CN102358896A - 一种耐热角质酶-cbd融合酶及其突变体和应用 - Google Patents
一种耐热角质酶-cbd融合酶及其突变体和应用 Download PDFInfo
- Publication number
- CN102358896A CN102358896A CN2011102953840A CN201110295384A CN102358896A CN 102358896 A CN102358896 A CN 102358896A CN 2011102953840 A CN2011102953840 A CN 2011102953840A CN 201110295384 A CN201110295384 A CN 201110295384A CN 102358896 A CN102358896 A CN 102358896A
- Authority
- CN
- China
- Prior art keywords
- cbd
- cutinase
- mutants
- enzyme
- thr
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 43
- 230000004927 fusion Effects 0.000 title claims abstract description 8
- 229920002678 cellulose Polymers 0.000 title description 5
- 239000001913 cellulose Substances 0.000 title description 5
- 239000000835 fiber Substances 0.000 claims abstract description 35
- 229920000728 polyester Polymers 0.000 claims abstract description 14
- 230000004048 modification Effects 0.000 claims abstract description 10
- 238000012986 modification Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 230000035772 mutation Effects 0.000 claims description 7
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- 125000000539 amino acid group Chemical group 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 102220542741 Ras and Rab interactor 1_W68Y_mutation Human genes 0.000 abstract description 11
- 102220401893 c.203G>T Human genes 0.000 abstract description 11
- 238000000034 method Methods 0.000 abstract description 9
- 230000007062 hydrolysis Effects 0.000 abstract description 7
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 7
- 238000001179 sorption measurement Methods 0.000 abstract description 6
- 241000203780 Thermobifida fusca Species 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 238000006467 substitution reaction Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 9
- 229920000742 Cotton Polymers 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- 239000005020 polyethylene terephthalate Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 235000011009 potassium phosphates Nutrition 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920004933 Terylene® Polymers 0.000 description 3
- 239000006035 Tryptophane Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 230000000869 mutational effect Effects 0.000 description 3
- 229940093916 potassium phosphate Drugs 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229960004799 tryptophan Drugs 0.000 description 3
- 241000235349 Ascomycota Species 0.000 description 2
- RAQMSGVCGSJKCL-FOHZUACHSA-N Asn-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(N)=O RAQMSGVCGSJKCL-FOHZUACHSA-N 0.000 description 2
- 229920004934 Dacron® Polymers 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 108010005400 cutinase Proteins 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 235000011008 sodium phosphates Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- HXUVTXPOZRFMOY-NSHDSACASA-N 2-[[(2s)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-phenylpropanoyl]amino]acetic acid Chemical compound NCC(=O)NCC(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 HXUVTXPOZRFMOY-NSHDSACASA-N 0.000 description 1
- QMOQBVOBWVNSNO-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(O)=O QMOQBVOBWVNSNO-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- XCVRVWZTXPCYJT-BIIVOSGPSA-N Ala-Asn-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N XCVRVWZTXPCYJT-BIIVOSGPSA-N 0.000 description 1
- ZIBWKCRKNFYTPT-ZKWXMUAHSA-N Ala-Asn-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZIBWKCRKNFYTPT-ZKWXMUAHSA-N 0.000 description 1
- ZIWWTZWAKYBUOB-CIUDSAMLSA-N Ala-Asp-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O ZIWWTZWAKYBUOB-CIUDSAMLSA-N 0.000 description 1
- BGNLUHXLSAQYRQ-FXQIFTODSA-N Ala-Glu-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O BGNLUHXLSAQYRQ-FXQIFTODSA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 description 1
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 1
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 1
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 1
- ZJLORAAXDAJLDC-CQDKDKBSSA-N Ala-Tyr-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O ZJLORAAXDAJLDC-CQDKDKBSSA-N 0.000 description 1
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 1
- SLQQPJBDBVPVQV-JYJNAYRXSA-N Arg-Phe-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O SLQQPJBDBVPVQV-JYJNAYRXSA-N 0.000 description 1
- VDCIPFYVCICPEC-FXQIFTODSA-N Asn-Arg-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O VDCIPFYVCICPEC-FXQIFTODSA-N 0.000 description 1
- IHUJUZBUOFTIOB-QEJZJMRPSA-N Asn-Gln-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N IHUJUZBUOFTIOB-QEJZJMRPSA-N 0.000 description 1
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 1
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- JWKDQOORUCYUIW-ZPFDUUQYSA-N Asn-Lys-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JWKDQOORUCYUIW-ZPFDUUQYSA-N 0.000 description 1
- IDUUACUJKUXKKD-VEVYYDQMSA-N Asn-Pro-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O IDUUACUJKUXKKD-VEVYYDQMSA-N 0.000 description 1
- KZYSHAMXEBPJBD-JRQIVUDYSA-N Asn-Thr-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KZYSHAMXEBPJBD-JRQIVUDYSA-N 0.000 description 1
- QRULNKJGYQQZMW-ZLUOBGJFSA-N Asp-Asn-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O QRULNKJGYQQZMW-ZLUOBGJFSA-N 0.000 description 1
- CJUKAWUWBZCTDQ-SRVKXCTJSA-N Asp-Leu-Lys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O CJUKAWUWBZCTDQ-SRVKXCTJSA-N 0.000 description 1
- DINOVZWPTMGSRF-QXEWZRGKSA-N Asp-Pro-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O DINOVZWPTMGSRF-QXEWZRGKSA-N 0.000 description 1
- 229920000832 Cutin Polymers 0.000 description 1
- NITLUESFANGEIW-BQBZGAKWSA-N Cys-Pro-Gly Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O NITLUESFANGEIW-BQBZGAKWSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- MWLYSLMKFXWZPW-ZPFDUUQYSA-N Gln-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCC(N)=O MWLYSLMKFXWZPW-ZPFDUUQYSA-N 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- QDXMSSWCEVYOLZ-SZMVWBNQSA-N Gln-Leu-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCC(=O)N)N QDXMSSWCEVYOLZ-SZMVWBNQSA-N 0.000 description 1
- QQLBPVKLJBAXBS-FXQIFTODSA-N Glu-Glu-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O QQLBPVKLJBAXBS-FXQIFTODSA-N 0.000 description 1
- BUAKRRKDHSSIKK-IHRRRGAJSA-N Glu-Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BUAKRRKDHSSIKK-IHRRRGAJSA-N 0.000 description 1
- PJBVXVBTTFZPHJ-GUBZILKMSA-N Glu-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N PJBVXVBTTFZPHJ-GUBZILKMSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-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
- YZACQYVWLCQWBT-BQBZGAKWSA-N Gly-Cys-Arg Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YZACQYVWLCQWBT-BQBZGAKWSA-N 0.000 description 1
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 1
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 1
- SCJJPCQUJYPHRZ-BQBZGAKWSA-N Gly-Pro-Asn Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O SCJJPCQUJYPHRZ-BQBZGAKWSA-N 0.000 description 1
- IXHQLZIWBCQBLQ-STQMWFEESA-N Gly-Pro-Phe Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IXHQLZIWBCQBLQ-STQMWFEESA-N 0.000 description 1
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- RAVLQPXCMRCLKT-KBPBESRZSA-N His-Gly-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RAVLQPXCMRCLKT-KBPBESRZSA-N 0.000 description 1
- HJUPAYWVVVRYFQ-PYJNHQTQSA-N His-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CN=CN1)N HJUPAYWVVVRYFQ-PYJNHQTQSA-N 0.000 description 1
- BFOGZWSSGMLYKV-DCAQKATOSA-N His-Ser-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC1=CN=CN1)N BFOGZWSSGMLYKV-DCAQKATOSA-N 0.000 description 1
- HDOYNXLPTRQLAD-JBDRJPRFSA-N Ile-Ala-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)O)N HDOYNXLPTRQLAD-JBDRJPRFSA-N 0.000 description 1
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 1
- PDTMWFVVNZYWTR-NHCYSSNCSA-N Ile-Gly-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O PDTMWFVVNZYWTR-NHCYSSNCSA-N 0.000 description 1
- YNMQUIVKEFRCPH-QSFUFRPTSA-N Ile-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)O)N YNMQUIVKEFRCPH-QSFUFRPTSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- NURNJECQNNCRBK-FLBSBUHZSA-N Ile-Thr-Thr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NURNJECQNNCRBK-FLBSBUHZSA-N 0.000 description 1
- NXRNRBOKDBIVKQ-CXTHYWKRSA-N Ile-Tyr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N NXRNRBOKDBIVKQ-CXTHYWKRSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 1
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 1
- KLSUAWUZBMAZCL-RHYQMDGZSA-N Leu-Thr-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(O)=O KLSUAWUZBMAZCL-RHYQMDGZSA-N 0.000 description 1
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 1
- LECIJRIRMVOFMH-ULQDDVLXSA-N Lys-Pro-Phe Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LECIJRIRMVOFMH-ULQDDVLXSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- QMMRHASQEVCJGR-UBHSHLNASA-N Phe-Ala-Pro Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 QMMRHASQEVCJGR-UBHSHLNASA-N 0.000 description 1
- NHCKESBLOMHIIE-IRXDYDNUSA-N Phe-Gly-Phe Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 NHCKESBLOMHIIE-IRXDYDNUSA-N 0.000 description 1
- UNBFGVQVQGXXCK-KKUMJFAQSA-N Phe-Ser-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O UNBFGVQVQGXXCK-KKUMJFAQSA-N 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 1
- SSSFPISOZOLQNP-GUBZILKMSA-N Pro-Arg-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSSFPISOZOLQNP-GUBZILKMSA-N 0.000 description 1
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 1
- TXPUNZXZDVJUJQ-LPEHRKFASA-N Pro-Asn-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O TXPUNZXZDVJUJQ-LPEHRKFASA-N 0.000 description 1
- ZCXQTRXYZOSGJR-FXQIFTODSA-N Pro-Asp-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZCXQTRXYZOSGJR-FXQIFTODSA-N 0.000 description 1
- QEWBZBLXDKIQPS-STQMWFEESA-N Pro-Gly-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QEWBZBLXDKIQPS-STQMWFEESA-N 0.000 description 1
- DSGSTPRKNYHGCL-JYJNAYRXSA-N Pro-Phe-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O DSGSTPRKNYHGCL-JYJNAYRXSA-N 0.000 description 1
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 1
- QUBVFEANYYWBTM-VEVYYDQMSA-N Pro-Thr-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O QUBVFEANYYWBTM-VEVYYDQMSA-N 0.000 description 1
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- RSTWKJFWBKFOFC-JYJNAYRXSA-N Pro-Trp-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O RSTWKJFWBKFOFC-JYJNAYRXSA-N 0.000 description 1
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 1
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 1
- DKKGAAJTDKHWOD-BIIVOSGPSA-N Ser-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)C(=O)O DKKGAAJTDKHWOD-BIIVOSGPSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- SOACHCFYJMCMHC-BWBBJGPYSA-N Ser-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N)O SOACHCFYJMCMHC-BWBBJGPYSA-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
- 241001647802 Thermobifida Species 0.000 description 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- UNURFMVMXLENAZ-KJEVXHAQSA-N Thr-Arg-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UNURFMVMXLENAZ-KJEVXHAQSA-N 0.000 description 1
- LXWZOMSOUAMOIA-JIOCBJNQSA-N Thr-Asn-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O LXWZOMSOUAMOIA-JIOCBJNQSA-N 0.000 description 1
- UZJDBCHMIQXLOQ-HEIBUPTGSA-N Thr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O UZJDBCHMIQXLOQ-HEIBUPTGSA-N 0.000 description 1
- XXNLGZRRSKPSGF-HTUGSXCWSA-N Thr-Gln-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O XXNLGZRRSKPSGF-HTUGSXCWSA-N 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- SIMKLINEDYOTKL-MBLNEYKQSA-N Thr-His-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C)C(=O)O)N)O SIMKLINEDYOTKL-MBLNEYKQSA-N 0.000 description 1
- XOWKUMFHEZLKLT-CIQUZCHMSA-N Thr-Ile-Ala 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)C(O)=O XOWKUMFHEZLKLT-CIQUZCHMSA-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
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- WTMPKZWHRCMMMT-KZVJFYERSA-N Thr-Pro-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WTMPKZWHRCMMMT-KZVJFYERSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 1
- CXUFDWZBHKUGKK-CABZTGNLSA-N Trp-Ala-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O)=CNC2=C1 CXUFDWZBHKUGKK-CABZTGNLSA-N 0.000 description 1
- RXEQOXHCHQJMSO-IHPCNDPISA-N Trp-His-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O RXEQOXHCHQJMSO-IHPCNDPISA-N 0.000 description 1
- RRVUOLRWIZXBRQ-IHPCNDPISA-N Trp-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N RRVUOLRWIZXBRQ-IHPCNDPISA-N 0.000 description 1
- ARKBYVBCEOWRNR-UBHSHLNASA-N Trp-Ser-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O ARKBYVBCEOWRNR-UBHSHLNASA-N 0.000 description 1
- WTRQBSSQBKRNKV-MNSWYVGCSA-N Trp-Thr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)[C@H](O)C)C(O)=O)C1=CC=C(O)C=C1 WTRQBSSQBKRNKV-MNSWYVGCSA-N 0.000 description 1
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 1
- XQYHLZNPOTXRMQ-KKUMJFAQSA-N Tyr-Glu-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O XQYHLZNPOTXRMQ-KKUMJFAQSA-N 0.000 description 1
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 1
- COSLEEOIYRPTHD-YDHLFZDLSA-N Val-Asp-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 COSLEEOIYRPTHD-YDHLFZDLSA-N 0.000 description 1
- APQIVBCUIUDSMB-OSUNSFLBSA-N Val-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](C(C)C)N APQIVBCUIUDSMB-OSUNSFLBSA-N 0.000 description 1
- AJNUKMZFHXUBMK-GUBZILKMSA-N Val-Ser-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N AJNUKMZFHXUBMK-GUBZILKMSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 1
- 108010036533 arginylvaline Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- OBNCKNCVKJNDBV-UHFFFAOYSA-N ethyl butyrate Chemical compound CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000003063 flame retardant Substances 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 235000011187 glycerol Nutrition 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
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000009991 scouring Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 108010084932 tryptophyl-proline Proteins 0.000 description 1
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种耐热角质酶-CBD融合酶及其突变体和其在涤纶纤维改性中的应用，属于基因工程和酶工程领域。本发明提供了一种嗜热子囊菌（Thermobifida fusca）角质酶-CBD及其突变体的制备方法，突变体包括一个位于嗜热子囊菌（Thermobifida fusca）角质酶-CBD融合酶中CBD上的结合位点的取代。突变体W68L和W68Y对涤纶纤维的吸附效率比角质酶-CBD分别提高14%和17%，而对涤纶纤维的水解能力是角质酶-CBD的1.4倍和1.5倍。这两种角质酶-CBD突变体更适合在涤纶纤维改性中应用。
Description
技术领域
本发明涉及一种耐热角质酶-CBD融合酶及其突变体和其在涤纶纤维改性中的应用，属于基因工程和酶工程领域。
背景技术
涤纶作为当今世界的三大合成纤维之一，与天然纤维相比，表现出良好的物理性能（如强度、弹性、韧性、硬度和耐磨性等）、染色性能和耐高温、耐化学品等特性。然而，由于涤纶纤维是疏水性纤维，其结晶度高，吸湿性差，且易起静电，不仅影响穿着舒适性，而且还限制了复合物改性剂、着色剂、阻燃剂等在加工处理过程中的应用。因此，为了达到优良的润湿性和染色效果，必须对涤纶纤维进行改性。目前，国内采用传统的强碱性或酸性剂处理方法不仅需要消耗大量的能源和化学品（粘合剂，偶联剂等），而且对生态环境造成了严重威胁。相比于化学改性，生物酶应用于纤维改性是提高合成纤维质量和加工性能的有效途径，顺应了纤维改性工业可持续发展的要求。
角质酶是一种多功能的水解酶，它不仅水解植物表皮的角质层，也能水解很多酯类结构包括甘油三酯和合成聚酯等。近来，角质酶在涤纶纤维改性中有潜在的应用价值。然而，角质酶处理大分子聚酯类底物（如涤纶）时存在亲和力差、可及度低的问题，大大降低角质酶的作用效果。国外对角质酶的研究主要集中于真菌来源角质酶，但真菌角质酶热稳定性差，也不适合于纺织工业中的应用。
本实验室研究开发的嗜热子囊菌（Thermobifida fusca）可产耐热角质酶，并能有效水解角质、PET等聚酯（陈坚, 吴敬, 陈晟. 一种耐热角质酶及其编码基因和表达. 申请号200710026074.2）。另外，我们采用一种新方法将纤维素结合域（CBD）融合到角质酶上，从而提高角质酶对棉纤维的结合效率及精练效果（吴敬, 陈坚, 张瑶, 陈晟. 一种耐热角质酶-CBD的制备及其在棉纤维精练中 的应用. 申请号 200910033667.0）。为了使角质酶-CBD能有效地作用于涤纶纤维，通过对CBM进行结构模拟，结合涤纶纤维结构特点，利用定点突变方法提高角质酶-CBD对涤纶纤维的改性效果。这种提高酶催化效率的结合位点改造策略目前国内外未见报道。
发明内容
本发明要解决的技术问题是提供一种耐热角质酶-CBD融合酶，其氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2 所示。
本发明要解决的另一个技术问题是提供一种耐热角质酶-CBD融合酶的突变体，包括一个相应于嗜热子囊菌（Thermobifida fusca）的角质酶-CBD结合位点的氨基酸残基的取代，突变成与涤纶聚酯结构类似的氨基酸，与角质酶-CBD融合酶相比具有更强的涤纶纤维水解效率。
所述角质酶-CBD突变体是在CBD上的第68位色氨酸Trp突变为亮氨酸Leu，命名为W68L；突变为酪氨酸Tyr，命名为W68Y。
本发明所要解决的另一个技术问题是提供上述突变体的制备方法。
为解决上述问题，本发明的技术方案是：其特征是在纤维素结合域模拟结构的基础上确定突变位点；设计定点突变引物，以携带角质酶-CBD基因的pET20b（+）载体为模板构建突变质粒W68L-pET20b和W68Y-pET20b；将突变质粒转化到大肠杆菌BL21(DE3)中，挑选阳性克隆进行发酵培养，30℃培养48 h；发酵上清液经硫酸铵沉淀、亲和色谱纯化角质酶-CBD突变体W68L和W68Y。
所述耐热角质酶-CBD融合酶及其突变体应用于涤纶纤维改性亦为本发明要求保护的范围。
本发明提供了一种耐热角质酶-CBD融合酶及两种具有涤纶纤维高水解效率的耐热角质酶-CBD突变体及其制备方法；突变体W68L和W68Y对涤纶纤维的吸附效率比角质酶-CBD分别提高14%和17%，而对涤纶纤维的水解效率分别是天然角质酶-CBD的1.4倍和1.5倍，非常适宜涤纶纤维改性中应用。
附图说明
图１耐热角质酶-CBD突变体分离纯化SDS-PAGE图谱
1, W68L发酵上清液; 2, W68Y发酵上清液; 3, W68L Ni柱纯化结果; 4, W68Y Ni柱纯化结果
图2耐热角质酶-CBD突变体50℃稳定性研究
图3耐热角质酶-CBD突变体对涤纶水解效率分析。
具体实施方式
实施例1 角质酶-CBD融合酶的制备方法
通过基因全合成的方法获得编码角质酶-CBD的融合基因，序列如SEQ ID NO: 1。角质酶-CBD基因以质粒pET20b(+)为表达载体，以E. coli BL21(DE3)为表达宿主，实现耐热角质酶-CBD基因的高效表达。其发酵和纯化方法同角质酶-CBD突变体。
实施例2 角质酶-CBD融合酶对棉纤维的作用效果
在10 mL反应体系中，分别加入1mL角质酶-CBD（或天然角质酶）的酶液(酶活力100U/mL)，1mL果胶酶液(酶活力100U/mL)，8mL 25mM磷酸钾（pH 8.0），0.5g原棉纤维和50μL渗透剂TX-10。对照与上述条件一致但处理液中不加棉纤维。在室温下反应，定时取样。取出的样品经离心3 min后按照测定酶活的方法测上清液中酶活。被吸附的酶量为对照溶液中的酶活减去上清液中的酶活。结果表明，在与果胶酶互作下，天然角质酶对棉纤维的吸附率为10%，而角质酶-CBD对棉纤维的吸附率为55%。
10mL反应体系包括0.5g原棉纤维、8mL 25 mM 磷酸钾缓冲液(pH 8.0)、1mL角质酶-CBD（或天然角质酶）酶液(酶活力100U/mL)，1mL果胶酶液(酶活力100U/mL) 和50μL渗透剂TX-10，在50℃保温。不同时间取样，用0.02mol/L NaOH溶液滴定生成的脂肪酸。结果显示，天然角质酶15h达到反应平衡，生产脂肪酸为160μmol；角质酶-CBD 6h达到反应平衡时，生成脂肪酸为180μmol。
实施例3 角质酶-CBD突变体突变位点的确定
纤维素结合域与纤维素结合的关键氨基酸是高度保守的色氨酸残基。通过对第二家族CBD多重序列比对，结合涤纶纤维的结构特点，从以上两方面将两个关键点突变成与涤纶聚酯结构类似的氨基酸，以增强酶与底物的疏水作用，从而提高催化效率。具体突变如下：（1）将色氨酸突变成亮氨酸，增强与聚酯结构中烷基链的疏水作用，以提高与底物的亲和力；（2）将色氨酸突变成酪氨酸，提高与聚酯结构中苯环的疏水作用，并且羟基产生氢键作用以增强与底物的结合力。
实施例4 角质酶-CBD突变体的制备方法
利用一步PCR定点突变方法构建W68L-pET20b和W68Y-pET20b突变质粒。
W68L-pET20b和W68Y-pET20b突变质粒是以cutinase-CBMCenA/pET20b质粒为模板（Yao Zhang, Sheng Chen, Meng Xu, Artur Cavaco-Paulo, Jing Wu, Jian Chen. Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring. Applied and Environmental Microbiology, 2010, 76(20): 6870-6876），分别用W68L和W68Y的突变引物通过PCR得到大小为5000bp产物，然后将PCR产物经过Dpn I处理后转化大肠杆菌JM109，再筛选转化子。
测序结果证明除所需位点外，无其他位点突变。因此，突变质粒W68L-pET20b和W68Y-pET20b构建成功。
定点突变互补引物如下所示（突变位点斜体加粗）：
1、W68Y突变
Primer-F: GTCACCAGCCTGCCC TAT AACGGCAGCATCCCG
Primer-R: CGGGATGCTGCCGTT ATA GGGCAGGCTGGTGAC
2、W68L突变
Primer-F: GTCACCAGCCTGCCC TTG AACGGCAGCATCCCG
Primer-R: CGGGATGCTGCCGTT CAA GGGCAGGCTGGTGAC
PCR反应体系（50 μL）如下：
| ddH2O | 37 |
| 10×Prime STARTMGC Buffer | 5 |
| dNTP Mixture （2.5 mM） | 4 |
| Mg2+（250 mM） | 1 |
| Primer-F | 1 |
| Primer-R | 1 |
| 质粒 | 0.5 |
| Prime STARTM HS DNA Polymerase（2.5 U/μL） | 0.5 |
PCR反应程序为：94 oC预变性5 min；95 oC变性30 s，55 oC退火60 s，72 oC延伸6 min；共循环30次；72 oC延伸10 min；4 oC保存。核酸电泳检测目的条带大小。
实施例5 角质酶-CBD突变体的表达及纯化
将突变质粒W68L-pET20b和W68Y-pET20b转化大肠杆菌BL21(DE3)。筛选出正确的阳性克隆，在TB（甘油5 g/L，蛋白胨12 g/L，酵母膏24 g/L，K2HPO4 12.54 g/L，KH2PO4 2.31 g/L）发酵液体培养基30℃培养48 h时产酶达40-45 U/mL。
将上述耐热角质酶-CBD突变体发酵液于4 ℃、10000 rpm 离心10 min 除菌体；上清液中加入50 %固体硫酸铵盐析过夜，所得沉淀用缓冲液A (含50 mM 磷酸钠、0.5 M氯化钠、10 mM咪唑，pH 7.4)溶解，并在缓冲液A中透析过夜后，通过0.22 μm膜过滤后制成上样样品；Ni亲和柱用缓冲液A平衡后，将上样样品吸入Ni柱，使之完全吸附后，用缓冲液(含50 mM 磷酸钠、0.5 M氯化钠、0 -500 mM咪唑，pH 7.4) 线性梯度洗脱，流速1 mL/ min，检测波长为280 nm，分部收集含角质酶酶活的洗脱液；活力组分用30000道尔顿膜离心浓缩，得纯化角质酶-CBD突变体制品；纯化后角质酶达到电泳纯，表观分子量45000道尔顿。纯化过程电泳图见图1。
实施例6 角质酶-CBD突变体的热稳定性
以可溶性酯对硝基苯丁酸酯（pNPB）为底物时，角质酶-CBD突变体在50℃下保温24 h，酶活仍可达70%以上（图2）。
实施例7 角质酶-CBD突变体对涤纶纤维的吸附效果测定
将涤纶纤维在60 oC水浴振荡清洗30 min后于室温晾干。在10 ml反应体系中，分别加入1ml角质酶-CBD突变体（或天然角质酶-CBD）的酶液(酶活力100U/ml)，9 ml 25mM磷酸钾（pH 8.0），0.5 g预处理的涤纶纤维。对照与上述条件一致但处理液中不加涤纶纤维。在25 oC，60 rpm下处理1 h后，取出的样品经离心3000 rpm离心2 min，测定上清液中的酶活。被吸附的酶量为对照溶液中的酶活减去上清液中的酶活。结果表明，突变体W68L和W68Y对涤纶纤维的吸附效率分别比角质酶-CBD提高14%和17%。
实施例8 角质酶-CBD突变体对涤纶纤维的改性效果
10ml反应体系包括0.5g预处理的涤纶纤维、9 ml 25 mM 磷酸钾缓冲液(pH 8.0)、1 ml角质酶-CBD突变体（或天然融合酶CBD）的酶液（酶活力100U/ml），在50℃，200 rpm下反应。用紫外分光光度计测试不同处理时间下240 nm处的紫外吸光度值。参比样为相同条件下不加入涤纶纤维的角质酶-CBD处理液。结果显示（图3），随着处理时间的延长，紫外吸光度逐渐上升，当处理时间达到20 h时，紫外吸光度的增加逐渐趋于平缓达到平衡。突变体W68L和W68Y释放的产物量比角质酶-CBD有了较明显的提高，分别提高了1.4倍和1.5倍。
序 列 表
<110> 江南大学
<120> 一种耐热角质酶-CBD融合酶及其突变体和应用
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 395
<212> PRT
<213> 人工合成序列
<400> 1
Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Ser Glu
1 5 10 15
Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg Leu
20 25 30
Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu Asn
35 40 45
Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Gly Thr Glu
50 55 60
Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser Arg
85 90 95
Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala Ser
100 105 110
Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met Gly
115 120 125
His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg Pro
130 135 140
Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys Asn
145 150 155 160
Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu Asp
165 170 175
Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser Leu
180 185 190
Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr His
195 200 205
Phe Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val Ala
210 215 220
Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe Leu
225 230 235 240
Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr Arg
245 250 255
Ser Thr Cys Pro Phe Met Ala Ile Ala Ala Gly Gly Thr Asn Pro Asn
260 265 270
Pro Asn Pro Asn Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Pro
275 280 285
Pro Ala Pro Gly Cys Arg Val Asp Tyr Ala Val Thr Asn Gln Trp Pro
290 295 300
Gly Gly Phe Gly Ala Asn Val Thr Ile Thr Asn Leu Gly Asp Pro Val
305 310 315 320
Ser Ser Trp Lys Leu Asp Trp Thr Tyr Thr Ala Gly Gln Arg Ile Gln
325 330 335
Gln Leu Trp Asn Gly Thr Ala Ser Thr Asn Gly Gly Gln Val Ser Val
340 345 350
Thr Ser Leu Pro Trp Asn Gly Ser Ile Pro Thr Gly Gly Thr Ala Ser
355 360 365
Phe Gly Phe Asn Gly Ser Trp Ala Gly Ser Asn Pro Thr Pro Ala Ser
370 375 380
Phe Ser Leu Asn Gly Thr Thr Cys Thr Gly Thr
385 390 395
<210> 2
<211> 1185
<212> DNA
<213>人工序列
<220>
<223>根据基因序列设计，用于表达。
<400> 2
gccaacccct acgagcgcgg ccccaacccg accgacgccc tgtccgaagc cagcagcggc 60
cccttctccg tcagcgagga gaacgtctcc cggttgagcg ccagcggctt cggcggcggc 120
accatctact acccgcggga gaacaacacc tacggtgcgg tggcgatctc ccccggctac 180
accggcactg aggcttccat cgcctggctg ggcgagcgca tcgcctccca cggcttcgtc 240
gtcatcacca tcgacaccat caccaccctc gaccagccgg acagccgggc agagcagctc 300
aacgccgcgc tgaaccacat gatcaaccgg gcgtcctcca cggtgcgcag ccggatcgac 360
agcagccgac tggcggtcat gggccactca atgggcggcg gcggcaccct gcgtctggcc 420
tcccagcgtc ccgacctgaa ggccgccatc ccgctcaccc cgtggcacct caacaagaac 480
tggagcagcg tcaccgtgcc gacgctgatc atcggggccg acctcgacac gatcgcgccg 540
gtcgccacgc acgcgaaacc gttctacaac agcctgccga gctccatcag caaggcctac 600
ctggagctgg acggcgcaac ccacttcgcc ccgaacatcc ccaacaagat catcggcaag 660
tacagtgtcg cctggctcaa gcggttcgtc gacaacgaca cccgctacac ccagttcctc 720
tgccccggac cgcgcgacgg actcttcggc gaggtcgaag agtaccgctc cacctgcccg 780
ttcatggcga tcgccgcggg cggcaccaac cccaacccga accccaaccc gacgcccacc 840
cccactccga cccccacgcc gcctcccgct cccggctgcc gcgtcgacta cgccgtcacc 900
aaccagtggc ccggcggctt cggcgccaac gtcacgatca ccaacctcgg cgaccccgtc 960
tcgtcgtgga agctcgactg gacctacacc gcaggccagc ggatccagca gctgtggaac 1020
ggcaccgcgt cgaccaacgg cggccaggtc tccgtcacca gcctgccctg gaacggcagc 1080
atcccgaccg gcggcacggc gtcgttcggg ttcaacggct cgtgggccgg gtccaacccg 1140
acgccggcgt cgttctcgct caacggcacc acctgcacgg gcacc 1185
<210> 3
<211> 33
<212> DNA
<213>人工序列
<220>
<223>根据基因序列设计，用于扩增。
<400> 3
gtcaccagcc tgccctataa cggcagcatc ccg 33
<210> 4
<211> 33
<212> DNA
<213>人工序列
<220>
<223>根据基因序列设计，用于扩增。
<400> 4
cgggatgctg ccgttatagg gcaggctggt gac 33
<210> 5
<211> 33
<212> DNA
<213>人工序列
<220>
<223>根据基因序列设计，用于扩增。
<400> 5
gtcaccagcc tgcccttgaa cggcagcatc ccg 33
<210> 6
<211> 33
<212> DNA
<213>人工序列
<220>
<223>根据基因序列设计，用于扩增。
<400> 6
cgggatgctg ccgttcaagg gcaggctggt gac 33
Claims (7)
1.一种耐热角质酶-CBD融合酶，其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的耐热角质酶-CBD的基因，其核苷酸序列如SEQ ID NO.2 所示。
3.权利要求1所述耐热角质酶-CBD融合酶的突变体，其特征是包括一个相应于融合酶中CBD结合位点的氨基酸残基突变成与涤纶聚酯结构类似的氨基酸。
4.权利要求3所述的突变体，其特征在于所述突变为CBD第68位氨基酸色氨酸。
5.权利要求4所述的突变体，其特征在于所述突变为CBD第68位氨基酸色氨酸突变为亮氨酸。
6.权利要求4所述的突变体，其特征在于所述突变为CBD第68位氨基酸色氨酸突变为酪氨酸。
7.权利要求1或3或4所述的耐热角质酶-CBD突变体应用于涤纶纤维改性。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110295384 CN102358896B (zh) | 2011-10-08 | 2011-10-08 | 一种耐热角质酶-cbd融合酶及其突变体和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110295384 CN102358896B (zh) | 2011-10-08 | 2011-10-08 | 一种耐热角质酶-cbd融合酶及其突变体和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102358896A true CN102358896A (zh) | 2012-02-22 |
| CN102358896B CN102358896B (zh) | 2013-01-30 |
Family
ID=45584273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110295384 Active CN102358896B (zh) | 2011-10-08 | 2011-10-08 | 一种耐热角质酶-cbd融合酶及其突变体和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102358896B (zh) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479999A (zh) * | 2016-09-30 | 2017-03-08 | 苏州埃德蒙生物技术有限公司 | 一种l‑谷氨酸氧化酶‑cbd融合酶及其表达基因和应用 |
| CN110776571A (zh) * | 2019-11-04 | 2020-02-11 | 福建省中医药研究院(福建省青草药开发服务中心） | 一种金属硫蛋白融合蛋白构建、固定化载体快速制备及其在重金属离子去除中的应用 |
| CN112159478A (zh) * | 2020-09-28 | 2021-01-01 | 江南大学 | 一种融合蛋白及其在降解聚合物中的应用 |
| CN112725302A (zh) * | 2021-01-15 | 2021-04-30 | 江南大学 | 一种融合蛋白的构建及其降解聚合物的方法和应用 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4370594A1 (en) * | 2021-07-15 | 2024-05-22 | Samsara Eco Pty Limited | Enzymes and uses thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168735A (zh) * | 2007-08-17 | 2008-04-30 | 江南大学 | 一种耐热角质酶及其编码基因和表达 |
| CN101591648A (zh) * | 2009-06-05 | 2009-12-02 | 江南大学 | 一种耐热角质酶-cbd的制备及其在棉纤维精练中的应用 |
| CN101775382A (zh) * | 2009-12-18 | 2010-07-14 | 江南大学 | 一种角质酶的突变体及其制备方法 |
| CN102146633A (zh) * | 2011-01-15 | 2011-08-10 | 江南大学 | 一种应用角质酶和cbd-碱性果胶酶进行棉织物精练的方法 |
-
2011
- 2011-10-08 CN CN 201110295384 patent/CN102358896B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168735A (zh) * | 2007-08-17 | 2008-04-30 | 江南大学 | 一种耐热角质酶及其编码基因和表达 |
| CN101591648A (zh) * | 2009-06-05 | 2009-12-02 | 江南大学 | 一种耐热角质酶-cbd的制备及其在棉纤维精练中的应用 |
| CN101775382A (zh) * | 2009-12-18 | 2010-07-14 | 江南大学 | 一种角质酶的突变体及其制备方法 |
| CN102146633A (zh) * | 2011-01-15 | 2011-08-10 | 江南大学 | 一种应用角质酶和cbd-碱性果胶酶进行棉织物精练的方法 |
Non-Patent Citations (6)
| Title |
|---|
| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20101031 Yao Zhang 等 Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring 第76卷, 第20期 * |
| GRAHAM,R.W.等: "L24367.1", 《GENBANK》, 8 May 1996 (1996-05-08) * |
| NEENA DIN 等: "The cellulose-binding domain of endoglucanase A(CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding", 《MOLECULAR MICROBIOLOGY》, vol. 11, no. 4, 31 December 1994 (1994-12-31) * |
| TERESA MATAMA 等: "Functionalization of Cellulose Acetate Fibers with Engineered Cutinases", 《BIOTECHNOL. PROG.》, vol. 26, no. 3, 15 December 2009 (2009-12-15) * |
| WONG,W.K.等: "AAA23084.1", 《GENBANK》, 26 April 1993 (1993-04-26) * |
| YAO ZHANG 等: "Characterization of Thermobifida fusca Cutinase–Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》, vol. 76, no. 20, 31 October 2010 (2010-10-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479999A (zh) * | 2016-09-30 | 2017-03-08 | 苏州埃德蒙生物技术有限公司 | 一种l‑谷氨酸氧化酶‑cbd融合酶及其表达基因和应用 |
| CN110776571A (zh) * | 2019-11-04 | 2020-02-11 | 福建省中医药研究院(福建省青草药开发服务中心） | 一种金属硫蛋白融合蛋白构建、固定化载体快速制备及其在重金属离子去除中的应用 |
| CN110776571B (zh) * | 2019-11-04 | 2021-08-10 | 福建省中医药研究院(福建省青草药开发服务中心） | 一种金属硫蛋白融合蛋白构建、固定化载体快速制备及其在重金属离子去除中的应用 |
| CN112159478A (zh) * | 2020-09-28 | 2021-01-01 | 江南大学 | 一种融合蛋白及其在降解聚合物中的应用 |
| CN112159478B (zh) * | 2020-09-28 | 2023-02-24 | 江南大学 | 一种融合蛋白及其在降解聚合物中的应用 |
| CN112725302A (zh) * | 2021-01-15 | 2021-04-30 | 江南大学 | 一种融合蛋白的构建及其降解聚合物的方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102358896B (zh) | 2013-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11884954B2 (en) | Protein complex based on DNA enzymes of E family of Escherichia coli and application thereof in artificial protein scaffolds | |
| CN101591648B (zh) | 一种耐热角质酶-cbd的制备及其在棉纤维精练中的应用 | |
| CN102358896A (zh) | 一种耐热角质酶-cbd融合酶及其突变体和应用 | |
| JP6330240B2 (ja) | 耐熱性β−グルコシダーゼ | |
| JP6340647B2 (ja) | 超耐熱性セロビオハイドロラーゼ | |
| JP6319907B2 (ja) | 耐熱性β―キシロシダーゼ | |
| US9580702B2 (en) | Thermostable cellobiohydrolase and amino acid substituted variant thereof | |
| JP6350987B2 (ja) | ＧＨファミリー３に属する耐熱性β―キシロシダーゼ | |
| CN109762799A (zh) | 一种κ-卡拉胶酶及其应用 | |
| JP6364662B2 (ja) | ＧＨファミリー３に属する耐熱性β―キシロシダーゼ | |
| CN101392242A (zh) | α-葡萄糖苷酶及其基因和制备方法以及载体和宿主细胞 | |
| CN107236719B (zh) | 耐热性纤维二糖水解酶 | |
| JP6286745B2 (ja) | 耐熱性β−グルコシダーゼ | |
| CN103667212B (zh) | 一种糖化酶及其应用 | |
| JP6429377B2 (ja) | 耐熱性セロビオハイドロラーゼ | |
| CN104911165A (zh) | 一种纤维素酶基因及其应用 | |
| EP3095858B1 (en) | Thermostable glycoside hydrolase | |
| EP2995687B1 (en) | Thermostable beta-glucosidase | |
| JP6531974B2 (ja) | 耐熱性セロビオハイドロラーゼ | |
| JP5062730B2 (ja) | 改良耐熱性セルラーゼ | |
| JP2017175957A (ja) | 耐熱性セロビオハイドロラーゼ | |
| Dung et al. | PURIFICATION OF RECOMBINANT ENDOGLUCANASE GH5-CBM72-CBM72 EXPRESSED IN Escherichia coli. | |
| KR101388460B1 (ko) | 동애등에 장 내 미생물 유래의 신규 베타-글루코시다제 | |
| JP6244597B2 (ja) | 超耐熱性エンドグルカナーゼ | |
| EP2982750B1 (en) | Hyperthermostable endoglucanase belonging to gh family 12 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |