Fumonisin-degrading enzyme mutant with improved specific activity and thermal stability, encoding gene and application thereof

Abstract

The application belongs to the technical field of protein engineering, and particularly relates to a fumonisin-degrading enzyme mutant with improved specific activity and thermal stability and application. The fumonisin-degrading enzyme FumDSB mutant A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K of the application show significantly improved relative activity and thermal stability compared with wild-type fumonisin-degrading enzyme. The enzyme is mainly applied to the industries of agricultural products, animal feed, food and the like to reduce the health hazards of fumonisin to animals and humans. The fumonisin mutant with improved activity and thermal stability obtained by the application is conducive to reducing the cost of the enzyme in industrial application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein engineering, and particularly relates to a fumonisin-degrading enzyme mutant with improved specific activity and thermal stability and application thereof. BACKGROUND

[0002] Fumonisin is a water-soluble mycotoxin produced by Fusarium verticillioides. Among the many fumonisins discovered so far, fumonisin B1 (FB1) accounts for the main component. FB1 contaminates many crops such as corn, wheat, soybean and related feed, and long-term exposure or ingestion of the mycotoxin will cause harm to the health of the body.

[0003] Studies have shown that fumonisin B1 can cause nerve dysfunction in horses, lung edema in piglets, damage to the structure and function of reproductive organs, and damage to the normal function of the immune system. In order to cope with the threat of fumonisin, many methods have been developed to eliminate fumonisin. Traditional physical and chemical methods have the limitations of high condition requirements and incomplete toxin removal, while the biological enzyme method has the advantages of high removal efficiency, no pollution residue, and little damage to feed nutrition.

[0004] Fungal toxin pollution is increasing globally, causing huge losses to global agriculture and animal husbandry. The biological enzyme method can effectively control pollution and recover some economic losses. SUMMARY

[0005] In order to be able to use the biological enzyme method to degrade fumonisin, the present application is proposed and completed.

[0006] The present application aims to provide a fumonisin-degrading enzyme mutant with improved specific activity and thermal stability.

[0007] The present application further aims to provide a fumonisin-degrading enzyme gene with improved specific activity and thermal stability.

[0008] The present application further aims to provide a recombinant expression vector comprising the above-mentioned gene.

[0009] The present application further aims to provide a recombinant strain comprising the above-mentioned gene.

[0010] The present application further aims to provide a method for preparing a fumonisin-degrading enzyme.

[0011] The present application further aims to provide a method for improving the specific activity and thermal stability of a fumonisin-degrading enzyme.

[0012] The fumonisin-degrading enzyme mutant A373R according to the specific embodiment of the present application has an amino acid sequence as shown in SEQ ID NO: 1.

[0013] SEQ ID NO: 1:

[0014] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0015] The present application provides a gene encoding the above fumonisin-degrading enzyme mutant A373R, the genomic sequence of the gene is shown as SEQ ID NO: 2

[0016] SEQ ID NO: 2

[0017] cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0018] The fumonisin-degrading enzyme mutant A373R / S248P according to the specific embodiment of the present application has an amino acid sequence as shown in SEQ ID NO: 3.

[0019] SEQ ID NO: 3:

[0020] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLET P TLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA RTGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0021] The present application provides a gene encoding the above-mentioned fumonisin-degrading enzyme mutant A373R / S248P, the genomic sequence of which is shown as SEQ ID NO: 4

[0022] SEQ ID NO: 4

[0023] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagacc ccgaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttcggtgaacagctgcgtgcagaactgggcgatttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0024] The fumonisin-degrading enzyme mutant A373R / Q259P according to the specific embodiment of the present application has an amino acid sequence as shown in SEQ ID NO: 5.

[0025] SEQ ID NO: 5:

[0026] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPP P ALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0027] The present application provides a gene encoding the above fumonisin-degrading enzyme mutant A373R / Q259P, the genomic sequence of the gene is shown as SEQ ID NO: 6

[0028] SEQ ID NO: 6

[0029] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccg ccgGCTCTGCTGACCAAACAAGCAGCAAGCGTGAGCCATCCGGTTATCGAACCGTGGCTGCTGCCGC GCACCCCTTTCGAAGTTTTCA GTGTGGGCCAACAGCACGGTGCAGACATTTTAGTGGGCTATAACGC AGAAGAGGGCCGTGCCTTTTTTGATGCCAGCAGTGTGACC GCACCAATTTCGGTGAACAGCTGC GTGCAGA ACTGGGCGATTTACC GCCGGCCATCATGGCCGCCTATCCGTTTGCAAGCGACGTGGAGGCTGGTCAAGCACGC GTGGCTTTAGAACGCATCTGC GTTTCGGCTGGAAATATGTGGACTTGGGCCAAACTGCATGCC cgt ACCGGTA AAAACGCCGTGCATGCATACTACTTCACCCACAAACC GCCGTTCCCTAGCGACAGTGTTCGCCGCAATTGGCAAGCTAGTCATTTTGCAGA ACTGTGGTATATGTTTGATCATT TAGGCCAAGAAGATTGGCAGTGGACCAAATTCGATCGTCAGATTGCCC GTACCATGAGTCGCTACTGGGTGAACTTTGCACGCAACGGTAACCCGAA TGGTCGC GGCTTACCTCATTGGCCGGCATATCGTACC GACCAACC GCTG GTTTTACAGATC GGCCAGCCGATTACACC GACCCCGGAACC GAATACC GGCAGCATTGGCGTGATTGATGCAGTGT TAGC GCAGTTCGC GGC GACACC

[0030] The fumonisin-degrading enzyme mutant A373R / T282D according to the specific embodiment of the present application has an amino acid sequence as shown in SEQ ID NO: 7.

[0031] SEQ ID NO: 7:

[0032] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPR D PFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0033] The present application provides a gene encoding the above fumonisin-degrading enzyme mutant A373R / T282D, the genomic sequence of the gene is shown as SEQ ID NO: 8

[0034] SEQ ID NO: 8

[0035] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgc gaccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttcggtgaacagctgcgtgcagaactgggcgatttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0036] The fumonisin-degrading enzyme mutant A373R / G321A according to the specific embodiment of the present application has an amino acid sequence as shown in SEQ ID NO: 9.

[0037] SEQ ID NO: 9:

[0038] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANF A EQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0039] The present application provides a gene encoding the above fumonisin-degrading enzyme mutant A373R / G321A, the genomic sequence of the gene is shown as SEQ ID NO: 10

[0040] SEQ ID NO: 10

[0041] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttc gcagaacagctgcgtgcagaactgggcgatttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgc tactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0042] The amino acid sequence of the fumonisin degrading enzyme mutant A373R / D330P according to a specific embodiment of the present invention is shown in SEQ ID NO:11.

[0043] SEQ ID NO: 11:

[0044] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELG P LPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0045] The present application provides a gene encoding the above fumonisin-degrading enzyme mutant A373R / D330P, the genomic sequence of the gene is shown as SEQ ID NO: 12

[0046] SEQ ID NO: 12

[0047] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttcggtgaacagctgcgtgcagaactgggc ccgttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0048] According to the fumonisin-degrading enzyme mutant A373R / A378K of the embodiment of the present application, the amino acid sequence is shown in SEQ ID NO: 13.

[0049] SEQ ID NO: 13:

[0050] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKN K VHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0051] The present application provides a gene encoding the fumonisin-degrading enzyme mutant A373R / A378K described above, the genomic sequence of which is shown as SEQ ID NO: 14

[0052] SEQ ID NO: 14

[0053] cgt accggtaaaaac a aa gtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0054] The present application also provides a recombinant vector comprising the fumonisin-degrading enzyme mutant-encoding gene described above, wherein the fumonisin-degrading enzyme mutant-encoding gene of the present application is inserted between appropriate restriction enzyme sites of an expression vector, and the nucleic acid sequence thereof is linked to an expression control sequence to express the mutant protein.

[0055] The method for improving the specific activity and thermal stability of a fumonisin-degrading enzyme according to the present application comprises the following steps:

[0056] The amino acid residue at position 373 of the fumonisin-degrading enzyme having the amino acid sequence shown in SEQ ID NO: 15 is mutated from alanine (A) to arginine (R).

[0057] The method for improving the specific activity and thermal stability of a fumonisin-degrading enzyme according to the present application, when the fumonisin-degrading enzyme having the amino acid sequence shown in SEQ ID NO: 3 is subjected to the A373R mutation, further comprises the step of subjecting the fumonisin-degrading enzyme to the S248P, Q259P, T282D, G321A, D330P and A378K mutations.

[0058] The fumonisin-degrading enzyme FumDSB according to the specific embodiment of the present application has the amino acid sequence shown in SEQ ID NO: 15.

[0059] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHAATGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0060] The present application provides a gene encoding the above fumonisin-degrading enzyme FumDSB, and the genomic sequence of the gene is shown as SEQ ID NO: 16.

[0061]

[0062] The application also provides a recombinant strain comprising the gene encoding the fumonisin-degrading enzyme mutant A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K, preferably the strain is Escherichia coli.

[0063] The application also provides a method for preparing fumonisin-degrading enzyme, comprising the following steps:

[0064] (1) transforming a host cell with a recombinant expression vector carrying a fumonisin-degrading enzyme mutant gene to obtain a recombinant strain;

[0065] (2) culturing the recombinant strain to the logarithmic growth phase and expressing the fumonisin-degrading enzyme under suitable conditions;

[0066] (3) isolating and purifying the fumonisin-degrading enzyme.

[0067] Preferably, the host cell is an Escherichia coli competent cell, and the Escherichia coli cell body is BL21 (DE3).

[0068] The application also provides the application of the above-mentioned fumonisin-degrading enzyme mutant, especially in the degradation of fumonisin.

[0069] The fumonisin-degrading enzyme mutants A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K of the application have higher relative activity and thermal stability than the fumonisin-degrading enzyme FumDSB. The method of the application can be used to 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

[0070] Figure 1 It is a protein electrophoretogram of fumonisin-degrading enzyme mutants, wherein M: Marker; 1: fumonisin-degrading enzyme wild type WT; 2: fumonisin-degrading enzyme mutant A373R; 3: fumonisin-degrading enzyme mutant A373R / S248P; 4: fumonisin-degrading enzyme mutant A373R / Q259P; 5: fumonisin-degrading enzyme mutant A373R / T282D; 6: fumonisin-degrading enzyme mutant A373R / G321A; 7: fumonisin-degrading enzyme mutant A373R / D330P; 8: fumonisin-degrading enzyme mutant A373R / A378K.

[0071] Figure 2 Showing the activity of fumonisin-degrading enzyme mutants.

[0072] Figure 3 Showing the thermostability of fumonisin-degrading enzyme mutants. DETAILED DESCRIPTION

[0073] The experimental materials and reagents involved in the following examples are:

[0074] 1. Strains and vectors: Escherichia coli expression vector pET28a(+) and strain BL21(DE3);

[0075] 2. Culture medium: Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

[0076] Example 1 Preparation of recombinant fumonisin degrading enzyme mutants A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K.

[0077] Using fumonisin-degrading enzyme FumDSB as a template, the recombinant plasmids pET28a(+)-FumDSB-A373R, pET28a(+)-FumDSB-A373R / S248P, pET28a(+)-FumDSB-A373R / Q259P, pET28a(+)-FumDSB-A373R / T282D, pET28a(+)-FumDSB-A373R / G321A, pET28a(+)-FumDSB-A373R / D330P, and pET28a(+)-FumDSB-A373R / A378K containing fumonisin-degrading enzyme mutants were obtained by PCR. The recombinant Escherichia coli strains BL21(DE3) / FumDSB-A373R, BL21(DE3) / FumDSB-A373R / S248P, BL21(DE3) / FumDSB-A373R / Q259P, BL21(DE3) / FumDSB-A373R / T282D, BL21(DE3) / FumDSB-A373R / G321A, BL21(DE3) / FumDSB-A373R / D330P, and BL21(DE3) / FumDSB-A373R / A378K were obtained.

[0078] The BL21(DE3) strain containing the recombinant plasmid was inoculated into 100 mL of LB culture medium, and cultured at 37°C with 220 rpm shaking for 2-3 h. When the OD 600 When the OD was 0.6-0.8, 0.5 mM IPTG was added to a final concentration, and the culture was induced at 25°C for 20 h. After induction, the bacterial cells were collected by centrifugation at 4°C. The supernatant was collected by ultrasonic disruption, and the recombinant fumonisin-degrading enzyme was expressed in E. coli, as shown in SDS-PAGE results Figure 1

[0079] 2.1 High-performance liquid chromatography was used to detect the enzyme activity of fumonisin-degrading enzyme mutants, and the specific method was as follows:

[0080] (1) FB1 standard stock solution: 1 mg of standard was dissolved in 20 mL of acetonitrile and water (1:1) to prepare a standard solution with a concentration of 50 μg / mL, which was stored at -4°C;

[0081] (2) Sample preparation: 45 μL of purified fumonisin-degrading enzyme mutant enzyme solution was added with 5 μL of FB1 standard stock solution to make the final concentration of FB1 5 μg / mL, and the reaction was carried out at 37°C in the dark for 20 min;

[0082] ​(3) Sample derivatization: Take 50 μL of the sample to be tested, add 200 μL of 50% aqueous acetonitrile, 250 μL of OPA derivatization solution, mix for 30 s, and inject within 2 min of derivatization. The filter membrane is used for testing. The enzyme activity of the fumonisin-degrading enzyme mutant is determined by comparing the peak pattern with that of the FB1 standard. As shown in Table 1, the relative enzyme activity of fumonisin-degrading enzyme FumDSB is 100%, the relative enzyme activity of mutant A373R is 178.26%, the relative enzyme activity of mutant A373R / S248P is 147.28%, the relative enzyme activity of mutant A373R / Q259P is 153.35%, the relative enzyme activity of mutant A373R / T282D is 129.97%, the relative enzyme activity of mutant A373R / G321A is 155.45%, the relative enzyme activity of mutant A373R / D330P is 190.15%, and the relative enzyme activity of mutant A373R / A378K is 130.23%.

[0083] Table 1

[0084]

[0085] 2.2 Determination of thermal stability of fumonisin-degrading enzyme mutants

[0086] An appropriate amount of wild-type and mutant fumonisin-degrading enzyme was treated at 45 and 55°C for 0 min, 30 min, and 60 min, respectively. After treatment, FB1 was used as a substrate, 5 uL of the substrate was taken and added to 45 uL of the treated enzyme solution, and the reaction was carried out in a citric acid-disodium hydrogen phosphate buffer (pH 7.0) buffer system for 20 min, followed by boiling for 10 min to inactivate the enzyme. After cooling to room temperature, the sample was filtered and subjected to high-performance liquid chromatography detection. Three parallel experiments were performed for each temperature gradient. As shown in Table 1, the fumonisin-degrading enzyme mutants still retained about 60% and 50% activity after being treated at 45 and 55°C for one hour, while the wild-type enzyme retained less than 20% and 10% activity under the same conditions. Figure 3

[0087] (1) Fumonisin degradation rate: represented by the ratio of the peak area m0 of hydrolyzed fumonisin HFB1 to the peak area m of fumonisin FB1 standard;

[0088]

[0089] (2) Relative activity of wild type: represented by the ratio of the degradation rate w o of the mutant to the degradation rate w of the wild type;

[0090]

[0091] The above examples are only for understanding the technical solutions of the present application and do not limit the protection scope of the present application.​

Claims

1. A fumonisin-degrading enzyme mutant with improved specific activity and 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 specific activity and 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, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:

13.

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

4. The fumonisin degrading enzyme gene according to claim 3, characterized in that The nucleotide sequence of the gene should be as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO:

14.

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

6. A recombinant strain comprising the fumonisin degrading enzyme gene according to claim 3.

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

8. A method for improving the specific activity and thermal stability of a fumonisin-degrading enzyme, characterized in that: The method comprises the following steps: performing A373R mutation on the fumonisin degrading enzyme with an amino acid sequence as shown in SEQ ID NO:

15.

9. The method for improving the specific activity and thermal stability of fumonisin-degrading enzyme according to claim 8, characterized in that: When the amino acid sequence of the equine toxin degrading enzyme FumDSB is mutated as shown in SEQ ID NO: 15, the method further mutates S248P, Q259P, T282D, G321A, D330P or A378K.

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