A cyanase mutant, nucleic acid, recombinant expression vector and their applications

Abstract

The present invention discloses a cyanase mutant, nucleic acid, recombinant expression vector and their applications, belonging to the field of bioengineering. The cyanase gene derived from Escherichia coli E. coli K-12 substr. MG1655 was subjected to site-directed mutagenesis, so that the encoded amino acid sequence was mutated from glycine G to alanine A at the 37th position, from lysine K to arginine R at the 154th position, and from glutamine Q to histidine H at the 51st position. The specific activity of the cyanase mutant G37A / K154R / Q51H of the present invention against potassium cyanate is 2.4 times that before mutation, and the half-lives at 30 °C and 40 °C are increased by 2.1 times and 2.6 times respectively. The cyanase parent of the present invention cannot catalyze potassium cyanide, while the specific activity of the mutant G37A / K154R / Q51H against potassium cyanide reaches 5.5 U / mg, and the half-lives at 30 °C and 40 °C are 2.5 h and 3.0 h respectively. The cyanase mutant of the present invention can efficiently catalyze the decomposition of the substrates potassium cyanate and potassium cyanide, so as to achieve the purpose of degrading cyanide-containing wastewater, and can be widely applied to industries such as chemical engineering, pharmaceuticals and metallurgy, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a cyanase mutant with high thermal stability and high activity, a nucleic acid encoding the cyanase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, the preparation of a cyanase mutant catalyst, and the application of the cyanase mutant catalyst in the degradation of cyanide-containing compounds. Background Art

[0002] Cyanide is a toxic compound that exists in various natural and man-made environments. Despite its high toxicity, it is widely used in electroplating, metallurgy, medicine and other fields due to the strong metal complexing force of the cyanide group, and cyanide-containing wastewater is inevitably generated. For example, in the process of treating lead, silver, copper, etc. in the electroplating industry, cyanide wastewater of different concentrations and compositions will be generated; some projects in the pharmaceutical industry require cyanide for analysis and inspection, which will also produce cyanide wastewater. Cyanide-containing wastewater has a wide range of pollution, a long pollution time, and high toxicity. It is internationally recognized as a difficult-to-degrade wastewater. Currently, biological, physical and chemical methods can be used to remove or decompose cyanide. Among them, biological treatment of cyanide has many advantages, such as: low toxicity of the product, further conversion, trace and high efficiency, etc. Biological treatment of cyanide can not only effectively treat cyanide-containing wastewater, but also reduce capital investment, and is environmentally friendly (Olaya-Abril, Alfonso., Karolina., Rodríguez-Caballero, Gema. et al. (2024). Bacterial tolerance and detoxification of cyanide, arsenic and heavy metals: Holistic approaches applied to bioremediation of industrial complexwastes. Microbial biotechnology.).

[0003] Cyanase (CYN) can decompose cyanate into carbon dioxide and ammonia without the presence of any coenzymes or cofactors. Due to this catalytic property, cyanase has been applied to the degradation of cyanide-containing wastewater (Cabello P, Luque-Almagro VM, Olaya-Abril A et al. Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: from omic approaches to biotechnological applications. FEMS Microbiol Lett, 2018, 365(6):fny032.).

[0004] Rational design in protein engineering is based on a certain understanding of the enzymatic information such as the sequence, structure, and catalytic mechanism of the target protein. With the help of various bioinformatics software, the effects of mutations at different sites on the stability and catalytic performance of the target protein are predicted. The whole-plasmid PCR method is one of the most widely used site-directed mutagenesis methods, with the advantages of simplicity, speed, and efficiency. Shaimaa S. Sobieh constructed an expression vector containing the CYN gene and transformed it into Chlamydomonas reinhardtii to achieve overexpression of the CYN gene (Sobieh, Shaimaa S., Abed El-Gammal, Rasha., El-Kheiretal. Heterologous Expression of Cyanobacterial Cyanase Gene in Microalga for Bioremediation of Cyanide Pollution. Biology, 2022, 11, 1420). Compared with the wild type, the transgenic algae showed significant resistance to cyanide stress, demonstrating its ability to repair high-concentration cyanide-containing wastewater.

[0005] However, the cyanase disclosed in the prior art mainly focuses on its ability to convert cyanate and its activity, but does not focus on its ability to degrade inorganic cyanide. There are also no reports on using genetic engineering methods to improve the thermal stability and activity of cyanase. Summary of the Invention

[0006] The purpose of the present invention is to provide a cyanase mutant, nucleic acid, recombinant expression vector and application thereof in order to solve the current situation that the prior art lacks cyanase with both thermal stability and activity.

[0007] More specifically, the present invention provides a highly thermostable and highly active cyanase mutant, a nucleic acid encoding the cyanase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, the preparation of a cyanase mutant catalyst, and the use of the cyanase mutant in the degradation of cyanide-containing compounds.

[0008] The present invention addresses the problem of thermal stability of cyanase. By using a rationally designed method, mutation sites of the enzyme are selected for site-directed mutagenesis, and the catalytic activity of the cyanase is further improved while improving the thermal stability. In particular, a high-thermal-stable and high-activity cyanase mutant based on cyanase derived from Escherichia coli K-12 substr. MG1655 is proposed for the first time. The multi-site mutant of the cyanase has high catalytic activity towards inorganic cyanide, laying a foundation for the application of cyanase in the field of cyanide-containing wastewater.

[0009] The present application obtains the gene sequence encoding cyanase (CYN) from the genome of E. coli K-12 substr. MG1655 reported in the NCBI database, and successfully clones it into E. coli BL21 (DE3) for expression. At the same time, through molecular modification, the gene sequence is improved in terms of its activity and thermal stability towards cyanide (organic cyanides such as potassium cyanate and inorganic cyanides such as potassium cyanide). The multi-site mutant can simultaneously hydrolyze cyanate and inorganic cyanide, thus expanding the application range of cyanase in the degradation of cyanide-containing wastewater.

[0010] The present invention is guided by surface amino acid (Gly-Ala, Lys-Arg) replacement and consensus theory, and with the assistance of bioinformatics software GetArea and Consensus Finder, obtains a cyanase mutant with high thermal stability and high activity.

[0011] The present invention first uses the principle that amino acids on the surface of thermophilic proteins have Gly replaced by Ala and Lys replaced by Arg, uses GetArea software to find a total of 4 Gly and 3 Lys on the surface of CYN, and performs site-directed mutagenesis on them respectively to obtain two mutant enzymes with improved thermal stability; then, based on the principle that conserved amino acids in homologous sequences are more stable than non-conserved amino acids, uses Consensus Finder software to perform multiple sequence alignment of CYN, and screens out a total of 3 amino acids with a threshold value greater than 80%. After site-directed mutagenesis of these amino acids into corresponding conserved amino acids, a mutant enzyme with improved cyanate enzyme activity and thermal stability is obtained.

[0012] After mutating the amino acids at the three sites mentioned above, a three-point mutant with significantly improved thermal stability and specific activity was obtained, which improved the existing thermal stability of CYN. It was found that it has the ability to degrade cyanate and inorganic cyanide at the same time, further improving the level of biological degradation of cyanide-containing wastewater.

[0013] The purpose of the present invention can be achieved by the following technical solutions:

[0014] One of the technical solutions of the present invention is to provide a highly thermostable and highly active cyanase mutant, wherein at least one of positions 37, 154 or 51 of the amino acid sequence shown in SEQ ID NO.1 is mutated, and the resulting mutant has higher thermostable and activity than the cyanase CYN corresponding to the amino acid sequence shown in SEQ ID NO.1.

[0015] In one embodiment of the present invention, the glycine at position 37 is mutated to alanine, the lysine at position 154 is mutated to arginine, and the glutamine at position 51 is mutated to histidine.

[0016] In one embodiment of the present invention, the highly thermostable and highly active cyanase mutant is a mutant obtained by mutating glycine at position 37, lysine at position 154, and glutamine at position 51 in the amino acid sequence shown in SEQ ID NO.1 to alanine, arginine, and histidine, respectively.

[0017] In one embodiment of the present invention, the highly thermostable and highly active cyanase mutant is obtained by subjecting the cyanase CYN from Escherichia coli K-12 substr.MG1655 (whose amino acid sequence is shown in NCBI Accession No. NP_414874.1 and its nucleotide sequence is shown in NCBI Accession No. NC_000913.3) to site-directed mutagenesis, and mutations are introduced into glycine G at position 37, lysine K at position 154, and glutamine Q at position 51 of CYN by whole-plasmid PCR technology.

[0018] The cyanase mutant G37A / K154R / Q51H was named using the abbreviation of the original amino acid + mutation position + the abbreviation of the replaced amino acid.

[0019] When the substrate is potassium cyanate, compared with the cyanase of E. coli K-12 substr. MG1655 (the specific activity of the pure enzyme is 4.2 U / mg), the specific activities of the cyanase mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H are 5.7 U / mg, 7.7 U / mg, 6.3 U / mg, 9.7 U / mg, 12.4 U / mg and 15.4 U / mg, respectively, among which G37A / K154R / Q51H shows the best specific activity, which is 2.4 times higher than that of the parent.

[0020] When the substrate was potassium cyanide, no enzymatic activity was detected in the cyanase of E. coli K-12 substr. MG1655, and the cyanase mutants K96R, K349R, and V305L were also unable to catalyze the degradation of potassium cyanide. The specific activities of the combined mutations G37A / Q51H, K154R / Q51H, and G37A / K154R / Q51H in degrading potassium cyanide were 2.8 U / mg, 3.6 U / mg, and 5.5 U / mg, respectively, among which G37A / K154R / Q51H showed the best specific activity.

[0021] When the substrate is potassium cyanate, the half-life t 1 / 2 The half-lives of the cyanase mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H at 30°C were 1.1 times, 1.3 times, 1.1 times, 1.3 times, 1.6 times and 2.1 times that of the parent, respectively (t 1 / 2 The half-lives of the two strains at 40°C were 1.6 times, 1.7 times, 1.3 times, 1.8 times, 2.0 times and 2.6 times higher than those of the parent strain (t 1 / 2 1.6h, 1.7h, 1.3h, 1.8h, 2.0h and 2.6h respectively).

[0022] When the substrate is potassium cyanide, the half-lives of the cyanase mutants G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H at 30°C are 1.9h, 2.0h and 2.5h, respectively, and the half-lives at 40°C are 2.3h, 2.5h and 3.0h, respectively.

[0023] It can be seen that the thermal stability and activity of the cyanase mutant provided by the present invention are improved compared with the cyanase of E. coli K-12 substr. MG1655.

[0024] The second technical solution of the present invention provides an isolated nucleic acid, which encodes the highly thermostable and highly active cyanase mutant as described in the first technical solution.

[0025] The method for preparing the nucleic acid of the hyperthermostable, highly active cyanase mutant described in the present invention adopts conventional preparation methods in the art. Preferred preparation methods include: obtaining the encoding DNA of the hyperthermostable, highly active cyanase mutant through gene cloning technology, or obtaining the encoding DNA of the hyperthermostable, highly active cyanase mutant through artificial full-sequence synthesis. The method of obtaining the nucleic acid molecule encoding the hyperthermostable, highly active cyanase mutant through gene cloning technology described in the present invention is to obtain the encoding DNA encoding the hyperthermostable, highly active cyanase mutant through polymerase chain reaction (PCR).

[0026] The PCR system and PCR reaction procedures involved can be obtained according to conventional biotechnology means.

[0027] The third technical solution of the present invention is to provide a recombinant expression vector comprising the cyanase mutant nucleic acid of the present invention.

[0028] In one embodiment of the present invention, the recombinant expression vector is a recombinant expression plasmid.

[0029] The plasmid can be constructed by conventional methods in the art by ligating the gene sequence encoding the cyanase mutant nucleic acid of the present invention to a commercially available empty plasmid. The plasmid can be any of the common plasmids in the art, including but not limited to expression vectors such as pET expression vectors, pRSF expression vectors, pUC expression vectors, or pBR expression vectors.

[0030] For different expression hosts, the preferred plasmid vector may be different. The cyanase mutant gene can be operatively cloned into the expression control sequence downstream of the selected vector to achieve constitutive or inducible expression of the cyanase mutant. For E. coli hosts, the pET28a plasmid is preferably used as the vector.

[0031] The fourth technical solution of the present invention is to provide a nucleic acid comprising the highly thermostable and highly active cyanase mutant or a recombinant expression transformant of the recombinant expression vector.

[0032] The recombinant expression transformant of the present invention can be prepared by transforming the recombinant expression plasmid of the present invention into a host cell.

[0033] The host cell is any conventional host cell in the art, including but not limited to any one of Escherichia coli, yeast, Bacillus, lactic acid bacteria, or filamentous fungi. The host cell of the present invention is preferably Escherichia coli. The target recombinant expression transformant can be obtained by transforming the recombinant expression plasmid of the present invention into the host cell.

[0034] Preferably, the recombinant plasmid containing the mutated gene is transformed into the host Escherichia coli BL21 (DE3) for expression to obtain a recombinant expression transformant containing the cyanase mutant plasmid of the present invention.

[0035] The fifth technical solution of the present invention is to provide a recombinant cyanase mutant catalyst, wherein the recombinant cyanase mutant catalyst is any one of the following forms:

[0036] (1) culturing the recombinant expression transformant of the present invention and isolating transformant cells containing the cyanase mutant;

[0037] (2) freeze-dried cells obtained by freeze-drying the transformant cells described in (1);

[0038] (3) disrupting transformant cells containing the cyanase mutant to obtain a crude enzyme solution;

[0039] (4) Purifying the cell disrupted liquid containing the cyanase mutant to obtain a pure enzyme liquid.

[0040] The sixth technical solution of the present invention is to provide a method for obtaining the recombinant cyanase mutant, which is expressed by isolating a transformant containing the cyanase mutant.

[0041] Preferably, using plasmid pET28a-CYN as a template, primers are designed, and a recombinant plasmid encoding the mutant is obtained by PCR. The Escherichia coli seed liquid containing the recombinant plasmid is inoculated into a fermentation medium, and the fermentation time is 12 hours at 37°C and 200 rpm. The fermentation medium 2YT is composed of: 16 g / L tryptone, 10 g / L yeast extract powder, 5 g / L NaCl, and a pH of 7.0.

[0042] Technical solution seven of the present invention: provides the use of the cyanase mutant or the recombinant cyanase mutant catalyst in degrading cyanide.

[0043] In one embodiment of the present invention, the cyanide is selected from cyanate or inorganic cyanide.

[0044] Preferably, the cyanide is potassium cyanate or potassium cyanide.

[0045] When potassium cyanate is used as a substrate, catalysis can produce carbon dioxide and ammonia. When potassium cyanide is used as a substrate, catalysis can produce formic acid and ammonia.

[0046] In one embodiment of the present invention, the application is specifically to use the recombinant cyanase mutant or the recombinant cyanase mutant catalyst to degrade cyanide-containing wastewater.

[0047] The cyanase mutant provided by the present invention is used to catalyze the degradation of the substrate potassium cyanate. At a substrate concentration of 19.2 mM, a reaction temperature of 30°C, a pH of 7.0, and a cell weight of 5 g / L, the degradation rate of potassium cyanate can reach 77% after 2 hours of conversion, while the conversion rate of the wild type under the same conditions is 58%, an increase of 33%. When applied to the degradation of potassium cyanide, at a substrate concentration of 5.8 mM, a reaction temperature of 30°C, a pH of 7.0, and a cell weight of 10 g / L, the degradation rate of potassium cyanide can reach 82% after 0.5 hours of conversion, while the wild type cannot catalyze the degradation of potassium cyanide under the same conditions.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] Guided by surface amino acid substitution and consensus theory, site-directed mutagenesis of the cyanase gene resulted in the generation of a CYN mutant enzyme, G37A / K154R / Q51H, with enhanced thermostability and specific activity. The specific activity for the substrate potassium cyanate was 2.4-fold higher than before the mutation, and the half-life at 30°C and 40°C was increased by 2.0-fold and 2.5-fold, respectively. Furthermore, this multi-site mutant catalyzed the degradation of potassium cyanide, achieving a specific activity of 5.5 U / mg and a half-life of 2.5 hours at 30°C and 3.0 hours at 40°C. This mutant enzyme can degrade both cyanate and inorganic cyanide using potassium cyanate and potassium cyanide as substrates. Industrial cyanide-containing wastewater primarily contains inorganic cyanide and / or cyanate-containing compounds, and this mutant enzyme has excellent potential for degradation of cyanide-containing wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Comparison of the enzyme activities of the mutants constructed in the present invention and wild-type CYN;

[0051] Figure 2 includes Figure 2a 、 2b , Figure 2a 、 2b Comparison of the inactivation half-life of the mutants constructed in the present invention and wild-type CYN at different temperatures. DETAILED DESCRIPTION

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] The culture medium formula involved in the embodiment is as follows:

[0054] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0055] LB solid medium: Add 15 g / L agar to the basic formula of LB liquid medium.

[0056] Fermentation medium: tryptone 16 g / L, yeast extract powder 10 g / L, NaCl 5 g / L, pH about 7.0.

[0057] When potassium cyanate is used as substrate, the enzyme activity determination and analysis method of cyanase is as follows:

[0058] The activity test reaction system is as follows (1 mL): 20 μg of pure enzyme is mixed with 100 mM sodium phosphate buffer solution (pH 7.7), preheated in a thermomixer for 5 min, and 500 mg / L potassium cyanate (19.2 mM CN - ), reacted on a thermomixer for 15 min (1000 rpm, 30°C), and immediately added 500 μL of NaOH (0.1 M) to terminate the reaction. After centrifugation, the supernatant was diluted with sterile water and analyzed spectrophotometrically using Nessler's reagent.

[0059] Enzyme activity definition: At 30°C, the amount of enzyme required to catalyze the production of 1 μmol of product (ammonia nitrogen) or consume 1 μmol of substrate (potassium cyanate) per minute is defined as one enzyme activity unit (U).

[0060] When potassium cyanide is used as substrate, the enzyme activity determination and analysis method of cyanase is as follows:

[0061] The activity test reaction system (1 mL) was as follows: 20 μg of pure enzyme was mixed with 100 mM sodium phosphate buffer solution (pH 7.7), preheated in a thermomixer for 5 min, and 150 mg / L potassium cyanide (5.8 mM CN - ), reacted on a thermostatic mixer for 15 min (1000 rpm, 30°C), and immediately added 500 μL of NaOH (0.1 M) to terminate the reaction. After centrifugation, the supernatant was diluted with sterile water and analyzed spectrophotometrically using Nessler's reagent.

[0062] Enzyme activity definition: At 30°C, the amount of enzyme required to catalyze the production of 1 μmol of product (ammonia nitrogen) or consume 1 μmol of substrate (potassium cyanide) per minute is defined as one enzyme activity unit (U).

[0063] Nessler's reagent spectrophotometric analysis.

[0064] Analytical method (Nessler's reagent spectrophotometry): Add 20 μL of potassium sodium tartrate to the above sample, mix well, then add 20 μL of Nessler's reagent, mix well, incubate in a water bath at 25°C for 10 min, and detect and record the OD using a spectrophotometer.420 The absorbance value.

[0065] Determination of thermal stability: The diluted pure enzyme was divided into several packages and placed in metal water baths at 30℃ and 40℃ for insulation respectively. Samples were taken at regular intervals to determine the residual enzyme activity. A linear relationship was fitted with the insulation time as the x-axis and the logarithm of the residual enzyme activity as the y-axis. The results were then compared based on t 1 / 2 =ln2 / k d Determine the half-life.

[0066] Whole cell catalytic hydrolysis of potassium cyanate: 5 g / L of cyanase and its mutant cells were taken and 500 mg / L of substrate potassium cyanate (19.2 mM CN - ) Under the condition of pH 7.0, the reaction temperature was 30°C and the reaction time was 0.5, 1, 2, 3, 4, 5, and 6 h to observe the changes in the ammonia nitrogen conversion rate.

[0067] Whole cell catalytic hydrolysis of potassium cyanide: 10 g / L cyanase and its mutant cells were mixed with 150 mg / L potassium cyanide (5.8 mM CN - ) Under the condition of pH 7.0, the reaction temperature was 30°C and the reaction time was 0.5, 1, 1.5, and 2 h to observe the changes in the ammonia nitrogen conversion rate.

[0068] The conversion rate of product ammonia nitrogen is calculated as follows:

[0069] Conversion rate α2 = 100% × [5.7269 × (A - A0) - 0.0429] × B / (C·D)

[0070] Where A represents the OD after the reaction 420 , A0 represents the OD after reaction without enzyme or cell addition 420 , B represents the dilution factor of the total reaction system, C represents the atomic mass of nitrogen, and D represents the concentration of the substrate potassium cyanide. The relationship y = 5.7269x - 0.0429 represents the external standard method standard curve for ammonia nitrogen.

[0071] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0072] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It is necessary to point out that the following specific embodiments are only used to more clearly describe the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements to the present invention are included within the scope of protection of the present invention.

[0073] Example 1

[0074] Cloning of cyanase gene and construction of recombinant engineered bacteria

[0075] According to the protein NCBI database, a cyanase gene was obtained from Escherichia coli K-12 substr.MG1655 (its amino acid sequence is shown in NCBI accession number NP_414874.1, and its nucleotide sequence is shown in NCBI accession number NC_000913.3), and was synthesized by Shanghai Jierui Bioengineering Co., Ltd. The amino acid sequence of this cyanase CYN is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.8. The upstream primer 5'-CGC CATATG ATGATTCAGTCACAAATTAACCGCAATATTCGT-3' (the underlined bases are the recognition sites of restriction endonuclease Nde I) and the downstream primer 5'-TCA GGATCC TCAGAACGGTTTGGTCGGCAGATATTTACCATCTA-3' (the underlined bases are the restriction endonuclease BamH I recognition site tag) to amplify the target gene. PCR amplification used PrimeSTAR Max high-fidelity polymerase from Takara Biotechnology (Beijing) Co., Ltd. (Takara China). The PCR reaction system was as follows: (primer concentration was 10 μmol / L):

[0076]

[0077] The PCR amplification program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 5 s, 30 cycles; extension at 72°C for 7 min; and storage at 4°C.

[0078] After the reaction, the PCR product was examined by 1% agarose gel electrophoresis, yielding a 507-bp band, consistent with the expected target length. Following the kit protocol, the target fragment was recovered and purified using gel electrophoresis. The recovered fragment and the pET28a plasmid were double-digested with restriction endonucleases Nde I and BamH I. Ligation Mix was then used for ligation. The ligation product was transformed into competent E. coli BL21(DE3) cells and plated on LB plates containing ampicillin (100 μg / mL). Plasmids from positive clones were isolated and sequenced. The results confirmed the correct sequence of the inserted CYN gene, and the recombinant strain was named E. coli BL21(DE3) / pET28a-CYN.

[0079] Example 2

[0080] Construction of cyanase mutants based on whole-plasmid PCR

[0081] The plasmid containing the mutant gene was amplified in vitro by PCR using the recombinant plasmid pET28a-CYN as a template.

[0082] The primers used for site-directed mutagenesis are (the mutation sites are underlined):

[0083] G37Aprimer-F: 5'-GATTGCCGACGGCACC GCA CTGGCAGAAGCCT-3'

[0084] G37A primer-R: 5'-AGGCTTTCTGCCAG TGC GGTGCCGTCGGCAATC-3'

[0085] K154R primer-F: 5'-TAAATATCTGCCGACC CGT CCGTTCTGAGGATCCG-3'

[0086] K154R primer-R: 5'-CGGATCCTCAGAACGG ACG GGTCGGCAGATATTTA-3'

[0087] Q51H primer-F: 5'-GCTTTGCTGGTCAG CAT GCGCTTCCTGCCGACG-3'

[0088] Q51H primer-R: 5'-CGTCGGCAGGAAGCGC ATG CTGACCCAGCAAAGC-3'

[0089] The PCR reaction system is as follows: (primer concentration is 10 μmol / L)

[0090]

[0091] The PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0092] After 10 μL of the PCR product was verified by nucleic acid electrophoresis, the reaction solution was recovered using a universal DNA purification and recovery kit. The DNA template was then digested with Dpn I endonuclease at a temperature of 37°C for 4 hours. The digested product was then transformed into competent E. coli BL21 (DE3) cells. LB resistant solid plates (containing 100 μg / mL Amp) were coated and cultured at 37°C for 12-14 hours. 1-3 transformants were picked and transferred to LB liquid culture medium (containing 100 μg / mL Amp) and cultured for 6 hours. After the liquid became turbid, the bacterial solution was sampled and sent for sequencing.

[0093] The strain E. coli BL21(DE3) / pET28a-CYN with improved thermal stability compared with the original strain was obtained by screening the dominant strains. G37A (The amino acid sequence of the cyanase mutant G37A is shown in SEQ ID NO. 2, and its nucleotide sequence is shown in SEQ ID NO. 9), E. coli BL21 (DE3) / pET28a-CYN K154R (The amino acid sequence of the cyanase mutant K154R is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.10), and the strain E. coli BL21 (DE3) / pET28a-CYN with improved enzyme activity compared to the original strain Q51H (The amino acid sequence of the cyanase mutant Q51H is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 11).

[0094] The second round was carried out with pET28a-CYN G37A The plasmid was used as a template, and Q51H-F and Q51H-R were used as primers. After whole-plasmid PCR, transformation, and plating, the superior strain E. coli BL21(DE3) / pET28a-CYN with further improved thermal stability was obtained. G37A / Q51H (The amino acid sequence of the cyanase mutant G37A / Q51H is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.12). K154R The plasmid was used as a template, and Q51H-F and Q51H-R were used as primers. After whole-plasmid PCR, transformation, and plating, the superior strain E. coli BL21(DE3) / pET28a-CYN with further improved thermal stability was obtained. K154R / Q51H (The amino acid sequence of the cyanase mutant K154R / Q51H is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.13). K154R / Q51H The plasmid was used as a template, G37A-F and G37A-R were used as primers, and after whole plasmid PCR, transformation, and plating, the dominant strain E. coli BL21(DE3) / pET28a-CYN was obtained, which had improved enzyme activity and thermal stability compared with the original strain. G37A / K154R / Q51H (The amino acid sequence of the cyanase mutant G37A / K154R / Q51H is shown in SEQ ID NO. 7, and its nucleotide sequence is shown in SEQ ID NO. 14).

[0095] Example 3

[0096] Culture expression of parental and mutant strains

[0097] Plate activation: Use an inoculation loop to dip the bacterial solution in the glycerol tube, streak four lines on an LB resistance solid plate (containing 100 μg / mL Amp), and culture upside down in a constant temperature incubator at 37°C for 12 hours.

[0098] Seed culture: Pick a single colony from the solid plate and inoculate it into LB liquid medium (containing 100 μg / mL Amp) with a volume of 50 mL / 250 mL. Incubate on a shaker at 37°C and 200 rpm for 12 h.

[0099] Shake flask fermentation: The seed liquid cultured for 12 hours was inoculated into the fermentation medium (containing 100 μg / mL Amp) at a 1% inoculum volume of 50 mL / 250 mL, and cultured at 37 ° C, 200 rpm until the bacterial concentration OD 600 = 0.6-0.8, and after the liquid cooled to room temperature, IPTG was added to a final concentration of 0.1 mM for induction, and expression was induced at 25°C for 12 hours. After fermentation, all cells were collected by centrifugation at 10,000 rpm for 10 minutes in a 4°C centrifuge, and the cells were washed twice with 0.8% saline.

[0100] Example 4

[0101] Purification of parent and mutant cyanase

[0102] Add the corresponding buffer to the collected bacteria to resuspend the cells to a concentration of 10g / L, place them in a beaker containing ice water, and use an ultrasonic disruptor to disrupt the cells. The disruption conditions are: ultrasonic 2s, rest 4s, power 38%, time 10min, a total of two times, and then centrifuge at 12000rpm for 30min. The supernatant is the crude enzyme solution. The wild type and positive mutants of cyanase G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H were purified using Suzhou Beaver His-tag protein purification magnetic beads. Ni 2+ It forms a coordination bond with the histidine in the protein's His tag, binding the target protein to the magnetic beads. Adsorbed contaminants are then removed with 100mM imidazole, followed by elution with 500mM imidazole. Finally, the target protein is concentrated and buffer exchanged in a Millipore ultrafiltration centrifuge tube. Following purification, the same amount of protein and volume are loaded for SDS-PAGE verification.

[0103] Example 5

[0104] Comparison of activities of different mutants and the parent enzyme

[0105] The concentrated pure enzyme was diluted and the enzyme activity was determined according to the above method and the protein concentration was determined by Bradford method. Figure 1 As shown, the substrate is potassium cyanate, the specific activity of CYN is 4.2 U / mg, and the specific activities of mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H are 5.7 U / mg, 7.7 U / mg, 6.3 U / mg, 9.7 U / mg, 12.4 U / mg and 15.4 U / mg, respectively, which are 1.4, 1.8, 1.5, 1.5, 2.0 and 2.4 times higher than those of the parent. The substrate was potassium cyanide. The CYN specific activities of the parent, mutants K96R, K349R and V305L were not detected, while the specific activities of the combined mutations G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H were 2.8 U / mg, 3.6 U / mg and 5.5 U / mg, respectively, among which G37A / K154R / Q51H showed the best specific activity.

[0106] Example 6

[0107] Comparison of thermal stability between different mutants and the parent enzyme

[0108] The thermal stability of different mutants and the parent enzyme is mainly determined by measuring the half-life at different temperatures. Figure 2a As shown, the substrate is potassium cyanate, and the half-life of the parent at 30°C is 1.5h, while the half-lives of the mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H are 1.7h, 2.0h, 1.6h, 2.0h, 2.4h and 3.1h, which are 1.1 times, 1.3 times, 1.1 times, 1.3 times, 1.6 times and 2.1 times that of the parent, respectively; the substrate is potassium cyanide, and the half-lives of the mutants G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H at 30°C are 1.9h, 2.0h and 2.5h, respectively. As shown in the attached figure, the half-life of the mutants G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H are 1.9h, 2.0h and 2.5h, respectively. Figure 2bAs shown, the substrate is potassium cyanate, and the half-life of the parent at 40°C is 1.0 h, while the half-lives of the mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H are 1.6 h, 1.7 h, 1.3 h, 1.8 h, 2.0 h and 2.6 h, respectively, which are 1.6 times, 1.7 times, 1.3 times, 1.8 times, 2.0 times and 2.6 times that of the parent, respectively; the substrate is potassium cyanide, and the half-lives of the mutants G37A / Q51H, K154R / Q51H and G37A / K154R / Q51H at 40°C are 2.3 h, 2.5 h and 3.0 h, respectively. It can be seen that the three-point mutant G37A / K154R / Q51H shows the best thermal stability at both low and high temperatures, regardless of whether the substrate is potassium cyanate or potassium cyanide, indicating that the thermal stability of the enzyme can be further improved after combined mutation.

[0109] Example 7

[0110] Comparison of potassium cyanate conversion efficiency between mutant G37A / K154R / Q51H and parent cyanase at different temperatures

[0111] In a 50 mL centrifuge tube, 5 g / L of cells (parent and mutant enzymes), 100 mM NaCl (pH 7.0), and 500 mg / L of the substrate potassium cyanate (19.2 mM CN-) were added to a 10 mL total system. The reaction was incubated at 30°C and 40°C at 180 rpm for 0.5, 1, 2, 3, 4, 5, and 6 h, respectively. An equal volume of 0.1 M NaOH was added to quench the reaction, followed by centrifugation and analysis by Nessler's reagent spectrophotometry. The results showed that the potassium cyanate conversion rate reached 100% for the mutant enzyme and parent enzyme at 30°C after 3 h and 4 h, respectively. At 40°C, the potassium cyanate conversion rate reached 100% for the mutant enzyme and parent enzyme after 3 h and 5 h, respectively. At 30°C, the potassium cyanate conversion rate of the mutant enzyme reached 77% after 2 h, a 33% increase over the parent enzyme (58% at 30°C).

[0112] Example 8

[0113] Comparison of potassium cyanide conversion efficiency between mutant G37A / K154R / Q51H and parent cyanase at different temperatures

[0114] In a 50 mL centrifuge tube, the total system was 10 mL, 10 g / L cells (parent and mutant), 150 mg / L potassium cyanide (5.8 mM CN -) and incubated at 30°C and 40°C at 180 rpm for 0.5, 1, 1.5, and 2 hours, respectively. After completion, the reaction was quenched by adding an equal volume of 0.1M NaOH. After mixing, the supernatant was aspirated and analyzed spectrophotometrically using Nessler's reagent. The results showed that the parent strain had no potassium cyanide degradation activity, while the mutant achieved a potassium cyanide conversion rate of 82% in 0.5 hour and nearly 100% in 1 hour at 30°C. At 40°C, the potassium cyanide conversion rate reached 66% in 0.5 hour and nearly 100% in 1.5 hours.

[0115] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

[0116] The sequence information involved in the present invention is as follows:

[0117] SEQ ID NO.1

[0118] Amino acid sequence of cyanase CYN

[0119] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTGLAE AFVTAALLGQ QALPADAARL 60

[0120] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKA LVHEKFGDGI 120

[0121] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTKPF 156

[0122] SEQ ID NO.2

[0123] Amino acid sequence of cyanase mutant G37A

[0124] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTALAE AFVTAALLGQ QALPADAARL 60

[0125] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKA LVHEKFGDGI 120

[0126] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTKPF 156

[0127] SEQ ID NO.3

[0128] Amino acid sequence of cyanase mutant K154R

[0129] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTGLAE AFVTAALLGQ QALPADAARL 60

[0130] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKA LVHEKFGDGI 120

[0131] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTRPF 156

[0132] SEQ ID NO.4

[0133] Amino acid sequence of cyanase mutant Q51H

[0134] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTGLAE AFVTAALLGQ HALPADAARL 60

[0135] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKA LVHEKFGDGI 120

[0136] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTKPF 156

[0137] SEQ ID NO.5

[0138] Amino acid sequence of cyanase mutant G37A / Q51H

[0139] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTALAE AFVTAALLGQ HALPADAARL 60

[0140] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKALVHEKFGDGI 120

[0141] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTKPF 156

[0142] SEQ ID NO.6

[0143] Amino acid sequence of cyanase mutant K154R / Q51H

[0144] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTGLAE AFVTAALLGQHALPADAARL 60

[0145] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKALVHEKFGDGI 120

[0146] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTRPF 156

[0147] SEQ ID NO.7

[0148] Amino acid sequence of the cyanase mutant G37A / K154R / Q51H

[0149] MIQSQINRNI RLDLADAILL SKAKKDLSFA EIADGTALAE AFVTAALLGQHALPADAARL 60

[0150] VGAKLDLDED SILLLQMIPL RGCIDDRIPT DPTMYRFYEM LQVYGTTLKALVHEKFGDGI 120

[0151] ISAINFKLDV KKVADPEGGE RAVITLDGKY LPTRPF 156

[0152] SEQ ID NO.8

[0153] Nucleotide sequence of cyanase CYN

[0154]

[0155] SEQ ID NO.9

[0156] Nucleotide sequence of cyanase mutant G37A

[0157] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgc actggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag caggcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgacca aaccgttctg a 471 SEQ ID NO.10

[0158] Nucleotide sequence of cyanase mutant K154R

[0159] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgg tctggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag caggcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgaccc gtccgttctg 471 SEQ ID NO.11

[0160] Nucleotide sequence of cyanate hydratase mutant Q51H

[0161] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgg tctggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag catgcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgacca aaccgttctg a 471 SEQ ID NO.12

[0162] Nucleotide sequence of cyanase mutant G37A / Q51H

[0163] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgc actggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag catgcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgacca aaccgttctg a 471 SEQ ID NO.13

[0164] Nucleotide sequence of cyanase mutant K154R / Q51H

[0165] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgg tctggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag catgcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgaccc gtccgttctg 471 SEQ ID NO.14

[0166] Nucleotide sequence of cyanase mutant G37A / K154R / Q51H

[0167] atgattcagt cacaaattaa ccgcaatatt cgtcttgatc ttgccgatgc cattttgctc 60 agcaaagcta aaaaagatct ctcatttgcc gagattgccg acggcaccgc actggcagaa 120 gcctttgtaa ccgcggcttt gctgggtcag catgcgcttc ctgccgacgc cgcccgcctg 180 gtcggggcga agctggatct cgacgaagac tccattctac tgttgcagat gattccactg 240 cgtggctgca ttgatgaccg tattccaact gacccaacga tgtatcgttt ctatgaaatg 300 ttgcaggtgt acggtacaac cctgaaagcg ttggttcatg agaaatttgg cgatggcatt 360 attagcgcga ttaacttcaa actcgacgtt aagaaagtgg cggacccgga aggtggcgaa 420 cgtgcggtca tcaccttaga tggtaaatat ctgccgaccc gtccgttctg a 471 SEQ ID NO.15

[0168] Upstream primer

[0169] cgccatatga tgattcagtc acaaattaac cgcaatattc gt 42SEQ ID NO.16

[0170] Downstream primer

[0171] tcaggatcct cagaacggtt tggtcggcag atatttacca tcta 44SEQ ID NO.17

[0172] Primer G37A primer-Fgattgccgac ggcaccgcac tggcagaagc ct 32SEQ ID NO.18

[0173] Primer G37A primer-Raggcttctgc cagtgcggtg ccgtcggcaa tc 32SEQ ID NO.19

[0174] Primer K154R primer-Ftaaatatctg ccgacccgtc cgttctgagg atccg 35SEQ IDNO.20

[0175] Primer K154R primer-Rcggatcctca gaacggacgg gtcggcagat attta 35SEQ IDNO.21

[0176] Primer Q51H primer-Fgctttgctgg gtcagcatgc gcttcctgcc gacg 34SEQ ID NO.22

[0177] Primer Q51H primer-Rcgtcggcagg aagcgcatgc tgacccagca aagc 34

Claims

1. A cyanase mutant, characterized in that: is selected from one of the mutants G37A, K154R, Q51H, G37A / Q51H, K154R / Q51H or G37A / K154R / Q51H, The mutant G37A is a mutant in which the glycine at position 37 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine; The mutant K154R is a mutant in which the lysine at position 154 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine; Mutant Q51H is a mutant in which the glutamine at position 51 of the amino acid sequence shown in SEQ ID NO.1 is mutated to histidine; The mutant G37A / Q51H is a mutant in which the glycine at position 37 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, and the glutamine at position 51 is mutated to histidine; The mutant K154R / Q51H is a mutant in which the lysine at position 154 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine, and the glutamine at position 51 is mutated to histidine; The mutant G37A / K154R / Q51H is a mutant in which the glycine at position 37 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to alanine, the lysine at position 154 is mutated to arginine, and the glutamine at position 51 is mutated to histidine.

2. A cyanase mutant according to claim 1, characterized in that: The cyanase mutant is a mutant obtained by mutating the 37th glycine, the 154th lysine and the 51st glutamine in the amino acid sequence shown in SEQ ID NO.1 to alanine, arginine and histidine respectively.

3. An isolated nucleic acid, characterized in that The nucleic acid is a nucleic acid encoding the cyanase mutant according to claim 1 or 2. A recombinant expression vector comprising the nucleic acid of claim 3.

5. A recombinant expression transformant comprising the nucleic acid according to claim 3 or the recombinant expression vector according to claim 4.

6. A recombinant cyanase mutant catalyst, characterized in that: The recombinant cyanase mutant catalyst is in any of the following forms: (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the cyanase mutant; (2) freeze-dried cells obtained by freeze-drying the transformant cells described in (1); (3) disrupting transformant cells containing the cyanase mutant to obtain a crude enzyme solution; (4) Purifying the cell lysate containing the cyanase mutant to obtain a pure enzyme solution.

7. The method for obtaining the cyanase mutant according to claim 1 or 2, characterized in that: The recombinant expression transformant according to claim 5 is isolated to express a cyanase mutant.

8. Use of the cyanase mutant according to claim 1 or 2 or the recombinant cyanase mutant catalyst according to claim 6 in degrading potassium cyanate.

9. Use of the cyanase mutant according to claim 1 or 2 or the recombinant cyanase mutant catalyst according to claim 6 in the degradation of potassium cyanide, characterized in that: The cyanase mutant is selected from mutant G37A / Q51H, mutant K154R / Q51H or mutant G37A / K154R / Q51H.

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