Cyanide dihydratase mutant and application thereof in degrading cyanide-containing compound

Abstract

The invention relates to the technical field of bioengineering, in particular to a cyanogen dihydratase mutant, the amino acid sequence of the cyanogen dihydratase mutant is mutated relative to the amino acid sequence of wild type cyanogen dihydratase, one or more amino acids in the 34th site, the 97th site, the 225th site, the 301st site and the 307th site are mutated, and the amino acid sequence of the wild type cyanogen dihydratase mutant is / are mutated relative to the amino acid sequence of wild type cyanogen dihydratase. The mutated amino acid is selected from arginine, aspartic acid, asparagine or lysine. Compared with a wild type cyanogen dihydratase mutant, the cyanogen dihydratase mutant disclosed by the invention has the advantages that the enzymatic activity of cyanogen dihydratase is remarkably improved, the catalytic activity, alkali resistance and high temperature resistance are relatively strong, relatively high enzymatic activity can still be kept in a slightly alkaline solvent reaction system, cyanide can also be degraded under a high-temperature condition, and the cyanogen dihydratase mutant can be used for degrading cyanide. Good industrial application prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a cyanide dihydratase mutant and application thereof in degrading cyanide-containing compounds. BACKGROUND

[0002] Cyanide is a kind of highly toxic compound with cyanide (CN-), and common forms include potassium cyanide, sodium cyanide and hydrogen cyanide. Cyanide is widely used in industry, such as mining, jewelry, nylon, plastic, pharmaceutical industry, etc., resulting in a large amount of cyanide-containing wastewater discharge, and the pollutants contained in cyanide-containing wastewater from different sources are also different. In the actual treatment process, the common removal of these pollutants is the key to improve the treatment efficiency and reduce the treatment steps, which helps to improve the economic benefits. At present, biological method, physical method and chemical method can be applied to treat and remove cyanide, and the chemical method and the physical method both treat and remove cyanide through acidification or chemical oxidation, which has a high investment cost and also produces toxic by-products, which undoubtedly increases the relatively high work task from the environmental protection point of view. However, the biological method for treating cyanide-containing wastewater does not require auxiliary factors, has high cost-effectiveness, and is more in line with the requirements of green environmental protection, energy saving and emission reduction and sustainable development.

[0003] Cyanide dihydratase can convert HCN into formic acid and ammonia. The product formic acid has small toxicity, and does not require auxiliary factors, and has friendly degradation characteristics.

[0004] However, one of the biggest problems of enzyme treatment is that cyanide-containing wastewater is usually kept at a high pH value, and cyanide is easy to volatilize to form hydrogen cyanide gas at neutral pH value, so cyanide-containing wastewater needs to be kept quite alkaline to limit the formation of hydrogen cyanide. Most of the cyanide-degrading wild bacteria have weak alkaline reaction conditions, and are easy to be inactivated under high pH conditions, and have poor thermal stability. For example, the cyanide dihydratase DN25 disclosed in patent CN201610342894.1 has a sharp decrease in enzyme activity when the pH is higher than 8 and the reaction temperature is higher than 30℃. At present, people solve this problem through protein engineering technology, and the kinetics, thermal stability and pH of cyanide dihydratase are studied. For example, the cyanide dihydratase mutant disclosed in patent CN202410788676.5 effectively improves the thermal stability of cyanide dihydratase to cyanide through molecular modification, but the problems of low pH alkali resistance and poor high-temperature resistance in the application of degrading cyanide-containing wastewater are not obviously improved.

[0005] Therefore, it is urgent to solve the problems of low pH alkali resistance and poor high-temperature resistance of the existing cyanide dihydratase, so as to better play a role in the actual application of degrading cyanide-containing wastewater. SUMMARY

[0006] In view of the above-mentioned disadvantages of the prior art, in order to solve the technical problems of low pH alkali resistance and poor high temperature resistance of cyanate dihydratase in the prior art, the purpose of the present application is to provide a cyanate dihydratase mutant and its application in degrading cyanide-containing compounds, and to obtain a cyanate dihydratase mutant capable of efficiently catalyzing inorganic cyanide such as potassium cyanide through protein engineering technology. The present application constructs a cyanate dihydratase mutant capable of catalyzing cyanide-containing compounds, including the mutant protein coding gene, the recombinant vector containing the gene, and the recombinant genetically engineered bacteria obtained by transformation of the recombinant vector. The problems of low pH alkali resistance and poor high temperature resistance of cyanate dihydratase in the application of degrading cyanide-containing wastewater are solved.

[0007] To achieve the above-mentioned purposes and other related purposes, the first aspect of the present application provides a cyanate dihydratase mutant, wherein the amino acid sequence of the cyanate dihydratase mutant is mutated at one or more of positions 34, 97, 225, 301 and 307 of the amino acid sequence of wild-type cyanate dihydratase, and the mutated amino acid is selected from arginine, aspartic acid, asparagine or lysine; the amino acid sequence of wild-type cyanate dihydratase is shown in SEQ ID NO. 1.

[0008] In some embodiments of the present application, the cyanate dihydratase mutant can improve the pH alkali resistance and high temperature resistance of cyanate dihydratase compared with wild-type cyanate dihydratase.

[0009] In some embodiments of the present application, the amino acid sequence of the cyanate dihydratase mutant is selected from any one or more of the following:

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

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

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

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

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

[0015] In some embodiments of the present application, the amino acid sequence of the cyanate dihydratase mutant is selected from any one of the following:

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

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

[0018] 3) 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;

[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 the present application, the amino acid sequence of the cyanide dihydratase mutant is selected from the group consisting of the sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Preferably, the amino acid sequence of the cyanide dihydratase mutant is shown in SEQ ID NO: 12.

[0023] The second aspect of the present application provides an isolated polynucleotide encoding the aforementioned cyanide dihydratase mutant.

[0024] The third aspect of the present application provides a construct comprising the aforementioned polynucleotide.

[0025] The fourth aspect of the present application provides a host cell comprising the aforementioned construct or having the aforementioned polynucleotide integrated into its genome.

[0026] The fifth aspect of the present application provides a method for producing a cyanide dihydratase, comprising culturing the aforementioned host cell under conditions suitable for expression of the aforementioned cyanide dihydratase mutant to obtain the cyanide dihydratase mutant.

[0027] The sixth aspect of the present application provides the use of the aforementioned cyanide dihydratase mutant, polynucleotide, construct, or host cell in degrading a cyanide-containing compound.

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

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The cyanide dihydratase mutant is obtained by random mutation and site-directed mutation screening of cyanide dihydratase, and compared with wild-type cyanide dihydratase CynD, the cyanide dihydratase mutant has significantly improved cyanide dihydratase enzyme activity, thereby shortening the reaction time and reducing the degradation cost; the cyanide dihydratase mutant has 4.89-9.06 times higher enzyme activity than the wild type at 45℃ and pH 9.0, that is, in an alkaline solvent reaction system, it can still retain high enzyme activity, and it can also degrade cyanide under high temperature conditions, and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The figure shows the comparison of enzyme activity of wild-type cyanide dihydratase and K97R, K225N, S301D, K97R / K225N and K97R / K225N / S301D mutants on substrate potassium cyanide in the range of 30-40℃.

[0032] Figure 2 The figure shows the comparison of enzyme activity of wild-type cyanide dihydratase and E34K, K97R, H307K, E34K / K97R and E34K / K97R / H307K mutants on substrate potassium cyanide in the range of pH 7.5-9.5. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application will be further described below in conjunction with examples. It should be understood that the examples are only used to explain the present application, and are not used to limit the scope of the present application. Unless otherwise specified, the test methods used in the following examples are conventional methods, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0034] After a large number of exploratory researches, the present application successfully found a mutant of cyanide dihydratase, which has obvious enhancement compared with the corresponding wild-type cyanide dihydratase, and has obvious high-temperature resistance and pH alkali resistance, based on which the present application is completed.

[0035] The present application provides a cyanide dihydratase mutant, the amino acid sequence of the cyanide dihydratase mutant is mutated at one or more of positions 34, 97, 225, 301 and 307 of the amino acid sequence of wild-type cyanide dihydratase, and the mutated amino acid is selected from arginine, aspartic acid, asparagine or lysine; the amino acid sequence of wild-type cyanide dihydratase is shown in SEQ ID NO. 1.

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

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

[0038] In some embodiments of the present application, the amino acid sequence of the cyanodi waterase mutant is selected from any one or more of the following:

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

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

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

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

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

[0044] In specific embodiments, when glutamic acid at position 34 is mutated to lysine, the amino acid sequence of the cyanodi waterase mutant is as set forth in SEQ ID NO. 3, specifically:

[0045] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0046] In specific embodiments, when lysine at position 97 is mutated to arginine, the amino acid sequence of the cyanodi waterase mutant is as set forth in SEQ ID NO. 4, specifically:

[0047] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

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

[0049] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

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

[0051] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

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

[0053] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0054] In some embodiments of the present application, the amino acid sequence of the cyanodiwaterase mutant is selected from any one of the following:

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

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

[0057] 3) 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;

[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 lysine at position 97 is mutated to arginine and lysine at position 225 is mutated to asparagine, the amino acid sequence of the cyanodi water hydratase mutant is shown in SEQ ID NO. 8, specifically:

[0062] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[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 cyanodi water hydratase mutant is shown in SEQ ID NO. 9, specifically:

[0064] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0065] In a specific embodiment, when 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, the amino acid sequence of the carbonic anhydrase mutant is shown in SEQ ID NO. 10, specifically:

[0066] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

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

[0068] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0069] In specific embodiments, when 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 amino acid sequence of the carbonic anhydrase mutant is shown as SEQ ID NO. 12, specifically:

[0070] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVHKFGVNKNKKISYEEINFSVGSKL.

[0071] In specific embodiments, when 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 carbonic anhydrase mutant is shown as SEQ ID NO. 13, specifically:

[0072] MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCKLIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARRNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKNKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQDPTDVVKKFGVNKNKKISYEEINFSVGSKL.

[0073] In some embodiments of the present application, the amino acid sequence of the cyanide dihydratase mutant is selected from any one 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 cyanide dihydratase mutant is shown in SEQ ID NO: 12.

[0074] In the cyanide dihydratase mutant provided by the present application, the cyanide dihydratase mutant can improve the enzyme activity, pH alkaline tolerance and high temperature tolerance of cyanide dihydratase compared with wild-type cyanide dihydratase.

[0075] The present application also provides an isolated polynucleotide encoding the aforementioned cyanide dihydratase mutant.

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

[0077] The nucleotide full-length sequence of the hydrolytic enzyme mutant of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is relatively short. Generally, a long fragment of sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them.

[0078] On the basis of the amino acid sequence of the cyanide dihydratase having been disclosed, the skilled person in the art can obtain the nucleotide sequence of the polynucleotide while keeping the amino acid sequence encoded unchanged due to the degeneracy of codons, which belongs to the routine techniques in the art.

[0079] In an embodiment of the application, the polynucleotide is selected from the group consisting of the nucleotide sequence of any one of SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO. 17.

[0080] SEQ ID NO.14: ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTGAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG.

[0081] SEQ ID NO.15: ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAAGAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGAGCCCGACCGATGTGGTGAAGAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG.

[0082] SEQ ID NO.16: ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGCATAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG.

[0083] SEQ ID NO.17: ATGGGAGTAATAATGAATTCATATCCCAAATATCGTGCAGCGGCGATCCAAGCGGCGCCAGTGTACCTCGATCTGGACGCAACCGTCGAGAAGTCTTGTAAATTGATCGCCGAGGCTGCGAGCAACGGCGCACGTTTGGTTGCGTTCCCGGAAGCGTTCCTGCCGGGTTACCCGTGGTTTGCATTCATCGGCCATCCGGAGTATACCCGTTCGTTCTATCATGAACTGTACAAAAATGCAGTTGAGATTCCGTCCTTGGCGATCCAGAAAATCTCTGAGGCCGCTCGCCGTAACAACACCTATGTTTGCATTAGTTGCAGCGAGAAGGACGGCGGTAGCTTGTACCTGACTCAGCTGTGGTTCAACTCTAACGGCGACCTGATTGGTAAACACAGAAAGATGAAGGCGAGCGTCGCAGAGCGCCTGACCTGGGGTGACGGCAACGGCTCGTTCATGCCGGTGTTTGAAACGGAGTTGGGAAACCTGGGTGGTCTGATGTGCTGGGAACACCAGGTTCCGCTGAATCTTCTGGCCATGAATTCGCAAAACGAGCAGGTACACGTGGCCAGCTGGCCTGGTTATTTTGATGATGAAATCTCTAGCCGTTATTACGCGATCTCCACCCAGACCTTCGTGATTATGACGAGCTCAATTTACTCCAAAGAAATGAAAAACAAAATCTGCCTGACGGCTGAGCAAGAAGTTTACTTTAACACCTTTAAAAGCGGTCACACCTGTATCTACGCTCCGAACGGCGAACCGATTAGCGATATGATTCTGGCGGAGACTGAGGGTATTGCGTATGCTGACATCGACATTGAAAAGATCATCGATTATAAATACTACATTGACCCGGCGGGTCACTACTCCAATCAAAGCTTAAGCATGAACTTTAACCAGGATCCGACCGATGTGGTGAAGAAATTCGGCGTTAATAAGAACAAGAAGATCTCCTATGAAGAAATTAATTTTAGCGTTGGTAGCAAGCTG.

[0084] The present application also provides a construct further comprising an additional expression regulatory element configured to be operably linked to the optional nucleotide sequence. The construct can be generally constructed by inserting the isolated polynucleotide into a suitable vector, which can be selected by one skilled in the art. For example, the vector can be an expression vector or a cloning vector. For another example, the vector can be a plasmid, a bacteriophage, a bacteriophage derivative, an animal virus, a cosmid, etc. Preferably, the vector is pET-28a.

[0085] The present application also provides a host cell containing the aforementioned construct or having the aforementioned polynucleotide integrated into its genome. The construct can be transformed, transduced or transfected into the host cell by a method conventional in the art, such as calcium chloride method chemical transformation, high-voltage electric shock transformation. Specifically, the host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli, Bacillus subtilis, yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae) or various animal and plant cells, more preferably the host 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 aforementioned Escherichia coli needs to be able to express the aforementioned cyanide dihydrase mutant, so as to provide conditions for the existence of the cyanide dihydrase mutant. Suitable methods for constructing the aforementioned Escherichia coli should be known to one skilled in the art.

[0086] The present application also provides a method for producing cyanide dihydrase, comprising the step of culturing the aforementioned host cell under conditions for expression of the aforementioned cyanide dihydrase mutant to obtain the cyanide dihydrase mutant. Suitable methods for inducing expression should be known to one skilled in the art. For example, the aforementioned host cell can be induced under suitable conditions to provide cyanide dihydrase. For another example, Escherichia coli monoclonal is inoculated in LB liquid medium and cultured at 37°C for 8-12 h; then inoculated into 2xYT culture solution at a ratio of 2-5% and continued to be cultured at 37°C, and the growth of the cells is continuously monitored. At the ideal stage of the logarithmic growth phase, IPTG is added for induction to promote the expression of cyanide dihydrase, and the induction time is controlled at 3-12 h, specifically, it can be 3-6 h, 6-8 h or 8-12 h, etc.

[0087] The present application also provides the aforementioned cyanide dihydrase mutant, polynucleotide, construct, or the aforementioned host cell for use in degrading cyanide-containing compounds or preparing catalysts.

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

[0089] The cyanide dihydratase mutant provided by the application can effectively improve the stability of cyanide dihydratase. Experimental data show that, compared with wild-type cyanide dihydratase, the cyanide dihydratase mutant provided by the application has higher enzyme activity, higher temperature resistance and alkali resistance, thereby shortening the reaction time, reducing the degradation cost, retaining higher enzyme activity in an alkaline solvent reaction system, and degrading cyanide under high temperature conditions, and has a good industrial application prospect.

[0090] The application will be further described below by examples, but the scope of the application is not limited thereto.

[0091] Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the application all adopt conventional techniques in the fields of 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 mutation library of cyanide dihydratase CynD

[0094] The pET-28a-CynD plasmid was constructed based on cyanide dihydratase CynD, the host bacteria E. coli BL21(DE3) was transformed, the engineering bacteria E. coli BL21(DE3) / pET-28b-CynD was constructed, and the amino acid sequence of cyanide dihydratase CynD was SEQ ID No. 1: MGVIMNSYPKYRAAAIQAAPVYLDLDATVEKSCELIAEAASNGARLVAFPEAFLPGYPWFAFIGHPEYTRSFYHELYKNAVEIPSLAIQKISEAARKNNTYVCISCSEKDGGSLYLTQLWFNSNGDLIGKHRKMKASVAERLTWGDGNGSFMPVFETELGNLGGLMCWEHQVPLNLLAMNSQNEQVHVASWPGYFDDEISSRYYAISTQTFVIMTSSIYSKEMKKKICLTAEQEVYFNTFKSGHTCIYAPNGEPISDMILAETEGIAYADIDIEKIIDYKYYIDPAGHYSNQSLSMNFNQSPTDVVHKFGVNKNKKISYEEINFSVGSKL.

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

[0096] The full plasmid PCR amplification technology was used for site-specific saturation mutation of cyanide dihydrase CynD gene sequence, and the template was recombinant plasmid pET28a-CynD of cyanide dihydrase CynD.

[0097] The conventional full plasmid PCR amplification technology was used, and the system of PCR amplification reaction was as follows: 0.5 μL of plasmid template, 1 μL of each pair of primers, 10 μL of PrimeSTAR HS (Preamix), and adding ddH2O to 20 μL. The primers were synthesized by a gene synthesis company, and the degenerate primers (the primers contained NNK codons) of the mutation sites were synthesized. The primer sequences of the six mutation sites were shown in Table 1.

[0098] Table 1 primer sequence

[0099]

[0100]

[0101] The system of PCR amplification reaction was 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) were performed for 20 cycles, finally 72 ℃ extension for 7 min, and the product was stored at 4 ℃.

[0102] 3 μL of the PCR amplification product was taken for nucleic acid electrophoresis detection of PCR bands. If there was a single bright band and the band position was greater than 5000 markers, it was considered that PCR was successful. Then, 2 μL of CutOne buffer and 1 μL of DpnI enzyme for rapid enzyme digestion to eliminate the template plasmid were added to the PCR product tube, and the mixture was uniformly oscillated and mixed, and the mixed liquid was placed in a 37 ℃ water bath for 1 h. The mutant expression plasmid was constructed. If it was two-site mutation, the second round of PCR connection was performed on the basis of the constructed first-site mutant plasmid using the second-site mutant primer to construct a double-mutant expression plasmid. If it was three-site mutation, the third round of PCR connection was performed on the basis of the constructed two-site mutant plasmid using the third-site mutant primer to construct a three-site mutant expression plasmid. Then, it was transformed into E. coli BL21 (DE3) competent cells, and uniformly coated on LB agar medium plates containing 50 μg / mL kanamycin, and cultured at 37 ℃ overnight. The random mutant library was constructed by site-specific saturation mutation.

[0103] The cyanodiwaterase mutant of the present application can be made by introducing mutations into the wild-type cyanodiwaterase CynD gene and verified to be correct. Based on the amino acid sequence and the coding gene which have been disclosed, the skilled in the art can also construct the expression vector of the cyanodiwaterase CynD mutant by synthesizing the corresponding gene, and transform E. coli to express the corresponding mutant. The amino acid sequence of the cyanodiwaterase mutant is shown in Table 2.

[0104] The monoclonal is selected for culture in a deep well plate, and each well of the primary plate contains 300 μL of LB medium (containing 50 μg / mL kanamycin) and is cultured at 37°C and 350 rpm overnight. 50 μL of the primary seed liquid is transferred to each well of the secondary plate containing 400 μL of LB medium (containing 50 μg / mL kanamycin), and is cultured at 37°C and 350 rpm for 3-4 h. 40 μL of 1 mM IPTG (final concentration 0.1 mM) is added to each well for induction, and the culture is continued at 25°C for 16-18 h. The deep well plate is centrifuged at 3500 rpm and 4°C for 10 min, and the supernatant is discarded. 400 μL of lysis solution (100 mM KPB solution, pH 7.5, containing 0.75 mg / L lysozyme) is added to each well, and the cells are fully suspended by shaking. The cells are lysed at 37°C for 2 h, and then centrifuged at 3500 rpm and 4°C for 10 min.

[0105] 50 μL of the cell lysate in each well is centrifuged, and the supernatant is transferred to a new deep well plate. 450 μL of phosphate buffer (0.1 M, pH 7.5) containing 500 mg / L potassium cyanide is added, and the reaction is carried out at 37°C and 350 rpm for 15 min. The reaction is terminated by adding 50 μL of 5 M sodium hydroxide solution to each well, and then centrifuged at 3500 rpm and 4°C for 10 min. 25 μL of the supernatant in each well is taken out and added to a deep well plate containing 475 μL of phosphate buffer (0.1 M, pH 7.5), and diluted 10-fold. The content of ammonia nitrogen is analyzed by the Nash reagent spectrophotometry. 10 μL of sodium tartrate and 10 μL of Nash reagent are added to the above sample, mixed well, and then allowed to stand at 25°C for 10 min. 200 μL in each well is transferred to an enzyme-labeled plate, and the absorbance is detected at OD 540 nm. Mutants with significantly higher ammonia nitrogen yield than the wild type are screened, and the corresponding primary deep well plate containing the bacterial solution is preserved at -80°C. 420

[0106] Table 2 Amino acid sequence of cyanodiwaterase mutant

[0107]

[0108]

[0109]

[0110] Example 2 ​

[0111] Culture expression of cyanide dihydratase CynD and its mutants

[0112] The glycerol bacteria of cyanide dihydratase mutants with significantly increased ammonia nitrogen yield than wild type in Example 1 were performed plate streaking, after overnight culture at 37℃, single colonies were picked and inoculated into 10 mL LB test tube containing kanamycin (50 μg / mL), and placed in a 37℃ shaker at 220 rpm for 12-16 h, then inoculated into a 500 mL flask containing 150 mL LB medium at 2% (v / v) inoculation amount, and placed in a 37℃ shaker at 220 rpm for culture, when the absorbance of the culture solution OD 600 reached 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM for induction, the induction temperature was 25℃, and after 18 hours of induction, the culture solution was centrifuged to obtain resting cells.

[0113] The obtained resting cells were suspended in potassium phosphate buffer (KPB, 100 mM, pH 7.5), and were ultrasonically broken in an ice water bath, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant enzyme, and the expression was verified. Based on the disclosure of the amino acid sequence and the encoding gene, a person skilled in the art can also construct an expression vector of cyanide dihydratase CynD mutant by synthesizing the corresponding gene, and transform Escherichia coli to express the corresponding mutant.

[0114] Example 3 - Construction and screening of cyanide dihydratase mutants with better high temperature tolerance

[0115] The crude enzyme solution of the recombinant enzyme obtained in Example 2 was used for the construction and screening of cyanide dihydratase mutants with high temperature tolerance.

[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 make the obtained mixture react in a device with constant stirring and constant temperature for 15 min at 30℃, accurately take 0.5 mL of the reaction solution and add 0.5 mL of 0.5 M sodium hydroxide, terminate the reaction, dilute the terminated reaction solution to a certain multiple, centrifuge to remove impurities, and detect the ammonia nitrogen content by the Nash reagent spectrophotometry.

[0117] Nash reagent spectrophotometry: take 1 mL of the above diluted sample, add 20 μL of sodium tartrate and 20 μL of Nash reagent, mix well, and detect the absorbance at OD 420 25℃ after 10 min.

[0118] Enzyme activity definition: the amount of enzyme required to catalyze the generation of 1 μM product (ammonia nitrogen) per minute at 30℃ and pH 7.5 is 1 enzyme activity unit (U).

[0119] The enzyme activity of these cyanide dihydratase mutants was determined at 30-50°C, and the high temperature tolerance of the cyanide dihydratase was determined. Mutants with enzyme activity greater than the wild type were sequenced and re-screened to determine the mutation position. The enzyme activity of cyanide dihydratase mutants K97R, K225N, and S301D was significantly improved compared to the wild type cyanide dihydratase.

[0120] The amino acid mutations at the three sites were combined, and a multi-site mutant expression plasmid was constructed using the plasmid construction method of Example 1, and then transformed into E. coli BL21(DE3) competent cells for expression and culture to obtain a crude enzyme solution of the recombinant enzyme. The enzyme activity of these cyanide dihydratase mutants was determined at 30-50°C, and the high temperature tolerance of the cyanide dihydratase was determined. Mutants with enzyme activity greater than the wild type were sequenced and re-screened to determine the mutation position.

[0121] The enzyme activity of single mutations and combined mutations was determined, and the results are shown in Table 1. Figure 1 The enzyme activity of cyanide dihydratase mutants K97R, K225N, S301D, K97R / K225N, and K97R / K225N / S301D was significantly improved compared to the wild type cyanide dihydratase at a temperature range of 30-50°C. In particular, cyanide dihydratase mutant K97R / K225N / S301D retained an enzyme activity of 0.94 U / mg at a relatively high reaction temperature of 50°C, which was 12.5 times higher than the wild type, showing excellent enzyme activity and high temperature tolerance.

[0122] Example 4 - Construction and screening of cyanide dihydratase mutants with good alkali resistance

[0123] The crude enzyme solution of the recombinant cyanide dihydratase obtained in Example 2 was used to construct and screen cyanide dihydratase mutants with alkali resistance. 10 mL of phosphate buffer (0.1 M, pH 7.5) containing 500 mg / L potassium cyanide was added with 0.1 mL of crude enzyme solution, and the resulting mixture was reacted at 30°C for 15 min in a device with constant stirring and constant temperature. 0.5 mL of the reaction solution was accurately removed and added to 0.5 mL of 0.5 M sodium hydroxide, and the reaction was terminated with solvent. After dilution, the terminated reaction solution was centrifuged to remove impurities, and the ammonia nitrogen content was determined by the Nash reagent spectrophotometry method.

[0124] Nash reagent spectrophotometry: 1 mL of the above diluted sample was taken, 20 μL of sodium tartrate and 20 μL of Nash reagent were added, and after mixing, it was placed at 25°C for 10 min. The absorbance was detected at OD 420

[0125] ​The alkali resistance of these cyanodiwaterase mutants was determined by measuring the enzyme activity of these cyanodiwaterase mutants at pH 7.5-9.5, and the mutants with enzyme activity greater than the wild type were sequenced and re-screened to determine the mutation position. The enzyme activity of cyanodiwaterase mutants E34K, K97R, and H307K was significantly improved compared with the wild type cyanodiwaterase. The amino acid mutations at the three sites were combined, and a multi-site mutant expression plasmid was constructed using the plasmid construction method of Example 1, and then transformed into E. coli BL21 (DE3) competent cells for culture and expression to obtain a crude enzyme solution of the recombinant enzyme. The enzyme activity of these cyanodiwaterase mutants at pH 7.5-9.5 was measured, and the mutants with enzyme activity greater than the wild type were sequenced and re-screened to determine the mutation position.

[0126] By detecting the enzyme activity of single mutations and combined mutations, the results are shown in Table 1. Figure 2 As shown in Table 1, under the catalytic reaction conditions of reaction pH 8.0-9.5, the enzyme activity of cyanodiwaterase mutants E34K, K97R, H307K, E34K / K97R, and E34K / K97R / H307K was significantly improved compared with the wild type cyanodiwaterase; the enzyme activity of the wild type cyanodiwaterase showed a downward trend at reaction pH 7.5-9.0; the optimal reaction pH of mutant H307K was 8.0; the optimal reaction pH of mutants E34K, K97R, E34K / K97R, and E34K / K97R / H307K was 8.5; when the reaction pH was 9.5, the wild type cyanodiwaterase was inactivated, while cyanodiwaterase mutant E34K / K97R / H307K still retained 0.62 U / mg enzyme activity, showing excellent alkali resistance.

[0127] Example 5

[0128] Construction and screening of cyanodiwaterase mutants with significantly improved high temperature resistance and alkali resistance

[0129] The partial amino acid mutations in Example 3 and Example 4 were combined, and a plurality of site mutants were constructed using the method for constructing the mutant plasmid in Example 1, and then transformed into E. coli BL21(DE3) competent cells for culture and expression to obtain a crude enzyme solution of the recombinant enzyme, in order to integrate cyanide dihydrase mutants with high temperature resistance and alkali resistance. Based on the disclosed amino acid sequence and encoding gene, a person skilled in the art can also synthesize the corresponding gene to construct an expression vector of cyanide dihydrase CynD mutant, and transform E. coli to express the corresponding mutant. According to the cyanide dihydrase enzyme activity determination method described in Example 3, the enzyme activity of these cyanide dihydrase mutants at a reaction pH of 9.0 and a reaction temperature of 45°C was determined, so as to determine the alkali resistance and high temperature resistance of these cyanide dihydrase mutants. The mutant with enzyme activity greater than the wild type was selected for sequencing and re-screening detection to determine the mutation position. The enzyme activity detection results are shown in Table 3.

[0130] Table 3 Cyanide dihydrase mutant enzyme activity detection results

[0131] Combination mutation site Protein type 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 can be seen from Table 2, compared with the wild type cyanide dihydrase, the enzyme activity of mutants K97R / K225N / S301D, E34K / K97R / H307K, E34K / K97R / K225N / S301D and E34K / K97R / K225N / S301D / H307K was significantly improved under the catalytic environment of reaction pH 9.0 and reaction temperature 45°C; in particular, cyanide dihydrase mutant E34K / K97R / K225N / S301D still retained an enzyme activity of 1.421 U / mg under the catalytic environment of reaction pH 9.0 and reaction temperature 45°C, which was 8.5 times higher than that of the wild type enzyme, showing extremely excellent alkali resistance and high temperature resistance.

[0133] Example 6

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

[0135] The wild type cyanide dihydrase E. coli expression strain was cultured and induced to produce enzyme according to the method described in Example 2, and the cells were broken to obtain a crude enzyme solution containing the wild type cyanide dihydrase.

[0136] In 250 ml three-necked flask, 100 mL cyanide-containing wastewater (initial cyanide concentration of 430 mg / L) with pH 8.0 or pH 9.0 was added, 1 mL cell crushing solution containing the wild-type cyanide dihydrase protein was added, and catalytic reaction was carried out in a 35-45°C water bath, respectively, samples were taken at intervals, and residual cyanide concentration in the degraded wastewater was detected by silver nitrate titration method, so as to test the time required for the wild-type cyanide dihydrase to degrade the cyanide concentration in the wastewater to below 5 mg / L at different temperatures and pH values, and the results are shown in Table 4.

[0137] Table 4

[0138]

[0139] As can be seen from Table 4, as the reaction temperature and pH increase, the wastewater degradation rate decreases; when the reaction pH is 8.0, in the reaction temperature range of 35-45°C, cyanide in the wastewater can be degraded by more than 99% as the reaction time is prolonged; when the reaction pH is 9.0, in the reaction temperature range of 35-45°C, the cyanide degradation effect in the wastewater is poor.

[0140] 2. Cyanide dihydrase mutant degrading cyanide-containing wastewater

[0141] The cyanide dihydrase mutant of strain No. M-3 in Example 5, i.e., E34K / K97R / K225N / S301D, Escherichia coli expression strain, was cultured and induced to produce enzyme in the manner described in Example 2, and the cells were crushed to obtain a crude enzyme solution containing the cyanide dihydrase mutant.

[0142] In 250 ml three-necked flask, 100 mL cyanide-containing wastewater (initial cyanide concentration of 430 mg / L) with pH 9.0 or pH 9.5 was added, 1 mL cell crushing solution containing the cyanide dihydrase mutant protein was added, and catalytic reaction was carried out in a 40-50°C water bath, respectively, samples were taken at intervals, and residual cyanide concentration in the degraded wastewater was detected by silver nitrate titration method, so as to test the time required for the cyanide dihydrase mutant to degrade the cyanide concentration in the wastewater to below 5 mg / L at different temperatures and pH values, and the results are shown in Table 5.

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

[0144] Table 5

[0145]

[0146]

[0147] As shown in Table 4, with the increase of reaction temperature and pH, the degradation rate of wastewater decreases; when the reaction pH is 9.0, in the range of 40-50℃, with the extension of reaction time, cyanide in wastewater can be degraded by more than 99%; when the reaction pH is 9.5, in the range of 40-45℃, with the extension of reaction time, cyanide in wastewater can also be degraded by more than 99%; when the reaction pH is 9.5, the reaction temperature is 50℃, and the reaction time is 4h, the degradation rate of cyanide in wastewater is 92.27%.

[0148] In summary, the cyanide dihydrase is improved by using random mutation and site saturation mutation technology, and the cyanide dihydrase mutant is successfully screened, which has high enzyme activity, high pH alkaline resistance, and obvious high temperature resistance compared with the wild type cyanide dihydrase. These cyanide dihydrase mutants with better enzyme activity exhibit more excellent performance in degrading cyanide compounds.

[0149] The above examples only illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought of the present application should be covered by the claims of the present application.

Claims

1. A cyanodiwaterase mutant, characterized in that, The amino acid sequence of the cyanide dihydratase mutant is mutated at one or more of positions 34, 97, 225, 301, 307 relative to the amino acid sequence of wild-type cyanide dihydratase, and the mutated amino acid is selected from arginine, aspartic acid, asparagine or lysine. The amino acid sequence of the wild-type cyanide dihydratase is shown in SEQ ID NO.

1.

2. The cyanodiwaterase mutant of claim 1, wherein, The amino acid sequence of the cyanide dihydratase mutant is selected from any one or more 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.

3. The cyanodiwaterase mutant of claim 1, wherein, The amino acid sequence of the cyanide dihydratase mutant is selected from any one of the following: 1) lysine at position 97 is mutated to arginine, and lysine at position 225 is mutated to asparagine; 2) glutamic acid at position 34 is mutated to lysine, and lysine at position 97 is mutated to arginine; 3) 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; 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; 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; 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.

4. The cyanodiwaterase mutant of claim 1, wherein, The amino acid sequence of the cyanide dihydratase mutant is selected from any one of the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

13.

5. An isolated polynucleotide encoding the cyanide dihydratase mutant of any one of claims 1-4.

6. A construct, characterized in that, containing the polynucleotide of claim 5.

7. A host cell, characterized in that, containing the construct or genome of claim 6 integrated with the polynucleotide of claim 5.

8. A method for preparing a cyanide dihydratase mutant, comprising culturing the host cell of claim 7 under conditions suitable for expression of the cyanide dihydratase mutant of any one of claims 1-4 to obtain the cyanide dihydratase mutant.

9. Use of the cyanide dihydratase mutant of any one of claims 1-4, the polynucleotide of claim 5, the construct of claim 6 or the host cell of claim 7 in degrading a cyanide-containing compound or in preparing a catalyst.

10. Use according to claim 9, wherein The cyanide-containing compound is selected from inorganic cyanide.

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