Method for improving thermal stability of amine oxidase ACAO, mutant and application

Abstract

The invention belongs to the technical field of protein engineering modification, and particularly relates to a method for improving thermal stability of amine oxidase ACAO and a mutant. Compared with a wild type enzyme, the temperature tolerance of the mutant enzyme in a certain temperature range is superior to that of the wild type enzyme, and the thermal stability is remarkably improved. Through molecular modification on a wild type, the thermal stability of amine oxidase ACAO mutant enzyme is remarkably improved. The enzyme is mainly applied to the industries of food production, livestock feed processing and the like so as to reduce the harm of fumonisins to the health of animals and human beings. The thermal stability improved amine oxidase ACAO mutant obtained by the invention is beneficial to improving the production efficiency of continuous production of enzyme in actual industrial application, has good industrial application prospects, and lays a foundation for further research on the structure of amine oxidase.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and particularly relates to an amine oxidase ACAO mutant with improved thermal stability and its application. Background Art

[0002] Fumonisin B1 (FB1) is a diester compound composed of a polyhydric alcohol and tricarboxylic acid. Its primary active functional groups are primary amines, tricarboxylic acid, hydroxyl groups, and an aliphatic backbone. Studies have shown that fumonisins can cause kidney and liver tumors in rats, leukoencephalomalacia in horses, and pulmonary edema in pigs. It is also closely associated with esophageal cancer in humans. FB1 accounts for approximately 70%-80% of the natural abundance and is the most toxic and widespread.

[0003] Compared with traditional physical and chemical detoxification methods, enzymatic degradation of fumonisin B1 exhibits significant biocatalytic advantages: first, the enzymatic reaction is atom-economical and can achieve specific cleavage of the FB1 amino group under mild conditions, avoiding the loss of nutrients caused by high temperature and high pressure; second, the degradation products are identified as non-cytotoxic products by mass spectrometry, completely eliminating their pathogenic mechanism of inhibiting sphingolipid synthesis; third, the enzyme preparation itself is biodegradable, with no risk of secondary pollution, which is in line with the concept of green manufacturing.

[0004] Amine oxidases (Amine Oxidase) can effectively degrade fumonisins. This enzyme converts fumonisins into non-toxic degradation products through a redox reaction mediated by the flavin adenine dinucleotide prosthetic group. However, the thermal stability deficiencies of naturally derived Amine Oxidase (ACAO) significantly hinder its industrial application. Addressing this bottleneck, enhancing the thermal stability of Amine Oxidase through protein engineering has become a key breakthrough and an effective approach. This improvement in thermal stability opens up potential applications for Amine Oxidase in diverse scenarios: in corn and other grain processing, thermostable Amine Oxidase preparations can withstand drying processes above 80°C, enabling simultaneous degradation of toxins. In the feed industry, the improved enzymes can maintain activity after the transient high temperatures of pelleting, ensuring terminal detoxification before livestock and poultry ingestion. In agriculture, immobilizing Amine Oxidase on nanocarriers could enable the development of UV-resistant and weather-resistant biocontrol agents for direct application to contaminated farmland. Therefore, this invention aims to enhance the thermal stability of Amine Oxidase (ACAO) through protein engineering, extending its shelf life. This could effectively expand the application of Amine Oxidase in mitigating fumonisin contamination in food, feed, and agriculture, thereby alleviating the harmful effects of fumonisin on animals and humans. Summary of the Invention

[0005] The present invention is proposed and completed in order to utilize biodegradation to degrade fumonisin.

[0006] The object of the present invention is to provide an amine oxidase ACAO mutant with improved thermal stability.

[0007] Another object of the present invention is to provide an amine oxidase ACAO mutant gene with improved 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 amine oxidase ACAO mutants.

[0011] Another object of the present invention is to provide a method for improving the thermal stability of amine oxidase ACAO.

[0012] The amino acid sequence of the amine oxidase ACAO mutant S162E according to a specific embodiment of the present invention is shown in SEQ ID NO: 1.

[0013] SEQ ID NO: 1:

[0014] MSITNSFPTPAAPKEVDVIVVGAGLSGLRAALDVQAAGLSCIVVEAVDRVGGKTLSVPSKLS

[0015] GPGVNDIGAAWINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEH

[0016] DQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTAEEYCSKTFQSDLVVGIVDTVTQSL

[0017] LGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALEPGTLFL

[0018] STPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPEDKTRLASDNILGY

[0019] YSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCFIVGKRGREWSLLSS

[0020] SERRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDAS

[0021] AAQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRGAKEVIDLLKG

[0022] The present invention provides a gene encoding the amine oxidase ACAO mutant S162E. The genomic sequence of the gene is shown in SEQ ID NO: 2.

[0023] As shown in SEQ ID NO: 2:

[0024] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttg

[0025] acgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccgg

[0026] agtcaacgacattggtgctgcttggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatccagag

[0027] ggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggtcgc

[0028] ctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgactgc

[0029] cGAGgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctctatct

[0030] ctatgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttagggag

[0031] ggaactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctcag

[0032] acttgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatcag

[0033] tttcgaccctcctcttccagaggacaagactagattggcctctgacaacattttgggttattactctaagatgatttacgtcttcgaccaaccttggtg

[0034] gagaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtcta

[0035] tcacttgttttatcgtcggaaaaaggggtagggagtggagtttgttgtctagtagtgagaggaggagaaccgcttgggatcagtttaggaccacct

[0036] tcgaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcggaggt

[0037] gctccatgcccagtctctccacccggattgcttagttctgttgatgccagtgccgcccaatgccctttcaaggatgtccacttcgtcggaactgaga

[0038] ccgctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggagccaaggaggttatcgatttgcttaagggt

[0039] The amino acid sequence of the amine oxidase ACAO mutant S162E-D33C / A472C according to a specific embodiment of the present invention is shown in SEQ ID NO: 3.

[0040] As shown in SEQ ID NO: 3:

[0041] MSITNSFPTPAAPKEVDVIVVGAGLSGLRAAL C VQAAGLSCIVVEAVDRVGGKTLSVPSKLS

[0042] GPGVNDIGAAWINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEH

[0043] DQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTA E EYCSKTFQSDLVVGIVDTVTQSL

[0044] LGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALEPGTLFL

[0045] STPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPEDKTRLASDNILGY

[0046] YSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCFIVGKRGREWSLLSS

[0047] SERRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDAS

[0048] AAQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRG C KEVIDLLKG

[0049] The present invention provides a gene encoding the above-mentioned amine oxidase ACAO mutant S162E-D33C / A472C, and the nucleotide sequence of this gene is as shown in SEQ ID NO:4.

[0050] SEQ ID NO:4:

[0051] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttT

[0052] GTgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccg

[0053] gagtcaacgacattggtgctgcttggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatccaga

[0054] gggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggtcg

[0055] cctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgactg

[0056] ccGAGgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctctatc

[0057] tctatgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttaggga

[0058] gggaactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctca

[0059] gacttgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatca

[0060] gtttcgaccctcctcttccagaggacaagactagattggcctctgacaacattttgggttattactctaagatgatttacgtcttcgaccaaccttggt

[0061] ggagaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtct

[0062] atcacttgttttatcgtcggaaaaaggggtagggagtggagtttgttgtctagtagtgagaggaggagaaccgcttgggatcagtttaggaccacc

[0063] ttcgaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcggaggt

[0064] gctccatgcccagtctctccacccggattgcttagttctgttgatgccagtgccgcccaatgccctttcaaggatgtccacttcgtcggaactgaga

[0065] ccgctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggaTGTaaggaggttatcgatttgcttaagggt

[0066] According to a specific embodiment of the present invention, the amino acid sequence of the amine oxidase ACAO mutant S162E-D68C / A72C is shown in SEQ ID NO:5.

[0067] SEQ ID NO: 5:

[0068] MSITNSFPTPAAPKEVDVIVVGAGLSGLRAALDVQAAGLSCIVVEAVDRVGGKTLSVPSKLS

[0069] GPGVN C IGA C WINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEH

[0070] DQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTA E EYCSKTFQSDLVVGIVDTVTQSL

[0071] LGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALEPGTLFL

[0072] STPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPEDKTRLASDNILGY

[0073] YSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCFIVGKRGREWSLLSS

[0074] SERRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDAS

[0075] AAQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRGAKEVIDLLKG

[0076] The present invention provides a gene encoding the amine oxidase ACAO mutant S162E-D68C / A72C, the genomic sequence of which is shown in SEQ ID NO:6.

[0077] SEQ ID NO: 6:

[0078] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttg

[0079] acgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccgg

[0080] agtcaacTGTattggtgctTGTtggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatcca

[0081] gagggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggt

[0082] cgcctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgac

[0083] tgccGAGgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctcta

[0084] tctctatgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttaggg

[0085] agggaactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctc

[0086] agacttgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatc

[0087] agtttcgaccctcctcttccagaggacaagactagattggcctctgacaacattttgggttattactctaagatgatttacgtcttcgaccaaccttggt

[0088] ggagaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtct

[0089] atcacttgttttatcgtcggaaaaaggggtagggagtggagtttgttgtctagtagtgagaggaggagaaccgcttgggatcagtttaggaccacc

[0090] ttcgaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcggaggt

[0091] gctccatgcccagtctctccacccggattgcttagttctgttgatgccagtgccgcccaatgccctttcaaggatgtccacttcgtcggaactgaga

[0092] ccgctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggagccaaggaggttatcgatttgcttaagggt

[0093] According to the amine oxidase ACAO mutant S162E-D300C / A438C of the specific embodiment of the present invention, its amino acid sequence is as shown in SEQ ID NO:7.

[0094] [[ID=X]]SEQ ID NO:7:

[0095] MSITNSFPTPAAPKEVDVIVVGAGLSGLRAALDVQAAGLSCIVVEAVDRVGGKTLSVPSKLS

[0096] GPGVNDIGAAWINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEH

[0097] DQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTA E EYCSKTFQSDLVVGIVDTVTQSL

[0098] LGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALEPGTLFL

[0099] STPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPE C KTRLASDNILGY

[0100] YSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCFIVGKRGREWSLLSS

[0101] SERRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDAS

[0102] C AQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRGAKEVIDLLKG

[0103] The present invention provides a gene encoding the amine oxidase ACAO mutant S162E-D300C / A438C. The genomic sequence of the gene is shown in SEQ ID NO:8.

[0104] As shown in SEQ ID NO: 8:

[0105] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttg

[0106] acgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccgg

[0107] agtcaacgacattggtgctgcttggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatccagag

[0108] ggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggtcgc

[0109] ctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgactgc

[0110] cGAGgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctctatct

[0111] ctatgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttagggag

[0112] ggaactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctcag

[0113] acttgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatcag

[0114] tttcgaccctcctcttccagagTGTaagactagattggcctctgacaacattttgggttattactctaagatgatttacgtcttcgaccaaccttggt

[0115] ggagaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtct

[0116] atcacttgttttatcgtcggaaaaaggggtagggagtggagtttgttgtctagtagtgagaggaggagaaccgcttgggatcagtttaggaccacc

[0117] ttcgaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcggaggt

[0118] gctccatgcccagtctctccacccggattgcttagttctgttgatgccagtTGTgcccaatgccctttcaaggatgtccacttcgtcggaactgag

[0119] accgctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggagccaaggaggttatcgatttgcttaagggt

[0120] According to the amine oxidase ACAO mutant S162E-L372C / E376C of the specific embodiment of the present invention, its amino acid sequence is as shown in SEQ ID NO:9.

[0121] SEQ ID NO:9:

[0122] MSITNSFPTPAAPKEVDVIVVGAGLSGLRAALDVQAAGLSCIVVEAVDRVGGKTLSVPSKLS

[0123] GPGVNDIGAAWINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEH

[0124] DQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTA E EYCSKTFQSDLVVGIVDTVTQSL

[0125] LGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALEPGTLFL

[0126] STPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPEDKTRLASDNILGY

[0127] YSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCFIVGKRGREWSL C SS

[0128] S C RRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDAS

[0129] AAQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRGAKEVIDLLKG

[0130] The present invention provides a gene encoding the amine oxidase ACAO mutant S162E-L372C / E376C, the genomic sequence of which is shown in SEQ ID NO:10.

[0131] SEQ ID NO: 10:

[0132] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttg

[0133] acgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccgg

[0134] agtcaacgacattggtgctgcttggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatccagag

[0135] ggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggtcgc

[0136] ctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgactgc

[0137] cGAGgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctctatct

[0138] ctatgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttagggag

[0139] ggaactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctcag

[0140] acttgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatcag

[0141] tttcgaccctcctcttccagaggacaagactagattggcctctgacaacattttgggttattactctaagatgatttacgtcttcgaccaaccttggtg

[0142] gagaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtcta

[0143] tcacttgttttatcgtcggaaaaaggggtagggagtggagtttgTGTtctagtagtTGTaggaggagaaccgcttgggatcagtttaggacca

[0144] ccttcgaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcgga

[0145] ggtgctccatgcccagtctctccacccggattgcttagttctgttgatgccagtgccgcccaatgccctttcaaggatgtccacttcgtcggaactg

[0146] agaccgctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggagccaaggaggttatcgatttgcttaagggt

[0147] The present invention also provides a recombinant vector comprising the above-mentioned amine oxidase ACAO mutant encoding gene, wherein the amine oxidase ACAO mutant encoding gene of the present invention is inserted between appropriate restriction enzyme cutting sites of the expression vector so that its nucleotide sequence can be operably connected to the expression regulatory sequence.

[0148] The method for improving the thermal stability of amine oxidase ACAO according to the present invention comprises the following steps:

[0149] A single point mutation S162E was performed on the amine oxidase ACAO with the amino acid sequence shown in SEQ ID NO: 11.

[0150] The amino acid residues at positions 33 and 472 of the amine oxidase ACAO shown in the amino acid sequence were mutated from aspartic acid (D) to cysteine ​​(C) and alanine (A) to cysteine ​​(C), respectively.

[0151] According to the method for improving the thermal stability of amine oxidase ACAO of the present invention, when the amine oxidase ACAO with an amino acid sequence as shown in SEQ ID NO: 11 is subjected to S162E, the method further includes the steps of performing S162E-D33C / A472C, S162E-D68C / A72C, S162E-D300C / A438C and S162E-L372C / E376C mutations.

[0152] The amino acid sequence of the amine oxidase ACAO according to a specific embodiment of the present invention is shown in SEQ ID NO: 11. MSITNSFPTPAAPKEVDVIVVGAGLSGLRAALDVQAAGLSCIVVEAVDRVGGKTLSVPSKLSGPGVNDIGAAWINDTSQSEMYGLLRKYGLHGEIQRAEGMSLSLTAEGVITHPYGTLPLDEHDQVLVASVLQTIQGLVDQIDLKNPAAGSIGKELDSMTASEYCSKTFQSDLVVGIVDTVTQSLLGVEAASISMLSLVHYIKAATGVDAALSDGKDGGQYLRVREGTQSFSRKMAEALE PGTLFLSTPVTSIEQSSQTCICTVRTSSPTNSTFHAKKVILSIPTPLYHKISFDPPLPEDKTRLASDNILGYYSKMIYVFDQPWWRTAGLSGVMETDLGPILFSRDTSIPDDDQWSITCF IVGKRGREWSLLSSSERRRTAWDQFRTTTFESASGLSGKLDVPEPINVLEIEWTKQEFFGGAPCPVSPPPGLLSSVDASAAQCPFKDVHFVGTETAFVWRGYMEGAVRSGIRGAKEVIDLLKG

[0153] The present invention provides a gene encoding the amine oxidase ACAO, the genomic sequence of which is shown in SEQ ID NO:12.

[0154] atgtctatcactaactctttcccaaccccagctgccccaaaggaggttgacgttatcgttgtcggagccggattgagtggtttgagagctgcccttg

[0155] acgtccaagctgctggtcttagttgcatcgttgtcgaagctgtcgatagggtcggaggtaaaaccttgtctgtcccatctaagttgtctggtcccgg

[0156] agtcaacgacattggtgctgcttggatcaacgatacctctcagtctgagatgtatggattgttgaggaagtatggacttcacggagagatccagag

[0157] ggccgaaggaatgagtctttctttgaccgccgagggtgtcatcactcatccatacggaaccttgccacttgacgaacacgatcaagttttggtcgc

[0158] ctctgttcttcagaccatccaaggattggtcgatcagatcgatttgaagaacccagccgctggaagtatcggtaaggagcttgactctatgactgc

[0159] cagtgaatattgttctaagactttccagtctgacttggtcgtcggaatcgttgataccgtcactcagtctttgcttggtgtcgaggctgcctctatctcta

[0160] tgctttctcttgtccactatatcaaagccgctaccggtgttgatgccgctttgagtgacggtaaagacggtggtcaatatttgagagttagggaggg

[0161] aactcagagtttcagtaggaagatggctgaggctcttgaacccggaacccttttcttgtctaccccagtcacctctatcgagcagagttctcagact

[0162] tgcatctgcactgttagaaccagtagtccaactaattctacttttcacgctaaaaaagttattctttctatcccaaccccattgtatcacaagatcagttt

[0163] cgaccctcctcttccagaggacaagactagattggcctctgacaacattttgggttatattactctaagatgatttacgtcttcgaccaaccttggtgga

[0164] gaaccgctggtttgtctggagtcatggagaccgaccttggacctatccttttcagtagggacaccagtatcccagacgacgaccagtggtctatc

[0165] acttgttttatcgtcggaaaaaggggtagggagtggagtttgttgtctagtagtgagaggaggagaaccgcttgggatcagtttaggaccaccttc

[0166] gaatctgccagtggtttgagtggaaagttggacgtcccagagcctatcaacgtcttggagatcgagtggaccaagcaagaatttttcggaggtgc

[0167] tccatgcccagtctctccacccggattgcttagttctgttgatgccagtgccgcccaatgccctttcaaggatgtccacttcgtcggaactgagacc

[0168] gctttcgtctggaggggatacatggagggagccgttaggtctggtattagaggagccaaggaggttatcgatttgcttaagggt

[0169] The present invention also provides a recombinant strain comprising the above-mentioned amine oxidase ACAO variants S162E, S162E-D33C / A472C, S162E-D68C / A72C, S162E-D300C / A438C and S162E-L372C / E376C encoding genes, preferably the strain is Escherichia coli.

[0170] The present invention also provides a method for preparing an amine oxidase ACAO mutant, comprising the following steps:

[0171] (1) transforming a host cell with a recombinant vector containing a gene encoding an amine oxidase ACAO mutation to obtain a recombinant strain;

[0172] (2) culturing the recombinant strain to induce expression of the amine oxidase ACAO mutant;

[0173] (3) Isolate and purify the obtained amine oxidase ACAO mutant enzyme.

[0174] Among them, the host cell is preferably a common microbial expression host cell such as a large intestine cell, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae cell or Pseudomonas aeruginosa cell, preferably Escherichia coli BL21 (DE3).

[0175] The present invention also provides applications of the amine oxidase ACAO mutant, especially applications in degrading fumonisin.

[0176] Compared to the thermal stability of amine oxidase ACAO against HFB1, the amine oxidase ACAO mutants S162E, S162E-D33C / A472C, S162E-D68C / A72C, S162E-D300C / A438C, and S162E-L372C / E376C of the present invention all exhibit higher thermal stability than amine oxidase ACAO. The method of the present invention can be used to obtain amine oxidase ACAO mutants with superior properties. Improving the thermal stability of amine oxidase ACAO for HFB1 degradation is intended to better address fumonisin contamination and overcome the limitations of natural enzymes in industrial applications.

[0177] Compared with the wild type, the mutants of the present invention have better temperature tolerance than the wild type enzyme within a certain temperature range, and their thermal stability has been significantly improved. After molecular modification of the wild type, the thermal stability of the amine oxidase ACAO mutant enzymes S162E, S162E-D33C / A472C, S162E-D68C / A72C, S162E-D300C / A438C, and S162E-L372C / E376C was significantly improved. The enzyme is mainly used in industries such as food production and animal feed processing to reduce the health hazards of fumonisins to animals and humans. The amine oxidase ACAO mutants with improved thermal stability obtained by the present invention are conducive to improving the production efficiency of continuous production of enzymes in actual industrial applications, have good industrial application prospects, and lay the foundation for further research on the structure of amine oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1The figure is an SDS-PAGE image of amine oxidase ACAO mutant, wherein M: Marker; lane 1: amine oxidase wild type ACAO; lane 2: amine oxidase ACAO mutant S162E; lane 3: amine oxidase ACAO mutant S162E-D33C / A472C; lane 4: amine oxidase ACAO mutant S162E-D68C / A72C; lane 5: amine oxidase ACAO mutant S162E-D300C / A438C; lane 6: amine oxidase ACAO mutant S162E-L372C / E376C.

[0179] Figure 2 The activity of the amine oxidase ACAO mutant is shown.

[0180] Figure 3 Showing the thermostability of the amine oxidase ACAO mutant. DETAILED DESCRIPTION

[0181] The experimental materials and reagents involved in the following examples are:

[0182] 1. Strains and vectors: Escherichia coli expression vector pET28a(+) and strain BL21(DE3).

[0183] 2. Culture medium: Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). Example 1 Preparation of recombinant amine oxidase ACAO mutant

[0184] Using amine oxidase ACAO as a template, the plasmids pET28a(+)-ACAO-S162E, pET28a(+)-ACAO-S162E-D33C / A472C, pET28a(+)-ACAO-S162E-D68C / A72C, pET28a(+)-ACAO-S162E-D300C / A438C and pET28a(+)-ACAO-S162E-L372C / E376C of amine oxidase ACAO mutants were obtained by site-directed mutagenesis. The recombinant Escherichia coli strains BL21(DE3) / ACAO-S162E, BL21(DE3) / ACAO-S162E-D33C / A472C, BL21(DE3) / ACAO-S162E-D68C / A72C, BL21(DE3) / ACAO-S162E-D300C / A438C and BL21(DE3) / ACAO-S162E-L372C / E376C were obtained.

[0185] 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 0.6-0.8, IPTG inducer was added at a final concentration of 0.5 mM, and protein expression was induced at 30°C for 24 hours. 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 amine oxidase ACAO mutant was expressed in E. coli. Figure 1 As shown, lane 1: amine oxidase wild type ACAO; lane 2: amine oxidase ACAO mutant S162E; lane 3: amine oxidase ACAO mutant S162E-D33C / A472C; lane 4: amine oxidase ACAO mutant S162E-D68C / A72C; lane 5: amine oxidase ACAO mutant S162E-D300C / A438C; lane 6: amine oxidase ACAO mutant S162E-L372C / E376C.

[0186] Example 2 Determination of HFB1 Degradation Activity of Amine Oxidase ACAO Mutant

[0187] 2.1 High performance liquid chromatography was used to detect the enzyme activity of the amine oxidase ACAO mutant enzyme. The specific method is as follows:

[0188] (1) HFB1 standard stock solution: Weigh an appropriate amount of HFB1 standard product and dissolve it in 50% acetonitrile water to prepare a HFB1 standard solution with a concentration of 50 μg / mL. Store at -4°C.

[0189] (2) Sample preparation: 45 μL of amine oxidase purified by nickel column was mixed with 5 μL of HFB1 standard stock solution and reacted in a light-proof water bath at 37°C for 20 min. After the reaction, the mixture was inactivated at 100°C for 10 min to terminate the reaction.

[0190] (3) Sample Derivatization: Add 200 μL of 50% acetonitrile-water and 250 μL of OPA derivatization solution to the reaction mixture to a final HFB1 concentration of 10 μg / mL. Mix thoroughly, filter through a 0.22 μm filter, and analyze by HPLC after 5 minutes. The enzymatic activity of the amine oxidase mutant was determined by comparing the peak profile with that of the HFB1 standard. Figure 2 As shown, the activity of the amine oxidase ACAO mutant was almost the same as that of the wild-type ACAO, and the mutant did not show a decrease in activity.

[0191] 2.2 Determination of the thermal stability of the amine oxidase ACAO mutant

[0192] Take appropriate amounts of wild-type and mutant amine oxidase ACAO and treat them at 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. Figure 3 As shown in the figure, the stability of the amine oxidase ACAO mutants was significantly improved compared with the wild type when treated at 55°C for 60 minutes, among which S162E was 28% higher than the wild type, S162E-D33C / A472C and S162E-D68C / A72C were about 40% more stable than ACAO, and S162E-D300C / A438C and S162E-L372C / E376C were 50% more stable than ACAO.

[0193] (1) Hydrolysis fumonisin degradation rate: expressed as the ratio of the peak area m0 of the sample hydrolyzed fumonisin HFB1 to the peak area m of the standard hydrolyzed fumonisin HFB1;

[0194]

[0195] (2) Relative activity: using the mutant degradation rate w o It is expressed as a ratio to the wild-type degradation rate w;

[0196]

[0197] 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. An amine oxidase ACAO mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ NO:

1.

2. An amine oxidase ACAO 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. An amine oxidase ACAO gene, characterized in that: The gene encodes the amine oxidase ACAO mutant with improved thermal stability as described in claim 1 or 2.

4. The amine oxidase ACAO gene according to claim 3, characterized in that The gene sequence of the mutant 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 amine oxidase ACAO mutant gene according to claim 3.

6. A recombinant strain comprising the amine oxidase ACAO mutant gene according to claim 3.

7. Use of the amine oxidase ACAO mutant with improved thermal stability according to claim 1 or 2.

8. Use of the amine oxidase ACAO mutant with improved thermal stability according to claim 1 or 2 for degrading fumonisin.

9. A method for improving the thermal stability of amine oxidase ACAO, characterized in that: The method comprises the following steps: mutating the amine oxidase ACAO with an amino acid sequence as shown in SEQ ID NO:

11.

10. The method for improving the thermal stability of amine oxidase ACAO according to claim 9, characterized in that: When the amine oxidase ACAO with an amino acid sequence as shown in SEQ ID NO: 11 is mutated, the method further comprises the steps of S162E, S162E-D33C / A472C, S162E-D68C / A72C, S162E-D300C / A438C and S162E-L372C / E376C mutations.

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