Method for improving thermal stability and acid stability of zearalenone hydrolase, R76Q series mutants and application

Abstract

The invention relates to the field of gene engineering, in particular to a method for improving thermal stability and acid stability of zearalenone hydrolase, R76Q series mutants and application. The zearalenone hydrolase ZHDM2 is subjected to site-directed mutagenesis to obtain a mutant with improved thermal stability and acid stability. The zearalenone hydrolase mutant disclosed by the invention has good enzymatic properties and can be applied to industries such as food, feed and the like.

Description

Technical Field

[0001] 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, an R76Q series mutant, and applications 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 livestock industry in production practice.

[0003] Microbial degradation (ZEN) involves the collaborative degradation of toxins through the use of one or more enzymes produced by microbial metabolism. This method, characterized by mild conditions, strong specificity, and high efficiency without damaging the original nutrients, is environmentally friendly, efficient, and highly practical. However, the activity of the zearalenone hydrolases currently discovered is low, hindering their large-scale industrial production and application. 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 gene encoding the above-mentioned zearalenone hydrolase mutant.

[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 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] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAAS

[0012] GFRVTTFDMPGMSRSRDAPPETY R DVTGHKLAGYVDTLLEELKIPIASVWGC

[0013] SSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVS

[0014] RSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPV

[0015] DWTVGGG T PTAMFFDNIVIAVKEGLNIGLLPG G HFPYVSHPEAFAEYVVETCRKYI.

[0016] According to a specific embodiment of the present invention, the 217th and 242nd amino acids of zearalenone hydrolase ZHDM2 are mutated from threonine and glycine to cysteine to obtain mutant T217C-G242C, or the 76th amino acid is mutated from arginine to glutamine to obtain mutant R76Q.

[0017] 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.

[0018] 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:

[0019] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAAS

[0020] GFRVTTFDMPGMSRSRDAPPETYRDVTGHKLAGYVDTLLEELKIPIASVWGC

[0021] SSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVS

[0022] RSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPV

[0023] DWTVGGG C PTAMFFDNIVIAVKEGLNIGLLPG C HFPYVSHPEAFAEYVVETCRKYI.

[0024] 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:

[0025] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAAS

[0026] GFRVTTFDMPGMSRSRDAPPETY Q DVTGHKLAGYVDTLLEELKIPIASVWGC

[0027] SSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVS

[0028] RSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPV

[0029] DWTVGGGTPTAMFFFDNIVIAVKEGLNIGLLPGGHFPYVSHPEAFAEYVVETCRKYI.

[0030] 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:

[0031] MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAAS

[0032] GFRVTTFDMPGMSRSRDAPPETY Q DVTGHKLAGYVDTLLEELKIPIASVWGC

[0033] SSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVS

[0034] RSYSGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPV

[0035] DWTVGGG C PTAMFFDNIVIAVKEGLNIGLLPG C HFPYVSHPEAFAEYVVETCRKYI.

[0036] 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:

[0037] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGC

[0038] ATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGG

[0039] TGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGT

[0040] CACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTA

[0041] CAGAGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAAT

[0042] TGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGG

[0043] CCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCA

[0044] ACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTC

[0045] TCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATG

[0046] CTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAG

[0047] GTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAG

[0048] AGACCAGTTGATTGGACAGTTGGTGGTGGTACCCCAACTGCTATGTTTTTTGATAAT

[0049] ATTGTCATCGCTGTCAAGGAAAGGTTTGAACATTGGTTTGTTGCCAGGTGGTCATTT

[0050] CCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATTTAA.

[0051] According to a specific embodiment of the present invention, the coding gene sequence of the zearalenone hydrolase mutant T217C-G242C is shown in SEQ ID NO: 6:

[0052] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACAGAGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTTGTCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTTGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATT。

[0053] According to the specific embodiments of the present invention, the coding gene sequence of the zearalenone hydrolase mutant R76Q is as shown in SEQ ID NO:7:

[0054] ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACCAGGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGCTTCTGTTACTGGTATGGAGGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCATATAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTACCCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTGGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATT。

[0055] According to the specific embodiments of the present invention, the coding gene sequence of the zearalenone hydrolase mutant T217C-G242C / R76Q is shown in SEQ ID NO:8:

[0056] .

[0057] The method for improving the thermal stability and acid-thermal stability of zearalenone hydrolase according to the present invention comprises the following steps:

[0058] The zearalenone hydrolase ZHDM2 was subjected to double point mutations, with the amino acids at positions 217 and 242 mutated from threonine and glycine to cysteine;

[0059] A single point mutation was performed on the zearalenone hydrolase ZHDM2, and the amino acid at position 76 was mutated from arginine to glutamine.

[0060] The present invention provides a recombinant vector comprising the gene encoding the zearalenone hydrolase mutant. Preferably, the starting vector of the recombinant expression vector is pPICZ(α)A.

[0061] The present invention also provides a recombinant strain comprising the coding gene of the zearalenone hydrolase mutant. Preferably, the starting strain of the recombinant bacteria is GS115 (pPICZ(α)A-zhdm2).

[0062] According to a specific embodiment of the present invention, a method for preparing zearalenone hydrolase with improved thermal stability and acid stability is as follows:

[0063] (1) transforming a host cell with a recombinant vector containing a gene encoding a mutant of zearalenone hydrolase to obtain a recombinant strain;

[0064] (2) culturing the recombinant strain to induce the expression of zearalenone hydrolase;

[0065] (3) Recover and purify the expressed zearalenone hydrolase.

[0066] Beneficial effects of the present invention:

[0067] The present invention improves thermal and acid stability by introducing a disulfide bond and a glutamine into the structure of zearalenone hydrolase. ZHDM2 exhibits 26.96% and 24.77% residual activity after treatment at 50°C for 5 and 10 minutes, respectively. The mutants T217C-G242C, R76Q, and T217C-G242C / R76Q exhibited 68.75% and 33.13% residual activity after treatment at 50°C for 5 and 10 minutes, respectively. These improvements represent increases of 155.01% and 33.75% relative to ZHDM2, 174.81% and 132.18% relative to ZHDM2, and 207.68% and 153.09% relative to ZHDM2. 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. After treatment at pH 5.0 and 37°C for 60 min, the T217C-G242C mutant retained about 96.41% of the enzyme activity, the R76Q mutant retained about 5.13% of the enzyme activity, and the T217C-G242C / R76Q mutant 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%). m The value is 51.54℃, and the T m The value is 52.49℃, and the T m The value is 59.82℃, T217C-G242 / R76Q 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

[0068] Figure 1 Showing the comparison of thermal stability of the mutants of the present application and ZHDM2 treated at 50°C;

[0069] Figure 2 Showing the comparison of thermal stability of the mutants of the present application and ZHDM2 treated at 55°C;

[0070] Figure 3 Shows the comparison of acid stability of the mutants of the present application and ZHDM2 treated at pH 5.0;

[0071] Figure 4 The mutants of the present application and the T of ZHDM2 are shown m Value comparison. DETAILED DESCRIPTION

[0072] Test materials and reagents:

[0073] Strains and vectors: The expression host is Pichiapastoris GS115, and the expression plasmid vector is pPICZ(α)A;

[0074] Enzymes and other biochemical reagents: endonucleases, ligases;

[0075] Culture medium:

[0076] Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH);

[0077] Pichia pastoris medium YPD (Yeast Extract 1%, Trytone 2%, Glucose 2%, pH natural);

[0078] BMGY medium (Yeast Extract 1%, Trytone 2%, YNB 10%, Biotin 0.1%, natural pH);

[0079] BMMY medium (Yeast Extract 1%, Trytone 2%, Methanol 0.5%, YNB 10%, Biotin 0.1%, natural pH).

[0080] Note: Molecular biology experimental methods not specifically described in the following examples were performed with reference to the specific methods listed in the book "Molecular Cloning Laboratory Manual" (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0081] Example 1. Preparation of recombinant strain GS115 (pPICZ(α)A-zhdm2)

[0082] (1) Amplify the nucleic acid sequence zhdm2 of zearalenone hydrolase ZHDM2

[0083] The zhdm2 gene fragment was amplified by PCR, and the vector pPICZ(α)A nucleic acid fragment was obtained by double enzyme digestion. The two were connected using a recombination kit to obtain the recombinant plasmid pPICZ(α)A-zhdm2, and then transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 (pPICZ(α)A-zhdm2). The primers used in PCR are as follows:

[0084] ZHDM2-pPICZ(α)AF(SEQ ID NO:9):

[0085] 5'GAGGCTGAAGCTGAATTCATGTCCACCACCAGAGCCACC3';

[0086] ZHDM2-pPICZ(α)AR(SEQ ID NO:10):

[0087] 5'CTAGAAAGCTGGCGGCCGCTTAAATGTACTTTCTACAAG3'.

[0088] ZHDM2-pPICZ(α)AF and ZHDM2-pPICZ(α)AR were used to amplify the gene coding sequence for the zearalenone hydrolase ZHDM2. The pPICZ(α)A vector was obtained by inoculating a preserved bacterial strain and extracting it. After amplification, the PCR products and extracted plasmids were analyzed by nucleic acid electrophoresis. The band sizes of zhdm2 and pPICZ(α)A were 835 bp and 3579 bp, respectively (with primers added). The vectors were digested with EcoRI and NotI, and the PCR and digestion products were recovered and purified.

[0089] (2) Construction of recombinant strain GS115 (pPICZ(α)A-zhdm2)

[0090] The recovered zhdm2 was recombined with the pPICZ(α)A gene fragment using the kit's recombinase. The recombinant product was then transformed into competent Escherichia coli Trans1-T1 cells and plated on LB (containing 100 μg / mL zeocin) for screening. After sequencing, the recombinant plasmid pPICZ(α)A-zhdm2 was digested with the Sac I restriction endonuclease and the recovered product was transformed into competent Pichia pastoris GS115 cells by electroporation for induced expression, generating the recombinant expression strain GS115 (pPICZ(α)A-zhdm2).

[0091] 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)

[0092] (1) Construction of recombinant plasmids pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, and pPICZ(α)A-zhdm2-T217C-G242C / R76Q

[0093] After optimization, the mutation scheme of zearalenone hydrolase ZHDM2 is as follows:

[0094] Mutation scheme 1: amino acid 217 was mutated from threonine to cysteine, and amino acid 242 was mutated from glycine to cysteine;

[0095] Mutation scheme 2: amino acid 76 is mutated from arginine to glutamine;

[0096] Mutation scheme 3: Based on mutation scheme 1, the amino acid at position 76 is further mutated from arginine to glutamine.

[0097] Mutation sites were introduced using a point mutagenesis kit and sequenced to obtain the zearalenone hydrolase mutant plasmids pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, and pPICZ(α)A-zhdm2-T217C-G242C / R76Q. The primers used are as follows:

[0098] T217C-F (SEQ ID No: 11)5'GACAGTTGGTGGTGGTTGTCCAACTGC3',

[0099] T217C-R (SEQ ID No: 12)5'ACAACCACCACCAACTGTCCAATCAACTG3';

[0100] G242C-F (SEQ ID No: 13)5'GGTTTGTTGCCAGGTTGTCATTTCCCAT3',

[0101] G242C-R (SEQ ID No: 14)5'ACAACCTGGCAACAAACCAATGTTCAAACC3';

[0102] R76Q-F (SEQ ID No: 15)5'CCTCCAGAAACTTACCAGGATGTTACCGG3',

[0103] R76Q-R (SEQ ID No: 16)5'CTGGTAAGTTTCTGGAGGAGCATCTCTGG3'.

[0104] First, primers R76Q-F and R76Q-R were used to construct the mutant plasmid pPICZ(α)A-zhdm2-R76Q. Primers T217C-F and T217C-R were used to construct the mutant plasmid pPICZ(α)A-zhdm2-T217C. After sequencing was confirmed, primers G242C-F and G242C-R were used to construct the mutant plasmid pPICZ(α)A-zhdm2-T217C-G242C. After sequencing was confirmed, primers R76Q-F and R76Q-R were used to construct the mutant plasmid pPICZ(α)A-zhdm2-T217C-G242C.

[0105] (2) Construction of recombinant strain GS115 (pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, pPICZ(α)A-zhdm2-T217C-G242C / R76Q)

[0106] The recombinant plasmids pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, and pPICZ(α)A-zhdm2-T217C-G242C / R76Q were digested with Sac I, and the recovered products were transformed into Pichia pastoris competent cells GS115 by electroporation for induced expression to obtain the recombinant expression strain GS115 (pPICZ(α)A-zhdm2-T217C-G242C, pPICZ(α)A-zhdm2-R76Q, and pPICZ(α)A-zhdm2-T217C-G242C / R76Q).

[0107] Example 3 Obtaining Zearalenone Hydrolase ZHDM2 and Mutants T217C-G242C, R76Q, and T217C-G242C / R76Q

[0108] 1. Inducible expression of ZHDM2 and its mutants T217C-G242C, R76Q, and T217C-G242C / R76Q

[0109] The obtained recombinant expression strains 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. After culturing at 200 rpm and 30°C for 48 h, they were transferred to BM GY medium with a 1% inoculum amount and cultured at 200 rpm and 30°C for 48 h. After sufficient bacterial cells were enriched, the bacterial cells were collected and added to BMMY medium containing 1% methanol for induced expression.

[0110] 2. Purification of ZHDM2 and its mutants T217C-G242C, R76Q, and T217C-G242C / R76Q

[0111] After induced expression, the bacterial solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and concentrated, desalted in 20 mM Tris-HCl buffer (pH 7.6), and then purified by hydrophobic chromatography. Solution A consisted of 20 mM Tris HCl (containing 2 M (NH₄)₂SO₄, pH 7.6) and Solution B consisted of 20 mM Tris HCl. The protein was eluted using a linear gradient of (NH₄)₂SO₄ (0 M to 2 M).

[0112] 3. Determination of properties of the mutants and parent ZHDM2 of the present application

[0113] 3.1 Thermal stability determination of mutants and parent ZHDM2 of the present application

[0114] 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) 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, 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 increased by 155.01% and 33.75%, 174.81% and 132.18%, and 207.68% and 153.09% compared with ZHDM2, respectively.

[0115] like Figure 2 As shown, 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 increased by 191.25% and 93.27%, 256.06% and 826.37%, and 256.61% and 894.67% compared with ZHDM2, respectively.

[0116] 3.2 Acid stability determination of the mutants and parent ZHDM2 of the present application

[0117] 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 measuring 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°C for 60 min, the mutant T217C-G242C retained approximately 96.41% of the enzyme activity, the mutant R76Q retained approximately 5.13% of the enzyme activity, and the mutant T217C-G242C / R76Q retained approximately 75.00% of the enzyme activity, which were increases of 3157.09%, 73.31% and 2433.78% respectively compared with ZHDM2 (2.96%).

[0118] 3.3 T of the mutants of this application and the parent ZHDM2 m Value determination

[0119] The purified mutant and ZHDM2 enzyme solutions were concentrated and desalted using a pre-packed desalting column and 20 mM Tris-HCl buffer, pH 9.0. The protein concentration after desalting was determined to achieve a uniform concentration of approximately 0.5 mg / mL. DSC was then used to determine T m The measurement temperature range was set to 25-100℃, the temperature rise rate was set to 120℃ / h, and each sample was scanned three times. The T value of the sample was obtained by processing the DSC scan results using Origin 2021. m Value. Figure 4 As shown, the T m The value is 51.54℃, and the T m The value is 52.49℃, and the T m The value is 59.82℃, T217C-G242 / R76Q mThe value is 60.12℃, which is 0.95℃, 8.28℃ and 8.58℃ higher than that of ZHDM2 respectively.

[0120] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection 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: 3 or SEQ ID NO:

4.

2. A method for improving the thermal stability and acid stability of zearalenone hydrolase, characterized in that: The method comprises the following steps: performing the following mutations on the zearalenone hydrolase ZHDM2 whose amino acid sequence is shown in SEQ ID NO: 1: The amino acid at position 76 is mutated from arginine to glutamine; or The amino acids at positions 217 and 242 were mutated from threonine and glycine to cysteine, and the amino acid at position 76 was mutated from arginine to glutamine.

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

4. The zearalenone hydrolase gene according to claim 3, characterized in that The nucleotide sequence of the zearalenone hydrolase gene is shown in SEQ ID NO: 7 or SEQ ID NO:

8.

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

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

7. A method for preparing zearalenone hydrolase with 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 was recovered and purified.

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

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

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