CN110760531A - 一种剪接果胶酶基因中内含子的方法 - Google Patents
一种剪接果胶酶基因中内含子的方法 Download PDFInfo
- Publication number
- CN110760531A CN110760531A CN201911089149.0A CN201911089149A CN110760531A CN 110760531 A CN110760531 A CN 110760531A CN 201911089149 A CN201911089149 A CN 201911089149A CN 110760531 A CN110760531 A CN 110760531A
- Authority
- CN
- China
- Prior art keywords
- seq
- pmea
- amplification
- gene
- template
- 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.)
- Pending
Links
- 108010059820 Polygalacturonase Proteins 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 29
- 108010093305 exopolygalacturonase Proteins 0.000 title claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 52
- 230000003321 amplification Effects 0.000 claims abstract description 48
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 48
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 230000004927 fusion Effects 0.000 claims abstract description 14
- 108091092195 Intron Proteins 0.000 claims abstract description 11
- 108020004410 pectinesterase Proteins 0.000 claims abstract description 9
- 230000004544 DNA amplification Effects 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims abstract description 5
- 241000228232 Aspergillus tubingensis Species 0.000 claims description 15
- 238000007865 diluting Methods 0.000 claims description 15
- 239000012154 double-distilled water Substances 0.000 claims description 13
- 238000004925 denaturation Methods 0.000 claims description 9
- 230000036425 denaturation Effects 0.000 claims description 9
- 238000000137 annealing Methods 0.000 claims description 6
- 238000012257 pre-denaturation Methods 0.000 claims description 6
- 108700024394 Exon Proteins 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 24
- 239000012634 fragment Substances 0.000 abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 19
- 230000015572 biosynthetic process Effects 0.000 description 18
- 238000003786 synthesis reaction Methods 0.000 description 18
- 239000000047 product Substances 0.000 description 17
- 230000000694 effects Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 229920001277 pectin Polymers 0.000 description 6
- 239000001814 pectin Substances 0.000 description 6
- 235000010987 pectin Nutrition 0.000 description 6
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 5
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 229920002230 Pectic acid Polymers 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000010318 polygalacturonic acid Substances 0.000 description 4
- 241000880493 Leptailurus serval Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- AEMOLEFTQBMNLQ-BKBMJHBISA-N alpha-D-galacturonic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-BKBMJHBISA-N 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 235000019990 fruit wine Nutrition 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 230000035939 shock Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108010061238 threonyl-glycine Proteins 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- CSMHMEATMDCQNY-DZKIICNBSA-N Gln-Val-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CSMHMEATMDCQNY-DZKIICNBSA-N 0.000 description 2
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 2
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 2
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 2
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 2
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 2
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 2
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000015203 fruit juice Nutrition 0.000 description 2
- 235000012055 fruits and vegetables Nutrition 0.000 description 2
- 108010037850 glycylvaline Proteins 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- IGXNPQWXIRIGBF-KEOOTSPTSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IGXNPQWXIRIGBF-KEOOTSPTSA-N 0.000 description 1
- CVGNCMIULZNYES-WHFBIAKZSA-N Ala-Asn-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CVGNCMIULZNYES-WHFBIAKZSA-N 0.000 description 1
- SHYYAQLDNVHPFT-DLOVCJGASA-N Ala-Asn-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SHYYAQLDNVHPFT-DLOVCJGASA-N 0.000 description 1
- FRFDXQWNDZMREB-ACZMJKKPSA-N Ala-Cys-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O FRFDXQWNDZMREB-ACZMJKKPSA-N 0.000 description 1
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 1
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 1
- IFKQPMZRDQZSHI-GHCJXIJMSA-N Ala-Ile-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O IFKQPMZRDQZSHI-GHCJXIJMSA-N 0.000 description 1
- IAUSCRHURCZUJP-CIUDSAMLSA-N Ala-Lys-Cys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CS)C(O)=O IAUSCRHURCZUJP-CIUDSAMLSA-N 0.000 description 1
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- AENHOIXXHKNIQL-AUTRQRHGSA-N Ala-Tyr-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H]([NH3+])C)CC1=CC=C(O)C=C1 AENHOIXXHKNIQL-AUTRQRHGSA-N 0.000 description 1
- YEBZNKPPOHFZJM-BPNCWPANSA-N Ala-Tyr-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O YEBZNKPPOHFZJM-BPNCWPANSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- 101710194180 Alcohol oxidase 1 Proteins 0.000 description 1
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- OLDOLPWZEMHNIA-PJODQICGSA-N Arg-Ala-Trp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O OLDOLPWZEMHNIA-PJODQICGSA-N 0.000 description 1
- OTUQSEPIIVBYEM-IHRRRGAJSA-N Arg-Asn-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OTUQSEPIIVBYEM-IHRRRGAJSA-N 0.000 description 1
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 1
- WAEWODAAWLGLMK-OYDLWJJNSA-N Arg-Trp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N WAEWODAAWLGLMK-OYDLWJJNSA-N 0.000 description 1
- FTMRPIVPSDVGCC-GUBZILKMSA-N Arg-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FTMRPIVPSDVGCC-GUBZILKMSA-N 0.000 description 1
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 1
- ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N Asn-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N 0.000 description 1
- HJRBIWRXULGMOA-ACZMJKKPSA-N Asn-Gln-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJRBIWRXULGMOA-ACZMJKKPSA-N 0.000 description 1
- ZMUQQMGITUJQTI-CIUDSAMLSA-N Asn-Leu-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZMUQQMGITUJQTI-CIUDSAMLSA-N 0.000 description 1
- GZXOUBTUAUAVHD-ACZMJKKPSA-N Asn-Ser-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GZXOUBTUAUAVHD-ACZMJKKPSA-N 0.000 description 1
- NCXTYSVDWLAQGZ-ZKWXMUAHSA-N Asn-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O NCXTYSVDWLAQGZ-ZKWXMUAHSA-N 0.000 description 1
- FMNBYVSGRCXWEK-FOHZUACHSA-N Asn-Thr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O FMNBYVSGRCXWEK-FOHZUACHSA-N 0.000 description 1
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 1
- NECWUSYTYSIFNC-DLOVCJGASA-N Asp-Ala-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 NECWUSYTYSIFNC-DLOVCJGASA-N 0.000 description 1
- TVVYVAUGRHNTGT-UGYAYLCHSA-N Asp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O TVVYVAUGRHNTGT-UGYAYLCHSA-N 0.000 description 1
- PXLNPFOJZQMXAT-BYULHYEWSA-N Asp-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O PXLNPFOJZQMXAT-BYULHYEWSA-N 0.000 description 1
- FTNVLGCFIJEMQT-CIUDSAMLSA-N Asp-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N FTNVLGCFIJEMQT-CIUDSAMLSA-N 0.000 description 1
- XDGBFDYXZCMYEX-NUMRIWBASA-N Asp-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N)O XDGBFDYXZCMYEX-NUMRIWBASA-N 0.000 description 1
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 description 1
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 1
- RTXQQDVBACBSCW-CFMVVWHZSA-N Asp-Ile-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RTXQQDVBACBSCW-CFMVVWHZSA-N 0.000 description 1
- PCJOFZYFFMBZKC-PCBIJLKTSA-N Asp-Phe-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PCJOFZYFFMBZKC-PCBIJLKTSA-N 0.000 description 1
- MGSVBZIBCCKGCY-ZLUOBGJFSA-N Asp-Ser-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MGSVBZIBCCKGCY-ZLUOBGJFSA-N 0.000 description 1
- GCACQYDBDHRVGE-LKXGYXEUSA-N Asp-Thr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC(O)=O GCACQYDBDHRVGE-LKXGYXEUSA-N 0.000 description 1
- MRYDJCIIVRXVGG-QEJZJMRPSA-N Asp-Trp-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O MRYDJCIIVRXVGG-QEJZJMRPSA-N 0.000 description 1
- KNOGLZBISUBTFW-QRTARXTBSA-N Asp-Trp-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O KNOGLZBISUBTFW-QRTARXTBSA-N 0.000 description 1
- USENATHVGFXRNO-SRVKXCTJSA-N Asp-Tyr-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 USENATHVGFXRNO-SRVKXCTJSA-N 0.000 description 1
- KNDCWFXCFKSEBM-AVGNSLFASA-N Asp-Tyr-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O KNDCWFXCFKSEBM-AVGNSLFASA-N 0.000 description 1
- AWPWHMVCSISSQK-QWRGUYRKSA-N Asp-Tyr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O AWPWHMVCSISSQK-QWRGUYRKSA-N 0.000 description 1
- XWKPSMRPIKKDDU-RCOVLWMOSA-N Asp-Val-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O XWKPSMRPIKKDDU-RCOVLWMOSA-N 0.000 description 1
- GGBQDSHTXKQSLP-NHCYSSNCSA-N Asp-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N GGBQDSHTXKQSLP-NHCYSSNCSA-N 0.000 description 1
- QOJJMJKTMKNFEF-ZKWXMUAHSA-N Asp-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O QOJJMJKTMKNFEF-ZKWXMUAHSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- VZKXOWRNJDEGLZ-WHFBIAKZSA-N Cys-Asp-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O VZKXOWRNJDEGLZ-WHFBIAKZSA-N 0.000 description 1
- WDQXKVCQXRNOSI-GHCJXIJMSA-N Cys-Asp-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WDQXKVCQXRNOSI-GHCJXIJMSA-N 0.000 description 1
- LHMSYHSAAJOEBL-CIUDSAMLSA-N Cys-Lys-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O LHMSYHSAAJOEBL-CIUDSAMLSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- DTCCMDYODDPHBG-ACZMJKKPSA-N Gln-Ala-Cys Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(O)=O DTCCMDYODDPHBG-ACZMJKKPSA-N 0.000 description 1
- UICOTGULOUGGLC-NUMRIWBASA-N Gln-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O UICOTGULOUGGLC-NUMRIWBASA-N 0.000 description 1
- OFPWCBGRYAOLMU-AVGNSLFASA-N Gln-Asp-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O OFPWCBGRYAOLMU-AVGNSLFASA-N 0.000 description 1
- DSPQRJXOIXHOHK-WDSKDSINSA-N Glu-Asp-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O DSPQRJXOIXHOHK-WDSKDSINSA-N 0.000 description 1
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 1
- WVTIBGWZUMJBFY-GUBZILKMSA-N Glu-His-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O WVTIBGWZUMJBFY-GUBZILKMSA-N 0.000 description 1
- ZSWGJYOZWBHROQ-RWRJDSDZSA-N Glu-Ile-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSWGJYOZWBHROQ-RWRJDSDZSA-N 0.000 description 1
- RXESHTOTINOODU-JYJNAYRXSA-N Glu-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCC(=O)O)N RXESHTOTINOODU-JYJNAYRXSA-N 0.000 description 1
- MRWYPDWDZSLWJM-ACZMJKKPSA-N Glu-Ser-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O MRWYPDWDZSLWJM-ACZMJKKPSA-N 0.000 description 1
- ZQNCUVODKOBSSO-XEGUGMAKSA-N Glu-Trp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O ZQNCUVODKOBSSO-XEGUGMAKSA-N 0.000 description 1
- HAGKYCXGTRUUFI-RYUDHWBXSA-N Glu-Tyr-Gly Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)O)N)O HAGKYCXGTRUUFI-RYUDHWBXSA-N 0.000 description 1
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 1
- VXKCPBPQEKKERH-IUCAKERBSA-N Gly-Arg-Pro Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N1CCC[C@H]1C(O)=O VXKCPBPQEKKERH-IUCAKERBSA-N 0.000 description 1
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 1
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 1
- HDNXXTBKOJKWNN-WDSKDSINSA-N Gly-Glu-Asn Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O HDNXXTBKOJKWNN-WDSKDSINSA-N 0.000 description 1
- PDAWDNVHMUKWJR-ZETCQYMHSA-N Gly-Gly-His Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CNC=N1 PDAWDNVHMUKWJR-ZETCQYMHSA-N 0.000 description 1
- HAXARWKYFIIHKD-ZKWXMUAHSA-N Gly-Ile-Ser Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HAXARWKYFIIHKD-ZKWXMUAHSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- WDEHMRNSGHVNOH-VHSXEESVSA-N Gly-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)CN)C(=O)O WDEHMRNSGHVNOH-VHSXEESVSA-N 0.000 description 1
- HFPVRZWORNJRRC-UWVGGRQHSA-N Gly-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN HFPVRZWORNJRRC-UWVGGRQHSA-N 0.000 description 1
- LBDXVCBAJJNJNN-WHFBIAKZSA-N Gly-Ser-Cys Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O LBDXVCBAJJNJNN-WHFBIAKZSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 1
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 1
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- NGBGZCUWFVVJKC-IRXDYDNUSA-N Gly-Tyr-Tyr Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NGBGZCUWFVVJKC-IRXDYDNUSA-N 0.000 description 1
- YDIDLLVFCYSXNY-RCOVLWMOSA-N Gly-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN YDIDLLVFCYSXNY-RCOVLWMOSA-N 0.000 description 1
- DFHVLUKTTVTCKY-PBCZWWQYSA-N His-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N)O DFHVLUKTTVTCKY-PBCZWWQYSA-N 0.000 description 1
- UPGJWSUYENXOPV-HGNGGELXSA-N His-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N UPGJWSUYENXOPV-HGNGGELXSA-N 0.000 description 1
- RGPWUJOMKFYFSR-QWRGUYRKSA-N His-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RGPWUJOMKFYFSR-QWRGUYRKSA-N 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- LQSBBHNVAVNZSX-GHCJXIJMSA-N Ile-Ala-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N LQSBBHNVAVNZSX-GHCJXIJMSA-N 0.000 description 1
- IIXDMJNYALIKGP-DJFWLOJKSA-N Ile-Asn-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N IIXDMJNYALIKGP-DJFWLOJKSA-N 0.000 description 1
- GLYJPWIRLBAIJH-FQUUOJAGSA-N Ile-Lys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N GLYJPWIRLBAIJH-FQUUOJAGSA-N 0.000 description 1
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 1
- CKRFDMPBSWYOBT-PPCPHDFISA-N Ile-Lys-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CKRFDMPBSWYOBT-PPCPHDFISA-N 0.000 description 1
- WYUHAXJAMDTOAU-IAVJCBSLSA-N Ile-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N WYUHAXJAMDTOAU-IAVJCBSLSA-N 0.000 description 1
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 1
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 1
- QQVXERGIFIRCGW-NAKRPEOUSA-N Ile-Ser-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)O)N QQVXERGIFIRCGW-NAKRPEOUSA-N 0.000 description 1
- JDCQDJVYUXNCGF-SPOWBLRKSA-N Ile-Ser-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N JDCQDJVYUXNCGF-SPOWBLRKSA-N 0.000 description 1
- HJDZMPFEXINXLO-QPHKQPEJSA-N Ile-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N HJDZMPFEXINXLO-QPHKQPEJSA-N 0.000 description 1
- QHUREMVLLMNUAX-OSUNSFLBSA-N Ile-Thr-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)O)N QHUREMVLLMNUAX-OSUNSFLBSA-N 0.000 description 1
- ZFWISYLMLXFBSX-KKPKCPPISA-N Ile-Trp-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N ZFWISYLMLXFBSX-KKPKCPPISA-N 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- DLCOFDAHNMMQPP-SRVKXCTJSA-N Leu-Asp-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DLCOFDAHNMMQPP-SRVKXCTJSA-N 0.000 description 1
- WQWSMEOYXJTFRU-GUBZILKMSA-N Leu-Glu-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O WQWSMEOYXJTFRU-GUBZILKMSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- JRJLGNFWYFSJHB-HOCLYGCPSA-N Leu-Gly-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JRJLGNFWYFSJHB-HOCLYGCPSA-N 0.000 description 1
- USLNHQZCDQJBOV-ZPFDUUQYSA-N Leu-Ile-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O USLNHQZCDQJBOV-ZPFDUUQYSA-N 0.000 description 1
- JKSIBWITFMQTOA-XUXIUFHCSA-N Leu-Ile-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O JKSIBWITFMQTOA-XUXIUFHCSA-N 0.000 description 1
- IFMPDNRWZZEZSL-SRVKXCTJSA-N Leu-Leu-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O IFMPDNRWZZEZSL-SRVKXCTJSA-N 0.000 description 1
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 1
- MVHXGBZUJLWZOH-BJDJZHNGSA-N Leu-Ser-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MVHXGBZUJLWZOH-BJDJZHNGSA-N 0.000 description 1
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 1
- LFSQWRSVPNKJGP-WDCWCFNPSA-N Leu-Thr-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(O)=O LFSQWRSVPNKJGP-WDCWCFNPSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- QUCDKEKDPYISNX-HJGDQZAQSA-N Lys-Asn-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QUCDKEKDPYISNX-HJGDQZAQSA-N 0.000 description 1
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 1
- LUTDBHBIHHREDC-IHRRRGAJSA-N Lys-Pro-Lys Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O LUTDBHBIHHREDC-IHRRRGAJSA-N 0.000 description 1
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 1
- RPWQJSBMXJSCPD-XUXIUFHCSA-N Lys-Val-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(O)=O RPWQJSBMXJSCPD-XUXIUFHCSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010047562 NGR peptide Proteins 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- WYPVCIACUMJRIB-JYJNAYRXSA-N Phe-Gln-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N WYPVCIACUMJRIB-JYJNAYRXSA-N 0.000 description 1
- FIRWJEJVFFGXSH-RYUDHWBXSA-N Phe-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 FIRWJEJVFFGXSH-RYUDHWBXSA-N 0.000 description 1
- YYKZDTVQHTUKDW-RYUDHWBXSA-N Phe-Gly-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N YYKZDTVQHTUKDW-RYUDHWBXSA-N 0.000 description 1
- ISYSEOWLRQKQEQ-JYJNAYRXSA-N Phe-His-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISYSEOWLRQKQEQ-JYJNAYRXSA-N 0.000 description 1
- QTDBZORPVYTRJU-KKXDTOCCSA-N Phe-Tyr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O QTDBZORPVYTRJU-KKXDTOCCSA-N 0.000 description 1
- KLSOMAFWRISSNI-OSUNSFLBSA-N Pro-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 KLSOMAFWRISSNI-OSUNSFLBSA-N 0.000 description 1
- HATVCTYBNCNMAA-AVGNSLFASA-N Pro-Leu-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O HATVCTYBNCNMAA-AVGNSLFASA-N 0.000 description 1
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 1
- OOKCGAYXSNJBGQ-ZLUOBGJFSA-N Ser-Asn-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OOKCGAYXSNJBGQ-ZLUOBGJFSA-N 0.000 description 1
- YMEXHZTVKDAKIY-GHCJXIJMSA-N Ser-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO)C(O)=O YMEXHZTVKDAKIY-GHCJXIJMSA-N 0.000 description 1
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-N 0.000 description 1
- YQQKYAZABFEYAF-FXQIFTODSA-N Ser-Glu-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQQKYAZABFEYAF-FXQIFTODSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- IXCHOHLPHNGFTJ-YUMQZZPRSA-N Ser-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N IXCHOHLPHNGFTJ-YUMQZZPRSA-N 0.000 description 1
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 1
- JIPVNVNKXJLFJF-BJDJZHNGSA-N Ser-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N JIPVNVNKXJLFJF-BJDJZHNGSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 description 1
- KKKVOZNCLALMPV-XKBZYTNZSA-N Ser-Thr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KKKVOZNCLALMPV-XKBZYTNZSA-N 0.000 description 1
- FLMYSKVSDVHLEW-SVSWQMSJSA-N Ser-Thr-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLMYSKVSDVHLEW-SVSWQMSJSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 1
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 description 1
- LYGKYFKSZTUXGZ-ZDLURKLDSA-N Thr-Cys-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)NCC(O)=O LYGKYFKSZTUXGZ-ZDLURKLDSA-N 0.000 description 1
- ZLNWJMRLHLGKFX-SVSWQMSJSA-N Thr-Cys-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZLNWJMRLHLGKFX-SVSWQMSJSA-N 0.000 description 1
- KGKWKSSSQGGYAU-SUSMZKCASA-N Thr-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KGKWKSSSQGGYAU-SUSMZKCASA-N 0.000 description 1
- CQNFRKAKGDSJFR-NUMRIWBASA-N Thr-Glu-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O CQNFRKAKGDSJFR-NUMRIWBASA-N 0.000 description 1
- LGNBRHZANHMZHK-NUMRIWBASA-N Thr-Glu-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O LGNBRHZANHMZHK-NUMRIWBASA-N 0.000 description 1
- VULNJDORNLBPNG-SWRJLBSHSA-N Thr-Glu-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O VULNJDORNLBPNG-SWRJLBSHSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- NWECYMJLJGCBOD-UNQGMJICSA-N Thr-Phe-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O NWECYMJLJGCBOD-UNQGMJICSA-N 0.000 description 1
- NYQIZWROIMIQSL-VEVYYDQMSA-N Thr-Pro-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O NYQIZWROIMIQSL-VEVYYDQMSA-N 0.000 description 1
- XZUBGOYOGDRYFC-XGEHTFHBSA-N Thr-Ser-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O XZUBGOYOGDRYFC-XGEHTFHBSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- KXFYAQUYJKOQMI-QEJZJMRPSA-N Trp-Ser-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 KXFYAQUYJKOQMI-QEJZJMRPSA-N 0.000 description 1
- SUGLEXVWEJOCGN-ONUFPDRFSA-N Trp-Thr-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)N)O SUGLEXVWEJOCGN-ONUFPDRFSA-N 0.000 description 1
- VNRTXOUAOUZCFW-WDSOQIARSA-N Trp-Val-His Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O VNRTXOUAOUZCFW-WDSOQIARSA-N 0.000 description 1
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 1
- JWGXUKHIKXZWNG-RYUDHWBXSA-N Tyr-Gly-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O JWGXUKHIKXZWNG-RYUDHWBXSA-N 0.000 description 1
- NXRGXTBPMOGFID-CFMVVWHZSA-N Tyr-Ile-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O NXRGXTBPMOGFID-CFMVVWHZSA-N 0.000 description 1
- ILTXFANLDMJWPR-SIUGBPQLSA-N Tyr-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N ILTXFANLDMJWPR-SIUGBPQLSA-N 0.000 description 1
- YYLHVUCSTXXKBS-IHRRRGAJSA-N Tyr-Pro-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YYLHVUCSTXXKBS-IHRRRGAJSA-N 0.000 description 1
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 1
- HZDQUVQEVVYDDA-ACRUOGEOSA-N Tyr-Tyr-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HZDQUVQEVVYDDA-ACRUOGEOSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- OQWNEUXPKHIEJO-NRPADANISA-N Val-Glu-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N OQWNEUXPKHIEJO-NRPADANISA-N 0.000 description 1
- WNZSAUMKZQXHNC-UKJIMTQDSA-N Val-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N WNZSAUMKZQXHNC-UKJIMTQDSA-N 0.000 description 1
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 description 1
- YDVDTCJGBBJGRT-GUBZILKMSA-N Val-Met-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)O)N YDVDTCJGBBJGRT-GUBZILKMSA-N 0.000 description 1
- XBJKAZATRJBDCU-GUBZILKMSA-N Val-Pro-Ala Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XBJKAZATRJBDCU-GUBZILKMSA-N 0.000 description 1
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 1
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 1
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 108010028939 alanyl-alanyl-lysyl-alanine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 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 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 108010009297 diglycyl-histidine Proteins 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010042598 glutamyl-aspartyl-glycine Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01011—Pectinesterase (3.1.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种剪接果胶酶基因DNA中内含子的方法。所述方法以果胶酶基因作为模板进行融合PCR扩增得到所需基因片段，所述果胶酶基因包括内切聚半乳糖醛酸酶基因PGII和果胶甲酯酶基因PmeA，其中PGII基因扩增引物包括序列SEQ ID NO:1～SEQ ID NO:4；PmeA基因扩增引物包括序列SEQ ID NO:5～SEQ ID NO:18。本发明设计了多对引物，将基因中内含子进行剪接，筛选的引物序列特异性优异，灵敏度高；本发明融合PCR操作简单、高效、快捷扩增片段DNA；涉及的PCR扩增反应体系较小，检测所需试剂量也少，经济适用，还能减轻实验废弃物对环境的污染。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种剪接果胶酶基因中内含子的方法。
背景技术
果胶酶是指分解植物主要成分—果胶质的酶类。果胶酶广泛分布于高等植物和微生物中，根据其作用底物的不同分为两类，一类能催化果胶解聚如内切聚半乳糖醛酸酶，另一类能催化果胶分子中酯水解如果胶酯酶。内切聚半乳糖醛酸酶以随机的方式切割底物，酶解产物主要是半乳糖醛酸三糖和四糖。果胶甲酯酶是一种专一催化果胶分子长链上甲酯基水解，生成低酯果胶的一种酶。它不会减小果胶聚合物的体积，能较好地保持产品的胶凝度。内切聚半乳糖醛酸酶及果胶酯酶具有重要的应用价值，其已被广泛应用于食品、饲料、造纸、纺织和果胶废液处理等领域。主要应用于食品领域，特别是果蔬汁、果酒的加工，其能够显著提高压榨果蔬汁的出汁率，降低果汁果酒的粘度，增加澄清效果，从而保证果汁果酒的品质。因而对内切聚半乳糖醛酸酶及果胶酯酶的研究具有较高的潜在应用价值。
发明内容
本发明解决的问题在于提供一种剪接塔宾曲霉果胶酶基因中内含子的方法，该方法是利用融合PCR将基因片段中的内含子去除。
本发明的目的通过如下技术方案实现：
一种剪接果胶酶基因中内含子的方法，所述方法以果胶酶基因作为模板进行融合PCR扩增得到所需基因片段，所述果胶酶基因包括内切聚半乳糖醛酸酶基因PGII和果胶甲酯酶基因PmeA，其中PGII基因扩增引物包括序列SEQ ID NO:1～SEQ ID NO:4；PmeA基因扩增引物包括序列SEQ ID NO:5～SEQ ID NO:18。
优选的，所述果胶酶基因为塔宾曲霉Aspergillus tubingensis GYC 501所含有的果胶酶基因，所述塔宾曲霉Aspergillus tubingensis GYC 501保藏号为CCTCC M2014425。
优选的，所述PGII基因由内含子分隔的两个外显子分别表示为PGII 1～2，其中：
PGII 1～2反应体系20μL：2×Phanta Max Master Mix 10μL,GYC 501 GenomeDNA 1μL,正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
反应程序：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，30个循环，延伸72℃X min；
PGII 1的扩增：反应体系中正向引物为SEQ ID NO:1，反向引物为SEQ ID NO:2；反应程序：延伸时间X为5min；
PGII 2的扩增：反应体系中正向引物为SEQ ID NO:3，反向引物为SEQ ID NO:4；反应程序：延伸时间X为5min；
PGII 1和PGII 2的连接序列PGII 3的扩增：反应体系50μL：2×Phanta MaxMaster Mix 25μL,PGII-1 1μL,PGII-2 1μL,SEQ ID NO:1 2μL,SEQ ID NO:4 2μL,ddH2O19μL，所述体系模板由PGII 1和PGII 2的扩增产物分别稀释500倍体积制得；反应程序：延伸时间X为1min。优选的，所述PmeA基因由内含子分隔的七个外显子分别表示为PmeA1～7，其中：
PmeA1～7反应体系20μL：2×Phanta Max Master Mix 10μL,GYC 501 Genome DNA1μL,正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
PmeA8～13反应体系20μL：2×Phanta Max Master Mix 10μL,模板A 0.5μL,模板B0.5μL，正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
反应程序：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，30个循环，延伸72℃5min；
PmeA1的扩增：反应体系中正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:6；
PmeA2的扩增：反应体系中正向引物为SEQ ID NO:7，反向引物为SEQ ID NO:8；
PmeA3的扩增：反应体系中正向引物为SEQ ID NO:9，反向引物为SEQ ID NO:10；
PmeA4的扩增：反应体系中正向引物为SEQ ID NO:11，反向引物为SEQ ID NO:12；
PmeA5的扩增：反应体系中正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:14；
PmeA6的扩增：反应体系中正向引物为SEQ ID NO:15，反向引物为SEQ ID NO:16；
PmeA7的扩增：反应体系中正向引物为SEQ ID NO:17，反向引物为SEQ ID NO:18；
PmeA 1和PmeA 2的连接序列PmeA 8的扩增：反应体系：模板A和模板B分别由PmeA1、PmeA2的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ IDNO:8；
PmeA 3和PmeA 4的连接序列PmeA 9的扩增：模板A和模板B分别由PmeA3、PmeA4的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:9，反向引物为SEQ ID NO:12；
PmeA 5和PmeA 6的连接序列PmeA 10的扩增：模板A和模板B分别由PmeA5、PmeA6的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:16；
PmeA 8和PmeA 9的连接序列PmeA 11的扩增：模板A和模板B分别由PmeA8、PmeA9的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:12；
PmeA 10和PmeA 7的连接序列PmeA 12的扩增：模板A和模板B分别由PmeA10、PmeA7的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:18；
PmeA 11和PmeA 12的连接序列PmeA 13的扩增：模板A和模板B分别由PmeA11、PmeA12的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:18。
本发明为了将基因中内含子进行剪接，设计了多对引物，筛选的引物序列特异性十分优异，灵敏度高。本发明PCR扩增反应体系较小，检测所需试剂量也少，经济适用，还能减轻实验废弃物对环境的污染。本发明融合PCR操作简单、高效、快捷扩增片段DNA。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是PGII-1～3的琼脂糖电泳图，条带1：583bp，条带2：500bp，条带3：1038bp。
图2是为PmeA1～13的琼脂糖电泳图，上半图中条带1～7分别为：88bp 167bp、110bp、177bp、91bp、225bp、281bp，下半图中条带8～13分别为：225bp、257bp、286bp、452bp、537bp、959bp。
图3为PGII酶切验证图，图中载体条带2：9276bp，目的片段条带1:1038bp；
图4为PmeA酶切验证图；图中载体条带2:9276bp，目的片段条带1：959bp；
图5为蛋白凝胶图像；
图6为rePGII的酶活力分析图。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料和试剂，如无特殊说明，均可从商业途径得到。
实施例1
1.基因的获得
塔宾曲霉菌株(Aspergillus tubingensis GYC 501)和基因组提取方法出自之前的研究(Zhenxing,Haorui,Hong,Zhihua,Lizheng,Yun,et al.)，菌株保藏号为CCTCC M2014425。针对内切聚半乳糖醛酸酶基因PGII和果胶甲酯酶基因PmeA分别设计的引物如下所示：
PGII：
TubPGII F(SEQ ID NO:1)：GGAATTCCATCACCATCACCATCACGGAAGCTGCACCTTCAAAA
TubPGIIIntron F(SEQ ID NO:2)：CCAGATGTTCTCGCCAGAATTGATCGCAAG
TubPGIIIntron R(SEQ ID NO:3)：ATTCTGGCGAGAACATCTGGTTCACCGGCG
TubPGII R(SEQ ID NO:4)：ATAAGAATGCGGCCGCGCTACAACCGACCAACCT
PmeA：
TubPmeA F(SEQ ID NO:5)：GGAATTCGCGAGCCGCATGACGGCT
TubPmeAIntron 1 F(SEQ ID NO:6)：AACGGCAGCGCTGATCGTGTCGTAGTCACC
TubPmeAIntron 1 R(SEQ ID NO:7)：GGTGACTACGACACGATCAGCGCTGCCGTT
TubPmeAIntron 2 F(SEQ ID NO:8)：GTGTAGGTAGTGGTGTCCTCAGTCTGACCG
TubPmeAIntron 2 R(SEQ ID NO:9)：CGGTCAGACTGAGGACACCACTACCTACAC
TubPmeAIntron 3 F(SEQ ID NO:10)：AGTTACGAAGGGTTGCAGTCTCATCGTCAT
TubPmeAIntron 3 R(SEQ ID NO:11)：ATGACGATGAGACTGCAACCCTTCGTAACT
TubPmeAIntron 4 F(SEQ ID NO:12)：CGGCCAGAAGGGTGTCCTGGTATCCGGTGA
TubPmeAIntron 4 R(SEQ ID NO:13)：TCACCGGATACCAGGACACCCTTCTGGCCG
TubPmeAIntron 5 F(SEQ ID NO:14)：CCGAAGATGAAGTCAACGGCACCCTCGATG
TubPmeAIntron 5 R(SEQ ID NO:15)：CATCGAGGGTGCCGTTGACTTCATCTTCGG
TubPmeAIntron 6 F(SEQ ID NO:16)：TGGGACCAGGGGCGGCCGAGGTAGTAGGTG
TubPmeAIntron 6 R(SEQ ID NO:17)：CACCTACTACCTCGGCCGCCCCTGGTCCCA
TubPmeA R(SEQ ID NO:18)：GGAATTCTTAGTTGATGTAGCTAGT
因为PGII有一个内含子，所以采用融合PCR的策略将基因片段内的内含子去除。TubPGII F在上游为目的基因引入了一个EcoR I酶切位点和六个组氨酸残基，TubPGII R在下游为目的基因引入了一个Not I酶切位点。PCR使用的聚合酶是
Max MasterMix(购于Vazyme biotech Co.,Ltd,Nanjing,China，型号P515-01，规格1mL)。PGII的融合PCR分为两步，第一步分别使用TubPGII F-TubPGIIIntron F和TubPGIIIntron R-TubPGIIR分别扩增出PGII基因内含子上游和下游的序列，反应体系20μL(2×Phanta Max MasterMix 10μL,GYC 501Genome DNA 1μL,Forward primer 0.8μL,Reverse primer 0.8μL,ddH2O 7.4μL)，反应条件：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，从变性到延伸重复30个循环，最后一步彻底延伸72℃5min。以上PCR步骤得到PGII-1与PGII-2，用无菌蒸馏水将它们分别稀释500倍，加入融合PCR体系(2×Phanta Max Master Mix 25μL,PGII-1 1μL,PGII-2 1μL,TubPGII F 2μL,TubPGII R 2μL,ddH2O 19μL)，反应条件于上一步一致，但延伸时间变为1min。这次PCR得到PGII-3，即去掉内含子的PGII基因片段。
TubPmeA F和TubPmeA R在基因上游与下游引入了EcoR I酶切位点。PmeA基因有6段内含子，利用融合PCR获得不含内含子的PmeA序列分为四步：第一步将7对引物TubPmeAF-TubPmeAIntron 1 F、TubPmeAIntron 1 R-TubPmeAIntron 2 F、TubPmeAIntron 2 R-TubPmeAIntron 3 F、TubPmeAIntron 3 R-TubPmeAIntron 4 F、TubPmeAIntron 4 R-TubPmeAIntron 5F、TubPmeAIntron 5 R-TubPmeAIntron 6 F、TubPmeAIntron 6 R-TubPmeA R按照PGII的第一次PCR方法进行，即反应体系20μL(2×Phanta Max Master Mix10μL,GYC 501 Genome DNA 1μL,Forward primer 0.8μL,Reverse primer 0.8μL,ddH2O7.4μL)，反应条件：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，从变性到延伸重复30个循环，最后一步彻底延伸72℃5min。分别得到片段PmeA1～7；第二步将第一步PCR产物稀释500倍，作为模板，各取0.5μL加入到与上一步相同的新的PCR体系中，反应条件不变，PmeA-1与PmeA-2作为模板的体系使用TubPmeA F-TubPmeAIntron 2 F的引物对，PmeA-3与PmeA-4使用TubPmeAIntron 2 R-TubPmeAIntron 4 F，PmeA-5与PmeA-6使用TubPmeAIntron 4 R-TubPmeAIntron 6 F，这一步分别得到PmeA-8～10；第三步将第二步PCR产物稀释500倍，作为模板，各取0.5μL加入到与上一步相同的新的PCR体系中，反应条件不变，第三步PCR模板PmeA-8与PmeA-9使用TubPmeA F-TubPmeAIntron 4 F的引物对，PmeA-10与PmeA-7使用TubPmeAIntron 4 R-TubPmeA R，这一步得到PmeA-11与PmeA-12；最后一步融合PCR以PmeA-11和PmeA-12为模板使用PmeA F-PmeA R作为引物，第四步将第三步PCR产物稀释500倍，作为模板，各取0.5μL加入到与上一步相同的新的PCR体系中，反应条件不变，得到PmeA 13，即去掉内含子的PmeA基因片段。
融合PCR产物纯化后于-20℃储藏待用。
图1是PGII-1～3是各外显子和融合基因的琼脂糖电泳图，图2是PmeA1～13各外显子和融合基因的琼脂糖电泳图。
rePGII(重组PGII)成熟蛋白对应的核酸序列(包括信号肽残留序列(GCT TACGTA)+EcoR I(GAA TTC)+组氨酸标签(CAT CAC CAT CAC CAT CAC))如SEQ ID NO:19所示。
rePGII成熟蛋白对应的氨基酸序列(包括信号肽残留序列(AYV)+EcoR I(EF)+组氨酸标签(HHHHHH))如SEQ ID NO:20所示。
rePmeA(重组PmeA)成熟蛋白对应的核酸序列(包括信号肽残留序列(GCT TACGTA)+EcoR I(GAA TTC))如SEQ ID NO:21所示。
rePmeA成熟蛋白对应的氨基酸序列(包括信号肽残留序列(AYV)+EcoR I(EF))如SEQ ID NO:22所示。
2.重组质粒的制作与处理
质粒为穿梭质粒pPIC9K(购于Invitrogen)，复制宿主为大肠杆菌DH-5α。提取质粒后，将质粒与去掉内含子的基因都用核酸限制性内切酶(购于Thermo Fisher FastDigest)消化，对于PGII使用EcoR I与Not I，而PmeA只使用EcoR I消化，纯化后用T4 Ligase(购于Takara,Dalian)连接，纯化后于-20℃储藏待用。
制作大肠杆菌DH-5α电击感受态细胞，将连接产物用电击转化法转入感受态细胞，使用卡那霉素抗性LB平板筛选转化子，使用菌落PCR的方法鉴定转化子的正确性：PCR用酶为CWBIOTECH(购于Beijing,China)的2×Es Taq MasterMix，事先在每个PCR小管中加入3μL ddH2O，在超净工作台中用灭菌牙签取少许菌落，并放入PCR小管中搅动数次，随后加入PCR体系(2×Es Taq MasterMix 7.5μL,α-Factor 0.6μL,TubPGII R或TubPmeA R 0.6μL,ddH2O 5μL)，反应条件：预变性94℃5min，变性94℃30s，退火56℃30s，延伸72℃30s或40s，从变性到延伸重复29个循环，最后一步彻底延伸72℃2min。用于涂平板的转化体系中有未进入细胞的正确质粒存在，因此PCR循环从30个循环减少到29个循环，以减少假阳性条带过亮而影响判断。选取最亮条带的克隆使用试剂盒(Plasmid Mini Kit I,OMEGA bio-tekInc.,U.S.)提取质粒，并用EcoR I(GS115-rePmeA)或EcoR I/Not I(GS115-rePGII)酶切验证，验证图如图3和图4所示。
质粒验证正确后以引物5’AOX1或α-Factor/3’AOX1测序质粒，确定质粒正确性后-80℃保藏质粒宿主。
3.表达载体的构建
制作毕赤酵母GS115(购于Invitrogen)电击感受态，将约4㎎重组质粒加入100μL感受态，按照参数(1.5kV,2.5μF,200Ω,5ms)使用电转仪(Bio-Rad)电击转化。涂布100-200μL转化体系至RDB或MD平板，2.5d后进行菌落PCR，步骤与方法同上一节，只是需要在加入PCR体系前使用-20℃/100℃各5min使酵母菌体裂解。选取条带最亮的编号进行下一步表达。
4.蛋白表达
挑取菌体入装有5mL BMGY的指形瓶中，恒温摇床30℃(180rpm)培养48h，遂取3mL5000rpm常温离心1min后弃去上清液，吸净残余培养液后重悬到装有50mL BMMY的500mL锥形瓶中，恒温摇床30℃(180rpm)培养7d，在BMGY、BMMY中培养时以12h为间隔取300μL酶液12000rpm 4℃离心5min取上清液测定酶活力，在BMMY中培养时每24h补加3mL BMMY，再与其错开12h每24h补加250μL甲醇至终浓度为0.5％。取1-7d以及-24h(转入BMMY前24h)、0d(BMGY培养48h)的培养液上清12000rpm 4℃离心5min，每40μL酶液加入10μL蛋白质电泳上样缓冲液，沸水浴5min，进行SDS-PAGE，蛋白凝胶图像如5所示。
5.酶活性检测
5.1 GS115-rePGII
rePGII属于内切聚半乳糖醛酸酶，可以分解聚半乳糖醛酸生成α-半乳糖醛酸，α-半乳糖醛酸与DNS形成的络合物在540nm处有最大吸收峰，遂采用DNS法测定此酶酶活。
配制半乳糖醛酸浓度梯度液，取各浓度半乳糖醛酸液体0.075mL，加入0.3mLddH2O，0.375mL DNS，沸水显色5min，放入凉水冷却，加入1.25mL ddH2O，混匀后测定540nm处的吸光值，制成半乳糖醛酸标准曲线。
将酶液稀释，取0.075mL加入到0.3mL 0.5％聚半乳糖醛酸溶液(使用pH 4.0的柠檬酸-柠檬酸钠缓冲体系配制)中，所述聚半乳糖醛酸底物(购于源叶生物，生物技术级，85％)，混匀后置于40℃水浴30min，遂放入冰浴暂停反应，加入0.375mL DNS溶液终止反应，空白对照在此步之后加入酶液(不需灭活)，沸水浴5min，加入1.25mL ddH2O，于540nm处测定吸光值，减去空白对照的吸光值即可真实反映酶活力。
酶活力定义：在上述反应条件下，每毫升酶液每小时分解聚半乳糖醛酸，生成1㎎半乳糖醛酸，视为一个聚半乳糖醛酸酶酶活(U)。酶活计算方法如下(半乳糖醛酸含量通过标准曲线公式计算)：
rePGII的酶活力从-24h(换入BMMY前24h)就已经呈现，并在此后持续增加。
rePGII(内切聚半乳糖醛酸酶)暂未检测出果胶酶活性，有聚半乳糖醛酸酶活性，十万级别。
rePmeA(果胶甲酯酶)暂未检测出果胶酶活性，但有一定聚半乳糖醛酸酶活性。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序 列 表
SEQUENCE LISTING
<110> 广西中烟工业有限责任公司
<120> 一种剪接果胶酶基因中内含子的方法
<130> HA201904304-L
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 44
<212> DNA
<213> 人工合成
<400> 1
ggaattccat caccatcacc atcacggaag ctgcaccttc aaaa 44
<210> 2
<211> 30
<212> DNA
<213> 人工合成
<400> 2
ccagatgttc tcgccagaat tgatcgcaag 30
<210> 3
<211> 30
<212> DNA
<213> 人工合成
<400> 3
attctggcga gaacatctgg ttcaccggcg 30
<210> 4
<211> 34
<212> DNA
<213> 人工合成
<400> 4
ataagaatgc ggccgcgcta caaccgacca acct 34
<210> 5
<211> 25
<212> DNA
<213> 人工合成
<400> 5
ggaattcgcg agccgcatga cggct 25
<210> 6
<211> 30
<212> DNA
<213> 人工合成
<400> 6
aacggcagcg ctgatcgtgt cgtagtcacc 30
<210> 7
<211> 30
<212> DNA
<213> 人工合成
<400> 7
ggtgactacg acacgatcag cgctgccgtt 30
<210> 8
<211> 30
<212> DNA
<213> 人工合成
<400> 8
gtgtaggtag tggtgtcctc agtctgaccg 30
<210> 9
<211> 30
<212> DNA
<213> 人工合成
<400> 9
cggtcagact gaggacacca ctacctacac 30
<210> 10
<211> 30
<212> DNA
<213> 人工合成
<400> 10
agttacgaag ggttgcagtc tcatcgtcat 30
<210> 11
<211> 30
<212> DNA
<213> 人工合成
<400> 11
atgacgatga gactgcaacc cttcgtaact 30
<210> 12
<211> 30
<212> DNA
<213> 人工合成
<400> 12
cggccagaag ggtgtcctgg tatccggtga 30
<210> 13
<211> 30
<212> DNA
<213> 人工合成
<400> 13
tcaccggata ccaggacacc cttctggccg 30
<210> 14
<211> 30
<212> DNA
<213> 人工合成
<400> 14
ccgaagatga agtcaacggc accctcgatg 30
<210> 15
<211> 30
<212> DNA
<213> 人工合成
<400> 15
catcgagggt gccgttgact tcatcttcgg 30
<210> 16
<211> 30
<212> DNA
<213> 人工合成
<400> 16
tgggaccagg ggcggccgag gtagtaggtg 30
<210> 17
<211> 30
<212> DNA
<213> 人工合成
<400> 17
cacctactac ctcggccgcc cctggtccca 30
<210> 18
<211> 25
<212> DNA
<213> 人工合成
<400> 18
ggaattctta gttgatgtag ctagt 25
<210> 19
<211> 1038
<212> DNA
<213> 塔宾曲霉菌（Aspergillus tubingensis）
<400> 19
gcttacgtag aattccatca ccatcaccat cacggaagct gcaccttcac cacggctgct 60
gctgccaaag cgggcaaggc gaaatgctct accatcaccc tcgacagcat caaagtgccc 120
gctggaacca ccctcgacct gaccggtctc accagtggta ccaaggtcat cttcgagggc 180
actacgacct tcgactatga agaatgggca ggccccttga tctccatgag tggcaaagac 240
atcaccgtca ctggtgcctc aggccacctc atcaactgcg acggttcgcg gtggtgggac 300
ggtaagggga ccagcggaaa gaagaagccc aagttcttct acgcccatgg ccttgactcc 360
tcgtccatta ctggattgaa tatcaagaac actcccctta tggcgtttag tgttgagtcg 420
gatgacatca ccctgactga cattaccatc aacaatgcgg acggtgatag cctgggtgga 480
cacaacactg atgcgtttga tgttggtaac tcggtcggtg taaatatcat caaaccatgg 540
gttcataacc aggatgactg tcttgcgatc aattctggcg agaacatctg gttcaccggc 600
ggcacctgca ttgggggcca cggtctctcc atcggctctg tcggcgaccg ctccaacaac 660
gtcgtcaaga atgtcacgat tgagcactcc accgtgagca attccgagaa cgccgttcgg 720
atcaagacta tctccggtgc cactggctcc gtgtctgaga tcacctactc caacattgtc 780
atgtccggca tctccgatta cggcgtcgtt atccagcagg attacgagga tggtaagcct 840
acgggtaagc ccacgaacgg tgtcactatt acggatgtca aactggagag cgttactggt 900
actgtggata gtaaggccac tgatatctat cttctttgcg gatctggtag ctgctcggac 960
tggacttggg acgatgtgaa ggtcactggg ggaaagaagt ctagcgcttg caagaactac 1020
ccttcggtgg cttcttgc 1038
<210> 20
<211> 346
<212> PRT
<213> 塔宾曲霉菌（Aspergillus tubingensis）
<400> 20
Ala Tyr Val Glu Phe His His His His His His Gly Ser Cys Thr Phe
1 5 10 15
Thr Thr Ala Ala Ala Ala Lys Ala Gly Lys Ala Lys Cys Ser Thr Ile
20 25 30
Thr Leu Asp Ser Ile Lys Val Pro Ala Gly Thr Thr Leu Asp Leu Thr
35 40 45
Gly Leu Thr Ser Gly Thr Lys Val Ile Phe Glu Gly Thr Thr Thr Phe
50 55 60
Asp Tyr Glu Glu Trp Ala Gly Pro Leu Ile Ser Met Ser Gly Lys Asp
65 70 75 80
Ile Thr Val Thr Gly Ala Ser Gly His Leu Ile Asn Cys Asp Gly Ser
85 90 95
Arg Trp Trp Asp Gly Lys Gly Thr Ser Gly Lys Lys Lys Pro Lys Phe
100 105 110
Phe Tyr Ala His Gly Leu Asp Ser Ser Ser Ile Thr Gly Leu Asn Ile
115 120 125
Lys Asn Thr Pro Leu Met Ala Phe Ser Val Glu Ser Asp Asp Ile Thr
130 135 140
Leu Thr Asp Ile Thr Ile Asn Asn Ala Asp Gly Asp Ser Leu Gly Gly
145 150 155 160
His Asn Thr Asp Ala Phe Asp Val Gly Asn Ser Val Gly Val Asn Ile
165 170 175
Ile Lys Pro Trp Val His Asn Gln Asp Asp Cys Leu Ala Ile Asn Ser
180 185 190
Gly Glu Asn Ile Trp Phe Thr Gly Gly Thr Cys Ile Gly Gly His Gly
195 200 205
Leu Ser Ile Gly Ser Val Gly Asp Arg Ser Asn Asn Val Val Lys Asn
210 215 220
Val Thr Ile Glu His Ser Thr Val Ser Asn Ser Glu Asn Ala Val Arg
225 230 235 240
Ile Lys Thr Ile Ser Gly Ala Thr Gly Ser Val Ser Glu Ile Thr Tyr
245 250 255
Ser Asn Ile Val Met Ser Gly Ile Ser Asp Tyr Gly Val Val Ile Gln
260 265 270
Gln Asp Tyr Glu Asp Gly Lys Pro Thr Gly Lys Pro Thr Asn Gly Val
275 280 285
Thr Ile Thr Asp Val Lys Leu Glu Ser Val Thr Gly Thr Val Asp Ser
290 295 300
Lys Ala Thr Asp Ile Tyr Leu Leu Cys Gly Ser Gly Ser Cys Ser Asp
305 310 315 320
Trp Thr Trp Asp Asp Val Lys Val Thr Gly Gly Lys Lys Ser Ser Ala
325 330 335
Cys Lys Asn Tyr Pro Ser Val Ala Ser Cys
340 345
<210> 21
<211> 960
<212> DNA
<213> 塔宾曲霉菌（Aspergillus tubingensis）
<400> 21
gcttacgtag aattcgcgag ccgcatgacg gctccttccg gtgcgattgt cgttgccaag 60
tccggaggtg actacgacac gatcagcgct gccgttgatg ctctgagcac tacttcgacc 120
gagacccaga ccatcttcat tgaggaggga tcctacgacg agcaggtgta cattcctgct 180
ctcagtggaa agctgattgt ctacggtcag actgaggaca ccactaccta cactagcaac 240
ctggtcaaca tcacccatgc catcgcgttg gccgatgtcg acaatgacga tgagactgca 300
acccttcgta actacgctga gggctcggcc atctacaacc ttaacattgc caacacctgc 360
ggtcaggcct gccaccaggc tctcgccgtg agcgcctatg ccagcgagca gggatactac 420
gcctgccagt tcaccggata ccaggacacc cttctggccg agaccggcta ccaggtttac 480
gccggaacct acatcgaggg tgccgttgac ttcatcttcg gacagcacgc ccgcgcctgg 540
ttccacgagt gcgacatccg cgtcctcgag ggccccagct ccgcctccat caccgccaac 600
ggtcgctcct ccgagtcgga cgactcttac tacgtgatcc acaagtccac cgtcgctgct 660
gctgatggca acgatgtttc ctccggcacc tactacctcg gccgcccctg gtcccagtac 720
gctcgcgtct gcttccagaa gacctccatg accgatgtga tcaaccacct cggctggact 780
gagtggtcga cctccactcc caacaccgag aacgtcacct tcgttgaata cggcaacacc 840
ggcactggcg ccaagggtcc ccgtgctaac ttctcttctg agctgactga gcccatcact 900
atctcttggc ttctcggatc tgactgggag gattgggttg atactagcta catcaactaa 960
<210> 22
<211> 319
<212> PRT
<213> 塔宾曲霉菌（Aspergillus tubingensis）
<400> 22
Ala Tyr Val Glu Phe Ala Ser Arg Met Thr Ala Pro Ser Gly Ala Ile
1 5 10 15
Val Val Ala Lys Ser Gly Gly Asp Tyr Asp Thr Ile Ser Ala Ala Val
20 25 30
Asp Ala Leu Ser Thr Thr Ser Thr Glu Thr Gln Thr Ile Phe Ile Glu
35 40 45
Glu Gly Ser Tyr Asp Glu Gln Val Tyr Ile Pro Ala Leu Ser Gly Lys
50 55 60
Leu Ile Val Tyr Gly Gln Thr Glu Asp Thr Thr Thr Tyr Thr Ser Asn
65 70 75 80
Leu Val Asn Ile Thr His Ala Ile Ala Leu Ala Asp Val Asp Asn Asp
85 90 95
Asp Glu Thr Ala Thr Leu Arg Asn Tyr Ala Glu Gly Ser Ala Ile Tyr
100 105 110
Asn Leu Asn Ile Ala Asn Thr Cys Gly Gln Ala Cys His Gln Ala Leu
115 120 125
Ala Val Ser Ala Tyr Ala Ser Glu Gln Gly Tyr Tyr Ala Cys Gln Phe
130 135 140
Thr Gly Tyr Gln Asp Thr Leu Leu Ala Glu Thr Gly Tyr Gln Val Tyr
145 150 155 160
Ala Gly Thr Tyr Ile Glu Gly Ala Val Asp Phe Ile Phe Gly Gln His
165 170 175
Ala Arg Ala Trp Phe His Glu Cys Asp Ile Arg Val Leu Glu Gly Pro
180 185 190
Ser Ser Ala Ser Ile Thr Ala Asn Gly Arg Ser Ser Glu Ser Asp Asp
195 200 205
Ser Tyr Tyr Val Ile His Lys Ser Thr Val Ala Ala Ala Asp Gly Asn
210 215 220
Asp Val Ser Ser Gly Thr Tyr Tyr Leu Gly Arg Pro Trp Ser Gln Tyr
225 230 235 240
Ala Arg Val Cys Phe Gln Lys Thr Ser Met Thr Asp Val Ile Asn His
245 250 255
Leu Gly Trp Thr Glu Trp Ser Thr Ser Thr Pro Asn Thr Glu Asn Val
260 265 270
Thr Phe Val Glu Tyr Gly Asn Thr Gly Thr Gly Ala Lys Gly Pro Arg
275 280 285
Ala Asn Phe Ser Ser Glu Leu Thr Glu Pro Ile Thr Ile Ser Trp Leu
290 295 300
Leu Gly Ser Asp Trp Glu Asp Trp Val Asp Thr Ser Tyr Ile Asn
305 310 315
Claims (4)
1.一种剪接果胶酶基因中内含子的方法，其特征在于，所述方法以果胶酶基因作为模板进行融合PCR扩增得到所需基因片段，所述果胶酶基因包括内切聚半乳糖醛酸酶基因PGII和果胶甲酯酶基因PmeA，其中PGII基因扩增引物包括序列SEQ ID NO:1～SEQ ID NO:4；PmeA基因扩增引物包括序列SEQ ID NO:5～SEQ ID NO:18。
2.根据权利要求1所述的剪接果胶酶基因中内含子的方法，其特征在于，所述PGII基因由内含子分隔的两个外显子分别表示为PGII 1～2，其中：
PGII 1～2反应体系20μL：2×Phanta Max Master Mix 10μL,GYC501Genome DNA 1μL,正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
反应程序：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，30个循环，延伸72℃X min；
PGII 1的扩增：反应体系中正向引物为SEQ ID NO:1，反向引物为SEQ ID NO:2；反应程序：延伸时间X为5min；
PGII 2的扩增：反应体系中正向引物为SEQ ID NO:3，反向引物为SEQ ID NO:4；反应程序：延伸时间X为5min；
PGII 1和PGII 2的连接序列PGII 3的扩增：反应体系50μL：2×Phanta Max MasterMix 25μL,PGII-1 1μL,PGII-2 1μL,SEQ ID NO:1 2μL,SEQ ID NO:4 2μL,ddH2O 19μL，所述体系模板由PGII 1和PGII 2的扩增产物分别稀释500倍体积制得；反应程序：延伸时间X为1min。
3.根据权利要求1所述的剪接果胶酶基因中内含子的方法，其特征在于，所述PmeA基因由内含子分隔的七个外显子分别表示为PmeA1～7，其中：
PmeA1～7反应体系20μL：2×Phanta Max Master Mix 10μL,GYC 501Genome DNA 1μL,正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
PmeA8～13反应体系20μL：2×Phanta Max Master Mix 10μL,模板A 0.5μL,模板B0.5μL，正向引物0.8μL,反向引物0.8μL,ddH2O 7.4μL；
反应程序：预变性95℃3min，变性95℃15s，退火56℃15s，延伸72℃30s，30个循环，延伸72℃5min；
PmeA1的扩增：反应体系中正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:6；
PmeA2的扩增：反应体系中正向引物为SEQ ID NO:7，反向引物为SEQ ID NO:8；
PmeA3的扩增：反应体系中正向引物为SEQ ID NO:9，反向引物为SEQ ID NO:10；
PmeA4的扩增：反应体系中正向引物为SEQ ID NO:11，反向引物为SEQ ID NO:12；
PmeA5的扩增：反应体系中正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:14；
PmeA6的扩增：反应体系中正向引物为SEQ ID NO:15，反向引物为SEQ ID NO:16；
PmeA7的扩增：反应体系中正向引物为SEQ ID NO:17，反向引物为SEQ ID NO:18；
PmeA 1和PmeA 2的连接序列PmeA 8的扩增：反应体系：模板A和模板B分别由PmeA1、PmeA2的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:8；
PmeA 3和PmeA 4的连接序列PmeA 9的扩增：模板A和模板B分别由PmeA3、PmeA4的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:9，反向引物为SEQ ID NO:12；
PmeA 5和PmeA 6的连接序列PmeA 10的扩增：模板A和模板B分别由PmeA5、PmeA6的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:16；
PmeA 8和PmeA 9的连接序列PmeA 11的扩增：模板A和模板B分别由PmeA8、PmeA9的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:12；
PmeA 10和PmeA 7的连接序列PmeA 12的扩增：模板A和模板B分别由PmeA10、PmeA7的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:13，反向引物为SEQ ID NO:18；
PmeA 11和PmeA 12的连接序列PmeA 13的扩增：模板A和模板B分别由PmeA11、PmeA12的扩增产物稀释500倍体积制得，正向引物为SEQ ID NO:5，反向引物为SEQ ID NO:18。
4.根据权利要求1-3任一所述的剪接果胶酶基因中内含子的方法，其特征在于，所述果胶酶基因为塔宾曲霉Aspergillus tubingensis GYC 501所含有的果胶酶基因，所述塔宾曲霉Aspergillus tubingensis GYC 501保藏号为CCTCC M 2014425。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911089149.0A CN110760531A (zh) | 2019-11-08 | 2019-11-08 | 一种剪接果胶酶基因中内含子的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911089149.0A CN110760531A (zh) | 2019-11-08 | 2019-11-08 | 一种剪接果胶酶基因中内含子的方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110760531A true CN110760531A (zh) | 2020-02-07 |
Family
ID=69336844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911089149.0A Pending CN110760531A (zh) | 2019-11-08 | 2019-11-08 | 一种剪接果胶酶基因中内含子的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110760531A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005278575A (ja) * | 2004-03-30 | 2005-10-13 | National Research Inst Of Brewing | ポリガラクツロナーゼを用いた酒類の製造法 |
| CN105907649A (zh) * | 2016-05-23 | 2016-08-31 | 广西中烟工业有限责任公司 | 一种降解烟草梗丝果胶和纤维素的真菌及其应用 |
| CN106635846A (zh) * | 2016-12-28 | 2017-05-10 | 青岛蔚蓝生物集团有限公司 | 一种高产果胶甲酯酶的黑曲霉菌株 |
| CN107041567A (zh) * | 2017-02-15 | 2017-08-15 | 广西中烟工业有限责任公司 | 一种柠檬发酵型烟用香料及其制备方法和应用 |
| CN107151631A (zh) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | 一种高产果胶酶菌株及其应用 |
-
2019
- 2019-11-08 CN CN201911089149.0A patent/CN110760531A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005278575A (ja) * | 2004-03-30 | 2005-10-13 | National Research Inst Of Brewing | ポリガラクツロナーゼを用いた酒類の製造法 |
| CN105907649A (zh) * | 2016-05-23 | 2016-08-31 | 广西中烟工业有限责任公司 | 一种降解烟草梗丝果胶和纤维素的真菌及其应用 |
| CN106635846A (zh) * | 2016-12-28 | 2017-05-10 | 青岛蔚蓝生物集团有限公司 | 一种高产果胶甲酯酶的黑曲霉菌株 |
| CN107041567A (zh) * | 2017-02-15 | 2017-08-15 | 广西中烟工业有限责任公司 | 一种柠檬发酵型烟用香料及其制备方法和应用 |
| CN107151631A (zh) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | 一种高产果胶酶菌株及其应用 |
Non-Patent Citations (4)
| Title |
|---|
| FUTAGAMI, T., ET AL: "Genbank accesion number:GAA90887.1", 《GENBANK》 * |
| FUTAGAMI,T., ET AL.: "Genbank accesion number:GAA90446.1", 《GENBANK》 * |
| YAMADA,O., ET AL.: "Genbank accesion number:GAT30967.1", 《GENBANK》 * |
| 刘亮伟等主编: "《基因工程原理与实验指导》", 30 September 2010, 中国轻工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3655303B2 (ja) | 新規酵素及びｄｎａ配列 | |
| CN111154781B (zh) | 重组真菌漆酶的制备方法及其应用 | |
| CN110760531A (zh) | 一种剪接果胶酶基因中内含子的方法 | |
| CN114350639A (zh) | 一种密码子优化的透明质酸水解酶基因及其表达 | |
| CA2866238A1 (en) | Solution containing components of starting plant raw material, beverage, and method relating thereto | |
| CN107164353A (zh) | 一种低温碱性果胶裂解酶及其编码基因和应用 | |
| ES2219752T3 (es) | Enzima con actividad pectina esterasa. | |
| CN109694859B (zh) | 一种嗜热果胶酶及其表达基因与应用 | |
| CN108424894B (zh) | 一种嗜热真菌角质酶及其编码基因与应用 | |
| CN110791508A (zh) | 密码子优化的片球菌素基因、表达载体及重组工程菌株 | |
| Sinha et al. | Production, purification and kinetic characterization of L-asparaginase from Pseudomonas fluorescens | |
| CN106755038B (zh) | 一种高枝化型右旋糖酐蔗糖酶工程菌的构建方法及其表达应用 | |
| CN112522125B (zh) | 一种透明质酸酶工程菌及其构建方法与应用 | |
| CN107604059A (zh) | 一种朗德鹅填饲后鹅肥肝增重的分子标记及应用 | |
| CN114231514A (zh) | 一种重组褐藻胶裂解酶AlyL7及其应用 | |
| CN110872582B (zh) | 一种适冷过氧化物还原酶及其编码基因与应用 | |
| Laratta et al. | Pectin methylesterase in Citrus bergamia R.: purification, biochemical characterisation and sequence of the exon related to the enzyme active site | |
| ES2223231B1 (es) | Construccion de una cepa de levadura enologica de saccharomyces cerevisiae recombinante que sobreexpresa una endopoligalacturonasa. | |
| CN105779419A (zh) | 一种耐温，酸稳定的内切多聚半乳糖醛酸酶基因的克隆、定义及应用 | |
| Oleas et al. | Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp | |
| CN112941049A (zh) | 一种脂肪酶及其在水解虾青素酯中的应用 | |
| CN114752613B (zh) | 果胶酶基因、果胶酶、重组载体及其在蓝莓加工中的应用 | |
| CN103789283A (zh) | 一种中性阿拉伯呋喃糖苷酶Abf43及其基因和应用 | |
| CN111254153A (zh) | 一种聚半乳糖醛酸酶编码基因及酶和制备与应用 | |
| CN103484455B (zh) | 一种转人血清白蛋白水稻品系114-7-2的侧翼序列 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200207 |