Method for improving the thermal stability and acid stability of corn zearalenone hydrolase and T217C-G242C series mutants and applications

Abstract

The present invention relates to the field of genetic engineering, and in particular to a method for improving the thermal stability and acid stability of zearalenone hydrolase, a T217C-G242C series mutant, and an application thereof. A mutant with improved thermal stability and acid stability is obtained by site-directed mutagenesis of zearalenone hydrolase ZHDM2. The zearalenone hydrolase mutant of the present invention has good enzymatic properties and can be applied to industries such as food and feed.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a method for improving the thermal stability and acid stability of zearalenone hydrolase, a T217C-G242C series mutant and an application thereof. Background Art

[0002] Zearalenone (ZEN) is a common non-steroidal mycotoxin with estrogen-like effects found in cereals and crops. It is also known as F-2 toxin and is mainly produced by fungi such as Fusarium graminearum, Fusarum culmorum and Fusarum crookwellense. It causes serious harm to the animal husbandry in production practice.

[0003] Microbial degradation ZEN is a method of degradation of toxins by the cooperation of one or more enzymes produced by microbial metabolism. It has mild conditions, strong specificity, does not destroy the original nutrients, and is highly efficient. It is an environmentally friendly, efficient, and practical treatment method. However, the activity of zearalenone hydrolases found so far is low, which hinders the large-scale industrial production and application of zearalenone hydrolases. Summary of the invention

[0004] The object of the present invention is to provide a zearalenone hydrolase mutant with improved thermal stability and acid stability.

[0005] Another object of the present invention is to provide a gene encoding the above-mentioned zearalenone hydrolase mutant.

[0006] Another object of the present invention is to provide a recombinant vector comprising the gene encoding the above-mentioned zearalenone hydrolase mutant.

[0007] Another object of the present invention is to provide a recombinant strain comprising the above-mentioned zearalenone hydrolase mutant encoding gene.

[0008] Another object of the present invention is to provide a method for preparing zearalenone hydrolase with improved thermal stability and acid stability.

[0009] Another object of the present invention is to provide the application of the above-mentioned zearalenone hydrolase mutant.

[0010] The present invention mutates the zearalenone hydrolase ZHDM2 to obtain a zearalenone hydrolase mutant with improved thermal stability and acid stability, wherein the amino acid sequence of the parent zearalenone hydrolase ZHDM2 is shown in SEQ ID NO: 1:

[0011] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETY R DVTGHKLAGYVDTLLEELKIPIASVWGCSSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVSRSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGG T PTAMFFDNIVIAVKEGLNIGLLPG G HFPYVSHPEAFAEYVVETCRKYI.

[0012] According to a specific embodiment of the present invention, the 217th and 242nd amino acids of the zearalenone hydrolase ZHDM2 are mutated from threonine and glycine to cysteine ​​to obtain the mutant T217C-G242C, or the 76th amino acid is mutated from arginine to glutamine to obtain the mutant R76Q.

[0013] According to a specific embodiment of the present invention, the 217th, 242nd and 76th amino acids of zearalenone hydrolase ZHDM2 are mutated from threonine, glycine and arginine to cysteine, cysteine ​​and glutamine to obtain the mutant T217C-G242C / R76Q.

[0014] According to a specific embodiment of the present invention, the amino acid sequence of the zearalenone hydrolase mutant T217C-G242C is shown in SEQ ID NO: 2:

[0015] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETYRDVTGHKLAGYVDTLLEELKIPIASVWGCSSGA TTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVSRSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGG C PTAMFFDNIVIAVKEGLNIGLLPG C HFPYVSHPEAFAEYVVETCRKYI.

[0016] According to a specific embodiment of the present invention, the amino acid sequence of the zearalenone hydrolase mutant R76Q is shown in SEQ ID NO: 3:

[0017] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETY Q DVTGHKLAGYVDTLLEELKIPIASVWGCSSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVSRSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGGTPTAMFFDNIVIAVKEGLNIGLLPGGHFPYVSHPEAFAEYVVETCRKYI.

[0018] According to a specific embodiment of the present invention, the amino acid sequence of the zearalenone hydrolase mutant T217C-G242C / R76Q is shown in SEQ ID NO:4:

[0019] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETY Q DVTGHKLAGYVDTLLEELKIPIASVWGCSSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVSRSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGG C PTAMFFDNIVIAVKEGLNIGLLPG C HFPYVSHPEAFAEYVVETCRKYI.

[0020] The present invention provides a gene encoding the above-mentioned zearalenone hydrolase ZHDM2. According to a specific embodiment of the present invention, the gene sequence of the zearalenone hydrolase ZHDM2 is shown in SEQ ID NO: 5:

[0021] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACAGAGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTACCCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTGGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATTTAA。

[0022] According to the specific embodiments of the present invention, the coding gene sequence of the zearalenone hydrolase mutant T217C-G242C is as shown in SEQ ID NO:6:

[0023] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACAGAGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTTGTCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTTGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATT。

[0024] According to the specific embodiments of the present invention, the coding gene sequence of the zearalenone hydrolase mutant R76Q is shown in SEQ ID NO:7:

[0025] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACCAGGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTACCCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTGGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATT。

[0026] According to the specific embodiments of the present invention, the coding gene sequence of the zearalenone hydrolase mutant T217C-G242C / R76Q is as shown in SEQ ID NO:8:

[0027] .

[0028] The method for improving the thermal stability and acid thermal stability of zearalenone hydrolase according to the present invention comprises the following steps:

[0029] The zearalenone hydrolase ZHDM2 was subjected to double-point mutation, and the amino acids at positions 217 and 242 were mutated from threonine and glycine to cysteine;

[0030] A single point mutation was performed on the zearalenone hydrolase ZHDM2, and the amino acid at position 76 was mutated from arginine to glutamine.

[0031] The present invention provides a recombinant vector comprising the coding gene of the above-mentioned zearalenone hydrolase mutant. Preferably, the starting vector of the recombinant expression vector is specifically pPICZ(α)A.

[0032] The present invention also provides a recombinant strain comprising the coding gene of the above-mentioned zearalenone hydrolase mutant. Preferably, the starting strain of the recombinant bacteria is specifically GS115 ( pPICZ(α)A -zhdm2) .

[0033] According to a specific embodiment of the present invention, the method for preparing zearalenone hydrolase with improved thermal stability and acid stability is as follows:

[0034] (1) transforming a host cell with a recombinant vector containing a gene encoding a zearalenone hydrolase mutant to obtain a recombinant strain;

[0035] (2) culturing the recombinant strain to induce the expression of zearalenone hydrolase;

[0036] (3) Recovering and purifying the expressed zearalenone hydrolase.

[0037] Beneficial effects of the present invention:

[0038] The present invention achieves the purpose of improving thermal stability and acid stability by introducing a disulfide bond and a glutamine into the structure of zearalenone hydrolase. After ZHDM2 is treated at 50°C for 5 min and 10 min, the residual activity is 26.96% and 24.77%; after mutants T217C-G242C, R76Q and T217C-G242C / R76Q are treated at 50°C for 5 min and 10 min, the residual enzyme activities are 68.75% and 33.13%, 74.09% and 57.51%, 82.95% and 62.69%, respectively, which are 155.01% and 33.75%, 174.81% and 132.18%, 207.68% and 153.09% higher than ZHDM2, respectively. After treatment at 55 ℃ for 2 min and 5 min, the residual activity of ZHDM2 was 23.53% and 7.13%; after treatment at 55 ℃ for 2 min and 5 min, the residual enzyme activities of mutants T217C-G242C, R76Q and T217C-G242C / R76Q were 68.53% and 13.78%, 83.78% and 66.05%, 83.91% and 70.92%, respectively, which were 191.25% and 93.27%, 256.06% and 826.37%, 256.61% and 894.67% higher than those of ZHDM2, respectively. After treatment at pH 5.0 and 37 °C for 60 min, the mutant T217C-G242C retained about 96.41% of the enzyme activity, the mutant R76Q retained about 5.13% of the enzyme activity, and the mutant T217C-G242C / R76Q retained about 75.00% of the enzyme activity, which were 3157.09%, 73.31% and 2433.78% higher than those of ZHDM2 (2.96%), respectively. T m The value is 51.54℃, R76Q T m The value is 52.49 ℃, T217C-G242C T m The value is 59.82 ℃, T217C-G242 / R76Q T m The value is 60.12 °C, which is 0.95 °C, 8.28 °C, and 8.58 °C higher than that of ZHDM2, respectively. Therefore, the zearalenone hydrolase mutant provided by the present invention can be well applied in the food and feed industries and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The thermal stability comparison between the mutant of the present application and ZHDM2 treated at 50°C is shown;

[0040] Figure 2 The thermal stability comparison between the mutant of the present application and ZHDM2 treated at 55°C is shown;

[0041] Figure 3 A comparison of the acid stability of the mutants of the present application and ZHDM2 treated at pH 5.0 is shown;

[0042] Figure 4 The mutants of the present application and ZHDM2 T m Value comparison. DETAILED DESCRIPTION

[0043] Experimental materials and reagents:

[0044] Strains and vectors: The expression host is Pichia pastoris GS115, the expression plasmid vector is pPICZ(α)A;

[0045] Enzymes and other biochemical reagents: endonucleases, ligases;

[0046] Culture medium:

[0047] Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH);

[0048] Pichia pastoris medium YPD (Yeast Extract 1 %, Trytone 2 %, Glucose 2 %, natural pH);

[0049] BMGY medium (Yeast Extract 1%, Trytone 2%, YNB10%, Biotin 0.1%, natural pH);

[0050] BMMY medium (Yeast Extract 1%, Trytone 2%, Methanol 0.5%, YNB 10%, Biotin 0.1%, natural pH).

[0051] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0052] Example 1. Preparation of recombinant strain GS115 (pPICZ(α)A-zhdm2)

[0053] (1) Amplification of the nucleic acid sequence of zearalenone hydrolase ZHDM2 zhdm2

[0054] Amplification by PCR zhdm2 The gene fragment was obtained by double enzyme digestion to obtain the vector pPICZ(α)A nucleic acid fragment, and the two were connected by a recombination kit to obtain the recombinant plasmid pPICZ(α)A -zhdm2 , and transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 (pPICZ(α)A -zhdm2 ). The primers used for PCR are as follows:

[0055] ZHDM2-pPICZ(α)AF (SEQ ID NO: 9): 5'GAGGCTGAAGCTGAATTCATGTCCACCACCAGAGCCACC3';

[0056] ZHDM2-pPICZ(α)AR (SEQ ID NO: 10): 5'CTAGAAAGCTGGCGGCCGCTTAAATGTACTTTCTACAAG3'.

[0057] Among them, ZHDM2-pPICZ(α)AF and ZHDM2-pPICZ(α)AR were used to amplify the gene coding sequence of zearalenone hydrolase ZHDM2; the vector pPICZ(α)A was obtained by inoculating the preserved strains and then extracting them. After the amplification, the PCR products and the extracted plasmids were subjected to nucleic acid electrophoresis detection. zhdm2 The band sizes of pPICZ(α)A and pPICZ(α)A were 835 bp and 3579 bp (with primers), respectively. EcoR I and Not After enzyme digestion, the PCR product and the enzyme digestion product are recovered and purified respectively.

[0058] (2) Construction of recombinant strains GS115 (pPICZ(α)A -zhdm2 )

[0059] The recycled zhdm2 The pPICZ(α)A gene fragment was recombined and connected using the kit recombinase, and then the recombinant product was transformed into E. coli Trans1-T1 competent cells and spread on LB (containing 100 μg / mL zeocin) for screening. Sac I restriction endonuclease to recombinant plasmid pPICZ (α) A -zhdm 2. Enzyme digestion was performed, and the recovered product was electroporated and transformed into Pichia pastoris competent cells GS115 for induced expression to obtain the recombinant expression strain GS115 (pPICZ(α)A - zhdm2 ).

[0060] Example 2. Preparation of recombinant strain GS115 (pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, pPICZ(α)A-zhdm2-T217C-G242C / R76Q)

[0061] (1) Recombinant plasmid pPICZ(α)A -zhdm2-T217C-G242C , pPICZ(α)A -zhdm2-R76Q , pPICZ(α)A -zhdm2-T217C-G242C / R76Q Construction

[0062] After optimization, the mutation scheme of corn zearalenone hydrolase ZHDM2 is:

[0063] Mutation scheme 1: The amino acid at position 217 is mutated from threonine to cysteine, and the amino acid at position 242 is mutated from glycine to cysteine;

[0064] Mutation scheme 2: amino acid 76 was mutated from arginine to glutamine;

[0065] Mutation scheme 3: Based on mutation scheme 1, the amino acid at position 76 is further mutated from arginine to glutamine.

[0066] The mutation site was introduced by the point mutation kit method and sequenced to obtain the zearalenone hydrolase mutant plasmid pPICZ(α)A. -zhdm2-T217C-G242C , pPICZ(α)A -zhdm2-R76Q , pPICZ(α)A - zhdm2-T217C-G242C / R76Q The primers used are as follows:

[0067] T217C-F (SEQ ID No: 11) 5'GACAGTTGGTGGTGGTTGTCCAACTGC3',

[0068] T217C-R (SEQ ID No: 12) 5'ACAACCACCACCAACTGTCCAATCAACTG3';

[0069] G242C-F (SEQ ID No: 13) 5'GGTTTGTTGCCAGGTTGTCATTTCCCAT3',

[0070] G242C-R (SEQ ID No: 14) 5'ACAACCTGGCAACAAACCAATGTTCAAACC3';

[0071] R76Q-F (SEQ ID No: 15) 5'CCTCCAGAAACTTACCAGGATGTTACCGG3',

[0072] R76Q-R (SEQ ID No: 16) 5'CTGGTAAGTTTCTGGAGGAGCATCTCTGG3'.

[0073] Among them, the mutant plasmid pPICZ(α)A was first constructed using primers R76Q-F and R76Q-R. -zhdm2-R76Q Primers T217C-F and T217C-R were used to construct the mutant plasmid pPICZ(α)A. -zhdm2-T217C After the sequencing was correct, the mutant plasmid pPICZ(α)A was constructed using primers G242C-F and G242C-R. -zhdm2-T217C-G242C. After sequencing was completed, the mutant plasmid pPICZ(α)A was constructed using primers R76Q-F and R76Q-R. -zhdm2-T217C-G242C / R76Q .

[0074] (2) Construction of recombinant strain GS115 (pPICZ(α)A -zhdm2-T217C-G242C , pPICZ(α)A -zhdm2- R76Q , pPICZ(α)A -zhdm2-T217C-G242C / R76Q )

[0075] use Sac I recombinant plasmid pPICZ(α)A -zhdm2-T217C-G242C , pPICZ(α)A -zhdm2-R76Q , pPICZ(α)A -zhdm2-T217C-G242C / R76Q The product was digested by enzymes, and then electroporated into Pichia pastoris competent cells GS115 for induced expression to obtain the recombinant expression strain GS115 (pPICZ(α)A -zhdm2-T217C-G242C , pPICZ(α)A -zhdm2-R76Q , pPICZ(α)A -zhdm2-T217C-G242C / R76Q ).

[0076] Example 3. Obtaining Zearalenone Hydrolase ZHDM2 and Mutants T217C-G242C, R76Q, T217C-G242C / R76Q

[0077] 1. Inducible expression of ZHDM2 and its mutants T217C-G242C, R76Q, and T217C-G242C / R76Q

[0078] The obtained recombinant expression strain GS115 ( pPIC9-zhdm2 ) and GS115 (pPICZ(α)A -zhdm2- T217C-G242C , pPICZ(α)A -zhdm2-R76Q , pPICZ(α)A -zhdm2-T217C-G242C / R76Q ) were inoculated into YPD medium for seed culture, cultured at 200 rpm and 30 ℃ for 48 h, and then transferred to BMGY medium with 1% inoculum, cultured at 200 rpm and 30 ℃ for 48 h. After sufficient bacterial cells were enriched, the cells were collected and added to BMMY medium containing 1% methanol for induction of expression.

[0079] 2. Purification of ZHDM2 and mutants T217C-G242C, R76Q, and T217C-G242C / R76Q

[0080] The induced bacterial solution was centrifuged at 12000 rpm for 10 min, the supernatant was collected and concentrated, desalted in 20 mMTris-HCl buffer (pH 7.6), and then purified by hydrophobic chromatography. Solution A was 20 mM TrisHCl (containing 2 M (NH4)2SO4, pH 7.6) and solution B was 20 mM Tris HCl. The protein was eluted with a linear gradient of (NH4)2SO4 (0 M-2M).

[0081] 3. Determination of properties of the mutant and parent ZHDM2 of the present application

[0082] 3.1 Determination of thermal stability of mutants and parent ZHDM2 of the present application

[0083] The thermal stability of the mutants and ZHDM2 of the present application was determined by treating them in a 20 mM Tris-HCl buffer (pH 9.0) buffer system at different temperatures (50°C or 55°C) for different times (5 min and 10 min at 50°C; 2 min and 5 min at 55°C), and then measuring the residual enzyme activity at 37°C. Figure 1 As shown in the data, after treatment at 50°C for 5 min and 10 min, the residual activity of ZHDM2 was 26.96% and 24.77%; after treatment at 50°C for 5 min and 10 min, the residual enzyme activities of mutants T217C-G242C, R76Q and T217C-G242C / R76Q were 68.75% and 33.13%, 74.09% and 57.51%, 82.95% and 62.69%, respectively, which were 155.01% and 33.75%, 174.81% and 132.18%, and 207.68% and 153.09% higher than those of ZHDM2, respectively.

[0084] like Figure 2As shown in the data, after treatment at 55 °C for 2 min and 5 min, the residual activity of ZHDM2 was 23.53% and 7.13%; after treatment at 55 °C for 2 min and 5 min, the residual enzyme activities of mutants T217C-G242C, R76Q and T217C-G242C / R76Q were 68.53% and 13.78%, 83.78% and 66.05%, 83.91% and 70.92%, respectively, which were 191.25% and 93.27%, 256.06% and 826.37%, and 256.61% and 894.67% higher than those of ZHDM2, respectively.

[0085] 3.2 Acid stability determination of the mutant and parent ZHDM2 of the present application

[0086] The acid stability of the mutants and ZHDM2 of the present application was determined by treating them at 37°C and 0.1 mol / L citric acid-sodium hydrogen phosphate buffer (pH 5.0) for 60 min, and then determining the residual enzyme activity at 20 mM Tris-HCl pH 9.0 and 37°C. Figure 3 As shown, after treatment at pH 5.0 and 37 ℃ for 60 min, the mutant T217C-G242C retained about 96.41% of the enzyme activity, the mutant R76Q retained about 5.13% of the enzyme activity, and the mutant T217C-G242C / R76Q retained about 75.00% of the enzyme activity, which were increased by 3157.09%, 73.31% and 2433.78% respectively compared with ZHDM2 (2.96%).

[0087] 3.3 Mutants and parental ZHDM2 of the present application T m Value determination

[0088] The purified mutant and ZHDM2 enzyme solution were concentrated and desalted using a pre-packed desalting column and pH 9.0, 20 mM Tris-HCl buffer. The protein concentration after desalting was determined to make the concentration of the sample to be tested uniform to about 0.5 mg / mL. Then DSC was used to measure T m The measurement temperature range was set to 25-100 °C, the temperature rise rate was set to 120 °C / h, and each sample was scanned three times. The DSC scan results were processed using Origin 2021 to obtain the sample T m Value. Figure 4 As shown, ZHDM2 T m The value is 51.54℃, R76Q T mThe value is 52.49 ℃, T217C-G242C T m The value is 59.82℃, T217C-G242 / R76Q T m The value is 60.12 ℃, which is 0.95 ℃, 8.28 ℃ and 8.58 ℃ higher than that of ZHDM2 respectively.

[0089] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A mutant of zearalenone hydrolase with improved thermal stability and acid stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

2.

2. A method for improving the thermal stability and acid stability of zearalenone hydrolase, characterized in that: The method comprises the following steps: subjecting the zearalenone hydrolase ZHDM2 with the amino acid sequence shown in SEQ ID NO:1 to the following mutations: The amino acids at positions 217 and 242 were mutated from threonine and glycine to cysteine.

3. A zearalenone hydrolase gene, characterized in that: The zearalenone hydrolase gene encodes the zearalenone hydrolase mutant with improved thermal stability and acid stability as described in claim 1.

4. The zearalenone hydrolase gene according to claim 3, characterized in that The nucleotide sequence of the corn zearalenone hydrolase gene is as shown in SEQ ID NO:

6.

5. A recombinant expression vector comprising the zearalenone hydrolase gene according to claim 3.

6. A recombinant expression strain comprising the zearalenone hydrolase gene of claim 3.

7. A method for preparing a zearalenone hydrolase having improved thermal stability and acid stability, characterized in that: The method comprises the following steps: Transforming a host cell with the recombinant expression vector according to claim 5 to obtain a recombinant strain; Cultivating the recombinant strain to induce expression of zearalenone hydrolase; The expressed zearalenone hydrolase is recovered and purified.

8. Use of the zearalenone hydrolase mutant with improved thermal stability and acid stability according to claim 1 for hydrolyzing zearalenone.

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