CN114561412B - 一种碱性木聚糖酶基因xynAI优化序列及其表达 - Google Patents
一种碱性木聚糖酶基因xynAI优化序列及其表达 Download PDFInfo
- Publication number
- CN114561412B CN114561412B CN202210234830.5A CN202210234830A CN114561412B CN 114561412 B CN114561412 B CN 114561412B CN 202210234830 A CN202210234830 A CN 202210234830A CN 114561412 B CN114561412 B CN 114561412B
- Authority
- CN
- China
- Prior art keywords
- xynai
- pichia pastoris
- expression
- gene
- sequence
- 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.)
- Active
Links
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 42
- 238000005457 optimization Methods 0.000 title description 5
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 108700010070 Codon Usage Proteins 0.000 claims abstract description 3
- 239000013598 vector Substances 0.000 claims description 7
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 16
- 108020004705 Codon Proteins 0.000 abstract description 10
- 239000013604 expression vector Substances 0.000 abstract description 5
- 101150091506 xynA gene Proteins 0.000 abstract description 5
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 241001086533 Camptothecium Species 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 101100157016 Aspergillus niger (strain CBS 513.88 / FGSC A1513) xlnC gene Proteins 0.000 abstract 1
- 101100317617 Paenibacillus sp. (strain JDR-2) xynA1 gene Proteins 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 18
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 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 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229920001221 xylan Polymers 0.000 description 6
- 150000004823 xylans Chemical class 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 2
- QPZMOUMNTGTEFR-ZKWXMUAHSA-N Val-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N QPZMOUMNTGTEFR-ZKWXMUAHSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000010893 paper waste Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- AWMAZIIEFPFHCP-RCWTZXSCSA-N Arg-Pro-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O AWMAZIIEFPFHCP-RCWTZXSCSA-N 0.000 description 1
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 1
- HZPSDHRYYIORKR-WHFBIAKZSA-N Asn-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O HZPSDHRYYIORKR-WHFBIAKZSA-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
- FMNBYVSGRCXWEK-FOHZUACHSA-N Asn-Thr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O FMNBYVSGRCXWEK-FOHZUACHSA-N 0.000 description 1
- IPPFAOCLQSGHJV-WFBYXXMGSA-N Asn-Trp-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O IPPFAOCLQSGHJV-WFBYXXMGSA-N 0.000 description 1
- DPWDPEVGACCWTC-SRVKXCTJSA-N Asn-Tyr-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O DPWDPEVGACCWTC-SRVKXCTJSA-N 0.000 description 1
- XACXDSRQIXRMNS-OLHMAJIHSA-N Asp-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)O XACXDSRQIXRMNS-OLHMAJIHSA-N 0.000 description 1
- RTXQQDVBACBSCW-CFMVVWHZSA-N Asp-Ile-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RTXQQDVBACBSCW-CFMVVWHZSA-N 0.000 description 1
- OZBXOELNJBSJOA-UBHSHLNASA-N Asp-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N OZBXOELNJBSJOA-UBHSHLNASA-N 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- XMPAXPSENRSOSV-RYUDHWBXSA-N Glu-Gly-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XMPAXPSENRSOSV-RYUDHWBXSA-N 0.000 description 1
- NEDQVOQDDBCRGG-UHFFFAOYSA-N Gly Gly Thr Tyr Chemical compound NCC(=O)NCC(=O)NC(C(O)C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 NEDQVOQDDBCRGG-UHFFFAOYSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- UMBDRSMLCUYIRI-DVJZZOLTSA-N Gly-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN)O UMBDRSMLCUYIRI-DVJZZOLTSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- QAMFAYSMNZBNCA-UWVGGRQHSA-N His-Gly-Met Chemical compound CSCC[C@H](NC(=O)CNC(=O)[C@@H](N)Cc1cnc[nH]1)C(O)=O QAMFAYSMNZBNCA-UWVGGRQHSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- LEDRIAHEWDJRMF-CFMVVWHZSA-N Ile-Asn-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 LEDRIAHEWDJRMF-CFMVVWHZSA-N 0.000 description 1
- NKRJALPCDNXULF-BYULHYEWSA-N Ile-Asp-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O NKRJALPCDNXULF-BYULHYEWSA-N 0.000 description 1
- WXLYNEHOGRYNFU-URLPEUOOSA-N Ile-Thr-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N WXLYNEHOGRYNFU-URLPEUOOSA-N 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 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
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- MGKFCQFVPKOWOL-CIUDSAMLSA-N Lys-Ser-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N MGKFCQFVPKOWOL-CIUDSAMLSA-N 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- ZWJKVFAYPLPCQB-UNQGMJICSA-N Phe-Arg-Thr Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)Cc1ccccc1)C(O)=O ZWJKVFAYPLPCQB-UNQGMJICSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- XROLYVMNVIKVEM-BQBZGAKWSA-N Pro-Asn-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O XROLYVMNVIKVEM-BQBZGAKWSA-N 0.000 description 1
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- XVAUJOAYHWWNQF-ZLUOBGJFSA-N Ser-Asn-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O XVAUJOAYHWWNQF-ZLUOBGJFSA-N 0.000 description 1
- WXWDPFVKQRVJBJ-CIUDSAMLSA-N Ser-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N WXWDPFVKQRVJBJ-CIUDSAMLSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- NNFMANHDYSVNIO-DCAQKATOSA-N Ser-Lys-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NNFMANHDYSVNIO-DCAQKATOSA-N 0.000 description 1
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-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
- 108091081024 Start codon Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- DCLBXIWHLVEPMQ-JRQIVUDYSA-N Thr-Asp-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DCLBXIWHLVEPMQ-JRQIVUDYSA-N 0.000 description 1
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 1
- SSNGFWKILJLTQM-QEJZJMRPSA-N Trp-Gln-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SSNGFWKILJLTQM-QEJZJMRPSA-N 0.000 description 1
- ULHASJWZGUEUNN-XIRDDKMYSA-N Trp-Lys-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O ULHASJWZGUEUNN-XIRDDKMYSA-N 0.000 description 1
- DDHFMBDACJYSKW-AQZXSJQPSA-N Trp-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O DDHFMBDACJYSKW-AQZXSJQPSA-N 0.000 description 1
- KEHKBBUYZWAMHL-DZKIICNBSA-N Tyr-Gln-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O KEHKBBUYZWAMHL-DZKIICNBSA-N 0.000 description 1
- QRCBQDPRKMYTMB-IHPCNDPISA-N Tyr-Trp-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N QRCBQDPRKMYTMB-IHPCNDPISA-N 0.000 description 1
- DJIJBQYBDKGDIS-JYJNAYRXSA-N Tyr-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(C)C)C(O)=O DJIJBQYBDKGDIS-JYJNAYRXSA-N 0.000 description 1
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 1
- UDNYEPLJTRDMEJ-RCOVLWMOSA-N Val-Asn-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N UDNYEPLJTRDMEJ-RCOVLWMOSA-N 0.000 description 1
- NMPXRFYMZDIBRF-ZOBUZTSGSA-N Val-Asn-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N NMPXRFYMZDIBRF-ZOBUZTSGSA-N 0.000 description 1
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- IRAUYEAFPFPVND-UVBJJODRSA-N Val-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 IRAUYEAFPFPVND-UVBJJODRSA-N 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940101006 anhydrous sodium sulfite Drugs 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229930192071 betulinan Natural products 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000002761 deinking Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010059898 glycyl-tyrosyl-lysine Proteins 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 229940074439 potassium sodium tartrate Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004076 pulp bleaching Methods 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 238000005406 washing Methods 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- 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)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/64—Paper recycling
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种按毕赤酵母偏爱密码子优化的碱性木聚糖酶基因xynAI及其表达。该基因xynAI来源于喜碱菌（Bacillussp.）,原始基因为xynA（GenBank Accession No.U51675.1），按照毕赤酵母密码子偏爱性进行优化，优化序列与原序列同源性为78%。通过采用连续延伸PCR方法合成得到优化序列，所述优化核苷酸序列如SEQ ID No 1，全长561bp，其编码的蛋白质序列如SEQ ID No 2，共186个氨基酸。通过将该优化序列构建到毕赤酵母表达载体并转化入毕赤酵母中表达，可用于木聚糖酶的生产。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种按毕赤酵母偏爱密码子优化的碱性木聚糖酶基因xynAI及其表达。
背景技术
木聚糖是植物半纤维素的重要组成部分，是自然界中含量仅次于纤维素的可再生资源。由于木聚糖结构复杂，要彻底降解木聚糖需要多种酶的共同参与，其中β-1,4-内切木聚糖酶（endo-1，4-β-Xylanases，EC 3.2.1.8）是极其关键的一步水解酶，可将木聚糖水解成低聚木糖和木糖。
木聚糖被木聚糖酶分解后，其分解产物广泛应用于食品、饲料、纺织、造纸等行业。因不同木聚糖酶对酸碱环境耐受能力的不同，可将木聚糖酶分为酸性木聚糖酶、中性木聚糖酶、碱性木聚糖酶，其中碱性木聚糖酶适用于造纸行业。在造纸的制浆、纸浆漂白、废纸脱墨、废纸水处理等过程中，碱性木聚糖酶的使用不仅可以提高纸张的生产质量，还可减少有毒有害化工产品的使用，从而降低造纸对周围环境的污染【田文卓等，碱性木聚糖酶研究进展，中国生物工程杂志，2022，1-16】。
随着生物技术的发展，人们已经从真菌、放线菌、细菌等多种微生物中发现并克隆和表达木聚糖酶。但大多数天然来源的木聚糖酶的最适温度以及最适反应PH，难以满足造纸工业生产所需。因此利用生物工程技术，对自然界分离克隆的碱性木聚糖酶基因进行分子优化，提高其产量及耐碱能力，进一步满足实际运用。目前，毕赤酵母（Pichia pastoris）的真核表达***相比于原核微生物的表达***，具有表达量高、背景蛋白少、产物易纯化且活性高等优点，是合适的木聚糖酶表达宿主。而且，若选用优良的酵母表达载体pYPX88，该载体含有经毕赤酵母密码子偏爱性优化后的信号肽Signal (GenBank 登录号：AY145833.1) ，可更高幅度的提升木聚糖酶基因的表达水平【熊爱生等，Influence ofsignal peptide sequences on the expression of heterogeneous proteins inPichia pastoris，生物化学与生物物理学报，2003, 35(2):154-160】。
发明内容
本发明所要解决的技术问题在于提供一种按毕赤酵母偏爱密码子优化的碱性木聚糖酶基因xynAI及其在毕赤酵母中的表达，使所述基因在毕赤酵母中高效表达，且表达的木聚糖酶在碱性条件下具有较高活性。
为了达到上述目的，本发明采用以下技术方案实现：
所述经密码子优化的碱性木聚糖酶基因xynAI的核苷酸序列，利用连续延伸PCR方法合成，其核苷酸序列SEQ ID NO 1所示。
所述经密码子优化的碱性木聚糖酶基因xynAI的核苷酸序列编码的蛋白质序列如SEQ ID NO 2。
本发明按照毕赤酵母偏爱密码子对来自喜碱菌（Bacillus sp.）原始的xynA基因进行改造，改造后的基因序列与原序列（GenBank Accession No. U51675.1）同源性为78%（参见图1），其编码的蛋白质共186个氨基酸。基因合成采用连续延伸PCR方法【熊爱生等，Asimple，rapid，high fidelity and cost-effective PCR-based two-step DNAsynthesis (PTDS) method for long gene sequences，核酸研究，2004，32:e98】。使用的引物如下：
P1. BSXYNAI-1:
CTC,GAG,AAA,AGA,GAG,GCT,GAA,GCT,GCT,GGT,ACT,GAC,TAC,TGG,CAG,AAC,TGG,ACT,GAT,GGT
P2. BSXYNAI-2:
AGT,TAC,CAC,CAG,AAC,CAT,TGA,CAG,CGT,TGA,CAG,TAC,CAC,CAC,CAT,CAG,TCC,AGT,TCT,GCC
P3. BSXYNAI-3:
CAA,TGG,TTC,TGG,TGG,TAA,CTA,CTC,TGT,CAA,CTG,GTC,CAA,TAC,TGG,TAA,CTT,CGT,CGT,TGG
P4. BSXYNAI-4:
GTT,GAT,GGT,TCT,GAA,TGG,AGA,ACC,GGT,GGT,CCA,GCC,TTT,ACC,AAC,GAC,GAA,GTT,ACC,AGT
P5. BSXYNAI-5:
CTC,CAT,TCA,GAA,CCA,TCA,ACT,ACA,ATG,CTG,GTG,TCT,GGG,CTC,CTA,ATG,GCA,ATG,GCT,ACT
P6. BSXYNAI-6:
TAC,TCG,ATG,AGT,GGT,GCT,CTG,GTC,CAG,CCA,TAC,AGA,GTC,AAG,TAG,CCA,TTG,CCA,TTA,GGA
P7. BSXYNAI-7:
AGA,GCA,CCA,CTC,ATC,GAG,TAC,TAT,GTT,GTT,GAC,TCT,TGG,GGT,ACT,TAC,AGA,CCA,ACT,GGT
P8. BSXYNAI-8:
CAT,AAG,TAC,CAC,CAT,CGG,ACT,TGA,CAG,TAC,CTT,TGT,AAG,TAC,CAG,TTG,GTC,TGT,AAG,TAC
P9. BSXYNAI-9:
GTC,CGA,TGG,TGG,TAC,TTA,TGA,CAT,CTA,CAC,CAC,TAC,CAG,ATA,CAA,TGC,TCC,ATC,CAT,CGA
P10. BSXYNAI-10:
TCT,GAC,AGA,CCA,GTA,CTG,AGT,GAA,AGT,GGT,GTT,GTC,ACC,ATC,GAT,GGA,TGG,AGC,ATT,GTA
P11. BSXYNAI-11:
CTC,AGT,ACT,GGT,CTG,TCA,GAC,AGT,CCA,AGA,GAC,CAA,CTG,GTT,CCA,ACG,CTG,CTA,TCA,CCT
P12. BSXYNAI-12:
TTC,ATA,CCA,TGA,GAC,TTC,CAA,GCA,TTG,ACA,TGA,TTG,GAG,AAG,GTG,ATA,GCA,GCG,TTG,GAA
P13. BSXYNAI-13:
TGG,AAG,TCT,CAT,GGT,ATG,AAT,CTT,GGC,TCC,AAC,TGG,GCT,TAC,CAA,GTC,TTG,GCT,ACT,GAA
P14. BSXYNAI-14:
GCC,AAA,CAG,TGA,CGT,TGG,AAG,AAC,CAG,AGG,ACT,TGT,AGC,CTT,CAG,TAG,CCA,AGA,CTT,GGT
P15. BSXYNAI-15:
GAG,CTC,TTA,ATG,GTG,ATG,GTG,ATG,GTG,CCA,AAC,AGT,GAC,GTT,GGA,A
回收PCR扩增产物，使其与pMD18-T载体相连（Takara），4℃连接过夜，高效转化DH5α感受态中，获得阳性克隆。提取该阳性克隆质粒，经XhoI和SacI双酶切后，通过T4 DNA连接酶与毕赤酵母表达载体pYPX88（invitrogen）连接，酶切鉴定和序列测定获得了含有目的基因的重组质粒pYPX88-xynAI（参见图2）。
包括所述碱性木聚糖酶基因xynAI序列的pYPX88-xynAI载体，用于毕赤酵母重组表达。
利用电击法将上述pYPX88-xynAI质粒转入毕赤酵母GS115菌中，筛选到的菌株经1wt%甲醇摇瓶诱导72 h后，上清中表达量达到3.98 mg/mL。最适pH为8.6，最适反应温度为55℃。Km值和最大反应速率分别为12.82mg/mL、4.84 μm mg-1 min-1。
有益效果：
本发明按毕赤酵母偏爱密码子优化合成的xynAI基因，可以在毕赤酵母中重组表达出高比活的碱性木聚糖酶，在造纸工业中有广泛的应用潜力。
附图说明
图1为本发明优化的木聚糖酶xynAI基因和原始xynA基因序列比对图。
图2为本发明的用于毕赤酵母表达的pYPX88-xynAI载体。
图3为本发明优化的木聚糖酶基因xynAI的SDS-PAGE分析，其中：M为蛋白分子量标准；1和2为表达的xynAI蛋白。
图4为pH值对本发明优化的木聚糖酶基因xynAI活性的影响。
图5为温度对本发明优化的木聚糖酶基因xynAI活性的影响。
具体实施方式
下面结合具体实施方式来进一步阐述本发明。
本发明实施中未注明的实验方法，如连接、转化、相关培养基的配制等参照分子克隆实验指南第三版【黄培堂等译，中国，科学出版社，2002】和毕赤酵母表达手册（invitrogen）中方法进行。未注明的化学药品为分析纯级，购自上海国药集团有限公司。
实施例1
密码子优化后的碱性木聚糖酶基因的合成
本发明根据来源于菌（Bacillus amyloliquefaciens strain CH51）的xynA基因序列（GenBank Accession No. U51675.1），采取连续延伸PCR方法合成，按照毕赤酵母偏爱密码子优化的xynAI基因。由上海生物工程有限公司合成15条引物，用于xynAI基因的合成。在50µL反应体系中，内侧引物（P2-P14）的添加量为10 ng，外侧引物（P1，P15）添加量为100ng，扩增条件为：94℃ 预热10 min；94℃，30 s，54℃，30 s，72℃，40 s，35个循环；最后72℃延伸10 min。使用KOD FX TaqDNA聚合酶（Toyobo公司日本）扩增目的基因。
按毕赤酵母偏爱密码子优化合成的碱性木聚糖酶基因xynAI序列开放阅读框为561碱基，不包含起始密码子、酶切位点、保护碱基和His标签。通过DNAMAN软件进行同源性比对，结果发现合成序列与原序列（GenBank Accession No. U51675.1）的同源性为78%（参见图1），其编码的蛋白质共186个氨基酸。
实施例 2
优化木聚糖酶xynAI基因在毕赤酵母中的分泌表达
一、毕赤酵母表达载体的构建
1.5% 琼脂糖胶回收实例1中的PCR扩增产物，取10 µL与pMD18-T载体相连。4℃连接过夜，高效转化DH5α感受态中，获得阳性克隆。抽取阳性克隆质粒，经XhoI和SacI双酶切后，以正确的阅读框***到毕赤酵母表达载体pYPX88中，测序验证，得到重组质粒pYPX88-xynAI（参见图 2）。相关的DNA回收试剂盒、载体、连接酶、核酸内切酶均购自Takara公司，DH5α感受态由本实验室保存。
二、 毕赤酵母的转化
挑取划线培养的GS115（mut+his-，invitrogen）单克隆接种到10 mL YPD，30℃，225rpm培养过夜，取1 mL转接到100 mL新鲜YPD中继续培养，继续培养3-5h至OD600=0.5，5000rpm离心收集菌体。用等体积重蒸水洗涤两次后，加入1 mL预冷的山梨醇悬浮菌体，即可作为毕赤酵母感受态。
大量抽取pYPX88-xynAI质粒DNA，经 bglII线性化后，用电击法转入感受态细胞。用Bio-Red GenePulser电击仪电击，参数2.5kV，25µF。电击结束后，立即加入1.0 mL 预冷的1.0 mol/L山梨醇，取200 µL涂布于SD-his培养基平板，30℃培养3-4天至转化子出现。由于受体酵母为His 缺陷型（His-），在不加His 的基本培养基上不生长，只有整合得到的重组酵母才能正常生长。通过这一标记，筛选得到各质粒大量的His+转化子。
三、 毕赤酵母的筛选与诱导表达
采用小规模发酵筛选活性高的转化子，具体操作方法为：接种单个转化子到含有50 μL BMMY（10 g/L 酵母提取物、20 g/L 蛋白胨、13.4 g/L YNB、0.4 mg/L 生物素、1wt%甲醇）的96孔板中，28℃，225 rpm振荡培养8h，加入反应底物桦木木聚糖（sigma），37℃反应30 min，然后用DNS法测定酶的活性。
选取活性相对较高的转化子，接种到50 mL BMGY（10 g/L酵母提取物、20 g/L蛋白胨、13.4 g/L YNB、0.4 mg/L 生物素、10 g/L甘油），培养至OD600=2-3时，离心收集菌体，然后加入等体积BMMY诱导72 h，其间每隔12 h补加甲醇至终浓度1wt%。表达的目的蛋白分泌在上清液中，因此诱导完成后，12000 rpm，离心10 min收集上清。吸取10 µL上清用12wt%SDS-PAGE 进行电泳检测（参见图3），分析得到蛋白质的表达量为3.98 mg/mL。上清用pH=5.5的柠檬酸-磷酸二氢钠稀释600倍后用于酶活力的测定。
实施例3
木聚糖酶xynAI基因在毕赤酵母表达产物的酶活测定
一、木聚糖酶xynAI酶活力测定：
酶活测定采用DNS法。具体实施方法为：取600倍稀释的酶液50 µL，加入1wt%桦木木聚糖50 µL，置50℃水浴保温10min，然后加入DNS试剂150 µL于沸水浴中显色反应8 min，迅速冷却至室温，使用infiniteM200多功能酶标仪（TECAN）测定540nm处比色测定光吸收值的变化。根据木糖标准曲线计算木聚糖酶的活力。
相关试剂的配制：
DNS试剂：称取酒石酸钾钠182.0g，溶于500mL蒸馏水中，加热（不超过50℃），于热溶液中依次加入3,5-二硝基水杨酸6.3g，NaOH 21.0g，苯酚5.0g，无水亚硫酸钠5.0g，搅拌至溶解完全，冷却后用蒸馏水定容至1000 mL，用玻璃漏斗过滤，滤液贮存于棕色瓶中，室温放置7天后使用，有效期为6个月。
木糖标准曲线：吸取1wt%木糖（sigma）溶液30 µL、40 µL、50 µL、60 µL、70 µL、80µL、90 µL、100 µL，用缓冲液定容到1 mL，配制成浓度为0.3 mg/mL、0.4 mg/mL、0.5 mg/mL、0.6 mg/mL、0.7 mg/mL、0.8 mg/mL、0.9 mg/mL、1.0 mg/mL木糖标准溶液。分别吸取上述浓度木糖标准溶液50 µL，代替酶液参与反应，反应完成后测定540 nm的光吸收值。以木糖浓度为Y轴，光吸收值为X轴，绘制标准曲线。
其中，酶活力单位（U）的定义：在该反应条件下，以每分钟产生1 µmol还原糖所需的酶量定义为1个单位。
二、木聚糖酶xynAI的酶学性质测定
最适pH值测定：在不同pH缓冲液下进行酶促反应，缓冲液分别为柠檬酸-磷酸二氢钠（pH 2.5-8.0），甘氨酸-氢氧化钠（pH>8.0），然后按照DNS法测定酶的活性。测得xynAI木聚糖酶的最适pH值为8.6（参见图4）。
最适反应温度：在最适pH，不同温度（20-90℃）下进行酶促反应，然后按照DNS法测定酶的活性。测得xynAI木聚糖酶的最适反应温度为55℃（参见图5）。
Km值和最大反应速率的测定：分别用不同浓度（5，7.5，10，12.5，15，17.5，20 mg/mL）的桦木木聚糖为底物，55℃、pH 5.5测定酶活性，利用米氏方程双倒数法求得Km值和最大反应速率，分别为12.82 mg/mL、4.84μm mg-1 min-1。
序列表
<110> 上海市农业科学院
<120> 一种碱性木聚糖酶基因xynAI优化序列及其表达
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 561
<212> DNA
<213> 芽孢杆菌(Bacillus sp.)
<400> 1
gctgctggta ctgactactg gcagaactgg actgatggtg gtggtactgt caacgctgtc 60
aatggttctg gtggtaacta ctctgtcaac tggtccaata ctggtaactt cgtcgttggt 120
aaaggctgga ccaccggttc tccattcaga accatcaact acaatgctgg tgtctgggct 180
cctaatggca atggctactt gactctgtat ggctggacca gagcaccact catcgagtac 240
tatgttgttg actcttgggg tacttacaga ccaactggta cttacaaagg tactgtcaag 300
tccgatggtg gtacttatga catctacacc actaccagat acaatgctcc atccatcgat 360
ggtgacaaca ccactttcac tcagtactgg tctgtcagac agtccaagag accaactggt 420
tccaacgctg ctatcacctt ctccaatcat gtcaatgctt ggaagtctca tggtatgaat 480
cttggctcca actgggctta ccaagtcttg gctactgaag gctacaagtc ctctggttct 540
tccaacgtca ctgtttggta a 561
<210> 2
<211> 186
<212> PRT
<213> 芽孢杆菌(Bacillus sp.)
<400> 2
Ala Ala Gly Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr
1 5 10 15
Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser
20 25 30
Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro
35 40 45
Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn
50 55 60
Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ala Pro Leu Ile Glu Tyr
65 70 75 80
Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys
85 90 95
Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr
100 105 110
Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Asn Thr Thr Phe Thr Gln
115 120 125
Tyr Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn Ala Ala
130 135 140
Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn
145 150 155 160
Leu Gly Ser Asn Trp Ala Tyr Gln Val Leu Ala Thr Glu Gly Tyr Lys
165 170 175
Ser Ser Gly Ser Ser Asn Val Thr Val Trp
180 185
Claims (4)
1.一种按照毕赤酵母的密码子偏爱性优化而来的碱性木聚糖酶基因xynAI，其特征在于，所述核苷酸序列如SEQ ID No1所示。
2.根据权利要求1所述的碱性木聚糖酶基因xynAI，其特征在于，其编码的蛋白质序列如SEQ ID No 2所示。
3.包括权利要求1所述的碱性木聚糖酶基因xynAI的pYPX88-xynAI载体。
4.权利要求3所述的pYPX88-xynAI载体，其用于毕赤酵母重组表达。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210234830.5A CN114561412B (zh) | 2022-03-11 | 2022-03-11 | 一种碱性木聚糖酶基因xynAI优化序列及其表达 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210234830.5A CN114561412B (zh) | 2022-03-11 | 2022-03-11 | 一种碱性木聚糖酶基因xynAI优化序列及其表达 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114561412A CN114561412A (zh) | 2022-05-31 |
| CN114561412B true CN114561412B (zh) | 2024-02-09 |
Family
ID=81717213
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210234830.5A Active CN114561412B (zh) | 2022-03-11 | 2022-03-11 | 一种碱性木聚糖酶基因xynAI优化序列及其表达 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561412B (zh) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10191971A (ja) * | 1997-01-08 | 1998-07-28 | Kagaku Gijutsu Shinko Jigyodan | キシラン分解酵素とその遺伝子 |
| WO2000032798A1 (en) * | 1998-12-01 | 2000-06-08 | Danisco A/S | Xylanase promoter from thermomyces lanuginosus |
| CN102108366A (zh) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | 按毕赤酵母偏爱密码子优化的木聚糖酶cxyI1基因及其表达 |
| CN102154361A (zh) * | 2011-01-17 | 2011-08-17 | 西北农林科技大学 | 一种木聚糖酶在枯草芽孢杆菌中高效表达载体及构建方法 |
| CN102586294A (zh) * | 2011-01-12 | 2012-07-18 | 上海市农业科学院 | 一种适于毕赤酵母表达的高比活植酸酶基因及其制备方法和表达 |
| CN102978188A (zh) * | 2012-08-06 | 2013-03-20 | 江南大学 | 一种宽pH作用范围木聚糖酶及其应用 |
| CN104130951A (zh) * | 2014-08-07 | 2014-11-05 | 新疆农业大学 | 一种重组毕赤酵母工程菌和代谢的重组木聚糖酶及其制备 |
| CN105219664A (zh) * | 2015-09-24 | 2016-01-06 | 新疆农业大学 | 一种重组基因工程菌构建及高活性β-D-1,4-内切木聚糖酶的制备与应用 |
| CN107090446A (zh) * | 2017-05-27 | 2017-08-25 | 安徽医科大学 | 一种耐热木聚糖酶及其编码基因 |
| CN109207498A (zh) * | 2018-11-08 | 2019-01-15 | 上海市农业科学院 | 一种来自芽孢杆菌耐高温饲料用木聚糖酶的制备 |
| CN111454929A (zh) * | 2019-01-22 | 2020-07-28 | 中国农业大学 | 一种耐高温木聚糖酶基因及其应用 |
-
2022
- 2022-03-11 CN CN202210234830.5A patent/CN114561412B/zh active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10191971A (ja) * | 1997-01-08 | 1998-07-28 | Kagaku Gijutsu Shinko Jigyodan | キシラン分解酵素とその遺伝子 |
| WO2000032798A1 (en) * | 1998-12-01 | 2000-06-08 | Danisco A/S | Xylanase promoter from thermomyces lanuginosus |
| CN102108366A (zh) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | 按毕赤酵母偏爱密码子优化的木聚糖酶cxyI1基因及其表达 |
| CN102586294A (zh) * | 2011-01-12 | 2012-07-18 | 上海市农业科学院 | 一种适于毕赤酵母表达的高比活植酸酶基因及其制备方法和表达 |
| CN102154361A (zh) * | 2011-01-17 | 2011-08-17 | 西北农林科技大学 | 一种木聚糖酶在枯草芽孢杆菌中高效表达载体及构建方法 |
| CN102978188A (zh) * | 2012-08-06 | 2013-03-20 | 江南大学 | 一种宽pH作用范围木聚糖酶及其应用 |
| CN104130951A (zh) * | 2014-08-07 | 2014-11-05 | 新疆农业大学 | 一种重组毕赤酵母工程菌和代谢的重组木聚糖酶及其制备 |
| CN105219664A (zh) * | 2015-09-24 | 2016-01-06 | 新疆农业大学 | 一种重组基因工程菌构建及高活性β-D-1,4-内切木聚糖酶的制备与应用 |
| CN107090446A (zh) * | 2017-05-27 | 2017-08-25 | 安徽医科大学 | 一种耐热木聚糖酶及其编码基因 |
| CN109207498A (zh) * | 2018-11-08 | 2019-01-15 | 上海市农业科学院 | 一种来自芽孢杆菌耐高温饲料用木聚糖酶的制备 |
| CN111454929A (zh) * | 2019-01-22 | 2020-07-28 | 中国农业大学 | 一种耐高温木聚糖酶基因及其应用 |
Non-Patent Citations (10)
| Title |
|---|
| Dawn Elizabeth Stephens ; Faez Iqbal Khan ; Parvesh Singh ; Krishna Bisetty ; Suren Singh ; Kugen Permaul.Creation of thermostable and alkaline stable xylanase variants by DNA shuffling.《Journal of Biotechnology》.2014,第187卷全文. * |
| Leya Thomas ; Raveendran Sindhu ; Parameswaran Binod ; Ashok Pandey.Production of an alkaline xylanase from recombinant Kluyveromyces lactis (KY1) by submerged fermentation and its application in bio-bleaching.《 Biochemical Engineering Journal》.2015,第102卷全文. * |
| 一种新型木聚糖酶基因在毕赤酵母中的表达;李琦;成莉凤;段盛文;冯湘沅;郑科;杨琦;刘志远;彭源德;;华北农学报(第04期);全文 * |
| 刘伟丰 ; 毛爱军 ; 祝令香 ; 于巍 ; 赵云 ; 董志扬.耐碱性木聚糖酶高产基因工程菌构建及应用.第四届中国酶工程学术交流讨论会.2003,全文. * |
| 来源于盐惰菌属Halopiger xanaduensis的木聚糖酶基因在毕赤酵母中诱导表达及其酶学性质的研究;于成野;韩红娟;付晓燕;游双红;孙淼;朱恒文;彭日荷;姚泉洪;;上海农业学报(第06期);全文 * |
| 碱性芽孢杆菌木聚糖酶基因的克隆及表达;陈娜;生吉萍;申琳;;食品科学(第05期);全文 * |
| 碱性芽饱杆菌木聚糖酶基因的克隆及表达;陈娜;生吉萍;申琳;食品科学;第032卷(第005期);全文 * |
| 耐碱性木聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质;包怡红;刘伟丰;何永志;董志杨;;微生物学报(第10期);全文 * |
| 耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究;刘伟丰;毛爱军;祝令香;赵云;董志扬;微生物学报(第004期);全文 * |
| 耐碱性木聚糖酶基因的克隆、表达及高产工程菌的构建;刘伟丰;《第四届中国酶工程学术交流讨论会论文集》;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561412A (zh) | 2022-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110607292B (zh) | 一种高比活木聚糖酶突变体 | |
| CN111454929B (zh) | 一种耐高温木聚糖酶基因及其应用 | |
| CN113755468B (zh) | 一种对胰蛋白酶抗性提高的玉米赤霉烯酮水解酶 | |
| CN115029334B (zh) | 一种高比活碱性木聚糖酶突变体 | |
| CN110029122B (zh) | 一种甘露聚糖酶高产菌株及其应用 | |
| CN117384889A (zh) | 耐高温木聚糖酶突变体及其应用 | |
| US6365390B1 (en) | Phenolic acid esterases, coding sequences and methods | |
| CN112322604B (zh) | 一种高比酶活木聚糖酶突变体及其应用 | |
| CN114561412B (zh) | 一种碱性木聚糖酶基因xynAI优化序列及其表达 | |
| US8383798B2 (en) | Polynucleotide encoding a cellulase enzyme and method for producing the enzyme | |
| CN113755473B (zh) | 分泌表达量提高的葡萄糖淀粉酶突变体m5及其基因和应用 | |
| CA2214425A1 (en) | Endo beta-1,4-glucanase from aspergillus | |
| US11371032B2 (en) | Beta glucosidase with high glucose tolerance, high thermal stability and broad PH activity spectrum | |
| CA2139099A1 (en) | Recombinant cellulases | |
| EP0939825A1 (en) | Truncated cbh i promoter from trichoderma reesei and use thereof | |
| CN111484988B (zh) | 一种具有木聚糖酶和阿魏酸酯酶活性的双功能酶及其编码基因和应用 | |
| CN111500558B (zh) | 具提升活性的葡萄糖苷酶 | |
| CN113817758A (zh) | 编码贝莱斯芽孢杆菌的壳聚糖酶基因、壳聚糖酶及其制备方法和应用 | |
| WO2010089302A1 (en) | Protein and dna sequence encoding a cold adapted xylanase | |
| CN109679935A (zh) | 快速水解木聚糖生成单一木糖的糖苷水解酶的编码基因及其应用 | |
| CN111757939A (zh) | 突变β-葡萄糖苷酶 | |
| CN114317495B (zh) | 一种热稳定性提高的葡聚糖酶突变体及其应用 | |
| CN114774385B (zh) | 一种对胰蛋白酶和胃蛋白酶抗性提高的玉米赤霉烯酮水解酶 | |
| CN113774045B (zh) | 分泌表达量提高的葡萄糖淀粉酶突变体m3及其基因和应用 | |
| US9034627B2 (en) | Method for producing glucosidase, enzyme composition, and method for hydrolyzing biomass |
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 |