exhibit higher enzyme activity, higher pH alkalinity tolerance, and significant heat resistance. These cyanidase mutants with improved enzyme activity demonstrate superior performance in degrading cyanide-containing compounds.

[0149] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A cyanidase mutant, characterized in that, The amino acid sequence of the cyanidase mutant, relative to the amino acid sequence of the wild-type cyanidase, has mutation sites selected from any of the following: 1) glutamic acid at position 34 is mutated to lysine; 2) lysine at position 97 is mutated to arginine; 3) lysine at position 225 is mutated to asparagine; 4) serine at position 301 is mutated to aspartic acid; 5) histidine at position 307 is mutated to lysine; 6) lysine at position 97 is mutated to arginine, and lysine at position 225 is mutated to asparagine; 7) glutamic acid at position 34 is mutated to lysine, and lysine at position 97 is mutated to arginine; 8) lysine at position 97 is mutated to arginine, lysine at position 225 is mutated to asparagine, and serine at position 301 is mutated to aspartic acid; 9) glutamic acid at position 34 is mutated to lysine, lysine at position 97 is mutated to arginine, and histidine at position 307 is mutated to lysine; 10) The glutamic acid at position 34 is mutated to lysine, the lysine at position 97 is mutated to arginine, the lysine at position 225 is mutated to asparagine; the serine at position 301 is mutated to aspartic acid; 11) The glutamic acid at position 34 is mutated to lysine, the lysine at position 97 is mutated to arginine, the lysine at position 225 is mutated to asparagine; the serine at position 301 is mutated to aspartic acid; the histidine at position 307 is mutated to lysine; The amino acid sequence of the wild-type dicyandihydrase is shown in SEQ ID NO.

1.

2. The cyanidase mutant as described in claim 1, characterized in that, The amino acid sequence of the cyanidase mutant is selected from any of the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

13.

3. An isolated polynucleotide encoding a cyanidase mutant as described in claim 1 or 2.

4. A construct, characterized in that, It contains the polynucleotide as described in claim 3.

5. A host cell, characterized in that, It contains the construct as described in claim 4 or the genome in which the polynucleotide as described in claim 3 is integrated.

6. The use of the cyanidase mutant of claim 1 or 2, the polynucleotide of claim 3, the construct of claim 4, or the host cell of claim 5 in the degradation of cyanide-containing compounds or the preparation of catalysts, wherein the cyanide-containing compound is selected from inorganic cyanides.

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