CN108546695A - A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application - Google Patents
A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application Download PDFInfo
- Publication number
- CN108546695A CN108546695A CN201810323783.5A CN201810323783A CN108546695A CN 108546695 A CN108546695 A CN 108546695A CN 201810323783 A CN201810323783 A CN 201810323783A CN 108546695 A CN108546695 A CN 108546695A
- Authority
- CN
- China
- Prior art keywords
- gene
- esta
- esterase
- coding albumen
- thermostable
- 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
- 108090000371 Esterases Proteins 0.000 title claims abstract description 56
- 241000894006 Bacteria Species 0.000 title claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 title abstract description 30
- 241000588724 Escherichia coli Species 0.000 claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 11
- 230000006798 recombination Effects 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 5
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 150000002148 esters Chemical class 0.000 claims description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000013604 expression vector Substances 0.000 claims 1
- 239000012847 fine chemical Substances 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 49
- 102000004190 Enzymes Human genes 0.000 abstract description 48
- 101710198555 Esterase EstA Proteins 0.000 abstract description 11
- 238000005215 recombination Methods 0.000 abstract description 7
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 abstract description 4
- 239000013613 expression plasmid Substances 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000000034 method Methods 0.000 abstract description 3
- 230000003197 catalytic effect Effects 0.000 abstract description 2
- 238000003259 recombinant expression Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 53
- 101001052533 Piromyces equi Feruloyl esterase B Proteins 0.000 description 27
- 101100162206 Aspergillus parasiticus (strain ATCC 56775 / NRRL 5862 / SRRC 143 / SU-1) aflJ gene Proteins 0.000 description 13
- 101150006505 estA gene Proteins 0.000 description 13
- 238000005457 optimization Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 239000000872 buffer Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 8
- 241000187747 Streptomyces Species 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 108091081024 Start codon Proteins 0.000 description 4
- 241001655322 Streptomycetales Species 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000003262 industrial enzyme Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 2
- IVZQNRPVAYGVJK-UHFFFAOYSA-N (2-hydroxy-5-nitrophenyl) butanoate Chemical compound CCCC(=O)OC1=CC([N+]([O-])=O)=CC=C1O IVZQNRPVAYGVJK-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 108010044087 AS-I toxin Proteins 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 241000203809 Actinomycetales Species 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- WYPUMLRSQMKIJU-BPNCWPANSA-N Ala-Arg-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WYPUMLRSQMKIJU-BPNCWPANSA-N 0.000 description 1
- VBRDBGCROKWTPV-XHNCKOQMSA-N Ala-Glu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N VBRDBGCROKWTPV-XHNCKOQMSA-N 0.000 description 1
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- CHFFHQUVXHEGBY-GARJFASQSA-N Ala-Lys-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N CHFFHQUVXHEGBY-GARJFASQSA-N 0.000 description 1
- RUXQNKVQSKOOBS-JURCDPSOSA-N Ala-Phe-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RUXQNKVQSKOOBS-JURCDPSOSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- QAODJPUKWNNNRP-DCAQKATOSA-N Arg-Glu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QAODJPUKWNNNRP-DCAQKATOSA-N 0.000 description 1
- QHVRVUNEAIFTEK-SZMVWBNQSA-N Arg-Pro-Trp Chemical compound N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O QHVRVUNEAIFTEK-SZMVWBNQSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- FIADUEYFRSCCIK-CIUDSAMLSA-N Cys-Glu-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FIADUEYFRSCCIK-CIUDSAMLSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- GHAXJVNBAKGWEJ-AVGNSLFASA-N Gln-Ser-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GHAXJVNBAKGWEJ-AVGNSLFASA-N 0.000 description 1
- OAGVHWYIBZMWLA-YFKPBYRVSA-N Glu-Gly-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)NCC(O)=O OAGVHWYIBZMWLA-YFKPBYRVSA-N 0.000 description 1
- ATVYZJGOZLVXDK-IUCAKERBSA-N Glu-Leu-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O ATVYZJGOZLVXDK-IUCAKERBSA-N 0.000 description 1
- DXVOKNVIKORTHQ-GUBZILKMSA-N Glu-Pro-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O DXVOKNVIKORTHQ-GUBZILKMSA-N 0.000 description 1
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 1
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 1
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 1
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 1
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 1
- RAVLQPXCMRCLKT-KBPBESRZSA-N His-Gly-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RAVLQPXCMRCLKT-KBPBESRZSA-N 0.000 description 1
- TWROVBNEHJSXDG-IHRRRGAJSA-N His-Leu-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O TWROVBNEHJSXDG-IHRRRGAJSA-N 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- IFMPDNRWZZEZSL-SRVKXCTJSA-N Leu-Leu-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O IFMPDNRWZZEZSL-SRVKXCTJSA-N 0.000 description 1
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 1
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- GCMWRRQAKQXDED-IUCAKERBSA-N Lys-Glu-Gly Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)N[C@@H](CCC([O-])=O)C(=O)NCC([O-])=O GCMWRRQAKQXDED-IUCAKERBSA-N 0.000 description 1
- DTUZCYRNEJDKSR-NHCYSSNCSA-N Lys-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN DTUZCYRNEJDKSR-NHCYSSNCSA-N 0.000 description 1
- SPNKGZFASINBMR-IHRRRGAJSA-N Lys-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCCN)N SPNKGZFASINBMR-IHRRRGAJSA-N 0.000 description 1
- PHWSCIFNNLLUFJ-NHCYSSNCSA-N Met-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N PHWSCIFNNLLUFJ-NHCYSSNCSA-N 0.000 description 1
- GGXZOTSDJJTDGB-GUBZILKMSA-N Met-Ser-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O GGXZOTSDJJTDGB-GUBZILKMSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- MQWISMJKHOUEMW-ULQDDVLXSA-N Phe-Arg-His Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 MQWISMJKHOUEMW-ULQDDVLXSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- DBALDZKOTNSBFM-FXQIFTODSA-N Pro-Ala-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DBALDZKOTNSBFM-FXQIFTODSA-N 0.000 description 1
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 1
- DIFXZGPHVCIVSQ-CIUDSAMLSA-N Pro-Gln-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DIFXZGPHVCIVSQ-CIUDSAMLSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- WSRWHZRUOCACLJ-UWVGGRQHSA-N Pro-Gly-His Chemical compound C([C@@H](C(=O)O)NC(=O)CNC(=O)[C@H]1NCCC1)C1=CN=CN1 WSRWHZRUOCACLJ-UWVGGRQHSA-N 0.000 description 1
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010025216 RVF peptide Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 1
- VXYQOFXBIXKPCX-BQBZGAKWSA-N Ser-Met-Gly Chemical compound CSCC[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N VXYQOFXBIXKPCX-BQBZGAKWSA-N 0.000 description 1
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 1
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 description 1
- DIPIPFHFLPTCLK-LOKLDPHHSA-N Thr-Gln-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O DIPIPFHFLPTCLK-LOKLDPHHSA-N 0.000 description 1
- JMGJDTNUMAZNLX-RWRJDSDZSA-N Thr-Glu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JMGJDTNUMAZNLX-RWRJDSDZSA-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
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- OBWQLWYNNZPWGX-QEJZJMRPSA-N Trp-Gln-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O OBWQLWYNNZPWGX-QEJZJMRPSA-N 0.000 description 1
- BARBHMSSVWPKPZ-IHRRRGAJSA-N Tyr-Asp-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BARBHMSSVWPKPZ-IHRRRGAJSA-N 0.000 description 1
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- MJOUSKQHAIARKI-JYJNAYRXSA-N Val-Phe-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 MJOUSKQHAIARKI-JYJNAYRXSA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 1
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- IUVHPPMKJSCSJR-UHFFFAOYSA-N butanoic acid;4-nitrophenol Chemical compound CCCC(O)=O.OC1=CC=C([N+]([O-])=O)C=C1 IUVHPPMKJSCSJR-UHFFFAOYSA-N 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 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
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- -1 p-nitrophenol ester Chemical class 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000002623 sporogenic effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 1
- 238000011144 upstream manufacturing 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and applications, esterase gene estA' base sequences are SEQ ID NO.1, gene estA' is connect to structure recombinant expression carrier pET28b estA' with plasmid, expression plasmid converts Escherichia coli structure recombination engineering Rosetta (DE3) pLysS/pET28b estA'；Esterase obtains heterogenous expression in Escherichia coli, using the zymologic property of para-nitrophenol method identification esterase EstA, with high heat stability, the purpose of the present invention is the productions for the esterase and its commercial Application in fields such as medicine, food, fine chemistry industries to provide service, especially when needing high-temperature catalytic, the enzyme is more suitable.
Description
Technical field
The invention belongs to technical field of bioengineering, are related to gene, coding protein engineering techniques field, and in particular to a kind of
Thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application.
Background technology
Esterase (Esterases, EC 3.1.1.X), which is one kind, can be catalyzed ester linkage hydrolyzing and the enzyme of formation, widely distributed
In animal, plant and microorganism.As important industrial enzymes, esterase has been widely used in medication chemistry, food work at present
The fields such as industry, fine chemistry industry, bioenergy, agricultural and environmental improvement.With constantly widening for esterase application range, existing oneself becomes
World industry enzyme preparation the third-largest industrial enzymes after protease, amylase in the market.But esterase industrial process conditions compared with
To be extensive, therefore in order to save production cost, seek high thermal stability, the esterase of high catalytic efficiency is always mesh that people make great efforts
Mark.
Streptomycete (Streptomyces) belongs to Actinomycetal Streptomycetaceae streptomyces, is a kind of be present in soil
Aerobic, Filamentous, sporogenic gram-positive bacterium.Streptomycete has abundant secondary metabolic pathways, can generate a large amount of
Secondary metabolites with Important Economic value, such as antibiotic, hydrolase, enzyme inhibitor, herbicide and antitumor drug,
It is extremely important industrial production bacterium.The size of streptomyces gene group is 5.5Mb-8.6Mb, about the 2 of genome of E.coli
Times, G+C contents are up to 70%-74%.Contain abundant esterase gene in streptomycete, these sequence informations carry for researchers
Abundant resource is supplied, but also fewer at present to their research.Researcher can clone corresponding according to these sequence informations
Esterase gene, and heterogenous expression and zymologic property research are carried out to them.But due to the high GC content of streptomyces gene and more
Complicated protein translation system, makes expression of the gene of streptomycete in heterologous host usually be limited.Only a small number of kinds at present
The esterase gene of class is in heterologous expression system such as Escherichia coli (Escherichia coli), muta lead mycillin, saccharomyces cerevisiae
Preferable expression is obtained in (Saccharomvcesc erevisiae), pichia pastoris yeast (Pichia pastoris).Ester
The non-translational region of enzyme gene, the presence of esterase propetide and signal skin, the difference of codon and different expression system expression recombinations
Glycosylation of esterase etc. can all influence the heterogenous expression of esterase gene.Especially Escherichia coli, as low prokaryotes,
Lack perfect posttranslational modification system, esterase is often beyond expression or exists with inclusion bodies wherein, few to obtain activity
Albumen.
Muta lead mycillin (Streptomyces lividands) TK24 full-length genomes (Complete Genome) nucleosides
For acid sequence in GenBank databases, accession number (Accession number) is CP009124, which is 8.3Mb,
G+C contents are up to 72.2%.The studied gene of the present invention is the locus (Locus tag) on chromosome (NZ_CP009124)
SLIV_RS27090, nucleotide sequence initiation site (Start) on chromosome is 6057885, and termination site (Stop) is
6058688.It is esterase, protein product to annotate protein product coded by this section of nucleotide sequence according to genome sequence
It is WP_003976692 that (Protein product), which numbers (Accession), and the protein accession numbers in GenBank are
AIJ16329.This section of sequence 804bp, initiation codon (Initiation codon) are TCA, terminator codon (Stop
Codon it is) CAC, G/C content 72.5% embodies the high GC content feature of streptomyces gene group.
Invention content
According to the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to propose a kind of thermostable esterases base
Cause, the engineering bacteria containing the gene and its coding albumen and application, in order to solve the above-mentioned technical problem, the technology that the present invention uses
Scheme is：
A kind of thermostable esterases gene, the thermostable esterases gene have the base sequence of SEQ ID NO.1, and can be in heterologous place
Successful expression in main Escherichia coli.
A kind of recombinant plasmid, the recombinant plasmid contain pET-28b (+) recombinant plasmid of the thermostable esterases gene.
A kind of thermostable esterases genetic recombination engineering bacteria, the engineering bacteria are Escherichia coli and contain the expression of recombinant plasmid
Carrier.
Preferably, the condition of the engineering bacterium expression thermostable esterases albumen is the IPTG induced concentrations and 16 of 0.01-0.5mM
DEG C -30 DEG C of inducing temperature.
A kind of coding albumen of thermostable esterases gene, the coding albumen have the amino acid sequence of SEQ ID NO.2.
Preferably, the reaction temperature of the coding albumen is 30-60 DEG C, and reaction pH is 7-9, active half-life at 100 DEG C >=
6h。
Preferably, the reaction temperature of the coding albumen is 55 DEG C, and reaction pH is that active half-life is 6h at 8.5,100 DEG C.
A kind of application of the coding albumen of thermostable esterases gene, the coding albumen are applied in medicine, food and fine chemistry industry
Field.
Compared with prior art, beneficial effects of the present invention：
1. the present invention carries out the locus SLIV_RS27090 nucleotide sequences on muta lead mycillin TK24 chromosomes
Optimization, obtains amino acid coded by nucleotide sequence and remains unchanged, but 609 nucleotide occur in 804 nucleotide sequences
Variation, accounts for the 75.7% of whole gene, G/C content is also dropped to by 72.5% of the original nucleic acid accounting in bacterial strain
53.4%, be conducive to expression of the optimization gene in Escherichia coli.
2. the gene estA' after optimization is connect by the present invention with pET28b plasmids, successfully the recombination table of structure tool Kan resistances
Up to carrier pET28b-estA'.
3. the present invention is by the recombinant plasmid transformed competent escherichia coli cell, the base that successfully structure is expressed for estA'
Because of engineering bacteria Rosetta (DE3) pLysS/pET28b-estA', which is provided simultaneously with Kan and Cam resistances.
4. the present invention uses Co2+Ion affinity chromatography method and SDS-PAGE electrophoresis obtain highly purified esterase protein EstA,
The esterase protein EstA of acquisition has high heat stability, can be applied to the fields such as medicine, food and fine chemistry industry.
Description of the drawings
1. Fig. 1 is the sequence alignment figure of esterase estA genes and the estA' after optimization；
2. Fig. 2 is the expression of SDS-PAGE analyses EstA；
3. Fig. 3 is EstA activity and react pH tendency charts；
4. Fig. 4 is EstA activity and reaction temperature tendency chart；
5. Fig. 5 is thermal stability tendency charts of the EstA at 55 DEG C；
6. Fig. 6 is thermal stability tendency charts of the EstA at 100 DEG C.
M, protein Marker, 1, the crude enzyme liquids of IPTG inductions, 2, the EstA albumen of purifying, ■, pH 6.0-7.0
Na-citrate buffer, ●, the Tris-HCl buffer of pH 7.0-9.0, ▲, the KH of pH 9.0-10.52PO4-NaOH
buffer。
Specific implementation mode
It below by the description to embodiment, is described in further detail, to help those skilled in the art to this hair
Bright inventive concept, technical solution have more complete, accurate and deep understanding.
It needs to illustrate：Locus (Locus tag) SLIV_RS27090 coding esterase genes are named as by the present invention
EstA, the esterase gene after optimization are named as estA', since the amino acid composition of protein coded by estA and estA' is identical,
Therefore the amino acid Uniform Name encoded is EstA.
The standard test system used in embodiment is 1mL, by the Tris-HCl, 10 μ L of 980 μ L 50mM pH 8.0
The enzyme solution of the p-nitrophenol butyrate of 0.5mM, 10 μ L is constituted.
Embodiment 1
1. a couple muta lead mycillin TK24 esterase genes estA is optimized, is synthesized
Theoretical direction：Muta lead mycillin TK24 esterase estA full length gene 804bp, initiation codon TCA are terminated close
Numeral is CAC；A bases 122, accounting 15.2%；T bases 99, accounting 12.3%；C bases 297, accounting 36.9%； G
Base 286, accounting 35.6%；G/C content is 72.5%, embodies the high GC content feature of muta lead mycillin genome.By
In the high G/C content of estA genes, expression of the albumen in Escherichia coli is seriously affected, and muta lead mycillin esterase is secondary
Raw metabolism protein, may need molecular chaperones to assist its folding, thus table of the gene in Escherichia coli during expression
Up to there is obstacle, the heterogenous expression in Escherichia coli can not be completed, in order to solve this problem, in expression same amino acid sequence
Under the premise of row, the nucleotide sequence of esterase estA genes is optimized in we, reduces the G/C content of the gene as possible, together
When its rare codon replaced with Escherichia coli preference codon, in addition, to make the gene be connect with carrier pET-28b (+),
Gene estA' Sequences upstreams and downstream after optimization add Nde I (CATATG) and Xho I (CTCGAG) corresponding digestions position respectively
The base of point.By Jin Sirui biotechnologies, company synthesizes.
2. the sequence analysis of esterase gene after optimization
Esterase gene estA' overall lengths after optimization are still 804bp, but initiation codon is shown as in terms of nucleotides feature
ATG, terminator codon TAA；A bases 162, accounting 20.1%；T bases 208, accounting 25.9%；C bases 201, accounting
25.0%；G bases 233, accounting 29.0%.After muta lead mycillin TK24 esterase estA gene nucleotide series and optimization
The gene nucleotide sequence estA' be compared as shown in Figure 1, in 804 bases only have 195 bases remain unchanged,
Only account for the 24.3% of whole amino acid sequences.In terms of G/C content, original series are compared, the bases G C content after optimization drops to
53.4%, to overcome the excessively high defect for causing the esterase to be beyond expression of G/C content, to make the target gene after optimization big
Heterogenous expression in enterobacteria provides guarantee.
Embodiment 2
Expression plasmid and the structure for expressing engineering bacteria：To the gene estA' sequences and carrier pET-28b (+) point after optimization
Not carry out Nde I and Xho I correspond to digestion, digestion condition is 37 DEG C, reacts 6h, and digestion system is：
The gene estA' of double digestion and carrier pET-28b (+) is attached, structure expression plasmid pET28b-estA'.
Condition of contact is that 16 DEG C of reactions are stayed overnight, and linked system is：
Expression plasmid pET28b-estA' is mixed with E.coli Rosetta (DE3) competent cell, ice bath 30min,
Intermediate jog for several times, prevents bacterial sediment, 42 DEG C of heat shock 90s from avoiding shaking, structure recombination engineering Rosetta (DE3)
PLysS/pET28b-estA', the engineering bacteria have Kan and Cam resistances, can be sieved on the LB culture mediums of tool Kan and Cam resistances
Select recombination engineering.
Embodiment 3
The expression of 1.EstA
Picking recombination engineering is in the test tube of the LB liquid medium containing 5mL, 37 DEG C, 225rpm, expands culture overnight；
Culture is with 1:100 are inoculated in the tap web bottle of the LB liquid medium containing 50mL, 37 DEG C, 225rpm, shaken cultivation to OD600
For 0.4-1.0 when (preferably 0.6)；The IPTG of low concentration 0.01-0.5mM is added, in 16 DEG C -30 DEG C of low temperature, 180rpm is lured
Lead expression for 24 hours；Then at 4 DEG C, under 5000rpm, 5min is centrifuged, collects thalline, ultrasonic disruption obtains esterase EstA.
2. the purifying of esterase EstA
Esterase EstA shares 267 amino acid compositions, and molecular weight (Molecular Weight) is 28.5kDa, isoelectric point
(isoelectric point) is 6.9, and Aliphatic index (Aliphatic index) is 96.8%, uses Co2+Ion
Affinity chromatography column purification, Co2+Ion affinity chromatography column purification uses the Co of Clontech companies2+Ion affinity column purified pool
Fusion protein EstA with 6 His-tag, protein after purification identify that the albumen is in corresponding esterase through 12%SDS-PAGE
The band such as Fig. 2 for having a specificity very high at EstA molecular weight, illustrates that heterologous table has successfully been obtained in the albumen in Escherichia coli
It reaches and passes through Co2+Ion affinity chromatography column purification obtains the higher albumen of purity, and protein concentration inspection is carried out to purifying protein
It surveys, using bovine serum albumin as standard, a concentration of 0.807mg/mL of esterase protein after purification.
Embodiment 4
1.EstA optimal pHs detect
The Enzyme activity assay of EstA uses standards system, uses Cary 300UV spectrophotometers that can be temperature automatically controlled, inspection
Survey wavelength is 410nm, as a contrast with not enzyme mixed liquor, 25 DEG C, reaction time 5min of reaction temperature, in corresponding pH models
It encloses using corresponding buffer solution, pH6.0-7.0,50mM sodium citrate buffer；PH7.0-9.0,50mM Tris-
HCl buffer；PH9.0-10.5,50mM K2HPO4- NaOH buffer, Detection wavelength OD410, experiment are independently repeated 3 times,
As a result show that the optimal pH of the enzyme is 8.5, Long-term change trend such as Fig. 3.
2.EstA optimum temperatures detect
Using standard test system, reaction pH is 8.5, and 10 DEG C -60 DEG C of reaction temperature is arranged, and experiment is independently repeated 3 times, takes
Average value, the results showed that the optimum temperature of the enzyme is 55 DEG C, Long-term change trend such as Fig. 4.
3. the most suitable substrates of esterase EstA measure
At 25 DEG C, under the conditions of pH8.5, using standard test system, different chain length C4-C16 p-nitrophenol esters are detected
The Rate activity of substrate, recombination enzyme activity are calculated by p-nitrophenol standard curve, and an enzyme-activity unit (U) is defined as 25 DEG C
Under the conditions of it is per minute from p-nitrophenol ester catalysis generate 1 μm of oL p-nitrophenol needed for enzyme amount, specific activity of enzyme unit is U/
Mg indicates enzyme activity possessed by every milligram of zymoprotein；As a result the Rate activity highest of display substrate p-nitrophenol butyric acid esterase,
Up to 1409.5U/mg, as shown in table 1 below：
The Rate activity of 1 esterase EstA of table
| Compounds | Specific activity(U/mg)±SD |
| pNPB4 | 1409.5±31.5 |
| pNPC6 | 760.5±29.6 |
| pNPC8 | 580.1±12.2 |
| pNPC10 | 566.0±6.7 |
| pNPL12 | 523.1±21.4 |
| pNPM14 | 393.2±11.1 |
| pNPP16 | 286.6±14.1 |
4. esterase EstA heat stability tests
The pure enzymes of EstA are incubated into certain time respectively in 55 DEG C and 100 DEG C, using standard test system, 25 DEG C of detections are remaining
Enzyme activity determines thermal stability, and 100% is set as with untreated pure enzymatic activity.The result shows that the enzyme places enzyme after 337h at 55 DEG C
Activity is still without significant change such as Fig. 5, and EstA places 5.5h at 100 DEG C, and enzyme activity, which still progressivelyes reach after 80% or more, 6h, partly to decline
Phase such as Fig. 6.
Embodiment 5
1. the influence of metal ion, EDTA and PMSF to EstA enzyme activity
At 25 DEG C, under the conditions of pH8.5, using standard test system, metal ion, EDTA and PMSF detections are added respectively
EstA enzyme activities, buffer Final concentration used are 50mM Tris-HCl, and the enzyme activity to be not added with chemical reagent is set as 100%,
Experiment is independently repeated 3 times, as shown in table 2, the K of 1mM+、Fe2+、Mn2+、Ca2+And Na+There is stronger activation effect to enzyme, can make
Enzyme activity improves 20% or more, especially K+、Fe2+And Mn2+ enzyme activity can be made to increase 53.3%, 48.6% and 44.2% respectively；Zn2+
And Mg2+Also there is certain activation to enzyme activity, can make enzyme activity that 13.7% and 11.3% be respectively increased；The K of 10mM+、Fe2+、
Mn2+、 Ca2+And Na+Still there is a degree of activation effect to enzyme, but removes Ca2+And Na+It is substantially unchanged to the activation effect of enzyme
Other outer three classes ions increase decrease to some degree with concentration；The Mg of 10mM2+To the activation effect of enzyme relative to 1mM's
Mg2+Without significant changes, opposite enzyme activity is 109.1%；The Zn of 10mM2+、Cu2+And Ni2+Strong inhibition enzyme activity, makes enzyme activity drop respectively
To 35.2%, 34.4% and 10.2%；The EDTA and PMSF of 1mM on esterase enzyme activity substantially without influence, when concentration increases to 10mM
When, enzyme activity is reduced to 86.0% and 72.6% respectively.
2 metal ion of table, EDTA and PMSF are on the active influences of EstA
2. influence of the organic solvent to EstA enzyme activity
At 25 DEG C, under the conditions of pH8.5, using standard test system, respectively different organic solvent detects EstA enzyme activities, institute
It is 50mM Tris-HCl with buffer Final concentration, the enzyme activity to be not added with chemical reagent is set as 100%, tests independent repetition 3
It is secondary, as shown in table 3, in all detected organic solvents, a concentration of 15% and 30% dimethyl sulfoxide (DMSO) and a concentration of
15% isopropanol all has no significant effect esterase enzyme activity, but when isopropyl alcohol concentration reaches 30%, can be generated to esterase enzyme activity
Strong inhibiting effect, makes enzyme activity be down to 51.6%；15% and 30% ethyl alcohol, methanol, dimethylformamide can be in certain journeys
Inhibitory enzyme activity on degree, and inhibit enzyme activity effect enhancing with the increase of its concentration, but amplification is little；15% and 30% acetonitrile and
Acetone has stronger inhibiting effect to enzyme activity, and inhibits enzyme activity effect also to enhance with the increase of its organic solvent concentration.
3 organic solvent of table is on the active influences of EstA
3. influence of the detergent to EstA enzyme activity
At 25 DEG C, under the conditions of pH8.5, using standard test system, respectively different organic solvent detects EstA enzyme activities, institute
It is 50mM Tris-HCl with buffer Final concentration, the enzyme activity to be not added with chemical reagent is set as 100%, tests independent repetition 3
Secondary, as shown in table 4, in all 0.1% detected detergents, Span-20 has stronger activation to the enzyme activity of esterase
Effect, makes which increase 37.2%；Tween-20 improves esterase enzyme activity to a certain extent, but function and effect are not very apparent；
SDS and Triton X-100 are on the activity of esterase substantially without influence, and in 1% detergent, Span-20, SDS and CTAB are to ester
The enzyme activity of enzyme has strong inhibiting effect, and enzyme activity is made to be down to 30.8%, 35.6% and 44.2% respectively.
4 detergent of table is on the active influences of EstA
The present invention is exemplarily described above in conjunction with specific embodiment, it is clear that the present invention implements not by upper
The limitation of mode is stated, if the improvement of the various unsubstantialities of inventive concept and technical scheme of the present invention progress is used, or
It is not improved by the present invention design and technical solution directly apply to other occasions, protection scope of the present invention it
It is interior.Protection scope of the present invention should be determined by the scope of protection defined in the claims.
Sequence table
<110>Anhui Normal University
<120>A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application
<130> 2018.04.12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 804
<212> DNA
<213>Muta lead mycillin (Streptomyces lividands)
<400> 1
atgtcagtgt taccgggtgc tgaaccattt cgtcatgaag gcggagatgt gggcgtttta 60
ctgtgtcatg gctttacggg ttctccacag tctctgcgtc cttgggctcg ctacttagcc 120
gcacgcggcc tgacagtttc actgccatta ttaccaggcc acggtacacg ttggcaggat 180
atgcaggtga ccggttggca ggattggtat gcagaagtgg atcgcgaact tagagccctg 240
cgcgaacgct gcgaacgagt gtttgttgcg ggtctgtcaa tgggcggcgc actggccctg 300
cgcttagcag ctaaacatgg ggatgccgtt tcaggcgtgg ttgtggttaa tccggctaac 360
aagatgcatg gcgttgctca acacgccctg cctgtgctgc gccatttggt tccagcgacc 420
aaaggcatcg caagcgatat tgcaaaacct ctgagtacgg aactgggcta tgatcgtgtt 480
ccgttacata gcgcacatag cttacgcgcc ttctttcgtc tggccgatgg ggatctccct 540
caggtgacac agccactgtt attattgcgc agtcctcagg atcatgttgt tcctccagcc 600
gatagtgctc gtatcttagg tcgcgtgtct tctacagatg tgacggaaat tctgctggaa 660
cagagctatc atgtggcaac cttagatcat gatgcagatc gtatctttgc tgaatcagtt 720
gccttcatcg gtcgcttagc tccgggtagt gtgggtgaac cagaaagcgg cttaggcaaa 780
gaaggaaccg ccgccggcgg ttaa 804
<210> 2
<211> 267
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 2
Met Ser Val Leu Pro Gly Ala Glu Pro Phe Arg His Glu Gly Gly Asp
1 5 10 15
Val Gly Val Leu Leu Cys His Gly Phe Thr Gly Ser Pro Gln Ser Leu
20 25 30
Arg Pro Trp Ala Arg Tyr Leu Ala Ala Arg Gly Leu Thr Val Ser Leu
35 40 45
Pro Leu Leu Pro Gly His Gly Thr Arg Trp Gln Asp Met Gln Val Thr
50 55 60
Gly Trp Gln Asp Trp Tyr Ala Glu Val Asp Arg Glu Leu Arg Ala Leu
65 70 75 80
Arg Glu Arg Cys Glu Arg Val Phe Val Ala Gly Leu Ser Met Gly Gly
85 90 95
Ala Leu Ala Leu Arg Leu Ala Ala Lys His Gly Asp Ala Val Ser Gly
100 105 110
Val Val Val Val Asn Pro Ala Asn Lys Met His Gly Val Ala Gln His
115 120 125
Ala Leu Pro Val Leu Arg His Leu Val Pro Ala Thr Lys Gly Ile Ala
130 135 140
Ser Asp Ile Ala Lys Pro Leu Ser Thr Glu Leu Gly Tyr Asp Arg Val
145 150 155 160
Pro Leu His Ser Ala His Ser Leu Arg Ala Phe Phe Arg Leu Ala Asp
165 170 175
Gly Asp Leu Pro Gln Val Thr Gln Pro Leu Leu Leu Leu Arg Ser Pro
180 185 190
Gln Asp His Val Val Pro Pro Ala Asp Ser Ala Arg Ile Leu Gly Arg
195 200 205
Val Ser Ser Thr Asp Val Thr Glu Ile Leu Leu Glu Gln Ser Tyr His
210 215 220
Val Ala Thr Leu Asp His Asp Ala Asp Arg Ile Phe Ala Glu Ser Val
225 230 235 240
Ala Phe Ile Gly Arg Leu Ala Pro Gly Ser Val Gly Glu Pro Glu Ser
245 250 255
Gly Leu Gly Lys Glu Gly Thr Ala Ala Gly Gly
260 265
Claims (8)
1. a kind of thermostable esterases gene, which is characterized in that the thermostable esterases gene has the base sequence of SEQ ID NO.1, and
Can in heterologous host Escherichia coli successful expression.
2. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid contains the pET- of thermostable esterases gene described in claim 1
28b (+) recombinant plasmid.
3. a kind of thermostable esterases genetic recombination engineering bacteria, which is characterized in that the engineering bacteria is Escherichia coli and contains claim 2
The recombinant plasmid expression vector.
4. thermostable esterases genetic recombination engineering bacteria according to claim 3, which is characterized in that the heat-resisting ester of the engineering bacterium expression
The condition of zymoprotein is the IPTG induced concentration and 16 DEG C -30 DEG C of inducing temperature of 0.01-0.5mM.
5. a kind of coding albumen of thermostable esterases gene described in claim 1, which is characterized in that the coding albumen has SEQ
The amino acid sequence of ID NO.2.
6. the coding albumen of thermostable esterases gene according to claim 5, which is characterized in that the reaction temperature of the coding albumen
Degree is 30-60 DEG C, and reaction pH is 7-9, active half-life >=6h at 100 DEG C.
7. the coding albumen of thermostable esterases gene according to claim 5, which is characterized in that the reaction temperature of the coding albumen
Degree is 55 DEG C, and reaction pH is that active half-life is 6h at 8.5,100 DEG C.
8. a kind of application of the coding albumen of any thermostable esterases genes of claim 5-7, which is characterized in that the coding
Albumen is applied in medicine, food and field of fine chemical.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810323783.5A CN108546695A (en) | 2018-04-12 | 2018-04-12 | A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application |
| CN201811488472.0A CN109554382B (en) | 2018-04-12 | 2018-12-06 | Heat-resistant esterase gene, engineering bacterium containing gene, encoding protein and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810323783.5A CN108546695A (en) | 2018-04-12 | 2018-04-12 | A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108546695A true CN108546695A (en) | 2018-09-18 |
Family
ID=63514579
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810323783.5A Pending CN108546695A (en) | 2018-04-12 | 2018-04-12 | A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application |
| CN201811488472.0A Active CN109554382B (en) | 2018-04-12 | 2018-12-06 | Heat-resistant esterase gene, engineering bacterium containing gene, encoding protein and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811488472.0A Active CN109554382B (en) | 2018-04-12 | 2018-12-06 | Heat-resistant esterase gene, engineering bacterium containing gene, encoding protein and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN108546695A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762832A (en) * | 2019-02-25 | 2019-05-17 | 安徽师范大学 | Carboxylesterase gene, recombinant plasmid, recombination engineering and coding albumen and application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621637A (en) * | 2021-08-16 | 2021-11-09 | 广西科学院 | Escherichia coli solubilizing expression plasmid and construction method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238811A1 (en) * | 2002-09-09 | 2009-09-24 | Mcdaniel C Steven | Enzymatic Antimicrobial and Antifouling Coatings and Polymeric Materials |
| CN101603037B (en) * | 2009-07-23 | 2011-02-02 | 安徽师范大学 | Thermostable carboxylesterase gene, coding protein and application thereof |
| CN102286441B (en) * | 2011-07-24 | 2012-12-12 | 国家***第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| MY184387A (en) * | 2013-03-13 | 2021-04-01 | Autodisplay Biotech Gmbh | Improved surface display of functional proteins in a broad range of gram negative bacteria |
-
2018
- 2018-04-12 CN CN201810323783.5A patent/CN108546695A/en active Pending
- 2018-12-06 CN CN201811488472.0A patent/CN109554382B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762832A (en) * | 2019-02-25 | 2019-05-17 | 安徽师范大学 | Carboxylesterase gene, recombinant plasmid, recombination engineering and coding albumen and application |
| CN109762832B (en) * | 2019-02-25 | 2022-11-29 | 夏盛（上海）生物科技有限公司 | Carboxylesterase gene, recombinant plasmid, recombinant engineering bacterium, encoding protein and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109554382B (en) | 2022-09-06 |
| CN109554382A (en) | 2019-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Coleman et al. | Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase | |
| CN109825484B (en) | Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using mutant | |
| Ogino et al. | Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability | |
| CN101603037B (en) | Thermostable carboxylesterase gene, coding protein and application thereof | |
| CN108546695A (en) | A kind of thermostable esterases gene, the engineering bacteria containing the gene and its coding albumen and application | |
| Zhang et al. | Purification, identification and characterization of an esterase with high enantioselectivity to (S)-ethyl indoline-2-carboxylate | |
| CN108285895A (en) | A kind of esterase EstC11 and its encoding gene and application | |
| CN109762832B (en) | Carboxylesterase gene, recombinant plasmid, recombinant engineering bacterium, encoding protein and application | |
| CN104109659A (en) | Carboxylesterase as well as coding gene and application thereof | |
| CN109929822B (en) | Aspergillus oryzae lipase mutant and application thereof | |
| CN104673809A (en) | Malate dehydrogenase gene and recombinant expression vector thereof | |
| CN103952385A (en) | Thermally stable lipase from marine actinomycetes and application thereof | |
| Lovell et al. | Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase | |
| CN116376871A (en) | Thermostable esterase EstF, mutant and application thereof | |
| CN103215238A (en) | Marine bacterial novel esterase, as well as preparation method and application thereof | |
| CN112430610B (en) | Low-temperature esterase functional gene DcaE and application thereof | |
| CN103131659A (en) | Organic solvent-resistant lipase, encoding gene thereof, producing strain and application | |
| CN110438176A (en) | With the gene estDR4 of esterase function and its application | |
| CN105176943B (en) | The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant and application | |
| CN108410843A (en) | A kind of new Pullulanase and its encoding gene and application | |
| CN106119224B (en) | Esterase EstP00714 and coding gene and application thereof | |
| CN105368802B (en) | A kind of salt tolerant esterase and its encoding gene and application | |
| CN109554383B (en) | Heat-resistant esterase encoding gene, vector, engineering bacterium and application of encoding protein thereof | |
| Wang et al. | Molecular cloning and characterization of a novel SGNH arylesterase from the goat rumen contents | |
| CN101457217B (en) | Thermophilic lipase with alpha-phenyl ethyl alcohol disassemble activity, coding gene and use 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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180918 |