CN107828763B - Endoglucanase, its encoding gene cel5A-h47 and its application - Google Patents
Endoglucanase, its encoding gene cel5A-h47 and its application Download PDFInfo
- Publication number
- CN107828763B CN107828763B CN201711369277.1A CN201711369277A CN107828763B CN 107828763 B CN107828763 B CN 107828763B CN 201711369277 A CN201711369277 A CN 201711369277A CN 107828763 B CN107828763 B CN 107828763B
- Authority
- CN
- China
- Prior art keywords
- endoglucanase
- cel5a
- gene
- seq
- ile
- 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.)
- Expired - Fee Related
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 title abstract description 25
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 14
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000001913 cellulose Substances 0.000 claims description 11
- 229920002678 cellulose Polymers 0.000 claims description 11
- 238000006731 degradation reaction Methods 0.000 claims description 7
- 230000015556 catabolic process Effects 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 108010089934 carbohydrase Proteins 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 25
- 102000004190 Enzymes Human genes 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 21
- 230000006698 induction Effects 0.000 abstract description 5
- 230000009465 prokaryotic expression Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 abstract 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 abstract 1
- 238000003766 bioinformatics method Methods 0.000 abstract 1
- 238000002525 ultrasonication Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 21
- 239000007788 liquid Substances 0.000 description 16
- 230000002255 enzymatic effect Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 102000018120 Recombinases Human genes 0.000 description 6
- 108010091086 Recombinases Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 210000004767 rumen Anatomy 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000004753 textile Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- -1 EC3.2.1.4) Proteins 0.000 description 2
- 101710112457 Exoglucanase Proteins 0.000 description 2
- 239000004606 Fillers/Extenders Substances 0.000 description 2
- FPQMQEOVSKMVMA-ACRUOGEOSA-N Lys-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CCCCN)N)O FPQMQEOVSKMVMA-ACRUOGEOSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000007852 inverse PCR Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 108010073969 valyllysine Proteins 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 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
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- XEXJJJRVTFGWIC-FXQIFTODSA-N Ala-Asn-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XEXJJJRVTFGWIC-FXQIFTODSA-N 0.000 description 1
- PXKLCFFSVLKOJM-ACZMJKKPSA-N Ala-Asn-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PXKLCFFSVLKOJM-ACZMJKKPSA-N 0.000 description 1
- MKZCBYZBCINNJN-DLOVCJGASA-N Ala-Asp-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MKZCBYZBCINNJN-DLOVCJGASA-N 0.000 description 1
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 1
- PUBLUECXJRHTBK-ACZMJKKPSA-N Ala-Glu-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O PUBLUECXJRHTBK-ACZMJKKPSA-N 0.000 description 1
- QJABSQFUHKHTNP-SYWGBEHUSA-N Ala-Ile-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QJABSQFUHKHTNP-SYWGBEHUSA-N 0.000 description 1
- GHBSKQGCIYSCNS-NAKRPEOUSA-N Ala-Leu-Asp-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O GHBSKQGCIYSCNS-NAKRPEOUSA-N 0.000 description 1
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- RAAWHFXHAACDFT-FXQIFTODSA-N Ala-Met-Asn Chemical compound CSCC[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CC(N)=O)C(O)=O RAAWHFXHAACDFT-FXQIFTODSA-N 0.000 description 1
- CYXCAHZVPFREJD-LURJTMIESA-N Arg-Gly-Gly Chemical compound NC(=N)NCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O CYXCAHZVPFREJD-LURJTMIESA-N 0.000 description 1
- RFXXUWGNVRJTNQ-QXEWZRGKSA-N Arg-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N RFXXUWGNVRJTNQ-QXEWZRGKSA-N 0.000 description 1
- DNUKXVMPARLPFN-XUXIUFHCSA-N Arg-Leu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DNUKXVMPARLPFN-XUXIUFHCSA-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
- YNSUUAOAFCVINY-OSUNSFLBSA-N Arg-Thr-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YNSUUAOAFCVINY-OSUNSFLBSA-N 0.000 description 1
- RZVVKNIACROXRM-ZLUOBGJFSA-N Asn-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N RZVVKNIACROXRM-ZLUOBGJFSA-N 0.000 description 1
- GNKVBRYFXYWXAB-WDSKDSINSA-N Asn-Glu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O GNKVBRYFXYWXAB-WDSKDSINSA-N 0.000 description 1
- BKDDABUWNKGZCK-XHNCKOQMSA-N Asn-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N)C(=O)O BKDDABUWNKGZCK-XHNCKOQMSA-N 0.000 description 1
- GYOHQKJEQQJBOY-QEJZJMRPSA-N Asn-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N GYOHQKJEQQJBOY-QEJZJMRPSA-N 0.000 description 1
- UYCPJVYQYARFGB-YDHLFZDLSA-N Asn-Phe-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O UYCPJVYQYARFGB-YDHLFZDLSA-N 0.000 description 1
- PLTGTJAZQRGMPP-FXQIFTODSA-N Asn-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(N)=O PLTGTJAZQRGMPP-FXQIFTODSA-N 0.000 description 1
- NSTBNYOKCZKOMI-AVGNSLFASA-N Asn-Tyr-Glu Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O NSTBNYOKCZKOMI-AVGNSLFASA-N 0.000 description 1
- VPPXTHJNTYDNFJ-CIUDSAMLSA-N Asp-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N VPPXTHJNTYDNFJ-CIUDSAMLSA-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
- VFUXXFVCYZPOQG-WDSKDSINSA-N Asp-Glu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VFUXXFVCYZPOQG-WDSKDSINSA-N 0.000 description 1
- DJCAHYVLMSRBFR-QXEWZRGKSA-N Asp-Met-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC(O)=O DJCAHYVLMSRBFR-QXEWZRGKSA-N 0.000 description 1
- FAUPLTGRUBTXNU-FXQIFTODSA-N Asp-Pro-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O FAUPLTGRUBTXNU-FXQIFTODSA-N 0.000 description 1
- OTKUAVXGMREHRX-CFMVVWHZSA-N Asp-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 OTKUAVXGMREHRX-CFMVVWHZSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- AVZHGSCDKIQZPQ-CIUDSAMLSA-N Glu-Arg-Ala Chemical compound C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AVZHGSCDKIQZPQ-CIUDSAMLSA-N 0.000 description 1
- RDDSZZJOKDVPAE-ACZMJKKPSA-N Glu-Asn-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDDSZZJOKDVPAE-ACZMJKKPSA-N 0.000 description 1
- UMIRPYLZFKOEOH-YVNDNENWSA-N Glu-Gln-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UMIRPYLZFKOEOH-YVNDNENWSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 1
- CIMULJZTTOBOPN-WHFBIAKZSA-N Gly-Asn-Asn Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CIMULJZTTOBOPN-WHFBIAKZSA-N 0.000 description 1
- FUTAPPOITCCWTH-WHFBIAKZSA-N Gly-Asp-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FUTAPPOITCCWTH-WHFBIAKZSA-N 0.000 description 1
- JUBDONGMHASUCN-IUCAKERBSA-N Gly-Glu-His Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O JUBDONGMHASUCN-IUCAKERBSA-N 0.000 description 1
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-N 0.000 description 1
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 1
- SCWYHUQOOFRVHP-MBLNEYKQSA-N Gly-Ile-Thr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SCWYHUQOOFRVHP-MBLNEYKQSA-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
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- KAFZDWMZKGQDEE-SRVKXCTJSA-N His-His-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KAFZDWMZKGQDEE-SRVKXCTJSA-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
- ATXGFMOBVKSOMK-PEDHHIEDSA-N Ile-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N ATXGFMOBVKSOMK-PEDHHIEDSA-N 0.000 description 1
- IDAHFEPYTJJZFD-PEFMBERDSA-N Ile-Asp-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N IDAHFEPYTJJZFD-PEFMBERDSA-N 0.000 description 1
- QZZIBQZLWBOOJH-PEDHHIEDSA-N Ile-Ile-Val Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)O QZZIBQZLWBOOJH-PEDHHIEDSA-N 0.000 description 1
- IALVDKNUFSTICJ-GMOBBJLQSA-N Ile-Met-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)O)N IALVDKNUFSTICJ-GMOBBJLQSA-N 0.000 description 1
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr 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)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 1
- YWCJXQKATPNPOE-UKJIMTQDSA-N Ile-Val-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YWCJXQKATPNPOE-UKJIMTQDSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- UCOCBWDBHCUPQP-DCAQKATOSA-N Leu-Arg-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O UCOCBWDBHCUPQP-DCAQKATOSA-N 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- XVSJMWYYLHPDKY-DCAQKATOSA-N Leu-Asp-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O XVSJMWYYLHPDKY-DCAQKATOSA-N 0.000 description 1
- RSFGIMMPWAXNML-MNXVOIDGSA-N Leu-Gln-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RSFGIMMPWAXNML-MNXVOIDGSA-N 0.000 description 1
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 1
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 1
- TWPCWKVOZDUYAA-KKUMJFAQSA-N Lys-Phe-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O TWPCWKVOZDUYAA-KKUMJFAQSA-N 0.000 description 1
- LMGNWHDWJDIOPK-DKIMLUQUSA-N Lys-Phe-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LMGNWHDWJDIOPK-DKIMLUQUSA-N 0.000 description 1
- DNWBUCHHMRQWCZ-GUBZILKMSA-N Lys-Ser-Gln Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DNWBUCHHMRQWCZ-GUBZILKMSA-N 0.000 description 1
- HUURTRNKPBHHKZ-JYJNAYRXSA-N Met-Phe-Val Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 HUURTRNKPBHHKZ-JYJNAYRXSA-N 0.000 description 1
- 241001465754 Metazoa Species 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
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- AYPMIIKUMNADSU-IHRRRGAJSA-N Phe-Arg-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O AYPMIIKUMNADSU-IHRRRGAJSA-N 0.000 description 1
- HTKNPQZCMLBOTQ-XVSYOHENSA-N Phe-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O HTKNPQZCMLBOTQ-XVSYOHENSA-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
- MMYUOSCXBJFUNV-QWRGUYRKSA-N Phe-Gly-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N MMYUOSCXBJFUNV-QWRGUYRKSA-N 0.000 description 1
- KRYSMKKRRRWOCZ-QEWYBTABSA-N Phe-Ile-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O KRYSMKKRRRWOCZ-QEWYBTABSA-N 0.000 description 1
- OQTDZEJJWWAGJT-KKUMJFAQSA-N Phe-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O OQTDZEJJWWAGJT-KKUMJFAQSA-N 0.000 description 1
- VXCHGLYSIOOZIS-GUBZILKMSA-N Pro-Ala-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 VXCHGLYSIOOZIS-GUBZILKMSA-N 0.000 description 1
- CJZTUKSFZUSNCC-FXQIFTODSA-N Pro-Asp-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 CJZTUKSFZUSNCC-FXQIFTODSA-N 0.000 description 1
- OQSGBXGNAFQGGS-CYDGBPFRSA-N Pro-Val-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OQSGBXGNAFQGGS-CYDGBPFRSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 1
- QWZIOCFPXMAXET-CIUDSAMLSA-N Ser-Arg-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O QWZIOCFPXMAXET-CIUDSAMLSA-N 0.000 description 1
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 1
- 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 1
- NLOAIFSWUUFQFR-CIUDSAMLSA-N Ser-Leu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O NLOAIFSWUUFQFR-CIUDSAMLSA-N 0.000 description 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- HKHCTNFKZXAMIF-KKUMJFAQSA-N Ser-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=C(O)C=C1 HKHCTNFKZXAMIF-KKUMJFAQSA-N 0.000 description 1
- SYCFMSYTIFXWAJ-DCAQKATOSA-N Ser-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N SYCFMSYTIFXWAJ-DCAQKATOSA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- UDQBCBUXAQIZAK-GLLZPBPUSA-N Thr-Glu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UDQBCBUXAQIZAK-GLLZPBPUSA-N 0.000 description 1
- XOTBWOCSLMBGMF-SUSMZKCASA-N Thr-Glu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOTBWOCSLMBGMF-SUSMZKCASA-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
- XFTYVCHLARBHBQ-FOHZUACHSA-N Thr-Gly-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XFTYVCHLARBHBQ-FOHZUACHSA-N 0.000 description 1
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 1
- WPAKPLPGQNUXGN-OSUNSFLBSA-N Thr-Ile-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WPAKPLPGQNUXGN-OSUNSFLBSA-N 0.000 description 1
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 1
- HUPLKEHTTQBXSC-YJRXYDGGSA-N Thr-Ser-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HUPLKEHTTQBXSC-YJRXYDGGSA-N 0.000 description 1
- NHQVWACSJZJCGJ-FLBSBUHZSA-N Thr-Thr-Ile 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@@H](C)CC)C(O)=O NHQVWACSJZJCGJ-FLBSBUHZSA-N 0.000 description 1
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 1
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 1
- IQGJAHMZWBTRIF-UBHSHLNASA-N Trp-Asp-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N IQGJAHMZWBTRIF-UBHSHLNASA-N 0.000 description 1
- CXPJPTFWKXNDKV-NUTKFTJISA-N Trp-Leu-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 CXPJPTFWKXNDKV-NUTKFTJISA-N 0.000 description 1
- UKWSFUSPGPBJGU-VFAJRCTISA-N Trp-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O UKWSFUSPGPBJGU-VFAJRCTISA-N 0.000 description 1
- GRSCONMARGNYHA-PMVMPFDFSA-N Trp-Lys-Phe Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GRSCONMARGNYHA-PMVMPFDFSA-N 0.000 description 1
- KSVMDJJCYKIXTK-IGNZVWTISA-N Tyr-Ala-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 KSVMDJJCYKIXTK-IGNZVWTISA-N 0.000 description 1
- AKLNEFNQWLHIGY-QWRGUYRKSA-N Tyr-Gly-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N)O AKLNEFNQWLHIGY-QWRGUYRKSA-N 0.000 description 1
- BCOBSVIZMQXKFY-KKUMJFAQSA-N Tyr-Ser-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O BCOBSVIZMQXKFY-KKUMJFAQSA-N 0.000 description 1
- MWUYSCVVPVITMW-IGNZVWTISA-N Tyr-Tyr-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 MWUYSCVVPVITMW-IGNZVWTISA-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
- XLDYBRXERHITNH-QSFUFRPTSA-N Val-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)C(C)C XLDYBRXERHITNH-QSFUFRPTSA-N 0.000 description 1
- QHFQQRKNGCXTHL-AUTRQRHGSA-N Val-Gln-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QHFQQRKNGCXTHL-AUTRQRHGSA-N 0.000 description 1
- JVYIGCARISMLMV-HOCLYGCPSA-N Val-Gly-Trp Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N JVYIGCARISMLMV-HOCLYGCPSA-N 0.000 description 1
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 1
- USXYVSTVPHELAF-RCWTZXSCSA-N Val-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](C(C)C)N)O USXYVSTVPHELAF-RCWTZXSCSA-N 0.000 description 1
- MIAZWUMFUURQNP-YDHLFZDLSA-N Val-Tyr-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N MIAZWUMFUURQNP-YDHLFZDLSA-N 0.000 description 1
- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 description 1
- ZLNYBMWGPOKSLW-LSJOCFKGSA-N Val-Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLNYBMWGPOKSLW-LSJOCFKGSA-N 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 108010072405 glycyl-aspartyl-glycine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 108010063431 methionyl-aspartyl-glycine Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
- 230000003462 zymogenic effect Effects 0.000 description 1
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
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- 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)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明属于基因工程领域，具体涉及一种内切葡聚糖酶、其编码基因cel5A‑h47及其应用。一种编码所述内切葡聚糖酶的基因cel5A‑h47，其核苷酸序列如SEQ ID No.1所示。生物信息学分析表明，该基因编码的内切葡聚糖酶属于糖苷水解酶第5家族。发明人通过设计引物成功将其克隆，并在原核表达系统中成功表达，经诱导超声纯化所得的重组内切葡聚糖酶的最适作用pH为5.0，最适温度为50°C，在50°C以下能保持相对稳定的酶活性，具有较强的pH耐受性，在pH4.0‑10.0下能保持较高的酶活性。该重组内切葡聚糖酶具有较大的研究和工业应用潜力。The invention belongs to the field of genetic engineering, and in particular relates to an endoglucanase, its encoding gene cel5A-h47 and applications thereof. A gene cel5A-h47 encoding the endoglucanase, the nucleotide sequence of which is shown in SEQ ID No.1. Bioinformatics analysis showed that the endoglucanase encoded by this gene belongs to the fifth family of glycoside hydrolases. The inventors successfully cloned it by designing primers, and successfully expressed it in a prokaryotic expression system. The optimum pH of the recombinant endoglucanase purified by induction ultrasonication was 5.0, and the optimum temperature was 50°C. Relatively stable enzyme activity can be maintained below °C, with strong pH tolerance, and high enzyme activity can be maintained at pH 4.0-10.0. The recombinant endoglucanase has great research and industrial application potential.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to a kind of endoglucanase, its encoding gene cel5A-h47 and
It is applied.
Background technique
Main constituents of the cellulose as plant cell wall are the most abundant renewable resources in current nature,
However its obstinate structure limits its utilization.Cellulose degradation needs endoglucanase at the process of glucose
(endoglucanase, EC3.2.1.4), exoglucanase (exoglucanase, E.C.3.2.1.91) and beta-glucosidase
The system same-action of enzyme (β-glucosidase, E.C.3.2.1.21) three kinds of cellulases.Wherein, endoglucanase is fine
The most important ingredient of plain enzyme system is tieed up, it mainly acts on the noncrystalline domain of cellulose, the β in random hydrolysis cellulose chain-Isosorbide-5-Nitrae sugar
Glycosidic bond, cut staple element long-chain are converted into the short chain of a large amount of different polymerization degrees, the degree of polymerization of cellulose are reduced, in degradation process
It is middle to play crucial effect.Therefore, its Commercial cultivation is most important for the degradation efficiency of cellulose.Currently, the enzyme is
It is widely used in the industries such as papermaking, weaving, food, bio-fuel, animal feeding.Being produced into using needs in these industries
This is low, the endoglucanase with different optimal pH and temperature and better stability.In the past few decades, it largely grinds
Study carefully the cost for concentrating on how reducing fungi production cellulase, the main random mutagenesis including bacterium, industry optimization, foreign protein
The methods of addition.However, fully understanding due to lacking to complicated enzyme system and zymogenic bacteria, this process is relatively slow.Therefore, it seeks
Look for a large amount of novel endoglucanases particularly important.
With group appearance learned, it is further that some microbial genome sequencings for producing lignocellulolyticenzymes are completed successively
Research provide clearly genetic background, more and more cellulose enzyme gene nucleotide sequences are determined, and in large intestine
It is expressed in bacillus and yeast, effectively supplements traditional enzyme system.And transcript profile sequencing is capable of providing gene cDNA
The expression of sequence and gene can really reflect the expression of albumen in vivo, therefore, from environmental microorganism transcript profile
It is relatively new that endo glucanase gene is excavated in information, while being also effective research direction.
Summary of the invention
The present invention provides a kind of novel endo glucanase gene cel5-h47, recombinase Cel5- after prokaryotic expression
H47 is thermophilic medium temperature enzyme, is able to maintain higher enzymatic activity at 50 DEG C；Optimal reactive temperature is 50 DEG C, optimal pH 5.0；With very
Strong pH tolerance is able to maintain high enzyme activity at pH4.0-10.0.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of endoglucanase, amino acid sequence is as shown in SEQ ID No.2.
A kind of endo glucanase gene cel5A-h47 encoding the endoglucanase, nucleotide sequence such as SEQ
Shown in ID No.1.Endo glucanase gene cel5A-h47 of the present invention comes from sheep rumen microorganism transcript profile data.
A kind of recombinant vector comprising the endo glucanase gene cel5A-h47.
A kind of recombinant bacterium comprising the xylanase gene cel5A-h47.
A method of endoglucanase is prepared, is recombinant bacterium described in fermented and cultured, obtains endoglucanase.
Expand the specific primer that the endo glucanase gene cel5A-h47 is used, the specific primer are as follows:
H47-F:5 'ACAGCAAATGGGTCGCGGATCCGTGACCATGAATTTTGTTACCGTAAG 3 ', nucleotide sequence such as SEQ
Shown in ID No.3；H47-R:5 'CTTGTCGACGGAGCTCGAATTCTTATGAAGCCGCTCCGTCCA3 ', nucleotide sequence
As shown in SEQ ID No.4.
The acquisition methods of endo glucanase gene cel5A-h47 of the present invention are as follows
A, the building of the clone of cel5A-h47 endo glucanase gene and recombinant plasmid pET-28a/cel5A-h47；
B, expression of the recombinant plasmid pET-28a/cel5A-h47 in e. coli bl21 (DE3)；
C, the induction, purifying of recombinant protein and enzymatic property analysis；
Preferably, clone and the recombinant plasmid pET-28a/ of step a cel5A-h47 endo glucanase gene
The building process of cel5A-h47 is specific as follows:
1. PCR amplification endo-glucanase enzyme coding gene cel5A-h47；
2. Inverse PCR amplification preparation linearisation pET-28a plasmid；
Experience 3. homologous recombination method constitutes recombinant plasmid pET-28a/cel5A-h47 and converts to e. coli bl21 (DE3)
State cell.
Application of the endoglucanase of the present invention in terms of the cellulose degradation under strong basicity environment, as alkalinity is washed
Wash the industrial productions such as agent, textile industry.The strong basicity environment is that pH is greater than 7.0.Further, the strong basicity environment is
pH7.0-8.0。
The present invention has following advantages and good effect:
1. the gene cloned in the present invention comes from sheep rumen microorganism transcript profile data, guarantee the novelty of gene.
2. select prokaryotic expression system in the present invention, there is genetic background to understand, is easy to operate and with short production cycle etc. excellent
Point.
3. what is taken when PCR amplification target fragment in the present invention is the specific primer of designed, designed, can be effectively ensured
Amplification efficiency and product specificities.
4. endoglucanase of the invention has at 50 DEG C or less compared with strong tolerance, it is able to maintain higher enzymatic activity.
5. there is endoglucanase of the invention wider pH adaptation range can protect within the scope of pH4.0-10.0
Greater activity is held (within the scope of pH4.0-8.0, to be able to maintain 80% or more enzymatic activity, be able to maintain within the scope of pH9.0-10.0
65% or more enzymatic activity).Therefore can be used under strong alkali environment cellulose industry degradation, such as detergent, weave industry,
Textile compared to convention acidic endo-glucanase enzymatic treatment can generate phenomenon of fading, and endoglucanase can be in the present invention
It is handled in alkaline environment, this phenomenon is avoided to occur.
Detailed description of the invention
Fig. 1 is clone and the expression strategy of cel5A-h47 endo glucanase gene；
Fig. 2 is clone's cel5A-h47PCR product；
Fig. 3 is the SDS-PAGE analysis chart of recombinant protein c el5A-H47；
Fig. 4 is influence of the pH to Cel5A-H47 enzymatic activity；
Fig. 5 is the pH stability of Cel5A-H47 enzyme；
Fig. 6 is influence of the temperature to Cel5A-H47 enzymatic activity；
Fig. 7 is the thermal stability of Cel5A-H47 enzyme.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.It should be appreciated that this hair
Bright implementation is not limited by the following examples, and the accommodation in any form made to the present invention and/or changed will all be fallen
Enter the scope of the present invention.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, used equipment and raw material etc.
It is commercially available or commonly used in the art.Method in following embodiments is unless otherwise instructed the normal of this field
Rule method.
Embodiment:
One, sheep rumen content Total RNAs extraction
Take rumen content sample extraction total serum IgE (the following lysate RL, adsorption column RA, deproteinized of -80 DEG C of freezen protectives
Liquid RE, rinsing liquid RW, RNase Free Water are purchased from Ai Delai Biotechnology Co., Ltd) it is ground into rapidly in liquid nitrogen
Powder, every 50~100mg sample are homogenized after adding the lysate RL of 1ml, and acutely concussion is mixed with lytic cell.It is incubated at 15-30 DEG C
Educating 5 minutes decomposes ribosome completely.12000rpm, 4 DEG C of centrifugation 10min remove insoluble matter.Supernatant is transferred to one to do
Net centrifuge tube, every 1ml lysate RL are added 0.2ml chloroform, acutely vibrate 15s, 12000rpm, 4 DEG C of centrifugation 10min.It takes
Layer colourless aqueous phase is transferred in new pipe, and the dehydrated alcohol of water phase volume half is added, and is added in adsorption column RA after mixing, at room temperature
12000rpm is centrifuged 45s, abandons supernatant.500 μ l protein liquid removal RE are added, 12000rpm is centrifuged 45s at room temperature, abandons supernatant.It is added
500 μ l rinsing liquid RW, 12000rpm is centrifuged 45s at room temperature, abandons supernatant.500 μ l rinsing liquid RW are added again, at room temperature
12000rpm is centrifuged 45s, abandons supernatant.13000rpm is centrifuged 2min and removes rinsing liquid as far as possible at room temperature.Adsorption column RA is put into
In RNase free centrifuge tube, position adds 50-80 μ l RNase Free Water among adsorbed film, is placed at room temperature for 2min,
12000rpm is centrifuged 1min, obtains total serum IgE, -80 DEG C of preservations.
Two, the clone of cel5A-h47 gene
The clone of cel5A-h47 endo glucanase gene and expression strategy are shown in Fig. 1.
(1) using rumen content total serum IgE as template, the first chain cDNA (following Random is synthesized using reverse transcription
Primer、RNase Free Water、5×RT Buffer、dNTP Mixture、RNase Inhibitor、ReverTra
Ace is purchased from mill Biotechnology Co., Ltd, Japan)
1. preparing following mixed liquor in microcentrifugal tube:
65 DEG C of incubation 5min make RNA thermal denaturation, are immediately placed on ice.
2. preparing following reverse transcription reaction mixed liquor:
3. successively by reaction solution in 30 DEG C of incubation 10min；42 DEG C of incubation 20min；99 DEG C of incubation 5min；4 DEG C of incubation 5min,
Then brief centrifugation obtains the first chain of cDNA.
(2) cel5A-h47 gene magnification
1. designing the primer containing pet28a Component Vectors homologous sequence and carrier Inverse PCR amplification primer:
H47-F:5 'ACAGCAAATGGGTCGCGGATCCGTGACCATGAATTTTGTTACCGTAAG 3 ', nucleotides sequence
It arranges as shown in SEQ ID No.3,
H47-R:5 'CTTGTCGACGGAGCTCGAATTCTTATGAAGCCGCTCCGTCCA3 ', nucleotide sequence such as SEQ
Shown in ID No.4.
2. using the cDNA of synthesis as template PCR amplifications cel5A-h47 gene, the following (wherein 2 × Hide- of amplification system
Fidelity Master Mix is purchased from Beijing Qing Kexin industry Bioisystech Co., Ltd):
Vortex oscillation mixes, and is put into PCR instrument after being slightly centrifuged.The amplification condition of cel5A-h47 gene is as follows:
PCR result is shown in Fig. 2, and amplification obtains the band of 1098bp, occurs in swimming lane without other miscellaneous bands, illustrate design primer
It is specific good.
Three, the building of recombinant plasmid pET-28a/cel5A-h47
(1) linearisation pET-28a carrier preparation
PCR reversely expands pET-28a vector plasmid, preparation linearisation cloning vector.PCR amplification system is as follows:
Vortex oscillation mixes, and is put into PCR instrument after being slightly centrifuged.The amplification condition for linearizing pET-28a carrier is as follows:
(2) target gene is connect with pET-28a carrier
Using the principle of homologous recombination, by SoSoo recombinant clone kit, (holding up the new industry biotechnology of section purchased from Beijing has
Limit company) quickly by target gene directed cloning into linearisation pET-28a carrier.Reaction system is as follows:
10 μ l systems, target gene and expression vector molar ratio are 5:1,50 DEG C of reaction 30min.
Four, expression of the recombinant plasmid in Escherichia coli
It is heat-shock transformed that 10 μ l connection products and E. coli competent BL21 (DE3) is taken carefully to mix, ice bath 30min；42
DEG C water-bath heat shock 50s takes out ice bath 2min immediately；The SOC solution of 37 DEG C of 500 μ l preheatings, 150rpm constant temperature incubation 1h is added；
Bacterium solution after drawing conversion, is coated in the LB screening and culturing medium containing kanamycins, 37 DEG C of constant temperature incubation 12-16h；Picking bacterial plaque
PCR identification is carried out, positive clone is confirmed as and is transferred to expansion culture in the LB liquid medium containing kanamycins.
Five, the induction, purifying of positive strain and enzymatic property analysis
(1) induction of positive strain
1. it draws 10 μ l positive bacterium solutions to be forwarded in LB liquid medium of the 5mL containing kanamycins (50 μ g/mL), 37 DEG C,
220rpm cultivates 12-16h；
2. 5mL bacterium solution is forwarded in 200mL LB liquid medium, 37 DEG C, 220rpm continues culture to OD=0.5-
1.0 (about 3-4 hours)；
3. being added 2ml IPTG (final concentration 1mmol/L), 20 DEG C, 150rpm low temperature induction culture 6h；
4. the bacterium solution of inducing expression is transferred in 50mL centrifuge tube, 12000rpm, 4 DEG C of centrifugation 15min discard supernatant,
30mL PBS buffer solution (pH7.4) is added, thallus is sufficiently resuspended；
5. be put into the beaker for filling ice cube, ultrasonic wave be crushed repeatedly 3 times (each ultrasound 15min, working time 3s,
Intermittent time 5s, No. 6 amplitude transformers are crushed power 195W)；
6. broken bacterium solution 12000rpm, 4 DEG C of centrifugation 15min, gained supernatant is crude protein liquid (CP), is used immediately or 4 DEG C
It saves.
(2) destination protein affinity chromatography
It takes and walks crude protein liquid obtained on 30mL, Ni-NTA resin extender 2mL is added, after mixing well, by mixed liquor
It is transferred to chromatography pillar, upper prop collection repeatedly penetrates liquid (flow-through), is denoted as FT.
When albumen sample liquid liquid level to be mixed is close to Ni-NTA resin extender, with the Washing of 40 times of bed volumes (40mL)
Buffer 1 (imidazoles containing 20mmol/L) washs pillar, removes foreigh protein removing, collects cleaning solution with 1.5mL centrifuge tube, is denoted as W1.
It is washed pillar 2 times, is used with the washing buffer 2 (imidazoles containing 50mmol/L) of 1 times of bed volume (1mL)
1.5mL centrifuge tube collects cleaning solution, is denoted as W2, W3.
Destination protein, elution 4 are eluted with the Elution buffer (imidazoles containing 250mmol/L) of 1 times of bed volume (1mL)
It is secondary, eluent is collected with 1.5mL centrifuge tube, is denoted as E1, E2, E3, E4.
Purified product is subjected to SDS-PAGE analysis (Fig. 3), as the result is shown: the albumen of recombinase Cel5A-H47 is a small amount of greatly
For 44kDa or so.
(3) enzymatic property is analyzed
The definition of endo-glucanase enzyme activity unit: the enzyme amount for generating 1 μm of ol reduced sugar per minute is 1 unit of activity (U),
Calculation formula: enzyme activity
A: the concentration (μm ol/mL) of reduced sugar is generated
K: pure enzyme extension rate
T: enzyme digestion reaction time (min)
C: pure enzyme protein concentration (mg/mL)
Influence of the pH to enzymatic activity
Optimal pH: be respectively adopted pH 3.0-10.0 buffer (wherein pH3.0-8.0 using McIlvaine ' s buffer
Liquid, pH8.0-9.0 use Tris-HCl buffer, and pH9.0-10.0 uses Glycine-NaOH buffer) 1% carboxymethyl of configuration
Sodium cellulosate (CMC) reaction substrate takes 450 μ l substrates in 50 DEG C of preheating 5min, 50 μ l enzyme solutions is added, react under the conditions of 50 DEG C
10min is added 500 μ l DNS solution, measures relative activity respectively using DNS method.As the result is shown (Fig. 4: recombinase Cel5A-
The optimal pH of H47 is 5.0.
PH stability: endoglucanase is incubated for 30min under the conditions of pH 3.0-10.0,37 DEG C respectively, then most
Enzyme activity is detected under the conditions of suitable.It is control with enzyme activity of the untreated endoglucanase under optimum condition, calculates remaining inscribe
The relative activity of dextranase.As the result is shown (Fig. 5): recombinase Cel5A-H47 has stronger pH between pH4.0-10.0
Tolerance is able to maintain high enzyme activity.
Influence of the temperature to enzyme activity
Optimum temperature: pH6.0 buffer 1%CMC substrate is used, endoglucanase is measured using DNS method respectively and is existed
Enzyme activity at 30 DEG C -70 DEG C calculates relative activity.As the result is shown (Fig. 6): the optimal reactive temperature of recombinase Cel5A-H47 is
50℃。
Thermal stability: endoglucanase being respectively placed under the conditions of 40,50 and 60 DEG C and is incubated for 50min respectively, every
10min sampling measures enzyme activity under optimum condition.It is control with enzyme activity of the untreated endoglucanase under optimum condition,
Calculate the relative activity of remaining endoglucanase.As the result is shown (Fig. 7): recombinase Cel5A-H47 is thermophilic medium temperature enzyme, at 50 DEG C
It is able to maintain greater activity below, but when temperature is higher than 60 DEG C, enzymatic activity is reduced rapidly.
Conclusion: according to above-mentioned experiment conclusion, it was demonstrated that endoglucanase of the invention is wide with preferable heat resistance and relatively
PH tolerance range, be still able to maintain under the particular surroundings of high ph-values good enzymatic activity (within the scope of pH4.0-8.0, equal energy
80% or more enzymatic activity is kept, 65% or more enzymatic activity is able to maintain within the scope of pH9.0-10.0), there is biggish industrialized production
And application potential, it can be applied to the cellulose degradation under strong basicity environment, such as alkaline detergent, textile industry industrial production.
Embodiment described above is the preferred version that the present invention excavates gene and external preparation endo-glucanase enzyme method,
It is not intended to limit the present invention in any form, there are also it on the premise of not exceeding the technical scheme recorded in the claims
Its variant and remodeling.
Sequence table
<110>Zhejiang University
<120>endoglucanase, its encoding gene cel5A-h47 and its application
<130> ZJDX-WJK007
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1098
<212> DNA
<213>endo glucanase gene cel5A-h47 (endoglucanase)
<400> 1
gtgaccatga attttgttac cgtaagatac agccacaagg atacacaatc acggcaggaa 60
cagatcggag atgatggaat tatagttgat atcggtttca gaaacgcaaa cgagatagta 120
gaggctatga atgtcggctg gaatctcggc aattcccttg acagctataa gacaggcctc 180
agcggtacag ctaccgagac aggctgggga aatcctgcaa ctactcctga tatggtaaaa 240
tctgtcaaaa gtgcaggatt caatactata cgcatccctg taacatgggg cgaacatctt 300
gatggtacta ctatcgatcc aagctggctc gcaagagtgc aggaagtagt agattatgct 360
tataatgaag gaatgttcgt catcctcgat atgcatcatg acgactatat atggctgact 420
cccgataatg aagtttacaa cgagaacagc gccaagctca gggctatatg gctccagata 480
gcaaacagat tcaaggatta cggcgacagg ctgatatttg agggcttgaa tgagccgagg 540
accataggaa gtgttactga atggagaggc ggtactacag aggaaagagc tgtagttaat 600
aactatgaag cggactttgt gaaaactatc agaagttcag gcggaaataa tcctgcaaga 660
acgctgataa tcacatccta tgctgcttct gccgaatctg ttgcactcga tgatgttatc 720
gttcctaaaa caggaaatat aatagtatcg gttcactatt acgctccatg gaaattctcc 780
gagggcaatg ctgataaatt tgatgacgaa ggcagagctg aaattgatgc aaagttcatt 840
gaactcagaa gcaaatttat tgacagaggc ataccggtta tcattgatga attcggctgt 900
gttgccaaag ctgataatga cgagagagct aagtactaca agtattacat agcatctgca 960
aaatcacagg gcataaaatg cgtggtctgg gataacggaa taacaacagg cgacggcggt 1020
tttggtatct tcacaagaag cagcaacgaa tggaataagg ctattcttaa agctataatg 1080
gacggagcgg cttcataa 1098
<210> 2
<211> 365
<212> PRT
<213>endoglucanase (endoglucanase)
<400> 2
Val Thr Met Asn Phe Val Thr Val Arg Tyr Ser His Lys Asp Thr Gln
1 5 10 15
Ser Arg Gln Glu Gln Ile Gly Asp Asp Gly Ile Ile Val Asp Ile Gly
20 25 30
Phe Arg Asn Ala Asn Glu Ile Val Glu Ala Met Asn Val Gly Trp Asn
35 40 45
Leu Gly Asn Ser Leu Asp Ser Tyr Lys Thr Gly Leu Ser Gly Thr Ala
50 55 60
Thr Glu Thr Gly Trp Gly Asn Pro Ala Thr Thr Pro Asp Met Val Lys
65 70 75 80
Ser Val Lys Ser Ala Gly Phe Asn Thr Ile Arg Ile Pro Val Thr Trp
85 90 95
Gly Glu His Leu Asp Gly Thr Thr Ile Asp Pro Ser Trp Leu Ala Arg
100 105 110
Val Gln Glu Val Val Asp Tyr Ala Tyr Asn Glu Gly Met Phe Val Ile
115 120 125
Leu Asp Met His His Asp Asp Tyr Ile Trp Leu Thr Pro Asp Asn Glu
130 135 140
Val Tyr Asn Glu Asn Ser Ala Lys Leu Arg Ala Ile Trp Leu Gln Ile
145 150 155 160
Ala Asn Arg Phe Lys Asp Tyr Gly Asp Arg Leu Ile Phe Glu Gly Leu
165 170 175
Asn Glu Pro Arg Thr Ile Gly Ser Val Thr Glu Trp Arg Gly Gly Thr
180 185 190
Thr Glu Glu Arg Ala Val Val Asn Asn Tyr Glu Ala Asp Phe Val Lys
195 200 205
Thr Ile Arg Ser Ser Gly Gly Asn Asn Pro Ala Arg Thr Leu Ile Ile
210 215 220
Thr Ser Tyr Ala Ala Ser Ala Glu Ser Val Ala Leu Asp Asp Val Ile
225 230 235 240
Val Pro Lys Thr Gly Asn Ile Ile Val Ser Val His Tyr Tyr Ala Pro
245 250 255
Trp Lys Phe Ser Glu Gly Asn Ala Asp Lys Phe Asp Asp Glu Gly Arg
260 265 270
Ala Glu Ile Asp Ala Lys Phe Ile Glu Leu Arg Ser Lys Phe Ile Asp
275 280 285
Arg Gly Ile Pro Val Ile Ile Asp Glu Phe Gly Cys Val Ala Lys Ala
290 295 300
Asp Asn Asp Glu Arg Ala Lys Tyr Tyr Lys Tyr Tyr Ile Ala Ser Ala
305 310 315 320
Lys Ser Gln Gly Ile Lys Cys Val Val Trp Asp Asn Gly Ile Thr Thr
325 330 335
Gly Asp Gly Gly Phe Gly Ile Phe Thr Arg Ser Ser Asn Glu Trp Asn
340 345 350
Lys Ala Ile Leu Lys Ala Ile Met Asp Gly Ala Ala Ser
355 360 365
<210> 3
<211> 48
<212> DNA
<213>artificial sequence (H47-F)
<400> 3
acagcaaatg ggtcgcggat ccgtgaccat gaattttgtt accgtaag 48
<210> 4
<211> 47
<212> DNA
<213>artificial sequence (H47-R)
<400> 4
cttgtcgacg gagctcgaat tcttatctta tgaagccgct ccgtcca 47
Claims (9)
1. a kind of endoglucanase, it is characterised in that: its amino acid sequence is as shown in SEQ ID No.2.
2. a kind of endo glucanase gene cel5A-h47 of endoglucanase described in coding claim 1, feature exist
In: its nucleotide sequence is as shown in SEQ ID No.1.
3. a kind of recombinant vector comprising endo glucanase gene cel5A-h47 described in claim 2.
4. a kind of recombinant bacterium comprising endo glucanase gene cel5A-h47 described in claim 2.
5. a kind of method for preparing endoglucanase is fermented and cultured recombinant bacterium as claimed in claim 4, it is poly- to obtain inscribe Portugal
Carbohydrase.
6. expanding the specific primer that endo glucanase gene cel5A-h47 described in claim 2 is used, it is characterised in that institute
The specific primer stated are as follows:
H47-F:5 'ACAGCAAATGGGTCGCGGATCCGTGACCATGAATTTTGTTACCGTAAG 3 ', nucleotides sequence
It arranges as shown in SEQ ID No.3,
H47-R:5 'CTTGTCGACGGAGCTCGAATTCIts nucleotide sequence of TTATGAAGCCGCTCCGTCCA 3 ' such as SEQ
Shown in ID No.4.
7. a kind of application of endoglucanase described in claim 1 in terms of the cellulose degradation under strong basicity environment.
8. application according to claim 7, it is characterised in that: the strong basicity environment is that pH is greater than 7.0.
9. application according to claim 7, it is characterised in that: the strong basicity environment is pH8.0-10.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711369277.1A CN107828763B (en) | 2017-12-18 | 2017-12-18 | Endoglucanase, its encoding gene cel5A-h47 and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711369277.1A CN107828763B (en) | 2017-12-18 | 2017-12-18 | Endoglucanase, its encoding gene cel5A-h47 and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107828763A CN107828763A (en) | 2018-03-23 |
| CN107828763B true CN107828763B (en) | 2019-06-11 |
Family
ID=61645170
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711369277.1A Expired - Fee Related CN107828763B (en) | 2017-12-18 | 2017-12-18 | Endoglucanase, its encoding gene cel5A-h47 and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107828763B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112195167B (en) * | 2020-11-02 | 2023-03-07 | 浙江万里学院 | A kind of β-glucanase, its encoding gene IDSGH5-26 and its application |
| CN116445457A (en) * | 2022-07-12 | 2023-07-18 | 佛山科学技术学院 | High-activity rumen cellulase and gene thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BRPI0714241A2 (en) * | 2006-07-13 | 2013-03-12 | Dsm Ip Assets Bv | use of a bacterial amylase, methods to prepare a composition, to increase the milk production of animals of the bovine subfamily, to increase the fat thickness on the back of animals of the bovine sfufamily, to increase the weight gain and / or conversion ratio animal feed of the bovinae subfamily, and to improve the apparent digestibility and / or disappearance of dry foodstuffs in animals of the bovinae subfamily, and, use of a bacterial amylase, methods to prepare a composition, to increase milk production of bovine subfamily animals, to increase the thickness of fat on the back of bovine subfamily animals, to increase weight gain and / or proportion of feed conversion of bovine subfamily animals, and to improve apparent digestibility and / or disappearance dry matter of foodstuffs in animals of the subfamily bovinae, and, composition |
-
2017
- 2017-12-18 CN CN201711369277.1A patent/CN107828763B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| hypothetical protein［Ruminococcus flavefaciens］;WP_080550482.1;《GenBank数据库》;20170330;参加序列部分 |
| Isolation and characterization of a novel endo-β-1,4-glucanase from a metagenomic library of the black-goat rumen;Song YH等;《Braz J Microbiol.》;20170626;第48卷(第4期);第801-808页 |
| Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora.;Ferrer M等;《Environmental Microbiology》;20051231;第7卷(第12期);第1996-2010页 |
| 宏基因组学揭示瘤胃微生物多样性及功能;吴鹏等;《动物营养学报》;20170615;第29卷(第5期);第1506-1514页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107828763A (en) | 2018-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105821020B (en) | A kind of β-mannanase mRmMan5A and its coding gene and application | |
| CN114410611B (en) | Laminarin degrading enzyme OUC-BsLam26 and its application | |
| CN106414728A (en) | Agarooligosaccharide hydrolase and method for producing 3,6-anhydro-L-galactose and galactose from agarose by using the same | |
| CN103555690A (en) | A novel fructosidase and its coding gene and application | |
| KR101864350B1 (en) | Enzyme Complex Containing Beta agarase, Kappa carrageenase and Anhydro-galactosidase and Use thereof | |
| CN107828763B (en) | Endoglucanase, its encoding gene cel5A-h47 and its application | |
| CN112553227A (en) | Heat-resistant multifunctional glycoside hydrolase as well as encoding gene and application thereof | |
| CN103114099A (en) | Beta-glucosaccharase gene for coding glycosyl hydrolase family 1 and application thereof | |
| CN107603967B (en) | A kind of chitosan enzyme CSN4 and its encoding gene and application | |
| CN102719458B (en) | Gene encoding alkaline beta-glucosidase and application thereof | |
| CN107828762B (en) | Endoglucanase, its encoding gene cel5A-h50 and its application | |
| CN107974442B (en) | Endoglucanase, its coding gene cel5A-h42 and its application | |
| CN107974441B (en) | Endoglucanase, its coding gene cel5A-h37 and its application | |
| CN107937371B (en) | Endoglucanase, its encoding gene cel5A-h31 and its application | |
| CN107964542B (en) | Endoglucanase, its encoding gene cel5A-h55 and its application | |
| CN114277043B (en) | A kind of thermostable mannosidase gene and its expression protein and application | |
| CN107779443B (en) | Cellobiohydrolase mutants and uses thereof | |
| CN107974440B (en) | Endoglucanase, its coding gene cel5A-h15 and its application | |
| Wang et al. | Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in Escherichia coli | |
| KR101190631B1 (en) | Novel gene encoding glycosyl hydrolase from cow rumen metagenome | |
| CN107964540B (en) | Endoglucanase, its coding gene cel5A-h28 and its application | |
| CN107964541B (en) | Endoglucanase, its coding gene cel5A-h38 and its application | |
| CN113493811B (en) | Method for preparing laminariae disaccharide by using cellulose as substrate | |
| CN118126988B (en) | Wide Wen Naqie beta-1, 4-glucanase and application thereof | |
| CN112195167B (en) | A kind of β-glucanase, its encoding gene IDSGH5-26 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190611 Termination date: 20211218 |