CN109907210A - Application of acyl-CoA thioesterase and its gene in degrading zearalenone, preparation for degrading zearalenone - Google Patents
Application of acyl-CoA thioesterase and its gene in degrading zearalenone, preparation for degrading zearalenone Download PDFInfo
- Publication number
- CN109907210A CN109907210A CN201910228804.XA CN201910228804A CN109907210A CN 109907210 A CN109907210 A CN 109907210A CN 201910228804 A CN201910228804 A CN 201910228804A CN 109907210 A CN109907210 A CN 109907210A
- Authority
- CN
- China
- Prior art keywords
- acyl
- amino acid
- coa thioesterase
- seq
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 107
- 101710120269 Acyl-CoA thioester hydrolase YbgC Proteins 0.000 title claims abstract description 94
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 title claims abstract description 92
- 230000000593 degrading effect Effects 0.000 title claims abstract description 66
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 108090000790 Enzymes Proteins 0.000 claims abstract description 85
- 102000004190 Enzymes Human genes 0.000 claims abstract description 84
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 13
- 239000002131 composite material Substances 0.000 claims abstract 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 44
- 238000003259 recombinant expression Methods 0.000 claims description 17
- 238000012217 deletion Methods 0.000 claims description 14
- 230000037430 deletion Effects 0.000 claims description 14
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 238000006467 substitution reaction Methods 0.000 claims description 14
- 125000000539 amino acid group Chemical group 0.000 claims description 10
- 238000005457 optimization Methods 0.000 claims description 8
- 108020004705 Codon Proteins 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims 2
- 241000193744 Bacillus amyloliquefaciens Species 0.000 abstract description 16
- 238000004458 analytical method Methods 0.000 abstract description 8
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 7
- 101150044508 key gene Proteins 0.000 abstract description 6
- 238000012163 sequencing technique Methods 0.000 abstract description 6
- 231100000678 Mycotoxin Toxicity 0.000 abstract description 5
- 230000004071 biological effect Effects 0.000 abstract description 5
- 239000002636 mycotoxin Substances 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 4
- 241000588724 Escherichia coli Species 0.000 abstract description 3
- 238000006065 biodegradation reaction Methods 0.000 abstract description 3
- 238000002965 ELISA Methods 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 17
- 230000015556 catabolic process Effects 0.000 description 16
- 238000006731 degradation reaction Methods 0.000 description 16
- 238000009472 formulation Methods 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 108020002982 thioesterase Proteins 0.000 description 8
- 240000008042 Zea mays Species 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 102000005488 Thioesterase Human genes 0.000 description 5
- 125000002252 acyl group Chemical group 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 150000002561 ketenes Chemical class 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- VCSABYLVNWQYQE-SRVKXCTJSA-N Ala-Lys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O VCSABYLVNWQYQE-SRVKXCTJSA-N 0.000 description 3
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- ASQKVGRCKOFKIU-KZVJFYERSA-N Arg-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ASQKVGRCKOFKIU-KZVJFYERSA-N 0.000 description 3
- DATSKXOXPUAOLK-KKUMJFAQSA-N Asn-Tyr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DATSKXOXPUAOLK-KKUMJFAQSA-N 0.000 description 3
- SPKRHJOVRVDJGG-CIUDSAMLSA-N Asp-Gln-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N SPKRHJOVRVDJGG-CIUDSAMLSA-N 0.000 description 3
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 3
- ATVYZJGOZLVXDK-IUCAKERBSA-N Glu-Leu-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O ATVYZJGOZLVXDK-IUCAKERBSA-N 0.000 description 3
- BXSZPACYCMNKLS-AVGNSLFASA-N Glu-Ser-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BXSZPACYCMNKLS-AVGNSLFASA-N 0.000 description 3
- UCZXXMREFIETQW-AVGNSLFASA-N Glu-Tyr-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O UCZXXMREFIETQW-AVGNSLFASA-N 0.000 description 3
- HQTDNEZTGZUWSY-XVKPBYJWSA-N Glu-Val-Gly Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)NCC(O)=O HQTDNEZTGZUWSY-XVKPBYJWSA-N 0.000 description 3
- COVXELOAORHTND-LSJOCFKGSA-N Gly-Ile-Val Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O COVXELOAORHTND-LSJOCFKGSA-N 0.000 description 3
- YLEIWGJJBFBFHC-KBPBESRZSA-N Gly-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 YLEIWGJJBFBFHC-KBPBESRZSA-N 0.000 description 3
- KBBFOULZCHWGJX-KBPBESRZSA-N Gly-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN)O KBBFOULZCHWGJX-KBPBESRZSA-N 0.000 description 3
- BRZQWIIFIKTJDH-VGDYDELISA-N His-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N BRZQWIIFIKTJDH-VGDYDELISA-N 0.000 description 3
- UIEZQYNXCYHMQS-BJDJZHNGSA-N Ile-Lys-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)O)N UIEZQYNXCYHMQS-BJDJZHNGSA-N 0.000 description 3
- FXJLRZFMKGHYJP-CFMVVWHZSA-N Ile-Tyr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FXJLRZFMKGHYJP-CFMVVWHZSA-N 0.000 description 3
- 108010065920 Insulin Lispro Proteins 0.000 description 3
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 3
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 3
- QBHGXFQJFPWJIH-XUXIUFHCSA-N Lys-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN QBHGXFQJFPWJIH-XUXIUFHCSA-N 0.000 description 3
- KNYPNEYICHHLQL-ACRUOGEOSA-N Phe-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 KNYPNEYICHHLQL-ACRUOGEOSA-N 0.000 description 3
- WPQKSRHDTMRSJM-CIUDSAMLSA-N Pro-Asp-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 WPQKSRHDTMRSJM-CIUDSAMLSA-N 0.000 description 3
- YXHYJEPDKSYPSQ-AVGNSLFASA-N Pro-Leu-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 YXHYJEPDKSYPSQ-AVGNSLFASA-N 0.000 description 3
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 3
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-N 0.000 description 3
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 3
- BPGDJSUFQKWUBK-KJEVXHAQSA-N Thr-Val-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BPGDJSUFQKWUBK-KJEVXHAQSA-N 0.000 description 3
- YTCNLMSUXPCFBW-SXNHZJKMSA-N Trp-Ile-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O YTCNLMSUXPCFBW-SXNHZJKMSA-N 0.000 description 3
- GIOBXJSONRQHKQ-RYUDHWBXSA-N Tyr-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O GIOBXJSONRQHKQ-RYUDHWBXSA-N 0.000 description 3
- KEANSLVUGJADPN-LKTVYLICSA-N Tyr-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N KEANSLVUGJADPN-LKTVYLICSA-N 0.000 description 3
- ZOBLBMGJKVJVEV-BZSNNMDCSA-N Tyr-Lys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O ZOBLBMGJKVJVEV-BZSNNMDCSA-N 0.000 description 3
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 3
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 3
- RFZFBOQPPFCOKG-BZSNNMDCSA-N Val-Trp-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCSC)C(=O)O)N RFZFBOQPPFCOKG-BZSNNMDCSA-N 0.000 description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 3
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 3
- 108010077245 asparaginyl-proline Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 3
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 3
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 3
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108010009298 lysylglutamic acid Proteins 0.000 description 3
- 235000009973 maize Nutrition 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 108010005942 methionylglycine Proteins 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 108010048818 seryl-histidine Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 2
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- 241000223195 Fusarium graminearum Species 0.000 description 2
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 2
- CRJZZXMAADSBBQ-SRVKXCTJSA-N Ser-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO CRJZZXMAADSBBQ-SRVKXCTJSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- WKWJJQZZZBBWKV-JYJNAYRXSA-N Val-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WKWJJQZZZBBWKV-JYJNAYRXSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- FPQFYIAXQDXNOR-QDKLYSGJSA-N alpha-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-QDKLYSGJSA-N 0.000 description 2
- 230000001925 catabolic effect Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 150000002148 esters Chemical group 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 235000019629 palatability Nutrition 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 229960002300 zeranol Drugs 0.000 description 2
- 101150076401 16 gene Proteins 0.000 description 1
- 101150024653 61 gene Proteins 0.000 description 1
- 101800000263 Acidic protein Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001669263 Bacillus amyloliquefaciens DSM 7 Species 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N Benzoic acid Natural products OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- SGIIOQQGLUUMDQ-IHRRRGAJSA-N Leu-His-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N SGIIOQQGLUUMDQ-IHRRRGAJSA-N 0.000 description 1
- XMQZLGBUJMMODC-AVGNSLFASA-N Met-His-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O XMQZLGBUJMMODC-AVGNSLFASA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005314 correlation function Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000036750 egg laying performance Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- -1 protocatechuic acid lactone Chemical class 0.000 description 1
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及酰基辅酶A硫酯酶及其基因在降解玉米赤霉烯酮中的应用、降解玉米赤霉烯酮的制剂，属于真菌毒素生物降解技术领域。本发明中通过微生物转录组测序分析发现解淀粉芽孢杆菌H6菌株中YBGC/FADM家族的酰基辅酶A硫酯酶基因是降解玉米赤霉烯酮的关键基因，将该酰基辅酶A硫酯酶基因在大肠杆菌中进行表达，发现该蛋白为可溶性蛋白，纯化后获得重组蛋白，经玉米赤霉烯酮ELISA检测试剂盒检测发现该重组蛋白具有高效降解ZEN的生物学活性。因此酰基辅酶A硫酯酶及其编码基因能够用于降解玉米赤霉烯酮，酰基辅酶A硫酯酶能够用于制备降解玉米赤霉烯酮的酶制剂或复合酶制剂。
The invention relates to the application of acyl-CoA thioesterase and its gene in degrading zearalenone, and a preparation for degrading zearalenone, belonging to the technical field of mycotoxins biodegradation. In the present invention, it is found that the acyl-CoA thioesterase gene of the YBGC/FADM family in the Bacillus amyloliquefaciens H6 strain is the key gene for degrading zearalenone through microbial transcriptome sequencing analysis, and the acyl-CoA thioesterase gene is in the It was expressed in Escherichia coli, and it was found that the protein was a soluble protein. After purification, the recombinant protein was obtained. The zearalenone ELISA detection kit found that the recombinant protein had the biological activity of efficiently degrading ZEN. Therefore, the acyl-CoA thioesterase and its encoding gene can be used to degrade zearalenone, and the acyl-CoA thioesterase can be used to prepare an enzyme preparation or a composite enzyme preparation for degrading zearalenone.
Description
Technical field
The application that the present invention relates to acyl-CoA thioesterase enzymes and its gene in degrading zearalenone, degrading maize
The preparation of zeranol belongs to mycotoxin biodegradation technique field.
Background technique
According to the statistics of Food and Agricultural Organization of the United Nations, there are about 25% crops in the annual whole world is polluted by mycotoxin,
There are about 2% crops, and nutrition and economic value are lost because seriously polluting, and causes hundreds billion of dollars of economic loss.Mycotoxin
It is exceeded to have become an important factor for endangering agricultural product quality and safety, huge economy is caused to grain, edible oil and feed industry
Loss, seriously threatens the health and safety of the mankind and livestock and poultry animal.Zearalenone (Zearalenone, ZEN) is a kind of
The oestrogen-like hormone mycotoxin generated by many Fusariums.Before and after corn, wheat and other grain harvests, it is highly prone to sickle
The pollution of knife Pseudomonas causes ZEN to have generally exceeded the standard if storing, processing, transporting improper.After edible ZEN exceeded feed, sow table
Reveal strong estrogenic activity, cause the serious diseases such as breast cancer, sow breeding difficulty, growth is shown after poultry food not
The decline of good and egg laying performance, causes massive losses to animal husbandry.Therefore, research and development are conveniently, safely malicious with effective degradative fungi
The technology of element is of great significance.
Currently, the poison-removing method of endotoxin contamination feed mainly has physics detoxification, chemical detoxication and biologic detoxication etc..Physics and chemistry is de-
The disadvantages of malicious method declines there are operating difficulties, nutritional quality decline, feed palatability, application effect is bad.Biologic detoxication tool
Have the advantages that reaction condition is mild, influences small, raising raw material nutritive value to raw material sensory properties and palatability, it is considered as
Best detoxification.Therefore, carry out removing toxic substances using biological enzyme formulation and have become the research hotspot of the world today, correlative study at
Fruit shows good development and application prospect.
Currently, ZEN Biodegradation Mechanism and the research of relevant functional gene are less.Publication No. CN105755023A's
Chinese invention patent application discloses a kind of zearalenone degrading enzyme gene, entitled zhd-101 gene, according to finishing red ferment
Gene order length after the optimization of female codon preference is 795bp, codon adaptation indexI 0.91%, and G/C content is
43.6%, mRNA stability height, long half time.It can by high density fermentation using the gene constructed recombinant pichia yeast strain
With efficient secretory expression zearalenone degrading enzyme, fermented supernatant fluid can quickly and efficiently degrade ZEN and its derivative.
The Chinese invention patent application of Publication No. CN106047749A discloses a kind of zearalenone degradation bacteria, should
Bacterial strain is the bacillus amyloliquefaciens H6(CCTCC NO of one plant of degradable corn zeranol of screening this research early period:
M2016 129), it is to screen to obtain by sole carbon source and the energy of ZEN, can be used for degrading zearalenone or preparation drop
Solve the microorganism formulation or feed addictive of zearalenone.But the mechanism of degradation of the bacterial strain and correlation function gene are also not
It is clear.
Summary of the invention
It is that corn is red the object of the present invention is to provide application of the acyl-CoA thioesterase enzyme in degrading zearalenone
The biodegrade of mould ketenes provides a kind of new approaches.
It is that corn is red the present invention also provides application of the acyl-CoA thioesterase enzyme gene in degrading zearalenone
The biodegrade of mould ketenes provides a kind of new approaches.
The present invention also provides the recombinant expression carriers containing acyl-CoA thioesterase enzyme gene in degrading zearalenone
In application, provide a kind of new approaches for the biodegrade of zearalenone.
The present invention also provides the answering in degrading zearalenone of the microorganism containing acyl-CoA thioesterase enzyme gene
With providing a kind of new approaches for the biodegrade of zearalenone.
The present invention also provides acyl-CoA thioesterase enzymes in the enzyme preparation or complex enzyme system for preparing degrading zearalenone
Application in agent provides a kind of new approaches for the biodegrade of zearalenone.
The present invention also provides the enzyme preparation of degrading zearalenone, main component is acyl-CoA in the enzyme preparation
Thioesterase is a kind of novel zearalenone catabolic enzyme preparation.
It include acyl-coenzyme in the complex enzyme formulation the present invention also provides the complex enzyme formulation of degrading zearalenone
A thioesterase is a kind of novel zearalenone catabolic enzyme preparation.
To achieve the goals above, the technical scheme adopted by the invention is that:
Application of the acyl-CoA thioesterase enzyme in degrading zearalenone, the acyl-CoA thioesterase enzyme have such as SEQ ID
Amino acid sequence shown in NO.2, or have the amino acid sequence as shown in SEQ ID NO.2 by one or more amino
The substitution and/or deletion and/or addition of sour residue are formed by amino acid sequence.
The acyl of YBGC/FADM family in bacillus amyloliquefaciens H6 bacterial strain is found in the present invention by transcript profile sequencing analysis
Base coacetylase thioesterase gene is the key gene of degrading zearalenone, by the acyl-CoA thioesterase enzyme gene through codon
It is expressed in Escherichia coli after optimization, discovery expression albumen is soluble protein, and purifying obtains recombinant protein, Gibberella zeae
The detection of ketenes ELISA kit finds its biological activity with efficient degradation ZEN, therefore acyl-CoA thioesterase enzyme can
For degrading zearalenone.
The acyl-CoA thioesterase enzyme has the amino acid sequence as shown in SEQ ID NO.4, or has such as SEQ ID
Amino acid sequence shown in NO.6.
The amino acid sequence as shown in SEQ ID NO.4, SEQ ID NO.6 is the bacillus amyloliquefaciens H6 in the present invention
The homologous sequence of the acyl-CoA thioesterase enzyme of YBGC/FADM family in bacterial strain equally also has degrading zearalenone
Function.
Application of the acyl-CoA thioesterase enzyme gene in degrading zearalenone, the acyl-CoA thioesterase enzyme gene
The albumen of coding has the amino acid sequence as shown in SEQ ID NO.2, or has the amino acid as shown in SEQ ID NO.2
Sequence is formed by amino acid sequence by the substitution and/or deletion and/or addition of one or more amino acid residues.
The acyl of YBGC/FADM family in bacillus amyloliquefaciens H6 bacterial strain is found in the present invention by transcript profile sequencing analysis
Base coacetylase thioesterase gene is the key gene of degrading zearalenone, by the acyl-CoA thioesterase enzyme gene through codon
After optimization in Escherichia coli solubility expression, and purify acquisition recombinant protein, there is efficiently drop through detection discovery recombinant protein
The biological activity of ZEN is solved, therefore acyl-CoA thioesterase enzyme and its encoding gene can be used in degrading zearalenone.
The acyl-CoA thioesterase enzyme gene have the nucleotide sequence such as SEQ ID NO.1 shown in, or with
The nucleotide sequence of 95% or more homology of nucleotide sequence shown in SEQ ID NO.1, or as shown in SEQ ID NO.1
It is obtained after the optimization of nucleotide sequence codon.
The nucleotide sequence as shown in SEQ ID NO.1 can encoding acyl CoA thioesterase enzyme, the sequence homologous with it and
The sequence of codon optimization also can encoding acyl CoA thioesterase enzyme, be used for degrading zearalenone.
Application of the recombinant expression carrier containing acyl-CoA thioesterase enzyme gene in degrading zearalenone, it is described
The albumen of acyl-CoA thioesterase enzyme gene coding has the amino acid sequence as shown in SEQ ID NO.2, or has such as SEQ
Amino acid sequence shown in ID NO.2 is formed by the substitution and/or deletion and/or addition of one or more amino acid residues
Amino acid sequence.
Recombinant expression carrier containing acyl-CoA thioesterase enzyme gene can express acyl-CoA thioesterase enzyme, for degrading
Zearalenone.
The recombinant expression carrier is that acyl-CoA thioesterase enzyme gene is cloned into expression vector, and building is recombinated
Expression vector.The recombinant expression carrier can be used conventional gene engineering method and obtain.
Application of the microorganism containing acyl-CoA thioesterase enzyme gene in degrading zearalenone, the acyl group are auxiliary
The albumen of enzyme A thioesterase gene coding has the amino acid sequence as shown in SEQ ID NO.2, or has such as SEQ ID
Amino acid sequence shown in NO.2 is formed by by the substitution and/or deletion and/or addition of one or more amino acid residues
Amino acid sequence.
Microorganism containing acyl-CoA thioesterase enzyme gene can express acyl-CoA thioesterase enzyme, red for degrading maize
Mould ketenes.
Application of the acyl-CoA thioesterase enzyme in the enzyme preparation or complex enzyme formulation for preparing degrading zearalenone, institute
Acyl-CoA thioesterase enzyme is stated with the amino acid sequence as shown in SEQ ID NO.2, or is had as shown in SEQ ID NO.2
Amino acid sequence be formed by amino acid sequence by the substitution and/or deletion and/or addition of one or more amino acid residues
Column.
Acyl-CoA thioesterase enzyme have the ability of degrading zearalenone can be consequently used for preparing degrading maize it is red mould
The enzyme preparation or complex enzyme formulation of ketenes.
The enzyme preparation of degrading zearalenone, the effective component of the enzyme preparation are acyl-CoA thioesterase enzyme；The acyl
Base coacetylase thioesterase has the amino acid sequence as shown in SEQ ID NO.2, or has the ammonia as shown in SEQ ID NO.2
Base acid sequence is formed by amino acid sequence by the substitution and/or deletion and/or addition of one or more amino acid residues.
Acyl-CoA thioesterase enzyme has very high degradation capability for zearalenone, is made with it for main component
Enzyme preparation also has zearalenone very high degradation capability.
The complex enzyme formulation of degrading zearalenone, the effective component of the complex enzyme formulation include acyl-CoA sulphur
Esterase；The acyl-CoA thioesterase enzyme has the amino acid sequence as shown in SEQ ID NO.2, or has such as SEQ ID
Amino acid sequence shown in NO.2 is formed by by the substitution and/or deletion and/or addition of one or more amino acid residues
Amino acid sequence.
Acyl-CoA thioesterase enzyme has very high degradation capability for zearalenone, includes acyl-CoA thioesterase enzyme
Complex enzyme formulation also there is very high degradation capability for zearalenone.
Detailed description of the invention
Fig. 1 is total ion current figure in experimental example of the present invention；
Fig. 2 is M1 first mass spectrometric figure in experimental example of the present invention；
Fig. 3 is M1 second order ms figure in experimental example of the present invention；
Fig. 4 is M2 first mass spectrometric figure in experimental example of the present invention；
Fig. 5 is M2 second order ms figure in experimental example of the present invention；
Fig. 6 be experimental example of the present invention in BL21(DE3) express ZTE138 albumen SDS-PAGE analysis chart；
Fig. 7 is SDS-PAGE the and Western Blot analysis chart of the ZTE138 albumen purified in experimental example of the present invention；
Fig. 8 is the structural formula schematic diagram of the M1 inferred in experimental example of the present invention.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below.It is each to implement in addition to specified otherwise
Equipment used in example and experimental example and reagent are commercially available.Hereinafter involved bacterial strain solution starch gemma
Bacillus H6 is disclosed in the Chinese invention patent application of Publication No. CN106047749A.
Bacillus amyloliquefaciens H6 preservation proves and survival proves explanation:
Preservation strain: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6, deposit number: CCTCC NO:
M2016129, preservation date: on March 17th, 2016, depositary institution: China typical culture collection center (CCTCC), preservation
Location: Wuhan, China Wuhan University (Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection).
The embodiment 1 of application of the acyl-CoA thioesterase enzyme in degrading zearalenone
The present embodiment acyl-CoA thioesterase enzyme acyl-CoA thioesterase used in the application in degrading zearalenone
Enzyme has the amino acid sequence as shown in SEQ ID NO.2.Acyl-CoA thioesterase enzyme also can have such as SEQ ID NO.4 or
Person's amino acid sequence as shown in SEQ ID NO.6.
The embodiment 1 of application of the acyl-CoA thioesterase enzyme gene in degrading zearalenone
Acyl-CoA thioesterase enzyme of the present embodiment acyl-CoA thioesterase enzyme gene in the application in degrading zearalenone
Gene has the nucleotide sequence as shown in SEQ ID NO.1.Acyl-CoA thioesterase enzyme gene also can have such as SEQ ID
NO.3 or the nucleotide sequence as shown in SEQ ID NO.5.
The implementation of application of the recombinant expression carrier containing acyl-CoA thioesterase enzyme gene in degrading zearalenone
Example 1
The present embodiment contains application of the recombinant expression carrier of acyl-CoA thioesterase enzyme gene in degrading zearalenone
In, inserted with acyl-CoA thioesterase enzyme gene in recombinant expression carrier, the acyl-CoA thioesterase enzyme gene has such as SEQ
ID NO.1, SEQ ID NO.3 or the nucleotide sequence as shown in SEQ ID NO.5.
The embodiment 1 of application of the microorganism containing acyl-CoA thioesterase enzyme gene in degrading zearalenone
The present embodiment contains the microorganism of acyl-CoA thioesterase enzyme gene in the application in degrading zearalenone, micro- life
It include recombinant expression carrier in object, inserted with acyl-CoA thioesterase enzyme gene, the acyl-CoA sulphur in recombinant expression carrier
Esterase gene has such as SEQ ID NO.1, SEQ ID NO.3 or the nucleotide sequence as shown in SEQ ID NO.5.It is described micro-
Biology can be recombinant bacterium, specifically have clone the recombinant expression carrier of the gene of coding zearalenone degrading enzyme to be transferred to
In host bacteria, building obtains recombinant bacterium.
The reality of application of the acyl-CoA thioesterase enzyme in the enzyme preparation or complex enzyme formulation for preparing degrading zearalenone
Apply example 1
Acyl-CoA thioesterase enzyme answering in the enzyme preparation or complex enzyme formulation for preparing degrading zearalenone in the present embodiment
It is using acyl-CoA thioesterase enzyme as the principle active component of the enzyme preparation of degrading zearalenone, or as drop in
Solve the part effective component of the complex enzyme formulation of zearalenone.
The embodiment 1 of the enzyme preparation of degrading zearalenone
Principle active component is acyl-CoA thioesterase enzyme, acyl-CoA in the enzyme preparation of the present embodiment degrading zearalenone
Thioesterase has the amino acid sequence as shown in SEQ ID NO.2.Acyl-CoA thioesterase enzyme also can have such as SEQ ID
NO.4 or the amino acid sequence as shown in SEQ ID NO.6.Also contain adjunct ingredient in the enzyme preparation.
The embodiment 1 of the complex enzyme formulation of degrading zearalenone
Effective component is acyl-CoA thioesterase enzyme and ZHD albumen in the complex enzyme formulation of the present embodiment degrading zearalenone
(as shown in CN105755023A), also contains adjunct ingredient；Acyl-CoA thioesterase enzyme has as shown in SEQ ID NO.2
Amino acid sequence.Acyl-CoA thioesterase enzyme also can have such as SEQ ID NO.4 or the amino as shown in SEQ ID NO.6
Acid sequence.
Experimental example
1. research process
1.1 experiment material therefors
1.1.1 bacterial strain: bacillus amyloliquefaciens H6 is the bacterial strain that an early screening provides zearalenone degradation characteristic
(as shown in CN106047749A).Express strain BL21(DE3) and prokaryotic expression carrier pet-30a be commercial strains and carrier.
1.1.2 main agents: Zebralenone Standard(Pribola), methanol, nutrient broth, qiaquick-
Pcr kit(Takara), rneasy mini kit(Takara), dntpstakara, RNase H(Takara), archaeal dna polymerase
I(Takara), buffer, IPTG and Trition X-100(Biotechnology BioEngineering are dissolved
(Shanghai) Co., Ltd.), plasmid extraction kit and DNA extraction kit (Tiangen Biochemical Te) science and technology
(Beijing) Co., Ltd, T4-DNA ligase, TAQ-DNA polymerase (precious bioengineering (Dalian) Co., Ltd).
1.2 experimental methods and result
1.2.1 sample preparation: selection single bacterium colony is seeded to 50mL nutrient solution (250 mL triangular flasks of the nutrient solution containing 50mL)
In, 180 r/min are cultivated 8 hours under the conditions of 37 DEG C, are then seeded to 6 bottles of 100mL nutrient solutions respectively with 6% inoculum concentration and (are contained
250 mL triangular flasks of 100mL nutrient solution), fermentation liquid is made in 180 r/min under the conditions of 37 DEG C, culture for 24 hours.Wherein three bottles of fermentations
The ZEN(1000 μ g/mL of 200 μ L is added in liquid), label are as follows: Z1, Z2, Z3(experimental group), 200 μ L methanol are added in excess-three bottle,
Labeled as C1, C2, C3(blank control group).After cultivating 12 h under the conditions of 37 DEG C, 180 r/min, in 12000 r/min high speed
It is centrifuged 6 minutes in refrigerated centrifuge, collects somatic cells, be frozen in liquid nitrogen, then stored at -80 DEG C until using.
1.2.2 Total RNAs extraction and quality testing: specification is extracted according to RNA, total serum IgE is extracted from bacterium.Using
Nanodrop and 2100 Biochemical Analyzer of Agilent measurement sample RNA concentration and OD260/OD280 ratio.Use Agilent
2100 detection Biochemical Analyzers measure total serum IgE quality, to determine RNA integrality.
1.2.3 the building of sequencing library: the DNA in RNA sample is removed with deoxyribonuclease DNA enzymatic method, with ribose core
RRNA in sour enzyme scavenger reagent box removal protokaryon total serum IgE, then destroys mRNA after purification with random enzyme digestion.With short-movie
The form of section, using random hexamer as template, using segment mRNA as first cDNA chain of templated synthesis.After removing dNTPs, pass through
Buffer, dATP, dGTP, dCTP, dUTP, RNAse H and DNA polymerase i is added and synthesizes Article 2 cDNA chain, uses
The double-strand cDNA that qiaquick-pcr kits obtain is added PolyA and is connected to sequencing connexon after end is repaired
On, then with UNG enzymic digestion Article 2 cDNA chain.After micro- elution PCR Purification Kit, PCR amplification is carried out.The survey of building
Preface library is sequenced using Illumina Hiseq 2500.
Total RNAs extraction and quality testing through 6 samples, establish 6 transcription sequence libraries altogether.The end transcript profile of pairing
It is sequenced using 2500 microarray dataset of Illummina Hiseq.Underproof reading is removed, and carries out data analysis.With
Bacillus amyloliquefaciens DSM7(NC014551.1 in ncbi database) full-length genome is refers to, to 6 groups of data progress genes
Matching, gene comparison rate are as a result excellent to carry out subsequent data analysis up to 84% or more.
1.2.4 the screening of difference expression gene: the gene of comparison is determined using the HTSEQ software in python kit
On position to exon, gene expression dose is then estimated by RPKM.Using DESeq R software, in fold differences >=1.5, p value
Difference expression gene is screened under≤0.05 screening conditions.77 difference expression genes are filtered out altogether, wherein 16 up-regulation genes,
61 down-regulated genes.
1.2.5 the triple TOF/MS systems of supernatant application UPLC of acquisition the analysis of ZEN catabolite: will be centrifuged in 1.2.1
System is analyzed, and is obtained total ion current figure (as shown in Figure 1).Experimental group catabolite is compared with blank control group 4.067
There are two apparent difference peaks when min and 14.786 min.
The first mass spectrometric result at 4.067 min difference peaks (M1) as shown in Fig. 2, wherein quasi-molecular ions m/z 399,381,416,
797,798 and 799 be respectively [M1+H]+、[M1+H]+、[M1+NH4]+、[M1+H]+、[2M1+H]+、[2M1+2H]+、[2M1+2H]+With
[2M1+3H]+.With second order ms method measure M1(4.067 min difference peak) molecular formula can be C18H23O8P or C25H18O5Or
C32H14(as shown in Figure 3).
The first mass spectrometric result at 14.786 min difference peaks (M2) as shown in figure 4, wherein quasi-molecular ions m/z 1036,1037,
1038,1058,1059 and 1060 be respectively [M2+H]+、[M2+2H]+、[M2+3H]+、[M2+H+Na]+、[M2+2H+Na]+[M2
+3H+Na]+.It can be C with second order ms method measurement M2 molecular formula64H93NO10、C52H97N3O7、C5993N3O12Or C71H895(as schemed
Shown in 5).Due to containing N element in M2 molecule, think that M2 may be a kind of polypeptide or protease, therefore, it is considered that M1 is beautiful
The catabolite of Zearlenone.
1.2.6 the prediction, expression and identification of functional gene
Analysis and Screening is carried out in the differential gene that 16 are raised, wherein there are two unknown genes that may drop with zearalenone
It solves related.The protein gene (being named as ZTE138) being wherein made of 138 amino acid comes the 2nd in up-regulation gene, can
It can be the key gene of zearalenone degradation, therefore this gene is cloned, functional gene ZTE138(nucleotide sequence is such as
Shown in SEQ ID NO.1) with YBGC/FADM family acyl-CoA thioesterase enzyme gene (as shown in SEQ ID NO.3) on NCBI
Comparison show 414 nucleotide sequences, discrimination 99%.The acidic protein average molecular weight for encoding 138 amino acid is
16974Da, only the 2nd amino acids are mismatched, and ZTE138 protein sequence is as shown in SEQ ID NO.2, YBGC/FADM family acyl
Base coacetylase thioesterase (accession number: wp017417881.1) protein sequence is as shown in SEQ ID NO.4.
ZTE138 gene is obtained using codon optimization software MaxCodonTM optimization program (V13), to ZTE138 albumen
The amino acid sequence of (individual point mutation) optimizes, egg of the gene order after optimization as shown in SEQ ID NO.5, after optimization
Bai Xulie is as shown in SEQ ID NO.6.Using plasmid pET-30a(+) in BL21(DE3) it expresses on engineering bacteria, identified expression
Albumen is soluble protein.
SDS-PAGE detection shows that ZTE138 albumen is expressed, as a result as shown in fig. 6, the road M: SDS-PAGE albumen
Maker；0: control；1: 15 DEG C induces 16 hours；2: 37 DEG C induce 16 hours；As can be seen from the figure relative to control
Group, inducing expression group all has apparent band (as shown by arrows) between 10-20 kDa, and control group does not have.
Then Western-Blot detection is carried out after purification, as a result as shown in fig. 7, M1:SDS-PAGE Maker(kDa)；
M2:Western-Blot Maker(kDa)；1: BSA；2: ZTE138 protein.The result shows that finally obtaining purity and being more than
90% soluble ZTE138 albumen.Final result shows recombinant expression carrier pet-30a(+)-ZTE138 is in Escherichia coli
BL21(DE3 correct expression has been obtained in), purifying has obtained soluble ZTE138 albumen.
Identification of its biological activity: with the identification ZTE138 recombinant protein degradation of zearalenone enzyme-linked immunologic detecting kit
The activity of zearalenone.
It is dissolved in 100mL nutrient solution from the recombinant protein obtained is purified in 100mL induction broth, the ZEN of 200 μ L is added
(1000 μ g/mL), reacts 12 h under the conditions of 37 DEG C, 180 r/min, and enzyme-linked immunologic detecting kit detects the content of ZEN, knot
Fruit shows that its degradation rate is 95.91%.
This result shows that, recombinant expression carrier pet-30a(+) soluble protein expressed by-ZTE138 has degradation ZEN
Ability.
It discusses
The key gene that zearalenone degradation is screened in the present invention, using high throughput sequencing technologies to Gibberella zeae alkene
The transcription product of the bacillus amyloliquefaciens H6 of ketone processing is sequenced.Transcript profile sequencing analysis shows zearalenone
77 genes of bacillus amyloliquefaciens are had a significant impact, wherein 16 gene upregulations, 61 gene deregulations.Think in jade
Under Zearlenone processing, the gene of bacterium up-regulation is mainly the enzyme that can promote bacterial growth using toxin, and is dropped using toxin
Solve or be converted into the related gene of other substances.Difference expression gene is screened, and the difference expression gene of prediction is cloned
And expression, degrading zearalenone Activity determination is carried out to expression albumen, verifies the biological activity of Gene degradation ZEN.
ZTE138 gene is YBGC/FADM family acyl-CoA thioesterase enzyme (bacillus amyloliquefaciens), zearalenone
For protocatechuic acid lactone.The lactone bond of zearalenone can be broken under the action of lactone hydrolase, and lactone hydrolase will
Ester ring structure is opened into linear chain structure, then spontaneous decarboxylation, the fracture not in conjunction with estrogen receptor is formed, to reduce poison
Property.
LC-MS analysis shows that the molecular formula of M1 is C18H23O8P or C25H18O5Or C32H14, molecular weight 398,
The molecular weight of zearalenone is C18H22O5, molecular weight 318.36.Point of this research zearalenone catabolite M1
Son amount is greater than the molecular weight of zearalenone, this may be to destroy ester under the action of ZTE138 due to zearalenone
Ring key, phosphite is instead of caused by the hydroxyl in phenyl ring branch.Therefore, the molecular formula for inferring M1 herein is C18H23O8P(is such as
Shown in Fig. 8), structure is (1e, 10s) -1-(3,5- dihydroxy -6- benzoic acid) -11 '-phosphorous acid -2 '-laurylene base -7 ' -
Ketone.
The expression of functional gene ZTE138 in vitro shows that expression product is primarily present in supernatant；Purified, mirror
Fixed, detection, obtains soluble protein, and enzyme linked immunosorbent assay testing result shows that the soluble protein has the energy of degradation ZEN
Power.These results indicate that functional gene ZTE138 is the key gene of degradation ZEN.The albumen of ZTE138 coding has degradation beautiful
The ability of Zearlenone.
<110>Agricultural University Of He'nan
<120>application in degrading zearalenone of acyl-CoA thioesterase enzyme and its gene, degrading zearalenone
Preparation
<160> 6
<170> SIPOSequenceListing 1.0
<211> 414
<212> DNA
<213>bacillus amyloliquefaciens
<221>acyl-CoA thioesterase enzyme gene
<400> 1
ttgcatgtgt caaaaaaaga aatagaagtg cgttatgcgg aaacagatca aatgggcatc 60
gtctatcatg ccaattactt agtctggatg gaggtcggca gaacggcgct gattaaggaa 120
ctcggattcc tttacaagga tatggaagat agaggagtgc tgtcccctgt attggatatc 180
agcatttctt ataaaaagcc gctccgctac ggagaaacgg ctgtcgtaca tacttggatt 240
gaagaatata acggatttaa aaccgtttac ggctaccaca tctataatcc ggatcaagaa 300
cttgccatca aagcgacatc ctcccatata tgcgttgata aagagagctt caaaccgatt 360
cagtttcgca aagcttttcc tgattggcat gccgcttacg aaaaggcaaa aaaa 414
<211> 138
<212> PRT
<213>bacillus amyloliquefaciens
<221>acyl-CoA thioesterase enzyme
<400> 2
Leu His Val Ser Lys Lys Glu Ile Glu Val Arg Tyr Ala Glu Thr
1 5 10 15
Asp Gln MET Gly Ile Val Tyr His Ala Asn Tyr Leu Val Trp MET
20 25 30
Glu Val Gly Arg Thr Ala Leu Ile Lys Glu Leu Gly Phe Leu Tyr
35 40 45
Lys Asp MET Glu Asp Arg Gly Val Leu Ser Pro Val Leu Asp Ile
50 55 60
Ser Ile Ser Tyr Lys Lys Pro Leu Arg Tyr Gly Glu Thr Ala Val
65 70 75
Val His Thr Trp Ile Glu Glu Tyr Asn Gly Phe Lys Thr Val Tyr
80 85 90
Gly Tyr His Ile Tyr Asn Pro Asp Gln Glu Leu Ala Ile Lys Ala
95 100 105
Thr Ser Ser His Ile Cys Val Asp Lys Glu Ser Phe Lys Pro Ile
110 115 120
Gln Phe Arg Lys Ala Phe Pro Asp Trp His Ala Ala Tyr Glu Lys
125 130 135
Ala Lys Lys
138
<211> 414
<212> DNA
<213>bacillus amyloliquefaciens
<221>acyl-CoA thioesterase enzyme gene
<400> 3
ttgcgtgtgt caaaaaaaga aatagaagtg cgttatgcgg aaacagatca aatgggcatc 60
gtctatcatg ccaattactt agtctggatg gaggtcggca gaacggcgct gattaaggaa 120
ctcggattcc tttacaaaga tatggaagat agaggagtgc tgtcccctgt attggatatc 180
agcatttctt ataaaaagcc gctccgctac ggagaaacgg ctgtcgtaca tacttggatt 240
gaagaatata acggatttaa aaccgtttac ggctaccaca tctataatcc ggatcaagaa 300
cttgccatca aagcgacatc ctctcatata tgcgttgata aagagagctt caaaccgatt 360
cagtttcgca aggcttttcc tgattggcat gccgcttacg aaaaggcaaa aaaa 414
<211> 303
<212> PRT
<213>bacillus amyloliquefaciens
<221>acyl-CoA thioesterase enzyme
<400> 4
MET Arg Val Ser Lys Lys Glu Ile Glu Val Arg Tyr Ala Glu Thr
1 5 10 15
Asp Gln MET Gly Ile Val Tyr His Ala Asn Tyr Leu Val Trp MET
20 25 30
Glu Val Gly Arg Thr Ala Leu Ile Lys Glu Leu Gly Phe Leu Tyr
35 40 45
Lys Asp MET Glu Asp Arg Gly Val Leu Ser Pro Val Leu Asp Ile
50 55 60
Ser Ile Ser Tyr Lys Lys Pro Leu Arg Tyr Gly Glu Thr Ala Val
65 70 75
Val His Thr Trp Ile Glu Glu Tyr Asn Gly Phe Lys Thr Val Tyr
80 85 90
Gly Tyr His Ile Tyr Asn Pro Asp Gln Glu Leu Ala Ile Lys Ala
95 100 105
Thr Ser Ser His Ile Cys Val Asp Lys Glu Ser Phe Lys Pro Ile
110 115 120
Gln Phe Arg Lys Ala Phe Pro Asp Trp His Ala Ala Tyr Glu Lys
125 130 135
Ala Lys Lys
138
<211> 414
<212> DNA
<213>artificial sequence
<221>the acyl-CoA thioesterase enzyme gene optimized
<400> 5
atgcacgtga gcaaaaaaga gatcgaggtt cgctacgcgg aaaccgatca gatgggtatt 60
gtctaccacg cgaactacct ggtttggatg gaagtaggtc gtaccgcact gattaaagag 120
ctgggcttcc tgtacaaaga catggaagat cgcggcgttc tgagtccggt tctggatatt 180
agtatcagct acaaaaaacc gctgcgttac ggcgaaaccg cagttgtcca tacctggatc 240
gaagagtaca acggcttcaa aaccgtctac ggttaccaca tctacaaccc ggatcaggaa 300
ctggcgatta aagcaaccag cagccatatc tgcgtcgaca aagagagctt caaaccgatc 360
cagtttcgca aagcgtttcc ggattggcac gcagcgtacg agaaagcgaa aaaa 414
<211> 138
<212> PRT
<213>artificial sequence
<221>the acyl-CoA thioesterase enzyme optimized
<400> 6
MET His Val Ser Lys Lys Glu Ile Glu Val Arg Tyr Ala Glu Thr
1 5 10 15
Asp Gln MET Gly Ile Val Tyr His Ala Asn Tyr Leu Val Trp MET
20 25 30
Glu Val Gly Arg Thr Ala Leu Ile Lys Glu Leu Gly Phe Leu Tyr
35 40 45
Lys Asp MET Glu Asp Arg Gly Val Leu Ser Pro Val Leu Asp Ile
50 55 60
Ser Ile Ser Tyr Lys Lys Pro Leu Arg Tyr Gly Glu Thr Ala Val
65 70 75
Val His Thr Trp Ile Glu Glu Tyr Asn Gly Phe Lys Thr Val Tyr
80 85 90
Gly Tyr His Ile Tyr Asn Pro Asp Gln Glu Leu Ala Ile Lys Ala
95 100 105
Thr Ser Ser His Ile Cys Val Asp Lys Glu Ser Phe Lys Pro Ile
110 115 120
Gln Phe Arg Lys Ala Phe Pro Asp Trp His Ala Ala Tyr Glu Lys
125 130 135
Ala Lys Lys
138
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910228804.XA CN109907210B (en) | 2019-03-25 | 2019-03-25 | acyl-CoA thioesterase, application of gene thereof in degradation of zearalenone and preparation for degradation of zearalenone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910228804.XA CN109907210B (en) | 2019-03-25 | 2019-03-25 | acyl-CoA thioesterase, application of gene thereof in degradation of zearalenone and preparation for degradation of zearalenone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109907210A true CN109907210A (en) | 2019-06-21 |
| CN109907210B CN109907210B (en) | 2021-12-31 |
Family
ID=66966653
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910228804.XA Active CN109907210B (en) | 2019-03-25 | 2019-03-25 | acyl-CoA thioesterase, application of gene thereof in degradation of zearalenone and preparation for degradation of zearalenone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109907210B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103958692A (en) * | 2010-12-23 | 2014-07-30 | 艾克森美孚研究与工程公司 | Genetically engineered microorganisms comprising 4-hydroxybenzoly-coa thioesterases and methods of using the same for producing free fatty acids and fatty acid derivatives |
| CN106047749A (en) * | 2016-05-30 | 2016-10-26 | 河南农业大学 | Zaralenone degrading bacteria and application thereof |
| US20180362597A1 (en) * | 2015-12-09 | 2018-12-20 | The Regents Of The University Of California | Novel Proteins from Anaerobic Fungi and Uses Thereof |
-
2019
- 2019-03-25 CN CN201910228804.XA patent/CN109907210B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103958692A (en) * | 2010-12-23 | 2014-07-30 | 艾克森美孚研究与工程公司 | Genetically engineered microorganisms comprising 4-hydroxybenzoly-coa thioesterases and methods of using the same for producing free fatty acids and fatty acid derivatives |
| US20180362597A1 (en) * | 2015-12-09 | 2018-12-20 | The Regents Of The University Of California | Novel Proteins from Anaerobic Fungi and Uses Thereof |
| CN106047749A (en) * | 2016-05-30 | 2016-10-26 | 河南农业大学 | Zaralenone degrading bacteria and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| GENPEPT: ""MULTISPECIES: YbgC/FadM family acyl-CoA thioesterase [Bacillus amyloliquefaciens group]，NCBI Reference Sequence: WP_017417881.1", 《GENPEPT》 * |
| HUI ZHOU ET AL.: ""A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae"", 《PNAS》 * |
| LAIPENG XU ET AL.: ""Identification of a Bacillus amyloliquefaciens H6 Thioesterase Involved in Zearalenone Detoxification by Transcriptomic Analysis"", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| LIU X. ET AL.: ""AOA1W6HIV1_BACVA"", 《EMBL-EBI》 * |
| 潘丽婷 等: ""玉米赤霉烯酮降解菌的分离鉴定及其降解特性研究"", 《中国粮油学报》 * |
| 魏单平 等: ""玉米赤霉烯酮降解菌的筛选及其活性检测"", 《畜牧兽医学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109907210B (en) | 2021-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chang et al. | Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1 | |
| KR101543501B1 (en) | Enzyme involved in synthesis of equol | |
| JP5340138B2 (en) | Cloning and expression of a novel phytase | |
| Zhou et al. | Dynamic changes of bacterial communities and nitrite character during northeastern Chinese sauerkraut fermentation | |
| KR19980074925A (en) | The strain E. coli JM83 / pKP2 transformed with the novel plasmid and the phytase produced therefrom | |
| CN102492645A (en) | Recombinant bacillus subtilis with high aminopeptidase yield, construction method thereof, and application thereof | |
| Ariyaei et al. | Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock | |
| Broda et al. | Clostridium algidixylanolyticum sp. nov., a psychrotolerant, xylan-degrading, spore-forming bacterium. | |
| KR100850640B1 (en) | A detoxifizyme having activity of transforming aflatoxin and the gene encodes thereof | |
| JP2011103863A (en) | Gene encoding protein having deoxynivalenol decomposing activity | |
| KR102778820B1 (en) | Strains capable of decomposing collagen and their applications | |
| CN109055417B (en) | Recombinant microorganism, preparation method thereof and application thereof in production of coenzyme Q10 | |
| CN113604445B (en) | A kind of tyrosinase and its preparation and application | |
| JP2011103864A (en) | Gene encoding protein having deoxynivalenol and nivalenol decomposing activity | |
| CN110791508B (en) | Codon-optimized pediocin gene, expression vector and recombinant engineering strain | |
| CN109907210A (en) | Application of acyl-CoA thioesterase and its gene in degrading zearalenone, preparation for degrading zearalenone | |
| Suresh et al. | Molecular cloning of alkaline phosphatase, acid phosphatase and phytase genes from Aspergillus fumigatus for applications in biotechnological industries | |
| CN114736880B (en) | Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof | |
| CN109370995A (en) | Manganese peroxidase CsMnP and its encoding gene and application | |
| CN110072881A (en) | Cabicidin streptomycete isolate of modification and application thereof | |
| CN108277210A (en) | Mould ketenes hydrolase ZEN214 and encoding gene and application | |
| CN100385001C (en) | Recombinant bacillus phytase and uses thereof | |
| EP3423474B1 (en) | A recombinant polynucleotide involved in lactone synthesis and process for synthesis of lactones thereof | |
| KR101785613B1 (en) | Novel Keratinase derived from thermophilic bacteria and Use thereof | |
| CN115044599B (en) | Streptococcus agalactiae strain delta ess C and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |