Fumonisins degrading enzyme mutant with improved specific activity and thermal stability as well as coding gene and application of fumonisins degrading enzyme mutant

Abstract

The invention belongs to the technical field of protein engineering modification, and particularly relates to a fumonisins degrading enzyme mutant with improved specific activity and thermal stability and application. According to the mutants of the fumonisins degrading enzyme FumDSB, which are provided by the invention, the relative activity and the thermal stability of the mutants A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K are obviously improved compared with those of a wild type fumonisins degrading enzyme. The enzyme is mainly applied to the industries of agricultural products, animal feed, food and the like so as to reduce the harm of fumonisins to the health of animals and human beings. The obtained fumonisins mutant with improved activity and thermal stability is beneficial to reducing the cost of the enzyme in industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a fumonisin degrading enzyme mutant with improved specific activity and thermal stability and an application thereof. Background Art

[0002] Fumonisin is a water-soluble mycotoxin produced by Fusarium verticillium. Among the many fumonisins discovered so far, fumonisin B1 (FB1) is the main component. FB1 contaminates a variety of crops such as corn, wheat, soybeans and their related feeds. Long-term contact or ingestion of this mycotoxin will cause harm to human health.

[0003] Studies have shown that fumonisin B1 can cause neurological dysfunction in horses, pulmonary edema in piglets, damage to reproductive organ structure and function, and disruption of the normal function of the immune system. In order to cope with the threat of fumonisin, a variety of methods for eliminating fumonisin have been developed. Traditional physical and chemical methods have limitations such as high conditions and incomplete toxin removal, while the bio-enzyme method has the advantages of high removal efficiency, no pollution residue, and little damage to feed nutrition.

[0004] The pollution of mycotoxins is getting worse all over the world, causing huge losses to agriculture and animal husbandry. The bio-enzyme method can effectively curb the pollution and recover certain economic losses. Summary of the invention

[0005] In order to utilize the biological enzyme method to degrade fumonisin, the present invention is proposed and completed.

[0006] The object of the present invention is to provide a fumonisin degrading enzyme mutant with improved specific activity and thermal stability.

[0007] Another object of the present invention is to provide a fumonisin degrading enzyme gene with improved specific activity and thermal stability.

[0008] Another object of the present invention is to provide a recombinant expression vector comprising the above gene.

[0009] Another object of the present invention is to provide a recombinant strain comprising the above gene.

[0010] Another object of the present invention is to provide a method for preparing fumonisin-degrading enzyme.

[0011] Another object of the present invention is to provide a method for improving the specific activity and thermal stability of fumonisin degrading enzyme.

[0012] According to a specific embodiment of the present invention, the amino acid sequence of the fumonisin degrading enzyme mutant A373R is shown in SEQ ID NO:1.

[0013] SEQ ID NO: 1:

[0014] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTL FGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

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

[0016] SEQ ID NO:2

[0017] cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0018] According to the fumonisin-degrading enzyme mutant A373R / S248P of the specific embodiment of the present invention, its amino acid sequence is as shown in SEQ ID NO:3.

[0019] SEQ ID NO:3:

[0020] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLET P TLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA RTGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0021] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant A373R / S248P, the genomic sequence of which is shown in 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 invention has an amino acid sequence as shown in SEQ ID NO:5.

[0025] SEQ ID NO:5:

[0026] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVV SVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPP P ALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

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

[0028] SEQ ID NO:6

[0029] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccg ccggctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttcggtgaacagctgcgtgcagaactgggcgatttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0030] According to the fumonisin degrading enzyme mutant A373R / T282D of the specific embodiment of the present invention, its amino acid sequence is as shown in SEQ ID NO:7.

[0031] SEQ ID NO:7:

[0032] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGF LAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPR D PFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0033] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant A373R / T282D, the genomic sequence of which is shown in 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 invention has an amino acid sequence as shown in SEQ ID NO:9.

[0037] SEQ ID NO:9:

[0038] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGL LDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANF A EQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

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

[0040] SEQ ID NO:10

[0041] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttc gcagaacagctgcgtgcagaactgggcgatttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgc tactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

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

[0043] SEQ ID NO:11:

[0044] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQI AALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELG P LPPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

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

[0046] SEQ ID NO:12

[0047] ggtccgagtgttgtgaccacagacgctggtcgcttacgtggcgcagtggaaggcgagctggatgtttttcgtggcgtgccttttgcagcagcccctattggccagctgcgttggcgtgagccgcagcgcattgcaccgtggaccgatattcgtgatgccagcaaatttgccccggcatgcatgcagagtggtgtgagcattcccggtgaaccggcaccgcagattagcgaagactgtttatatttaaacatttgggcaccgcgtcacagcggccgtgccaaactgccggtgatgatctttatccatggcggcggttggcagaatggtgccaccgcactgccgctgtattggggtgatcgtctggcacagcaaggtgcagttgtggtgagcgtgagctatcgtctgggcgcactgggctttctggcacacccggaactgacagccgaaagcccgcaccataccagcggcaactatggtttactggatcagatcgccgctttaaattgggtgcagcgtaacatcgcagcctttggcggcgatccggccaatgttactttattcggtcaaagcgccggcagcagcagcattgccattctgatggccagcccgctggcaaagggtttatttcatcgcgtgatcggtcagagcggtggcttcttcgaaccgctgcagctggccccgcattacgaactggccttagcagagaaacaaggtgtggcctttgcccactctttagagaccagtaccttagccgatttacgcagcttaccgccgcaagctctgctgaccaaacaagcagcaagcgtgagccatccggttatcgaaccgtggctgctgccgcgcacccctttcgaagttttcagtgtgggccaacagcacggtgcagacattttagtgggctataacgcagaagagggccgtgccttttttgatgccagcagtgtgaccgcagccaatttcggtgaacagctgcgtgcagaactgggc ccgttaccgccggccatcatggccgcctatccgtttgcaagcgacgtggaggctggtcaagcacgcgtggctttagaacgcgatctgcgtttcggctggaatatgtggacttgggccaaactgcatgcc cgt accggtaaaaacgccgtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgc tactgggtgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0048] The amino acid sequence of the fumonisin degrading enzyme mutant A373R / A378K according to a specific embodiment of the present invention is shown in SEQ ID NO:13.

[0049] SEQ ID NO:13:

[0050] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTL FGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHA R TGKN K VHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

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

[0052] SEQ ID NO:14

[0053] cgt accggtaaaaac a aa gtgcatgcatactacttcacccacaaaccgccgttccctagcgacagtgttcgccgcaattggcaagctagtcattttgcagaactgtggtatatgtttgatcatttaggccaagaagattggcagtggaccaaattcgatcgtcagattgcccgtaccatgagtcgctactggg tgaactttgcacgcaacggtaacccgaatggtcgcggcttacctcattggccggcatatcgtaccgaccaaccgctggttttacagatcggccagccgattacaccgaccccggaaccgaataccggcagcattggcgtgattgatgcagtgtttagcgcagttcgcggcgacacc

[0054] The present invention also provides a recombinant vector comprising the above-mentioned fumonisin degrading enzyme mutant encoding gene, which is inserted between appropriate restriction enzyme cutting sites of the expression vector to connect its nucleic acid sequence with the expression regulatory sequence to express the mutant protein.

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

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

[0057] According to the method for improving the specific activity and 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: 3 is subjected to A373R mutation, the method further comprises the steps of performing S248P, Q259P, T282D, G321A, D330P and A378K mutations.

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

[0059] GPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQ QGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLET STLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLH AATGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

[0060] The present invention provides a gene encoding the fumonisin degrading enzyme FumDSB. The genome sequence of the gene is shown in SEQ ID NO:16.

[0061]

[0062] The present invention also provides a recombinant strain comprising the above-mentioned fumonisin degrading enzyme mutants A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K encoding genes, preferably the strain is Escherichia coli.

[0063] The present invention also provides a method for preparing a 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 appropriate conditions;

[0066] (3) Isolate and purify the fumonisin-degrading enzyme.

[0067] Among them, it is preferred that the host cell is an Escherichia coli competent cell, and the Escherichia coli cell is preferably BL21 (DE3).

[0068] The present invention also provides the application of the fumonisin degrading enzyme mutant, especially in the application of degrading fumonisin.

[0069] Compared with the fumonisin degrading enzyme FumDSB, the fumonisin degrading enzyme mutants A373R, A373R / S248P, A373R / Q259P, A373R / T282D, A373R / G321A, A373R / D330P and A373R / A378K of the present invention have higher relative activity and thermal stability than the fumonisin degrading enzyme FumDSB. The method of the present invention can be used to obtain fumonisin degrading enzyme mutants with excellent properties, and the enzyme can be applied to agriculture, feed, food and other industries to reduce the harm of fumonisin to animal and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The protein electrophoresis diagram 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 The activities of the fumonisin-degrading enzyme mutants were shown.

[0072] Figure 3 Showing the thermal stability 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 PCR method was used to obtain 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. Transformed into Escherichia coli BL21(DE3) to obtain 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.

[0078] Take the BL21 (DE3) strain containing the recombinant plasmid and inoculate it into 100 mL LB culture medium. After shaking culture at 37°C and 220 rpm for 2-3 hours, the OD 600 When the pH value was 0.6-0.8, IPTG was added at a final concentration of 0.5 mM and the cells were 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 purified by nickel column. The results of SDS-PAGE showed that the recombinant fumonisin degrading enzyme was expressed in Escherichia coli. Figure 1 As shown, lane 1: fumonisin degrading enzyme wild type WT; lane 2: fumonisin degrading enzyme mutant A373R; lane 3: fumonisin degrading enzyme mutant A373R / S248P; lane 4: fumonisin degrading enzyme mutant A373R / Q259P; lane 5: fumonisin degrading enzyme mutant A373R / T282D; lane 6: fumonisin degrading enzyme mutant A373R / G321A; lane 7: fumonisin degrading enzyme mutant A373R / D330P; lane 8: fumonisin degrading enzyme mutant A373R / A378K; Example 2 Determination of FB1 activity of fumonisin degrading enzyme mutants

[0079] 2.1 High performance liquid chromatography was used to detect the enzymatic activity of the fumonisin degrading enzyme mutant enzyme. The specific method is as follows:

[0080] (1) FB1 standard stock solution: weigh 1 mg of the standard product and dissolve it in 20 mL of acetonitrile and water (1:1) to prepare a standard solution with a concentration of 50 μg / mL and store at -4°C.

[0081] (2) Sample preparation: Take 45 μL of the purified fumonisin degrading enzyme mutant enzyme solution, add 5 μL of FB1 standard stock solution to make the final concentration of FB1 5 μg / mL, and react at 37°C in a dark water bath for 20 min;

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

[0083] Table 1

[0084]

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

[0086] Take appropriate amount of wild-type and mutant fumonisin degrading enzyme and treat them at 45 and 55℃ for 0 minutes, 30 minutes and 60 minutes respectively. After the treatment, take 5uL of FB1 as substrate and add 45uL of treated enzyme solution, react for 20min in citric acid-disodium hydrogen phosphate buffer (pH7.0) buffer system, and then boil for 10 min to inactivate the enzyme. After cooling to room temperature, pass through a membrane and perform high performance liquid chromatography detection. Three parallel experiments were performed for each temperature gradient. Figure 3 As shown, the fumonisin degrading enzyme mutants still retained about 60% and 50% of their activities after being treated at 45 and 55°C for one hour, while the wild type only retained less than 20% and 10% of its activities under the same conditions.

[0087] (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;

[0088]

[0089] (2) Wild-type relative activity: mutant degradation rate w o It is expressed as a ratio to the wild-type degradation rate w;

[0090]

[0091] 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 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 as claimed in claim 1 or 2.

4. The fumonisin degrading enzyme gene according to claim 3, characterized in that: The nucleotide sequence of the gene needs to 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 as claimed in claim 1 or 2.

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

9. 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 equine toxin degrading enzyme with an amino acid sequence as shown in SEQ ID NO:

15.

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

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