Method for improving thermal stability of fumonisin-degrading enzyme and application thereof

Abstract

The present application belongs to the technical field of protein engineering, and particularly relates to a method for improving the thermal stability of fumonisin-degrading enzyme by forming disulfide bonds. The fumonisin-degrading enzyme FumD mutant according to the present application shows a significant improvement effect on the thermal stability compared with the wild-type fumonisin-degrading enzyme. The enzyme is mainly applied to the livestock feed processing and food production industries to reduce the health hazards of fumonisin to animals and humans. The fumonisin-degrading enzyme mutant with improved thermal stability obtained by the present application is beneficial to maintaining the activity of the enzyme at a higher temperature, adapting to a more high-temperature production environment, reducing the use amount and replacement frequency of the enzyme, and the improvement in stability facilitates transportation and storage, so that the industrial cost input can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and particularly relates to a fumonisin degrading enzyme mutant with improved thermal stability and applications thereof. Background Art

[0002] Fumonisins are primarily produced by Clostridium verticillium and Clostridium prolifera, as well as other Fusarium species. Their structure consists of two methyl (-CH3), one amino (-NH2), one to four hydroxyl (-OH), and two tricarboxylate groups, located at various positions within a linear polyketide-derived backbone. Currently, there are over 15 known fumonisin homologues, namely fumonisins A, B, C, and P. Fumonisins FB1 is the most toxic and has the highest concentration. The most common agricultural crops affected are cereals such as corn, wheat, barley, and soybeans. Fumonisins pose a serious threat to food safety. Consuming fumonisin-contaminated food can lead to liver disease, immune system suppression, neural tube defects, and nephrotoxicity. Numerous studies have focused on the degradation of fumonisins, but traditional physical and chemical methods only mitigate the harmful effects of fumonisins, fail to completely eliminate them, and may even result in other adverse effects. Enzymatic methods offer advantages such as environmental friendliness, high efficiency, zero residue, and minimal nutrient loss. Fumonisin-degrading enzymes with good thermal stability can withstand high-temperature treatment conditions during the enzyme preparation process and increase the use time of the enzyme, thereby reducing production costs and improving enzyme utilization efficiency. Summary of the Invention

[0003] In order to utilize the bioenzymatic method to efficiently degrade fumonisin, the present invention is proposed and completed.

[0004] The object of the present invention is to provide a fumonisin degrading enzyme mutant with improved thermal stability.

[0005] Another object of the present invention is to provide a fumonisin degrading enzyme gene with improved thermal stability.

[0006] Another object of the present invention is to provide a recombinant expression vector comprising the above gene.

[0007] Another object of the present invention is to provide a recombinant strain comprising the above gene.

[0008] Another object of the present invention is to provide a method for preparing fumonisin-degrading enzyme.

[0009] Another object of the present invention is to provide a method for improving the thermal stability of fumonisin-degrading enzyme.

[0010] The amino acid sequence of the fumonisin degrading enzyme mutant T470C / L481C according to a specific embodiment of the present invention is shown in SEQ ID NO: 1.

[0011] SEQ ID NO: 1:

[0012] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSGAVP YYDGSALAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEA NGLELGADISALRRADAGELTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDRLPVKTVLEYRAYLTEQFGDEADAWERCYPANSDAD VPAAVARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIM C FGSQVGSGEG C GVSPSKACQPSK

[0013] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T470C / L481C, the genomic sequence of which is shown in SEQ ID NO: 2

[0014] SEQ ID NO:2

[0015] tgt ttcggatcacaagttggttctggtgagggt tgt ggagtttctccttctaaagcatgtcaaccttcaaaa

[0016] The amino acid sequence of the fumonisin degrading enzyme mutant T470C / L481C / P44C / A168C according to a specific embodiment of the present invention is shown in SEQ ID NO: 3.

[0017] SEQ ID NO: 3:

[0018] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRP C APPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSGAVPYYDGSALAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDML C AFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISALRRADAGELTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDRLP VKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAAVARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIM C FGSQVGSGEG C GVSPSKACQPSK

[0019] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T470C / L481C / P44C / A168C, the genomic sequence of which is shown in SEQ ID NO: 4

[0020] SEQ ID NO:4

[0021] caaacagatgatccaaaattagttagacatacacaatctggtgcagttgaaggagttgaaggtgatgttgaaacttttttaggtattccttttgctgctcctccagtaggtgacttacgttggcgtcca tgt gcaccacctcgtgcttgggctggtacaagagatggtcgtcgttttgctcccgattgcattggaaacgagcgtcttcgtgaaggttcacgtgctgctggtacatcagaggactgtttatatttaaatatttggtcaccaaaacaagttggaaaaggtggtttaccagttatgatttgggtttacggaggaggtttctctggtggttctggtgctgttccttactacgacggatctgcacttgctcagaagggagttgttgttgttacatttaattatcgtgctggtattttaggatttttagcacatccagctttatctaaggaatcacctaatggagtatctggtaattatggacttttagatatgtta tgtgcttttaagtgggttcaaaataatatacgtgaatttggaggagatccaaaccgtgtaacagttttcggtgagtcagctggtgcttcagcattaggattattattaacatcaccactttcagaatctgcatttaatcaagctattcttcaatctcccggtcttgcacgtccacttgctacactttcagagtcagaagcaaatggtttagaacttggtgctgacatttctgctcttcgtcgtgctgatgctggagaattaactaaaattgctcaatcccgtattcctatgtctcgtcaatttacaaaaccacgtccaatgggacctattttagatggatatgttttacgtactcttgatgttgatgcatttgcaaaaggagcatttagaaaaattccagttcttgttggtggaaatgctgacgagggtcgtgcatttactgatcgtcttccagttaaaactgttttagaatatcgtgcatatttaacagaacaatttggagacgaagctgacgcttgggaacgttgttatcccgctaattcagatgctgacgttcccgctgctgttgcacgtttattcggagattctcaatttaataatggtattgaattattatccgctgcttttgcaaaatggcgtacaccattatggcgttatcgtttcactggtattcccggtgctggtagacgtcccgctacacatggtgatgaaattccttatgttttcgcaaatttaggtccatcttcagtttctatgttcggttcattagaaggaggagctggagcttctgatattaagttagcaacagaaatgtctgctgcttgggtttcattcgctgttcacggtgttcccgatcaaggtacaaaatcacattggccacgttttgaacgtagaggagaaattatg tgt ttcggatcacaagttggttctggtgagggt tgt ggagtttctccttctaaagcatgtcaaccttcaaaa

[0022] The amino acid sequence of the fumonisin degrading enzyme mutant T470C / L481C / F59C / A115C according to a specific embodiment of the present invention is shown in SEQ ID NO: 5.

[0023] SEQ ID NO: 5:

[0024] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRR C APDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSG C VPYYDGSALAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISALRRADAGELTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFA KGAFRKIPVLVGGNADEGRAFTDRLPVKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAAVARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIM C FGSQVGSGEG C GVSPSKACQPSK

[0025] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T470C / L481C / F59C / A115C, the genomic sequence of which is shown in SEQ ID NO: 6

[0026] SEQ ID NO:6

[0027] caaacagatgatccaaaattagttagacatacacaatctggtgcagttgaaggagttgaaggtgatgttgaaacttttttaggtattccttttgctgctcctccagtaggtgacttacgttggcgtccaccagcaccacctcgtgcttgggctggtacaagagatggtcgtcgt tgt gctcccgattgcattggaaacgagcgtcttcgtgaaggttcacgtgctgctggtacatcagaggactgtttatatttaaatatttggtcaccaaaacaagttggaaaaggtggtttaccagttatgatttgggtttacggaggaggtttctctggtggttctggt tgt tgt ttcggatcacaagttggttctggtgagggt tgt ggagtttctccttctaaagcatgtcaaccttcaaaa

[0028] The amino acid sequence of the fumonisin degrading enzyme mutant T470C / L481C / L140C / A258C according to a specific embodiment of the present invention is shown in SEQ ID NO: 7.

[0029] SEQ ID NO: 7:

[0030] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSGAVPYYDGSALAQKGVVVVTFNYRAG C LGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISALRRADAGELTKI C QSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDRLPVKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIM C FGSQVGSGEG C GVSPSKACQPSK

[0031] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T470C / L481C / L140C / A258C, the genomic sequence of which is shown in SEQ ID NO: 8

[0032] SEQ ID NO:8

[0033] caaacagatgatccaaaattagttagacatacacaatctggtgcagttgaaggagttgaaggtgatgttgaaacttttttaggtattccttttgctgctcctccagtaggtgacttacgttggcgtccaccagcaccacctcgtgcttgggctggtacaagagatggtcgtcgttttgctcccgattgcattggaaacgagcgtcttcgtgaaggttcacgtgctgctggtacatcagaggactgtttatatttaaatatttggtcaccaaaacaagttggaaaaggtggtttaccagttatgatttgggtttacggaggaggtttctctggtggttctggtgctgttccttactacgacggatctgcacttgctcagaagggagttgttgttgttacatttaattatcgtgctggt tgt ttaggatttttagcacatccagctttatctaaggaatcacctaatggagtatctggtaattatggacttttagatatgttagctgcttttaagtgggttcaaaataatatacgtgaatttggaggagatccaaaccgtgtaacagttttcggtgagtcagctggtgcttcagcattaggattattattaacatcaccactttcagaatctgcatttaatcaagctattcttcaatctcccggtcttgcacgtccacttgctacactttcagagtcagaagcaaatggtttagaacttggtgctgacatttctgctcttcgtcgtgctgatgctggagaattaactaaaatt tgtcaatcccgtattcctatgtctcgtcaatttacaaaaccacgtccaatgggacctattttagatggatatgttttacgtactcttgatgttgatgcatttgcaaaaggagcatttagaaaaattccagttcttgttggtggaaatgctgacgagggtcgtgcatttactgatcgtcttccagttaaaactgttttagaatatcgtgcatatttaacagaacaatttggagacgaagctgacgcttgggaacgttgttatcccgctaattcagatgctgacgttcccgctgctgttgcacgtttattcggagattctcaatttaataatggtattgaattattatccgctgcttttgcaaaatggcgtacaccattatggcgttatcgtttcactggtattcccggtgctggtagacgtcccgctacacatggtgatgaaattccttatgttttcgcaaatttaggtccatcttcagtttctatgttcggttcattagaaggaggagctggagcttctgatattaagttagcaacagaaatgtctgctgcttgggtttcattcgctgttcacggtgttcccgatcaaggtacaaaatcacattggccacgttttgaacgtagaggagaaattatg tgt ttcggatcacaagttggttctggtgagggt tgt ggagtttctccttctaaagcatgtcaaccttcaaaa

[0034] 根据本发明具体实施方式的伏马毒素降解酶突变体T470C / L481C / T188C / A446C,其氨基酸序列如SEQ ID NO:9所示。

[0035] SEQ ID NO:9:

[0036] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGSGGSGAVPYYDGSALAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRV C VFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISALRRADAGELTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDR LPVKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAAVARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSF C VHGVPDQGTKSHWPRFERRGEIM C FGSQVGSGEG C GVSPSKACQPSK

[0037] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T470C / L481C / T188C / A446C, the genomic sequence of which is shown in SEQ ID NO: 10

[0038] SEQ ID NO: 10

[0039] caaacagatgatccaaaattagttagacatacacaatctggtgcagttgaaggagttgaaggtgatgttgaaacttttttaggtattccttttgctgctcctccagtaggtgacttacgttggcgtccaccagcaccacctcgtgcttgggctggtacaagagatggtcgtcgttttgctcccgattgcattggaaacgagcgtcttcgtgaaggttcacgtgctgctggtacatcagaggactgtttatatttaaatatttggtcaccaaaacaagttggaaaaggtggtttaccagttatgatttgggtttacggaggaggtttctctggtggttctggtgctgttccttactacgacggatctgcacttgctcagaagggagttgttgttgttacatttaattatcgtgctggtattttaggatttttagcacatccagctttatctaaggaatcacctaatggagtatctggtaattatggacttttagatatgttagctgcttttaagtgggttcaaaataatatacgtgaatttggaggagatccaaaccgtgta tgtgttttcggtgagtcagctggtgcttcagcattaggattattattaacatcaccactttcagaatctgcatttaatcaagctattcttcaatctcccggtcttgcacgtccacttgctacactttcagagtcagaagcaaatggtttagaacttggtgctgacatttctgctcttcgtcgtgctgatgctggagaattaactaaaattgctcaatcccgtattcctatgtctcgtcaatttacaaaaccacgtccaatgggacctattttagatggatatgttttacgtactcttgatgttgatgcatttgcaaaaggagcatttagaaaaattccagttcttgttggtggaaatgctgacgagggtcgtgcatttactgatcgtcttccagttaaaactgttttagaatatcgtgcatatttaacagaacaatttggagacgaagctgacgcttgggaacgttgttatcccgctaattcagatgctgacgttcccgctgctgttgcacgtttattcggagattctcaatttaataatggtattgaattattatccgctgcttttgcaaaatggcgtacaccattatggcgttatcgtttcactggtattcccggtgctggtagacgtcccgctacacatggtgatgaaattccttatgttttcgcaaatttaggtccatcttcagtttctatgttcggttcattagaaggaggagctggagcttctgatattaagttagcaacagaaatgtctgctgcttgggtttcattc tgt gttcacggtgttcccgatcaaggtacaaaatcacattggccacgttttgaacgtagaggagaaattatg tgt ttcggatcacaagttggttctggtgagggt tgt ggagtttctccttctaaagcatgtcaaccttcaaaa

[0040] The present invention also provides a recombinant vector comprising the above-mentioned fumonisin degrading enzyme mutant encoding gene, wherein the fumonisin degrading enzyme mutant encoding gene of the present invention is inserted between appropriate restriction enzyme cutting sites of the expression vector, so that its nucleic acid sequence is connected to the expression regulatory sequence to express the mutant protein.

[0041] The method for improving the thermal stability of fumonisin degrading enzyme according to the present invention comprises the following steps:

[0042] The amino acid residues at positions 470 and 481 of the equine toxin degrading enzyme with the amino acid sequence shown in SEQ ID NO: 11 are mutated from glutamine (T) to cysteine ​​(C) and leucine (L) to cysteine ​​(C), respectively.

[0043] According to the method for improving the thermal stability of fumonisin degrading enzyme of the present invention, when the amino acid sequence of the fumonisin degrading enzyme shown in SEQ ID NO: 11 is subjected to T470C / L481C mutation, the method further includes the step of performing P44C / A168C, F59C / A115C, L140C / A258C, or T188C / A446C mutation.

[0044] The amino acid sequence of the fumonisin degrading enzyme FumD according to a specific embodiment of the present invention is shown in SEQ ID NO: 11.

[0045] QTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSGAVPYYDGSA LAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISAL RRADAGELTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDRLPVKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAAVARLFGDSQFNNGI ELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIMTFGSQVGSGEGLGVSPSKACQPSK

[0046] The present invention provides a gene encoding the fumonisin degrading enzyme FumD. The genomic sequence of the gene is shown in SEQ ID NO: 12.

[0047]

[0048] The present application also provides a recombinant strain comprising the gene encoding the above-mentioned fumonisin-degrading enzyme mutants T470C / L481C, T470C / L481C / P44C / A168C, T470C / L481C / F59C / A115C, T470C / L481C / L140C / A258C, and T470C / L481C / T188C / A446C, preferably the strain is Escherichia coli.

[0049] The present application also provides a method for preparing fumonisin-degrading enzyme, comprising the following steps:

[0050] (1) transforming a host cell with a recombinant expression vector carrying a fumonisin-degrading enzyme mutant gene to obtain a recombinant strain;

[0051] (2) culturing the recombinant strain to the logarithmic growth phase and expressing the fumonisin-degrading enzyme under suitable conditions;

[0052] (3) isolating and purifying the fumonisin-degrading enzyme.

[0053] Preferably, the host cell is Escherichia coli competent cell, and preferably the Escherichia coli cell body is BL21(DE3).

[0054] The present application also provides the application of the above-mentioned fumonisin-degrading enzyme mutants, especially in the degradation of fumonisin.

[0055] The fumonisin-degrading enzyme mutants T470C / L481C, T470C / L481C / P44C / A168C, T470C / L481C / F59C / A115C, T470C / L481C / L140C / A258C, and T470C / L481C / T188C / A446C of the present application have higher thermal stability than fumonisin-degrading enzyme FumD. The method of the present application can obtain fumonisin-degrading enzyme mutants with excellent properties, which can be applied in the industries of agriculture, feed and food, etc., to reduce the harm of fumonisin to animal and human health. BRIEF DESCRIPTION OF DRAWINGS

[0056] 图1The protein electrophoresis diagram of fumonisin degrading enzyme mutants, where M is Marker; 1 is wild-type fumonisin degrading enzyme FumD; 2 is fumonisin degrading enzyme mutant T470C / L481C; 3 is fumonisin degrading enzyme mutant T470C / L481C / P44C / A168C; 4 is fumonisin degrading enzyme mutant T470C / L481C / F59C / A115C; 5 is fumonisin degrading enzyme mutant T470C / L481C / L140C / A258C; 6 is fumonisin degrading enzyme mutant T470C / L481C / T188C / A446C;

[0057] 图2 Showing the activity of fumonisin-degrading enzyme mutants.

[0058] 图3 Showing the thermostability of fumonisin-degrading enzyme mutants. DETAILED DESCRIPTION

[0059] The experimental materials and reagents involved in the following examples are:

[0060] 1. Strains and vectors: Escherichia coli expression vector pET28a(+) and strain BL21(DE3);

[0061] 2. Culture medium: Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

[0062] Example 1 Preparation of recombinant fumonisin degrading enzyme mutants T470C / L481C, T470C / L481C / P44C / A168C, T470C / L481C / F59C / A115C, T470C / L481C / L140C / A258C, and T470C / L481C / T188C / A446C.

[0063] Using fumonisin-degrading enzyme FumD as a template, the plasmids pET28a(+)-FumD-T470C / L481C, pET28a(+)-FumD-T470C / L481C / P44C / A168C, pET28a(+)-FumD-T470C / L481C / F59C / A115C, pET28a(+)-FumD-T470C / L481C / L140C / A258C, and pET28a(+)-FumD-T470C / L481C / T188C / A446C, which express fumonisin-degrading enzyme mutants, were obtained by site-directed mutagenesis. The recombinant Escherichia coli strains BL21(DE3) / FumD-T470C / L481C, BL21(DE3) / FumD-T470C / L481C / P44C / A168C, BL21(DE3) / FumD-T470C / L481C / F59C / A115C, BL21(DE3) / FumD-T470C / L481C / L140C / A258C, and BL21(DE3) / FumD-T470C / L481C / T188C / A446C were obtained.

[0064] The BL21 (DE3) strain carrying the recombinant plasmid was activated overnight and then inoculated into 100 mL of LB liquid medium. The culture was shaken at 37°C and 220 rpm until the bacterial concentration reached OD 600 When the pH value reached about 0.6, IPTG inducer was added with a final concentration of 0.5 mM, and protein expression was induced at 25°C for 20 h. After induction, the cells were collected by centrifugation at 4°C. The supernatant was collected by ultrasonic disruption and the target protein was purified by nickel column. SDS-PAGE results showed that the recombinant fumonisin degrading enzyme was expressed in E. coli. 图1 As shown, lane 1: fumonisin degrading enzyme wild type FumD; lane 2: fumonisin degrading enzyme mutant T470C / L481C; lane 3: fumonisin degrading enzyme mutant T470C / L481C / P44C / A168C; lane 4: fumonisin degrading enzyme mutant T470C / L481C / F59C / A115C; lane 5: fumonisin degrading enzyme mutant T470C / L481C / L140C / A258C; lane 6: fumonisin degrading enzyme mutant T470C / L481C / T188C / A446C;

[0065] Example 2 Determination of FB1 activity by fumonisin degrading enzyme mutation

[0066] 2.1 The enzymatic activity of the fumonisin-degrading enzyme mutant was detected by high performance liquid chromatography. The specific method is as follows:

[0067] (1) FB1 standard stock solution: Weigh an appropriate amount of FB1 standard product and dissolve it in 50% acetonitrile water to prepare a 50 μg / mL FB1 standard solution and store at -4°C.

[0068] (2) Sample preparation: 45 μL of fumonisin-degrading enzyme purified by nickel column was mixed with 5 μL of FB1 standard stock solution, and reacted in a dark water bath at 37°C for 20 min. After the reaction, the reaction was terminated by inactivation at 100°C for 10 min.

[0069] (3) Sample Derivatization: Add 200 μL of 50% acetonitrile water and 250 μL of P-A derivatization solution to the reaction mixture to a final concentration of 10 μg / mL of FB1. Mix thoroughly, filter through a 0.22 μm filter, and analyze by HPLC after 5 minutes. The activity of the fumonisin-degrading enzyme mutant was determined by comparing the peak profile with that of the FB1 standard. 图2 As shown, the activity of the fumonisin degrading enzyme mutant was almost the same as that of the wild-type FumD, and the mutant did not show any decrease in activity.

[0070] 2.2 Determination of the thermal stability of fumonisin-degrading enzyme mutants

[0071] Take appropriate amounts of wild-type and mutant fumonisin-degrading enzymes and treat them at 45 and 55°C for 0 minutes, 30 minutes, and 60 minutes, respectively. After treatment, the enzyme activity was detected in the same manner as in Example 2.1. Three parallel experiments were performed for each temperature gradient. 图3 As shown, the stability of the fumonisin degrading enzyme mutants was significantly improved compared with the wild type when treated at 45 and 55°C for one hour, among which T470C / L481C was about 2.5 times higher than the wild type, the stability of T470C / L481C / P44C / A168C and T470C / L481C / F59C / A115C was 20% higher than that of T470C / L481C, and the stability of T470C / L481C / L140C / A258C and T470C / L481C / T188C / A446C was 10% higher than that of T470C / L481C.

[0072] (1) Fumonisin degradation rate: expressed as the ratio of the peak area m0 of the hydrolyzed fumonisin HFB1 to the peak area m of the fumonisin FB1 standard;

[0073]

[0074] (2) Relative activity: The degradation rate of the mutant w o It is expressed as a ratio to the wild-type degradation rate w;

[0075]

[0076] 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 fumonisin-degrading enzyme mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ NO:

1.

2. A fumonisin-degrading enzyme mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO:

9.

3. A gene encoding a fumonisin-degrading enzyme, characterized in that: The gene encodes the fumonisin-degrading enzyme mutant with improved thermal stability according to claim 1 or 2.

4. The gene encoding the fumonisin degrading enzyme according to claim 3, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:

10.

5. A recombinant vector comprising the gene encoding the fumonisin degrading enzyme according to claim 3. A recombinant strain comprising the gene encoding the fumonisin degrading enzyme according to claim 3 .

7. Use of the fumonisin-degrading enzyme mutant with improved thermal stability according to claim 1 or 2 for degrading fumonisin.

8. A method for improving the thermal stability of fumonisin-degrading enzyme FumD, characterized in that: The steps include: The fumonisin degrading enzyme FumD shown in SEQ ID NO: 11 is mutated, and the amino acid sequence after the mutation is shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

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