CN114292832A - Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof - Google Patents
Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof Download PDFInfo
- Publication number
- CN114292832A CN114292832A CN202210097396.0A CN202210097396A CN114292832A CN 114292832 A CN114292832 A CN 114292832A CN 202210097396 A CN202210097396 A CN 202210097396A CN 114292832 A CN114292832 A CN 114292832A
- Authority
- CN
- China
- Prior art keywords
- ser
- lys
- seq
- gly
- asn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 108010006654 Bleomycin Proteins 0.000 claims abstract description 8
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 8
- 229960001561 bleomycin Drugs 0.000 claims abstract description 8
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims abstract description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 4
- 239000013604 expression vector Substances 0.000 claims abstract description 4
- 239000003292 glue Substances 0.000 claims abstract description 3
- 239000013612 plasmid Substances 0.000 claims abstract description 3
- 238000000746 purification Methods 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 108010059820 Polygalacturonase Proteins 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 description 39
- 102000004190 Enzymes Human genes 0.000 description 39
- 230000000694 effects Effects 0.000 description 32
- 229920002230 Pectic acid Polymers 0.000 description 11
- 235000010987 pectin Nutrition 0.000 description 11
- 239000001814 pectin Substances 0.000 description 11
- 229920001277 pectin Polymers 0.000 description 11
- 239000010318 polygalacturonic acid Substances 0.000 description 11
- 239000000758 substrate Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 7
- 241001149947 Mucor circinelloides f. lusitanicus Species 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 239000007857 degradation product Substances 0.000 description 5
- SEKBHZJLARBNPB-GHCJXIJMSA-N Asn-Ile-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O SEKBHZJLARBNPB-GHCJXIJMSA-N 0.000 description 4
- NRVQLLDIJJEIIZ-VZFHVOOUSA-N Cys-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N)O NRVQLLDIJJEIIZ-VZFHVOOUSA-N 0.000 description 4
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 4
- 241000251121 Mustelus Species 0.000 description 4
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 4
- 108010091086 Recombinases Proteins 0.000 description 4
- 102000018120 Recombinases Human genes 0.000 description 4
- TYYBJUYSTWJHGO-ZKWXMUAHSA-N Ser-Asn-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O TYYBJUYSTWJHGO-ZKWXMUAHSA-N 0.000 description 4
- 108010087924 alanylproline Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 108010089804 glycyl-threonine Proteins 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 2
- AAXVGJXZKHQQHD-LSJOCFKGSA-N Ala-His-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCSC)C(=O)O)N AAXVGJXZKHQQHD-LSJOCFKGSA-N 0.000 description 2
- LBYMZCVBOKYZNS-CIUDSAMLSA-N Ala-Leu-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O LBYMZCVBOKYZNS-CIUDSAMLSA-N 0.000 description 2
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 2
- CJQAEJMHBAOQHA-DLOVCJGASA-N Ala-Phe-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CJQAEJMHBAOQHA-DLOVCJGASA-N 0.000 description 2
- JAQNUEWEJWBVAY-WBAXXEDZSA-N Ala-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 JAQNUEWEJWBVAY-WBAXXEDZSA-N 0.000 description 2
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 2
- AAWLEICNDUHIJM-MBLNEYKQSA-N Ala-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C)N)O AAWLEICNDUHIJM-MBLNEYKQSA-N 0.000 description 2
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 2
- TTXYKSADPSNOIF-IHRRRGAJSA-N Arg-Asp-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O TTXYKSADPSNOIF-IHRRRGAJSA-N 0.000 description 2
- LKDHUGLXOHYINY-XUXIUFHCSA-N Arg-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N LKDHUGLXOHYINY-XUXIUFHCSA-N 0.000 description 2
- KSUALAGYYLQSHJ-RCWTZXSCSA-N Arg-Met-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSUALAGYYLQSHJ-RCWTZXSCSA-N 0.000 description 2
- DAPLJWATMAXPPZ-CIUDSAMLSA-N Asn-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O DAPLJWATMAXPPZ-CIUDSAMLSA-N 0.000 description 2
- XXAOXVBAWLMTDR-ZLUOBGJFSA-N Asn-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N XXAOXVBAWLMTDR-ZLUOBGJFSA-N 0.000 description 2
- QNJIRRVTOXNGMH-GUBZILKMSA-N Asn-Gln-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(N)=O QNJIRRVTOXNGMH-GUBZILKMSA-N 0.000 description 2
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 2
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 2
- RCFGLXMZDYNRSC-CIUDSAMLSA-N Asn-Lys-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O RCFGLXMZDYNRSC-CIUDSAMLSA-N 0.000 description 2
- NLDNNZKUSLAYFW-NHCYSSNCSA-N Asn-Lys-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O NLDNNZKUSLAYFW-NHCYSSNCSA-N 0.000 description 2
- NJSNXIOKBHPFMB-GMOBBJLQSA-N Asn-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N)N NJSNXIOKBHPFMB-GMOBBJLQSA-N 0.000 description 2
- REQUGIWGOGSOEZ-ZLUOBGJFSA-N Asn-Ser-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N REQUGIWGOGSOEZ-ZLUOBGJFSA-N 0.000 description 2
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 2
- HPASIOLTWSNMFB-OLHMAJIHSA-N Asn-Thr-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O HPASIOLTWSNMFB-OLHMAJIHSA-N 0.000 description 2
- AECPDLSSUMDUAA-ZKWXMUAHSA-N Asn-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N AECPDLSSUMDUAA-ZKWXMUAHSA-N 0.000 description 2
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 2
- BUVNWKQBMZLCDW-UGYAYLCHSA-N Asp-Asn-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BUVNWKQBMZLCDW-UGYAYLCHSA-N 0.000 description 2
- SPWXXPFDTMYTRI-IUKAMOBKSA-N Asp-Ile-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SPWXXPFDTMYTRI-IUKAMOBKSA-N 0.000 description 2
- DPNWSMBUYCLEDG-CIUDSAMLSA-N Asp-Lys-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O DPNWSMBUYCLEDG-CIUDSAMLSA-N 0.000 description 2
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 2
- ZUNMTUPRQMWMHX-LSJOCFKGSA-N Asp-Val-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O ZUNMTUPRQMWMHX-LSJOCFKGSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- UWXFFVQPAMBETM-ZLUOBGJFSA-N Cys-Asp-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O UWXFFVQPAMBETM-ZLUOBGJFSA-N 0.000 description 2
- NXQCSPVUPLUTJH-WHFBIAKZSA-N Cys-Ser-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O NXQCSPVUPLUTJH-WHFBIAKZSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- ZPDVKYLJTOFQJV-WDSKDSINSA-N Gln-Asn-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O ZPDVKYLJTOFQJV-WDSKDSINSA-N 0.000 description 2
- TWTWUBHEWQPMQW-ZPFDUUQYSA-N Gln-Ile-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWTWUBHEWQPMQW-ZPFDUUQYSA-N 0.000 description 2
- JNENSVNAUWONEZ-GUBZILKMSA-N Gln-Lys-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O JNENSVNAUWONEZ-GUBZILKMSA-N 0.000 description 2
- ZVQZXPADLZIQFF-FHWLQOOXSA-N Gln-Phe-Tyr Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 ZVQZXPADLZIQFF-FHWLQOOXSA-N 0.000 description 2
- LXAUHIRMWXQRKI-XHNCKOQMSA-N Glu-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O LXAUHIRMWXQRKI-XHNCKOQMSA-N 0.000 description 2
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 description 2
- SUIAHERNFYRBDZ-GVXVVHGQSA-N Glu-Lys-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O SUIAHERNFYRBDZ-GVXVVHGQSA-N 0.000 description 2
- DLISPGXMKZTWQG-IFFSRLJSSA-N Glu-Thr-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O DLISPGXMKZTWQG-IFFSRLJSSA-N 0.000 description 2
- LSYFGBRDBIQYAQ-FHWLQOOXSA-N Glu-Tyr-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LSYFGBRDBIQYAQ-FHWLQOOXSA-N 0.000 description 2
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 2
- 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 2
- 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 2
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 2
- PDAWDNVHMUKWJR-ZETCQYMHSA-N Gly-Gly-His Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CNC=N1 PDAWDNVHMUKWJR-ZETCQYMHSA-N 0.000 description 2
- QITBQGJOXQYMOA-ZETCQYMHSA-N Gly-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QITBQGJOXQYMOA-ZETCQYMHSA-N 0.000 description 2
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 2
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 description 2
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 2
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 2
- 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 2
- MYXNLWDWWOTERK-BHNWBGBOSA-N Gly-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN)O MYXNLWDWWOTERK-BHNWBGBOSA-N 0.000 description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 2
- 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 2
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 2
- MWXBCJKQRQFVOO-DCAQKATOSA-N His-Cys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CN=CN1)N MWXBCJKQRQFVOO-DCAQKATOSA-N 0.000 description 2
- MKWFGXSFLYNTKC-XIRDDKMYSA-N His-Trp-Asp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC3=CN=CN3)N MKWFGXSFLYNTKC-XIRDDKMYSA-N 0.000 description 2
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 2
- UAVQIQOOBXFKRC-BYULHYEWSA-N Ile-Asn-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O UAVQIQOOBXFKRC-BYULHYEWSA-N 0.000 description 2
- LPFBXFILACZHIB-LAEOZQHASA-N Ile-Gly-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)O)N LPFBXFILACZHIB-LAEOZQHASA-N 0.000 description 2
- BBQABUDWDUKJMB-LZXPERKUSA-N Ile-Ile-Ile Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C([O-])=O BBQABUDWDUKJMB-LZXPERKUSA-N 0.000 description 2
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 2
- FIJMQLGQLBLBOL-HJGDQZAQSA-N Leu-Asn-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FIJMQLGQLBLBOL-HJGDQZAQSA-N 0.000 description 2
- QLDHBYRUNQZIJQ-DKIMLUQUSA-N Leu-Ile-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QLDHBYRUNQZIJQ-DKIMLUQUSA-N 0.000 description 2
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 2
- YRRCOJOXAJNSAX-IHRRRGAJSA-N Leu-Pro-Lys Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N YRRCOJOXAJNSAX-IHRRRGAJSA-N 0.000 description 2
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 2
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 2
- BGGTYDNTOYRTTR-MEYUZBJRSA-N Leu-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(C)C)N)O BGGTYDNTOYRTTR-MEYUZBJRSA-N 0.000 description 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 2
- SLQJJFAVWSZLBL-BJDJZHNGSA-N Lys-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN SLQJJFAVWSZLBL-BJDJZHNGSA-N 0.000 description 2
- PRSBSVAVOQOAMI-BJDJZHNGSA-N Lys-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN PRSBSVAVOQOAMI-BJDJZHNGSA-N 0.000 description 2
- NCZIQZYZPUPMKY-PPCPHDFISA-N Lys-Ile-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NCZIQZYZPUPMKY-PPCPHDFISA-N 0.000 description 2
- SBQDRNOLGSYHQA-YUMQZZPRSA-N Lys-Ser-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SBQDRNOLGSYHQA-YUMQZZPRSA-N 0.000 description 2
- DIBZLYZXTSVGLN-CIUDSAMLSA-N Lys-Ser-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O DIBZLYZXTSVGLN-CIUDSAMLSA-N 0.000 description 2
- XYLSGAWRCZECIQ-JYJNAYRXSA-N Lys-Tyr-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 XYLSGAWRCZECIQ-JYJNAYRXSA-N 0.000 description 2
- SMVTWPOATVIXTN-NAKRPEOUSA-N Met-Ser-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SMVTWPOATVIXTN-NAKRPEOUSA-N 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 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 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- AKJAKCBHLJGRBU-JYJNAYRXSA-N Phe-Glu-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N AKJAKCBHLJGRBU-JYJNAYRXSA-N 0.000 description 2
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 2
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 2
- YMEXHZTVKDAKIY-GHCJXIJMSA-N Ser-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO)C(O)=O YMEXHZTVKDAKIY-GHCJXIJMSA-N 0.000 description 2
- SWSRFJZZMNLMLY-ZKWXMUAHSA-N Ser-Asp-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O SWSRFJZZMNLMLY-ZKWXMUAHSA-N 0.000 description 2
- KMWFXJCGRXBQAC-CIUDSAMLSA-N Ser-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N KMWFXJCGRXBQAC-CIUDSAMLSA-N 0.000 description 2
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 2
- HDBOEVPDIDDEPC-CIUDSAMLSA-N Ser-Lys-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O HDBOEVPDIDDEPC-CIUDSAMLSA-N 0.000 description 2
- LRZLZIUXQBIWTB-KATARQTJSA-N Ser-Lys-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LRZLZIUXQBIWTB-KATARQTJSA-N 0.000 description 2
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 2
- 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 2
- PZHJLTWGMYERRJ-SRVKXCTJSA-N Ser-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N)O PZHJLTWGMYERRJ-SRVKXCTJSA-N 0.000 description 2
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 2
- 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 2
- 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 2
- HPQHHRLWSAMMKG-KATARQTJSA-N Thr-Lys-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N)O HPQHHRLWSAMMKG-KATARQTJSA-N 0.000 description 2
- JLNMFGCJODTXDH-WEDXCCLWSA-N Thr-Lys-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O JLNMFGCJODTXDH-WEDXCCLWSA-N 0.000 description 2
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 2
- CSNBWOJOEOPYIJ-UVOCVTCTSA-N Thr-Thr-Lys Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O CSNBWOJOEOPYIJ-UVOCVTCTSA-N 0.000 description 2
- IJKNKFJZOJCKRR-GBALPHGKSA-N Thr-Trp-Ser Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 IJKNKFJZOJCKRR-GBALPHGKSA-N 0.000 description 2
- VYVBSMCZNHOZGD-RCWTZXSCSA-N Thr-Val-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O VYVBSMCZNHOZGD-RCWTZXSCSA-N 0.000 description 2
- COLXBVRHSKPKIE-NYVOZVTQSA-N Trp-Trp-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O COLXBVRHSKPKIE-NYVOZVTQSA-N 0.000 description 2
- FFCRCJZJARTYCG-KKUMJFAQSA-N Tyr-Cys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N)O FFCRCJZJARTYCG-KKUMJFAQSA-N 0.000 description 2
- DBOXBUDEAJVKRE-LSJOCFKGSA-N Val-Asn-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N DBOXBUDEAJVKRE-LSJOCFKGSA-N 0.000 description 2
- BZMIYHIJVVJPCK-QSFUFRPTSA-N Val-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N BZMIYHIJVVJPCK-QSFUFRPTSA-N 0.000 description 2
- XXWBHOWRARMUOC-NHCYSSNCSA-N Val-Lys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N XXWBHOWRARMUOC-NHCYSSNCSA-N 0.000 description 2
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 2
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 2
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 2
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 108010009297 diglycyl-histidine Proteins 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010353 genetic engineering Methods 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
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108010018625 phenylalanylarginine Proteins 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 108010073969 valyllysine Proteins 0.000 description 2
- MJYQFWSXKFLTAY-OVEQLNGDSA-N (2r,3r)-2,3-bis[(4-hydroxy-3-methoxyphenyl)methyl]butane-1,4-diol;(2r,3r,4s,5s,6r)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O.C1=C(O)C(OC)=CC(C[C@@H](CO)[C@H](CO)CC=2C=C(OC)C(O)=CC=2)=C1 MJYQFWSXKFLTAY-OVEQLNGDSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 241001507865 Aspergillus fischeri Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100225553 Caenorhabditis elegans epg-4 gene Proteins 0.000 description 1
- 241001033162 Caldicellulosiruptor bescii DSM 6725 Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical group OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- GFLNKSQHOBOMNM-AVGNSLFASA-N Gln-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCC(=O)N)N GFLNKSQHOBOMNM-AVGNSLFASA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241001110277 Penicillium occitanis Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000204652 Thermotoga Species 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 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
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000004426 flaxseed Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a thermophilic high-temperature-resistant polygalacturonaseMlPG28BThe amino acid sequence is shown as SEQ ID NO. 1; the DNA sequence of the coding gene is shown as SEQ ID NO. 2; the preparation method comprises the following steps in sequence: constructing the gene shown as SEQ ID NO.3 on pPICZ alpha A eukaryotic expression vector, amplifying and culturing in Escherichia coli DH5 alpha, extracting plasmid and usingSacI, linearization is carried out, a linearization product is recovered from glue and is converted into pichia pastoris X-33 competent cells by electric shock, and then the pichia pastoris X-33 competent cells are coated on a solid YPDSZ culture medium containing 100 mu g/mL bleomycin and are cultured for 5 days at the temperature of 30 ℃; selecting single colony strain on the plate, culturing on YPD solid culture medium containing 300 microgram/mL bleomycin, selecting single colony strain, performing induced expression and purification to obtain thermophilic heat-resistant polygalactoseGlucuronidaseMlPG28B。
Description
Technical Field
The invention belongs to the field of genetic engineering and genetic engineering, and particularly relates to thermophilic high-temperature-resistant polygalacturonaseMlPG28BCoding gene and preparation method.
Background
Pectin is a high molecular weight carbohydrate polymer with a complex structure, is widely present in plant cell walls as a structural polysaccharide, is crosslinked with cellulose, hemicellulose and lignin to form a compact network structure, and needs to be degraded and removed in a plurality of industrial productions such as food, feed, papermaking, textile, pectin waste liquid treatment and the like.
The pectin polysaccharide backbone is mainly linked by alpha-1, 4-glycosidic linkages through the O-1 and O-4 positions of D-GalA, i.e., homogalacturonic acid, which constitutes about 70% of pectin. The alpha-1, 4-glycosidic bond in the pectin backbone can be cleaved by hydrolysis using Polygalacturonase (PG) from glycoside hydrolase 28 family (GH 28), which in turn depolymerizes the highly polymerized pectin. According to NCBI and CAZy database statistics, registered PG reaches 9000 pieces, wherein nearly 200 PG is cloned and characterized from bacteria and eukaryotes, but the optimal temperature is mainly concentrated at 30-60 ℃.
For many years, people have actively mined thermophilic, high temperature resistant PGs, e.g.Thermotoga maritimaPelB (TM 0437), the optimal temperature is 80 ℃; the optimal temperature of a soil metagenome sample (PecJKR 01) is 70 ℃;Caldicellulosiruptor besciiDSM 6725 (CbPelA, optimum temperature 72 ℃);Neosartorya fischeri p1 (Nf PG 5) optimum temperature 70 ℃;Penicillium occitanis(PG 1) optimum temperature 70 ℃;Penicillium oxalicumthe optimum temperature of CZ1028 (EPG 4) is 70 ℃;Bisporasp, MEY-1 (BiPG 28A) optimum temperature 70 ℃ andTalaromyces leycettanus JCM12802（TlPGA、TePG28a andTePG28 b) optimum temperature of 70 ℃. Although these PGs are thermophilic enzymes, they are not resistant to high temperatures,that is, the temperature (optimum temperature) at which the enzyme activity is highest is at least 70 ℃, but the catalytic enzyme activity is lost in a short time in a high-temperature environment of more than 60 ℃, and the industrial application of the catalytic enzyme in feed, papermaking, textile and the like with high-temperature requirements cannot be met.
Disclosure of Invention
The present invention provides a thermophilic high temperature resistant polygalacturonase, which aims to solve the technical problems in the prior artMlPG28BCoding gene and preparation method.
The technical solution of the invention is as follows: thermophilic high-temperature-resistant polygalacturonaseMlPG28BThe method is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.
A thermophilic high temperature resistant polygalacturonase as claimed in claim 1MlPG28BThe coding gene of (1), wherein: the DNA sequence is shown in SEQ ID NO. 2.
A thermophilic high temperature resistant polygalacturonase as claimed in claim 1MlPG28BThe preparation method is characterized by comprising the following steps in sequence: constructing the gene shown as SEQ ID NO.3 on pPICZ alpha A eukaryotic expression vector, amplifying and culturing in Escherichia coli DH5 alpha, extracting plasmid and usingSacI, linearization is carried out, a linearization product is recovered from glue and is converted into pichia pastoris X-33 competent cells by electric shock, and then the pichia pastoris X-33 competent cells are coated on a solid YPDSZ culture medium containing 100 mu g/mL bleomycin and are cultured for 5 days at the temperature of 30 ℃; selecting single colony strain on the plate, streaking YPD solid culture medium containing bleomycin 300 μ g/mL, culturing, selecting single colony strain, performing induced expression and purification, and obtaining recombinant thermophilic high temperature resistant polygalacturonase with amino acid sequence shown as SEQ ID NO.4MlPG28B。
Polygalacturonase of the present inventionMlPG28BNot only is thermophilic and high-temperature resistant, but also has wide pH tolerance, and the temperature (optimum temperature) when the enzyme activity is highest is 60 ℃; after heat treatment at 60 ℃ for 2 h, more than 30% of relative enzyme activity can still be kept, and the enzyme activity still remains after heat treatment at 100 ℃ for 60 min; the relative enzyme activity is maintained at 90% or more at a pH of 2.0 to 11.0. Can satisfy the pectin enzymolysis at medium and low temperature even higher than 60 DEG CAs needed.
Drawings
FIG. 1 shows thermophilic high temperature resistant polygalacturonase of the present inventionMlPG28B induced expression for 96h on SDS-PAGE.
FIG. 2 shows a thermophilic high temperature resistant polygalacturonase enzyme according to an embodiment of the present inventionMlTemperature-dependent activity curves of PG28B are shown.
FIG. 3 shows a thermophilic high temperature resistant polygalacturonase enzyme according to an embodiment of the present inventionMlpH-dependent activity curves of PG28B are shown schematically.
FIG. 4 shows a thermophilic high temperature resistant polygalacturonase enzyme according to an embodiment of the present inventionMlTemperature stability schematic of PG 28B.
FIG. 5 shows a thermophilic high temperature resistant polygalacturonase enzyme according to an embodiment of the present inventionMlpH stability of PG 28B.
FIG. 6 shows a thermophilic high temperature resistant polygalacturonase enzyme according to an embodiment of the present inventionMlPG28B was fitted to the enzyme kinetics.
FIG. 7 shows thermophilic high temperature resistant polygalacturonase of an embodiment of the present inventionMlESI-MS analysis of PG28B on polygalacturonic acid degradation products.
Detailed Description
The invention is derived from Mucor rasteus (Mucor rasteus) (III)Mucor lusitanicus) The gene sequence of (2) was analyzed by Basic Local Alignment Search Tool (BLAST) in GenBank database, DNAMAN software for multiple sequence Alignment, and Vector NTI for sequence information. His label and codon optimization are added, and the gene shown as SEQ ID NO.3 is obtained through artificial synthesis and belongs to glycoside hydrolase 28 th family (GH 28). The gene coding region is 1098bp in length, single-chain and the sequence is from 1 st to 1077 th to code thermophilic high-temperature-resistant polygalacturonaseMlPG28B (shown in SEQ ID NO. 2), His-Tag label at position 1078-1098 and a DNA sequence of stop codon. The gene sequence is constructed on pPICZ alpha A eukaryotic expression vector, amplified and cultured in Escherichia coli DH5 alpha, extracted and usedSacI, linearization, gel recovery of linearization product and electric shock transformation into Pichia pastoris X-33 competent cells, and then coating in 100-containing solutionCulturing on solid YPDSZ culture medium of bleomycin at 30 deg.C for 5 days; and selecting a single colony strain on the plate, streaking the single colony strain into a YPD solid culture medium containing 300 mu g/mL bleomycin, carrying out high-concentration antibiotic screening, and selecting a strain with a large single colony growth, and carrying out induction expression according to a pichia pastoris expression manual method.
After the induction expression for 96h, detecting the expression by polyacrylamide gel electrophoresis, and obtaining an electrophoretogram as shown in figure 1, wherein the thermophilic high temperature resistant polygalacturonaseMlPG28BA single band with a molecular weight of about 38 kDa was present on the gel. Finally crushing, cracking and purifying the thalli, and collecting the purified liquid to obtain the recombinant expression thermophilic high-temperature-resistant polygalacturonaseMlPG 28B. The amino acid sequence is shown as SEQ ID NO.4, and the 1 st-365 th amino acid residue sequence from the amino terminal is single chain, wherein the 1 st-359 th amino acid residue sequence has thermophilic high temperature resistant polygalacturonaseMlPG28BActive amino acid sequence (shown as SEQ ID NO. 1), and amino acid Tag sequence of His-Tag at position 360-365.
Experiment:
1. temperature vs. thermophilic thermostable polygalacturonase of the examples of the inventionMlPG28B (recombinant enzyme)MlPG28B, the same applies hereinafter) Activity influence experiment
Under the condition of pH 5.0 (HAc-NaAc), 200. mu.L of 0.5% (w/v) polygalacturonic acid (PGA) was used as a substrate, and 10. mu.L of recombinase was addedMlPG28B, reacting for 30 min, determining activity by 3, 5-dinitrosalicylic acid (DNS) method, and determining polygalacturonase at 20-100 deg.C and 10 deg.C respectivelyMlPG28B activity. And (3) taking the inactivated enzyme as a reference, calculating the relative enzyme activity by taking the highest enzyme activity of the reaction as 100%, and drawing a curve according to the relative activity of the enzyme at different temperatures. The results are shown in FIG. 2, showing that the recombinant enzymeMlThe optimal reaction temperature of PG28B was 60 ℃, indicating that it is a thermophilic enzyme.
2. pH vs. recombinant enzymeMlPG28B Activity Effect test
At 60 deg.C, 200. mu.L of 0.5% (w/v) PGA with pH of 2.0-11.0, respectively, as substrate, 10. mu.L of recombinase was addedMlPG28B, reaction for 30 min, using 3, 5-dinitrosalicylic acid (DNS)The activity of the compound is measured by the method. And (3) taking the inactivated enzyme as a reference, calculating the relative enzyme activity by taking the highest enzyme activity of the reaction as 100%, and drawing a curve according to the relative activity of the enzyme at different temperatures. The results are shown in FIG. 3, showing that the recombinant enzymeMlThe optimal reaction pH for PG28B was 5.0.
3. Recombinant enzymeMlTemperature stability test of PG28B
Recombination enzymeMlPG28B was incubated at different temperatures (60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C) for different periods of time, a certain amount of heat-treated enzyme solution was taken out at different time points of the above-mentioned different temperature treatments and placed at 4 deg.C for use, and then 200. mu.L of 0.5% (w/v) PGA was added as a substrate at 60 deg.C, 10. mu.L of the above-mentioned heat-treated recombinant enzyme was addedMlPG28B, reacted for 30 min, then the enzyme activity was measured by DNS method, the residual enzyme activity was detected and compared with the untreated enzyme activity, and the relative enzyme activity was calculated. The results are shown in FIG. 4, showing that the recombinant enzymeMlAfter heat treatment is carried out on PG28B at 60 ℃ for 2 hours, the relative enzyme activity of more than 30 percent can still be kept; the enzyme still has enzyme activity after heat treatment for 60 min at 100 ℃, which indicates that the enzyme is thermophilic high-temperature resistant enzyme.
4. Recombinant enzymeMlpH stability test of PG28B
Recombination enzymeMlPG28B is respectively placed in different pH buffer solutions (pH 2.0-11.0) to be diluted by 5 times, and is placed for 24 hours at the temperature of 4 ℃; subsequently, 10. mu.L of the enzyme solution was incubated with 200. mu.L of 5 mg/mL PGA substrate under optimum reaction conditions (60 ℃ C., pH 5.0) for 30 min, followed by determination of the enzyme activity by the DNS method and comparison with the untreated enzyme activity to calculate the relative enzyme activity. The results are shown in FIG. 5, showing that the recombinant enzymeMlPG28B maintained a relative enzyme activity of 90% or more at pH 2.0 to 11.0, indicating that the recombinant enzyme was present MlPG28B has a wide range of pH tolerance.
5. Recombinant enzymeMlSubstrate specificity assay for PG28B
Selecting 6 substrates of PGA, low ester pectin, high ester pectin, sodium alginate and flax seed gum to examine recombinaseMlSubstrate specificity of PG 28B. Reacting at optimum temperature of 60 deg.C and pH of 5.0 for 30 min, and detecting recombinase by standard methodMlThe relative enzymatic activity of PG 28B. The results are shown in Table 1Recombinant enzymeMlPG28B has high degrading enzyme activity to PGA and low ester pectin.
TABLE 1
6. Recombinant enzymeMlDetermination of enzymatic kinetic parameters of PG28B
ApPel1 (0.0159 mg/mL) enzyme solution (10. mu.l) was mixed with 200. mu.l of PGA substrates (pH 5.0) at different concentrations (0.5 mg/mL, 1.5 mg/mL, 2.5 mg/mL, 3.5 mg/mL, 4.5 mg/mL and 6.5 mg/mL), reacted at 60 ℃ for 30 min, and the activity thereof was measured by the DNS method. The obtained data are fitted by a Michaelis equation in GraphPad Prism 9.0.0 software to obtain a fitted curve of the reaction rate and the concentration of the substrate, and then relevant enzyme kinetic parameters can be obtained. The results are shown in FIG. 6 and are obtained by calculationVmax1.47 plus or minus 0.24 mg/L/s, Km is 3055 plus or minus 1104 mg/L, Kcat11.667 +/-1.905 s-1，Kcat/KmIt was 4.655. + -. 2.305 mL/mg/s.
7. Recombinant enzymeMlESI-MS analysis of PG28B on polygalacturonic acid degradation products
mu.L of recombinase was added to 500. mu.L of 0.5% (w/v) PGA with pH =5.0 as a substrate at 35 ℃MlPG28B was mixed well, shaken at 700 rpm for 21h, and ESI-MS analysis was performed on its degradation products. The results are shown in FIG. 7: when ESI-MS was performed for anion mode analysis of the degradation product, it was found that the degradation product was oligogalacturonic acid, mainly consisting of DP1, DP2, DP3, DP4, DP5, DP6, and DP 7. Indicating the recombinant enzymeMlPG28B can effectively degrade pectin polysaccharide-rich raw materials to prepare oligogalacturonan.
Sequence listing
<110> university of Dalian ocean
<120> thermophilic high-temperature-resistant polygalacturonase MlPG28B, coding gene and preparation method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 359
<212> PRT
<213> Mustelus Lasitanus (Mucor Lusitanicus)
<400> 1
Ser Lys Thr Cys Thr Ala Lys Ser Gly Thr Ser Asp Asp Ala Val Ser
1 5 10 15
Ile Ala Glu Ala Phe Asn Ser Cys Lys Ser Gly Gly Thr Val Val Phe
20 25 30
Thr Lys Gly Ala Thr Tyr Asn Leu Lys Thr Leu Ile Asn Ile Ser Gly
35 40 45
Leu Lys Asn Val Asn Val Gln Phe Tyr Gly Lys Val Asn Leu Pro Ala
50 55 60
Leu Asn Thr Lys Tyr Glu Gly Ala Thr Ala Phe Phe Lys Ile Ala Gly
65 70 75 80
Asp Asn Ile His Trp Asp Gly Ala Gly Ile Gly Glu Phe Val Gly Gly
85 90 95
Gly Gln Lys Trp Trp Asp Val Lys Asn Asn Lys Ala Pro Thr Val Phe
100 105 110
Arg Met Thr Ala Thr His Ser Val Phe Arg Asp Phe Lys Ile Thr Ala
115 120 125
Ala Pro Arg Ala His Met Ala Leu Thr Ser Ser Asp Asp Val Val Leu
130 135 140
Glu Lys Val Val Leu Tyr Thr Lys Ser Ser Asn Ser Asn Leu Pro Lys
145 150 155 160
Asn Thr Asp Ala Leu Asp Ile Ser Ser Ser Lys Asn Leu Ile Phe Arg
165 170 175
Asp Ser Asp Leu Thr Val Gly Asp Asp Cys Thr Ala Ile Asn Gly Gly
180 185 190
Val Ser Asn Val Thr Leu Ser Asn Ile His Cys Val Gly Gly His Gly
195 200 205
Phe Ser Val Gly Ser Leu Gly Lys Gly Gly Ser Thr Glu Thr Val Lys
210 215 220
Gln Ile Arg Val Thr Gly Ser Thr Cys Thr Lys Cys Gln Asn Gly Ile
225 230 235 240
Arg Ile Lys Thr Trp Ser Gly Gly Lys Gly Ser Val Ser Asp Val Arg
245 250 255
Phe Glu His Val Lys Leu Asn Lys Val Glu Asn Pro Ile Ile Ile Asp
260 265 270
Glu Tyr Tyr Cys Asp Asn Asn Gln Lys Ser Tyr Cys Gln Lys Asn Ser
275 280 285
Asp Lys Ser Leu Thr Ile Ser Asn Val Val Ile Asn Asp Ile Thr Gly
290 295 300
Ser Val Ser Glu Lys Lys Asn Pro Ile Met Ser Ile Asn Cys Ala Pro
305 310 315 320
Gly Thr Pro Cys Ser Gly Phe Ser Val Ser Asn Val Asn Ile Ser Lys
325 330 335
Asn Ser Lys Thr Thr Lys Asn Val Cys Asn Asn Leu Lys Gly Ser Asp
340 345 350
Lys Ile Ser Tyr Cys Lys Gln
355
<210> 2
<211> 1077
<212> DNA
<213> Mustelus Lasitanus (Mucor Lusitanicus)
<220>
<221> misc_feature
<222> (1)..(1077)
<400> 2
tccaagactt gcacagctaa gtccggtact tctgacgacg cagtttctat tgccgaagct 60
ttcaactctt gcaagtccgg aggtacagtc gttttcacca agggagctac ctacaacctg 120
aagaccctga tcaacatctc cggtctgaag aacgtcaacg tccagttcta cggtaaggtc 180
aacctgccag ctttgaacac caagtacgag ggtgctacag ctttcttcaa gattgccgga 240
gacaacatcc attgggacgg agcaggtatt ggagaatttg ttggaggagg tcaaaagtgg 300
tgggacgtta agaacaacaa ggctccaacc gttttcagaa tgaccgctac tcactccgtc 360
ttcagagact tcaagatcac cgcagctcca agagcccata tggctttgac ttcctccgac 420
gacgttgtct tggagaaggt tgtcctgtac accaagtcct ccaactctaa cctgccaaag 480
aacaccgacg ctttggatat ttcctcctcc aagaacctga tcttcagaga ctccgacttg 540
accgttggag acgattgtac cgctatcaac ggaggtgttt ccaacgttac cttgtccaac 600
atccattgcg ttggaggtca cggtttttcc gttggttctt tgggtaaggg aggttctacc 660
gaaaccgtta agcagatcag agtcaccggt tctacttgca ccaagtgcca gaacggtatc 720
agaatcaaga cttggtccgg aggaaagggt tccgtttccg acgttagatt cgagcacgtt 780
aagctgaaca aggtcgagaa cccaatcatc atcgacgagt actattgcga caacaaccag 840
aagtcctatt gccagaagaa ctccgacaag tccttgacca tctccaacgt cgtcatcaac 900
gacatcacag gttccgtttc cgagaagaag aacccaatca tgtccatcaa ttgcgcccca 960
ggtactcctt gttcaggttt ctccgtttcc aacgtcaaca tctccaagaa ctccaagacc 1020
accaagaacg tttgcaacaa cctgaaggga tccgacaaga tctcctattg caagcag 1077
<210> 3
<211> 1098
<212> DNA
<213> Mustelus Lasitanus (Mucor Lusitanicus)
<220>
<221> misc_feature
<222> (1)..(1098)
<400> 3
tccaagactt gcacagctaa gtccggtact tctgacgacg cagtttctat tgccgaagct 60
ttcaactctt gcaagtccgg aggtacagtc gttttcacca agggagctac ctacaacctg 120
aagaccctga tcaacatctc cggtctgaag aacgtcaacg tccagttcta cggtaaggtc 180
aacctgccag ctttgaacac caagtacgag ggtgctacag ctttcttcaa gattgccgga 240
gacaacatcc attgggacgg agcaggtatt ggagaatttg ttggaggagg tcaaaagtgg 300
tgggacgtta agaacaacaa ggctccaacc gttttcagaa tgaccgctac tcactccgtc 360
ttcagagact tcaagatcac cgcagctcca agagcccata tggctttgac ttcctccgac 420
gacgttgtct tggagaaggt tgtcctgtac accaagtcct ccaactctaa cctgccaaag 480
aacaccgacg ctttggatat ttcctcctcc aagaacctga tcttcagaga ctccgacttg 540
accgttggag acgattgtac cgctatcaac ggaggtgttt ccaacgttac cttgtccaac 600
atccattgcg ttggaggtca cggtttttcc gttggttctt tgggtaaggg aggttctacc 660
gaaaccgtta agcagatcag agtcaccggt tctacttgca ccaagtgcca gaacggtatc 720
agaatcaaga cttggtccgg aggaaagggt tccgtttccg acgttagatt cgagcacgtt 780
aagctgaaca aggtcgagaa cccaatcatc atcgacgagt actattgcga caacaaccag 840
aagtcctatt gccagaagaa ctccgacaag tccttgacca tctccaacgt cgtcatcaac 900
gacatcacag gttccgtttc cgagaagaag aacccaatca tgtccatcaa ttgcgcccca 960
ggtactcctt gttcaggttt ctccgtttcc aacgtcaaca tctccaagaa ctccaagacc 1020
accaagaacg tttgcaacaa cctgaaggga tccgacaaga tctcctattg caagcagcat 1080
catcatcatc atcattga 1098
<210> 4
<211> 365
<212> PRT
<213> Mustelus Lasitanus (Mucor Lusitanicus)
<400> 4
Ser Lys Thr Cys Thr Ala Lys Ser Gly Thr Ser Asp Asp Ala Val Ser
1 5 10 15
Ile Ala Glu Ala Phe Asn Ser Cys Lys Ser Gly Gly Thr Val Val Phe
20 25 30
Thr Lys Gly Ala Thr Tyr Asn Leu Lys Thr Leu Ile Asn Ile Ser Gly
35 40 45
Leu Lys Asn Val Asn Val Gln Phe Tyr Gly Lys Val Asn Leu Pro Ala
50 55 60
Leu Asn Thr Lys Tyr Glu Gly Ala Thr Ala Phe Phe Lys Ile Ala Gly
65 70 75 80
Asp Asn Ile His Trp Asp Gly Ala Gly Ile Gly Glu Phe Val Gly Gly
85 90 95
Gly Gln Lys Trp Trp Asp Val Lys Asn Asn Lys Ala Pro Thr Val Phe
100 105 110
Arg Met Thr Ala Thr His Ser Val Phe Arg Asp Phe Lys Ile Thr Ala
115 120 125
Ala Pro Arg Ala His Met Ala Leu Thr Ser Ser Asp Asp Val Val Leu
130 135 140
Glu Lys Val Val Leu Tyr Thr Lys Ser Ser Asn Ser Asn Leu Pro Lys
145 150 155 160
Asn Thr Asp Ala Leu Asp Ile Ser Ser Ser Lys Asn Leu Ile Phe Arg
165 170 175
Asp Ser Asp Leu Thr Val Gly Asp Asp Cys Thr Ala Ile Asn Gly Gly
180 185 190
Val Ser Asn Val Thr Leu Ser Asn Ile His Cys Val Gly Gly His Gly
195 200 205
Phe Ser Val Gly Ser Leu Gly Lys Gly Gly Ser Thr Glu Thr Val Lys
210 215 220
Gln Ile Arg Val Thr Gly Ser Thr Cys Thr Lys Cys Gln Asn Gly Ile
225 230 235 240
Arg Ile Lys Thr Trp Ser Gly Gly Lys Gly Ser Val Ser Asp Val Arg
245 250 255
Phe Glu His Val Lys Leu Asn Lys Val Glu Asn Pro Ile Ile Ile Asp
260 265 270
Glu Tyr Tyr Cys Asp Asn Asn Gln Lys Ser Tyr Cys Gln Lys Asn Ser
275 280 285
Asp Lys Ser Leu Thr Ile Ser Asn Val Val Ile Asn Asp Ile Thr Gly
290 295 300
Ser Val Ser Glu Lys Lys Asn Pro Ile Met Ser Ile Asn Cys Ala Pro
305 310 315 320
Gly Thr Pro Cys Ser Gly Phe Ser Val Ser Asn Val Asn Ile Ser Lys
325 330 335
Asn Ser Lys Thr Thr Lys Asn Val Cys Asn Asn Leu Lys Gly Ser Asp
340 345 350
Lys Ile Ser Tyr Cys Lys Gln His His His His His His
355 360 365
Claims (3)
1. Thermophilic high-temperature-resistant polygalacturonaseMlPG28BThe method is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.
2. A thermophilic high temperature resistant polygalacturonase as claimed in claim 1MlPG28BThe coding gene of (1), wherein: the DNA sequence is shown in SEQ ID NO. 2.
3. A thermophilic high temperature resistant polygalacturonase as claimed in claim 1MlPG28BThe preparation method is characterized by comprising the following steps in sequence: constructing the gene shown as SEQ ID NO.3 on pPICZ alpha A eukaryotic expression vector, amplifying and culturing in Escherichia coli DH5 alpha, extracting plasmid and usingSacI, linearization is carried out, a linearization product is recovered from glue and is converted into pichia pastoris X-33 competent cells by electric shock, and then the pichia pastoris X-33 competent cells are coated on a solid YPDSZ culture medium containing 100 mu g/mL bleomycin and are cultured for 5 days at the temperature of 30 ℃; single colony strains on selection plates were scored at 300. mu.g-mL bleomycin YPD solid culture medium, selecting single colony strain for induced expression and purification, and obtaining recombinant thermophilic high temperature resistant polygalacturonase with amino acid sequence shown as SEQ ID NO.4MlPG28B。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210097396.0A CN114292832A (en) | 2022-01-27 | 2022-01-27 | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210097396.0A CN114292832A (en) | 2022-01-27 | 2022-01-27 | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114292832A true CN114292832A (en) | 2022-04-08 |
Family
ID=80976758
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210097396.0A Pending CN114292832A (en) | 2022-01-27 | 2022-01-27 | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114292832A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997032014A1 (en) * | 1996-03-01 | 1997-09-04 | Novo Nordisk A/S | An enzyme with galactanase activity |
| CN1294633A (en) * | 1998-02-10 | 2001-05-09 | Dsm公司 | Novel endo-xylogalacturonase |
| WO2013114149A1 (en) * | 2012-01-31 | 2013-08-08 | University Of Calcutta | Thermostable enzymes and methods of making and using the same |
| CN103773700A (en) * | 2014-02-12 | 2014-05-07 | 广西科学院 | Penicillium oxalicum strain for producing high-temperature polygalacturonase and application of penicillium oxalicum strain |
| CN104818260A (en) * | 2015-04-17 | 2015-08-05 | 中国农业科学院饲料研究所 | High temperature resistant chaetomium polygalacturonase mutant and encoding gene and application thereof |
| CN106047840A (en) * | 2016-06-10 | 2016-10-26 | 北京盛拓达生物技术有限公司 | Acid exo-polygalacturonase and gene and application thereof |
| CN106566818A (en) * | 2016-10-28 | 2017-04-19 | 中国农业科学院饲料研究所 | Acidic thermophilic polygalacturonase TePG28A, and coding gene and application thereof |
-
2022
- 2022-01-27 CN CN202210097396.0A patent/CN114292832A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997032014A1 (en) * | 1996-03-01 | 1997-09-04 | Novo Nordisk A/S | An enzyme with galactanase activity |
| CN1294633A (en) * | 1998-02-10 | 2001-05-09 | Dsm公司 | Novel endo-xylogalacturonase |
| WO2013114149A1 (en) * | 2012-01-31 | 2013-08-08 | University Of Calcutta | Thermostable enzymes and methods of making and using the same |
| CN103773700A (en) * | 2014-02-12 | 2014-05-07 | 广西科学院 | Penicillium oxalicum strain for producing high-temperature polygalacturonase and application of penicillium oxalicum strain |
| CN104818260A (en) * | 2015-04-17 | 2015-08-05 | 中国农业科学院饲料研究所 | High temperature resistant chaetomium polygalacturonase mutant and encoding gene and application thereof |
| CN106047840A (en) * | 2016-06-10 | 2016-10-26 | 北京盛拓达生物技术有限公司 | Acid exo-polygalacturonase and gene and application thereof |
| CN106566818A (en) * | 2016-10-28 | 2017-04-19 | 中国农业科学院饲料研究所 | Acidic thermophilic polygalacturonase TePG28A, and coding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| MONDO,S.J. ET AL.: "glycoside hydrolase family 28 protein [Mucor lusitanicus]，GenBank: KAF1796673.1" * |
| 李烨青等: "Bispora sp． MEY－1 来源的新型嗜热多聚半乳糖醛酸酶的酶学性质研究及其应用评估" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108285900B (en) | Recombinant alginate lyase and construction method and application thereof | |
| CN112481240B (en) | GH16 family heat-resistant glucanase mutant and construction method and application thereof | |
| CN109355275B (en) | Beta-glucosidase mutant with high thermal stability and application thereof | |
| WO2017197546A1 (en) | Β-mannanase mrmman5a and encoding gene and application thereof | |
| Li et al. | Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH | |
| CN111041017A (en) | Chitosanase mutant and application thereof | |
| CN116410960B (en) | Beta-xylosidase mutant D41G with cold and pH adaptability improved halophilic suitability and application thereof | |
| CN109929859B (en) | Kappa-carrageenan enzyme coding gene and preparation and application thereof | |
| CN110656098B (en) | Application of acetyl esterase in promoting degradation of beta-mannase on acetylated mannan | |
| CN114480350B (en) | Application of carrageenase in degrading kappa-carrageenan and furcellaran | |
| US6087149A (en) | Starch conversion process | |
| Kobayashi et al. | Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1 | |
| CN112795556A (en) | beta-N-acetylglucosaminidase 159 and cloning expression and application thereof | |
| CN114292832A (en) | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof | |
| CN113755473B (en) | Glucoamylase mutant M5 with improved secretion expression level as well as gene and application thereof | |
| CN113337528B (en) | Engineering strain of mannosidase and application thereof | |
| CN110760532B (en) | Starch branching enzyme and gene thereof, engineering bacterium containing gene and application of engineering bacterium | |
| Antranikian et al. | Biodegradation of polymers at temperatures up to 130 C | |
| CN108165540B (en) | Rhizomucor miehei alpha-amylase and coding gene and application thereof | |
| CN112175974A (en) | Chitin deacetylase gene, chitin deacetylase and preparation method and application thereof | |
| CN113528486B (en) | Method for improving xylanase thermal stability by introducing disulfide bond | |
| CN109762798A (en) | The preparation method and application of a kind of balun Pueraria lobota hereby series bacillus chitosan enzyme | |
| CN113774045B (en) | Glucoamylase mutant M3 with improved secretion expression level as well as gene and application thereof | |
| CN111647582B (en) | Burkholderia pyrrocinia endoglucanase, recombinant expression method and application thereof | |
| CN114958810A (en) | Ultrahigh-temperature-resistant polygalacturonase McPG28A and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20220408 |
|
| WD01 | Invention patent application deemed withdrawn after publication |