CN105543192A - Esterase BSE01701 and encoding gene and application thereof - Google Patents
Esterase BSE01701 and encoding gene and application thereof Download PDFInfo
- Publication number
- CN105543192A CN105543192A CN201610101577.0A CN201610101577A CN105543192A CN 105543192 A CN105543192 A CN 105543192A CN 201610101577 A CN201610101577 A CN 201610101577A CN 105543192 A CN105543192 A CN 105543192A
- Authority
- CN
- China
- Prior art keywords
- esterase
- bse01701
- gene
- enzyme
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000371 Esterases Proteins 0.000 title claims abstract description 54
- 108090000623 proteins and genes Proteins 0.000 title abstract description 13
- LPEKGGXMPWTOCB-UHFFFAOYSA-N methyl 2-hydroxypropionate Chemical compound COC(=O)C(C)O LPEKGGXMPWTOCB-UHFFFAOYSA-N 0.000 claims abstract description 14
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 108010088545 esterase B Proteins 0.000 claims description 76
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 19
- LPEKGGXMPWTOCB-GSVOUGTGSA-N methyl (R)-lactate Chemical class COC(=O)[C@@H](C)O LPEKGGXMPWTOCB-GSVOUGTGSA-N 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 12
- -1 TritonX-100 Substances 0.000 claims description 11
- 238000006555 catalytic reaction Methods 0.000 claims description 10
- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
- 230000007062 hydrolysis Effects 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 238000005194 fractionation Methods 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- 230000032050 esterification Effects 0.000 claims description 4
- 238000005886 esterification reaction Methods 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 238000005809 transesterification reaction Methods 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 34
- 239000003960 organic solvent Substances 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 7
- 230000003287 optical effect Effects 0.000 abstract description 6
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 239000003054 catalyst Substances 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000004094 surface-active agent Substances 0.000 abstract description 2
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 abstract 1
- 239000002537 cosmetic Substances 0.000 abstract 1
- 238000010931 ester hydrolysis Methods 0.000 abstract 1
- 239000012847 fine chemical Substances 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 48
- 102000004190 Enzymes Human genes 0.000 description 48
- 230000000694 effects Effects 0.000 description 27
- 238000000034 method Methods 0.000 description 22
- 239000000758 substrate Substances 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 230000009471 action Effects 0.000 description 11
- 230000029087 digestion Effects 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- OBNCKNCVKJNDBV-UHFFFAOYSA-N ethyl butyrate Chemical compound CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 description 9
- 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 8
- 239000013543 active substance Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 229910021645 metal ion Inorganic materials 0.000 description 7
- 229940057867 methyl lactate Drugs 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 6
- 238000013016 damping Methods 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000000452 restraining effect Effects 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 150000004702 methyl esters Chemical class 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 108091008146 restriction endonucleases Proteins 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
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- ODQWQRRAPPTVAG-GZTJUZNOSA-N doxepin Chemical compound C1OC2=CC=CC=C2C(=C/CCN(C)C)/C2=CC=CC=C21 ODQWQRRAPPTVAG-GZTJUZNOSA-N 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 229960000448 lactic acid Drugs 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- AQIHDXGKQHFBNW-ZCFIWIBFSA-N (2r)-2-(4-hydroxyphenoxy)propanoic acid Chemical compound OC(=O)[C@@H](C)OC1=CC=C(O)C=C1 AQIHDXGKQHFBNW-ZCFIWIBFSA-N 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000019832 sodium triphosphate Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 101150033426 2.5 gene Proteins 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 229930182843 D-Lactic acid Natural products 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710194948 Protein phosphatase PhpP Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000012069 chiral reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940022769 d- lactic acid Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960001680 ibuprofen Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- HWGNBUXHKFFFIH-UHFFFAOYSA-I pentasodium;[oxido(phosphonatooxy)phosphoryl] phosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O HWGNBUXHKFFFIH-UHFFFAOYSA-I 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001432 poly(L-lactide) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 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
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 239000006273 synthetic pesticide Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 238000009941 weaving 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01001—Carboxylesterase (3.1.1.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an esterase BSE01701 and an encoding gene and application thereof. A novel esterase gene bse 01701 is obtained from development from Bacillus sp. SCSIO15121, the nucleotide sequence of the esterase gene bse 01701 is shown in SEQ ID No.1, the overall length is 768bp, the nucleotide sequence of the esterase BSE01701 encoded by the esterase gene is shown in SEQ ID No.2, and the esterase contains 255 amino acids. The esterase BSE01701 is used as a catalyst and can catalyze an ester hydrolysis reaction at room temperature, DL-methyl lactate can be split selectively, the optical purity of the obtained DL-methyl lactate can exceed 99%, and the yield can reach 61.79%. The esterase has the good stability, has the good tolerance for some surface active agents and organic solvents, and can be used for the fields of washing agents, biological medicines, cosmetics, fine chemicals and the like.
Description
Technical field
The invention belongs to biochemical industry and biological technical field, be specifically related to a kind of esterase B SE01701 and encoding gene thereof and application.
Background technology
Esterase (EC3.1.1.1) is extensively present in animal, plant and microorganism, is a class catalytic hydrolysis or forms the enzyme of ester bond, and the substrate of effect normally aliphatic chain is less than the ester class of ten carbon atoms.Esterase belongs to α/β and folds lytic enzyme superfamily, and catalytic center is made up of Serine, aspartic acid/L-glutamic acid and Histidine, and conserved sequence is pentapeptide (GXSXG) sequence near Serine.Esterase energy catalytic hydrolysis, esterification, the number of chemical such as transesterification reaction, be a kind of very important industrial biocatalytic agent, be widely used in fields such as fine chemistry industry, washing, medicine, food, papermaking, leather processing, weaving, wastewater treatment and fodder industries.From catalysis characteristics, esterase has chemo-selective and the stereoisomerism selectivity of height, and reaction does not need, and coenzyme, reaction conditions are gentle, by product is few.Another distinguishing feature of esterase in production application is that it can act in outphasing system (i.e. oil-water interface) or organic phase.In aqueous phase, the usual catalytic hydrolysis reaction of esterase, and in organic phase, it but can catalytic esterification and transesterification.Prepare newtype drug intermediate by the bio-transformation of microorganism esterase or remove the non-active ingredients of medicine raceme, it is a kind of important chiral technology, there is boundless application prospect, it can provide new platform for synthesis of chiral medicine, for preparing the method that optical pure compound provides new in a large number.Enzyme process selectivity resolving racemic compound, have stereospecificity high, side reaction is few, and productive rate is high, the advantage that product optical purity is good and reaction conditions is gentle, so be a kind of by the method for splitting extensively approved.
The starting monomer of high optically pure lactic acid normally biodegradable polylactic acid and polymer product.Poly (l-lactic acid) and poly-D-ALPHA-Hydroxypropionic acid have higher fusing point, thus improve the performance of plastics.Optically pure lactic acid and its ester roughly the same time be also the important chiral precurser reagent of pharmaceutical industry.Pfansteihl and ester class are the precursor chiral reagents of synthesis non-steroidal anti-inflammatory analgesics Ibuprofen BP/EP.D-ALPHA-Hydroxypropionic acid is usually used in asymmetric synthesis D-amino acid.Higher plant lacks the relevant enzymes utilizing D-ALPHA-Hydroxypropionic acid, can not synthesize thus or decomposing D-lactic acid ester, therefore conventional D-ALPHA-Hydroxypropionic acid ester class synthetic pesticide.D-ALPHA-Hydroxypropionic acid methyl esters is the precursor substance of (R)-2-(4-hydroxyphenoxy) propionic acid, and (R)-2-(4-hydroxyphenoxy) propionic acid is the important intermediate of synthesis aryloxyphenoxypropanoates class weedicide, market has openings is large.D-ALPHA-Hydroxypropionic acid methyl esters can be hydrolyzed further prepares optical purity D-ALPHA-Hydroxypropionic acid.But industrial production D-ALPHA-Hydroxypropionic acid is more difficult than Pfansteihl, most of optically pure lactic acid product is mainly raw material with Pfansteihl.Therefore, need to develop a kind of technique and more simply can be used for the industrial novel method preparing optical purity D-ALPHA-Hydroxypropionic acid methyl esters.
Summary of the invention
The object of this invention is to provide a kind of new esterase B SE01701 and encoding gene thereof and application.
The present invention obtains a kind of esterase B SE01701 and encoding gene esterase gene bse01701 thereof from a strain from exploitation genus bacillus (Bacillussp.) SCSIO15121 of ocean, construct the recombinant expression vector containing esterase gene bse01701 and genetic engineering bacterium, esterase B SE01701 is obtained after culturing gene engineering bacteria, it can be applicable to catalysis ester reaction, and can be applicable to prepare D-ALPHA-Hydroxypropionic acid methyl esters.
First object of the present invention is to provide a kind of esterase B SE01701, and its aminoacid sequence is as shown in SEQIDNO.2.
Second object of the present invention is to provide the esterase gene bse01701 of a kind of described esterase B SE01701 that encodes.
Preferably, the nucleotide sequence of described esterase gene bse01701 is as shown in SEQIDNO.1.
The present invention also provides a kind of recombinant expression vector containing described esterase gene bse01701.Described expression vector, is preferably pET-28a (+) carrier.
The present invention also provides a kind of genetic engineering bacterium containing described esterase gene bse01701.Described genetic engineering bacterium, is preferably e. coli bl21 (DE3).
3rd object of the present invention is to provide the application of described esterase B SE01701 in catalysis ester-type hydrolysis, esterification or transesterification.
Preferably, described application is esterase B SE01701 is obtained by reacting in D-ALPHA-Hydroxypropionic acid methyl esters application at catalysis fractionation DL-LACTIC ACID methyl esters.
4th object of the present invention is to provide described esterase B SE01701 at tolerance Ca
2+, Mn
2+, Tween-20, Tween-80, TritonX-100, TPP, carry out the application of catalysis under normal hexane and/or normal heptane environment.
(its source is genus bacillus (Bacillussp.) SCSIO15121 that esterase gene bse01701 of the present invention originates from deep-sea: 89 ° of 29.22 ' E, 10 ° of 00.12 ' N ,-3400m, pH7.8,2 DEG C), be kept at Chinese Academy of Science Nanhai Ocean Research Institute.The present invention utilizes bioinformatic analysis method, from genus bacillus (Bacillussp.) SCSIO15121 of gene order-checking, clone obtains esterase gene bse01701, total length is 768bp (from initiator codon to terminator codon), and the esterase B SE01701 of its coding contains 255 amino acid.Transformation of E. coli BL21 (DE3) after being connected with expression vector pET-28a (+) by esterase gene bse01701, cultivates and after abduction delivering, obtains Recombinant esterase BSE01701.Using the esterase B SE01701 of purifying as the reaction of catalyst ester-type hydrolysis, there is good stability, and to part surface promoting agent and organic solvent, there is good tolerance, and to fractionation, there is good promoter action.Esterase B SE01701 can react by catalysis ester-type hydrolysis as under catalyst normal temperature, and can optionally split DL-LACTIC ACID methyl esters, and the optical purity of the D-ALPHA-Hydroxypropionic acid methyl esters prepared can more than 99%, and productive rate can reach 61.79%.The demand that the characteristic conforms detergent additives of esterase B SE01701, greenization are industrial, can be used for the fields such as washing composition, biological medicine, makeup and fine chemistry industry.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of esterase B SE01701.Wherein, M is albumen marker, swimming lane 1 is e. coli bl21 (DE3) crude protein containing pET-28 α (+)-bse01701 before IPTG induction, swimming lane 2 and 3 is e. coli bl21 (DE3) crude protein containing pET-28 α (+)-bse01701 after IPTG induction, and swimming lane 4 is the esterase B SE01701 albumen of purifying.
Fig. 2 is the specificity of esterase B SE01701 to the p-nitrophenyl phenolic ester of different lengths acyl side-chain.
Fig. 3 is the impact of pH on esterase B SE01701 activity.Wherein, PBS is Na
2hPO
4/ NaH
2pO
4damping fluid.
Fig. 4 is the stability influence of different pH to esterase B SE01701.
Fig. 5 is the impact of temperature on esterase B SE01701 activity.
Fig. 6 is the impact of differing temps on esterase B SE01701 stability.
Fig. 7 is the impact of metal ion on esterase B SE01701 activity
Fig. 8 is the impact of tensio-active agent on esterase B SE01701 activity
Fig. 9 is the impact of organic solvent on esterase B SE01701 activity
Figure 10 is that D-ALPHA-Hydroxypropionic acid methyl esters and Pfansteihl methyl esters standard specimen GC scheme.
Figure 11 is that BSE01701 splits DL-LACTIC ACID methyl esters and prepares D-ALPHA-Hydroxypropionic acid methyl esters GC and scheme.
Embodiment
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: esterase gene bse01701 open reading frame border is determined and design of primers
Extract the genomic dna of genus bacillus (Bacillussp.) SCSIO15121, after genome sequencing, information biology means are utilized to carry out gene annotation to genome, analyze esterase gene wherein, determine the open reading frame of wherein esterase gene bse01701, its gene order is as shown in SEQIDNO.1, total length is 768bp (from initiator codon to terminator codon), the aminoacid sequence of esterase B SE01701 of its coding as shown in SEQIDNO.2, totally 255 amino acid.According to analyzing the esterase gene bse01701 sequence obtained, design total length amplimer is as follows: upstream primer: 5 '-TGCTAGC
cATATGaTACAAATTGAGAATCAAGC-3 ' (underscore is NdeI restriction enzyme site); Downstream primer: 5 '-AGG
aAGCTTtTATAAGTACGTTTCAAACCATT-3 ' (underscore is HindIII restriction enzyme site).
Embodiment 2: the clone of esterase gene bse01701 and vector construction
2.1PCR amplification
Primer (the upstream primer: 5 '-TGCTAGC that embodiment 1 is designed
cATATGaTACAAATTGAGAATCAAGC-3 '; Downstream primer: 5 '-AGG
aAGCTTtTATAAGTACGTTTCAAACCATT-3 ') deliver to the synthesis of Shanghai biotechnology company limited, the primer of synthesis uses TE buffer solution to become final concentration to be 10 μMs, using genus bacillus (Bacillussp.) the SCSIO15121 STb gene extracted as DNA profiling, set up reaction system as shown in table 1:
Table 1PCR reaction system
Use following pcr amplification program amplification esterase gene bse01701:94 DEG C of sex change 5min; 94 DEG C of sex change 1min, 62 DEG C of annealing 30s, 72 DEG C extend 1.5min, carry out 30 circulations; 72 DEG C extend 10min, are cooled to 18 DEG C.
By PCR primer in 0.8% sepharose, electrophoresis 20min under 120V voltage, is placed in gel imaging system and observes, and reclaims the band of about 750bp.The method that PCR primer reclaims test kit according to glue reclaims, and uses 20 μ L sterilized water wash-outs, obtains the PCR primer that purifying reclaims.
2.2 enzymes are cut
PCR primer uses following system to carry out enzyme and cuts, and enzyme cuts time 1.5h.The enzyme system of cutting is: NdeI1 μ L, HindIII1 μ L, DNA<0.3 μ g, and the distilled water of sterilizing adds to 50 μ L.Enzyme reclaims according to the method for glue recovery test kit after cutting, and obtains the PCR primer after double digestion.
The double digestion of plasmid pET-28a (+): picking contains the bacillus coli DH 5 alpha list bacterium colony of this plasmid, incubated overnight.Use plasmid extraction kit to extract plasmid, with NdeI and HindIII by following system double digestion, enzyme cuts time 1.5h.The enzyme system of cutting is: NdeI1 μ L, HindIII1 μ L, DNA<0.3 μ g, and the distilled water of sterilizing adds to 50 μ L.Enzyme cut after in 0.8% sepharose electrophoresis, according to glue reclaim kit method reclaim linear pET-28a (+) carrier obtained through double digestion.
The quick restriction endonuclease that the restriction enzyme that above-mentioned double digestion uses is produced for Thermo company, enzyme cut after purifying reclaim and use nucleic acid purification to reclaim test kit (Magen, HipureGelPureDNAMicroKit), plasmid extraction kit is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and working method is by its working instructions.
2.3 connect
Be connected through the PCR primer of double digestion by following system with linear pET-28a (+) carrier of double digestion: double digestion PCR primer 5 μ L, linear pET-28a (+) the carrier 0.5 μ L of double digestion, T4 ligase enzyme (5U/ μ L) 0.5 μ L, connect damping fluid (5 ×) 2 μ L, supply 10 μ L with deionized water; Connecting temperature is 25 DEG C, 20min.Obtain thus connecting product.
2.4 transform and screening
Get 10 μ L connect products add in 50 μ L bacillus coli DH 5 alpha competent cells, ice bath 20 ~ 30min, after in 42 DEG C of water-bath heat shock 90s, add 500 μ LLB liquid nutrient mediums after ice bath 2min, under 37 DEG C of 200rpm rotating speeds, hatch cultivate 30min.Get the LB that a certain amount of bacterium liquid coats containing 50 μ g/mL kantlex dull and stereotyped, cultivate picking individual colonies after 20h.Single bacterium colony extracts plasmid after incubated overnight in 5mLLB substratum, and carry out double digestion checking, what endonuclease bamhi was identical with gene size is positive colony.
2.5 gene nucleotide series measure
The positive colony of screening is delivered to Shanghai Mei Ji biological medicine company limited to check order, sequencing result and esterase gene sequence are compared, be confirmed to be further and esterase gene bse01701 (its nucleotide sequence is as shown in SEQIDNO.1) is inserted in pET-28a (+) plasmid, the entirely true rear confirmation of result obtains pET-28a (+) plasmid (called after pET-28a (+)-bse01701) with esterase gene bse01701, can be used for carrying out next step test.
The high expression of embodiment 3: esterase B SE01701 in e. coli bl21 (DE3)
Prepared by 3.1 e. coli bl21s (DE3) competent cell
1. by a small amount of e. coli bl21 (DE3) bacterial classification access 5mLLB test tube liquid, 200rpm, 37 DEG C of incubated overnight;
2. be inoculated in 200mLLB shaking flask by the inoculum size of 1% volume ratio by the bacterium liquid in test tube, 200rpm, 25 DEG C of incubated overnight, obtain stock culture;
3. cultured shaking flask is cooled to rapidly 0 DEG C in frozen water, stock culture is divided the centrifuge tube being filled to ice precooling (50mL), ice puts several minutes;
4.4 DEG C, the centrifugal 15min of 4000rpm reclaims cell, abandons supernatant;
5. the CaCl of ice-cold 10mL0.1M
2re-suspended cell, 4 DEG C, the centrifugal 10 ~ 15min of 4000rpm reclaims cell;
6. repeat 5, with the CaCl of 10mL0.1M
2re-suspended cell, more than ice bath 1h;
7.4 DEG C, the centrifugal 10min of 4000rpm reclaims cell;
8. cell 2 ~ 3mL that every 50mL stock culture obtains contains 15%DMSO+CaCl
2come resuspended, be sub-packed in 1.5mL centrifuge tube, 50 ~ 100 μ L often manage.-80 DEG C of preservations.Obtain e. coli bl21 (DE3) competent cell thus.
3.2 transform
PET-28a (+)-bse01701 plasmid 0.5 ~ 1 μ L obtained in Example 2 mixes with 50 μ L e. coli bl21 (DE3) competent cells, ice bath 30min, in 42 DEG C of water-bath heat shock 90s, add 500 μ LLB liquid nutrient mediums after ice bath 2min, 37 DEG C of 200rpm cultivate 1h.The kantlex LB of the centrifugal rear coating 50 μ g/mL of culture is dull and stereotyped, selects single bacterium after overnight incubation 20h.Obtain the e. coli bl21 (DE3) containing pET-28 α (+)-bse01701 thus.
The expression and purification of embodiment 4: esterase B SE01701
4.1 protein induced
E. coli bl21 (DE3) containing pET-28a (+)-bse01701 is cultured to OD in LB substratum
600be about 0.85, add IPTG to final concentration 0.2mM, cultivate 18h for 22 DEG C.300mL bacterium liquid 4000rpm, 4 DEG C of centrifugal 20min, collect thalline, and with the resuspended thalline of 30mL (50mM, pH7.4) PBS damping fluid, ultrasonic 400w, super 5s, stops 5s, broken 10min minute, centrifugal, collects supernatant.
The purifying of 4.2 esterase B SE01701 and SDS-PAGE electrophoresis
With the supernatant collected in nickel ion affinity chromatograph column purification 4.1, specific embodiments is as follows: imidazoles wash-out 5 column volumes using 20mM, 40mM imidazoles wash-out 20 ~ 30 column volumes, finally use 3.5mL300mM imidazoles wash-out, collect last 2.5mL elutriant.Carry out desalination with desalting column SephadexG25, concrete operation method carries out with reference to the operational manual of GE company.The expression product of purifying is carried out SDS-PAGE gel electrophoresis, and obtain the esterase B SE01701 (Fig. 1) of purifying, the albumen size of purifying is about 30.5kD, and coincidence theory is expected.
4.3 esterase B SE01701 determinations of activity
Esterase B SE01701 vitality test adopts p-nitrophenyl phenolic ester, and concrete grammar is as follows: the p-NP ester solution 1. preparing 200mM with DMSO; 2. in 0.5mL reaction system, add 495 μ LTris-HClbuffer (100mM, pH8.5), 2 μ L p-NP ester solutions, 3 μ L dilute the pure enzyme liquid of esterase B SE01701 (final concentration 0.0428 μ g/ μ L) of 50 times; 3., at 35 DEG C, after reaction 2min, add 0.5mL acetonitrile termination reaction, measure absorbancy in 405nm.
Enzyme is lived, and unit definition: 1min is interior is hydrolyzed p-nitrophenyl phenolic ester, and the enzyme amount discharged needed for 1 μM of p-NP is defined as a Ge Meihuo unit.
The zymologic property of embodiment 5: esterase B SE01701
The p-nitrophenyl phenolic ester of the different side chain lengths of 5.1 hydrolysis
According to the condition determination of 4.3, compare the p-nitrophenyl phenolic ester C that esterase B SE01701 is hydrolyzed different lengths acyl group
2-C
10, result is as Fig. 2.Illustrate that esterase B SE01701 is to long-chain p-nitrophenyl phenolic ester poor specificity, and better for the action effect of the p-nitrophenyl phenolic ester of short chain, and best substrate is C
4, i.e. p-NP butyric ester.
5.2 optimal pHs and pH stability
Prepare different buffered soln, these buffered soln have different pH, as shown in table 2, and its concentration is 100mM.
The buffer system of the different pH of table 2
Replaced respectively according to the buffered soln in table 2 by damping fluid (Tris-HCl damping fluid) in 4.3 described in condition determination, measure the enzyme activity of Recombinant esterase BSE01701 in the buffered soln of different pH, substrate is p-NP butyric ester.The impact of pH on restructuring esterase B SE01701 activity the results are shown in Figure 3.Time in the Tris-HCl buffered soln of pH8.5, the enzymic activity of esterase B SE01701 is the highest, has higher enzymic activity in the buffered soln between pH7.5-9.0.The stability of esterase B SE01701 in the buffered soln of 4 DEG C of different pH is shown in Fig. 4.When pH7.0-8.0, enzymic activity is more stable, and after process 12h, residual activity still keeps more than 65%.Although enzyme is lived the highest when pH8.5, after process 12h, loss of activity is then comparatively obvious, higher than 9.0 time, along with time lengthening, enzyme is unstable.
5.3 optimum temperutures and temperature stability
Use the Tris-HClpH8.5 of 100mM as damping fluid, p-NP butyric ester, as substrate, according to the reaction system in 4.3, measures enzyme at different temperatures and lives.The optimum temperuture recording esterase B SE01701 catalytic hydrolysis p-NP butyric ester is 35 DEG C (Fig. 5).Enzyme activity between 20-50 DEG C reaches more than 60%, and when temperature is greater than 50 DEG C, enzymic activity sharply declines.Processed at different temperatures by esterase B SE01701, separated in time taking-up is pressed measuring method survey enzyme in 4.3 and is lived, and the results are shown in Figure 6.Esterase B SE01701 is when lower than 4 DEG C, and enzyme is lived and kept higher, and after process 12h, remnant enzyme activity still keeps more than 50%; Along with the rising for the treatment of temp, esterase residual enzyme activity declines gradually, and when temperature sharply reduces higher than enzyme when 45 DEG C is alive, after 55 DEG C of process 1h, residual enzyme activity is lost substantially (Fig. 6), illustrates that esterase B SE01701 stability when low temperature is better.
5.4 esterase B SE01701 are hydrolyzed p-nitrophenyl phenolic ester kinetic parameter
Under optimum temperuture 35 DEG C and optimal pH 8.5 condition, change concentration of substrate with different p-nitrophenyl phenolic ester for substrate, the method according to 4.3 measures the change of the speed of reaction of enzyme, adopt Lineweaver-Burk double-reciprocal plot, obtain Km and Vmax of reaction.Measurement result is in table 3.
Table 3 esterase B SE01701 is hydrolyzed the kinetic parameter of p-nitrophenyl phenolic ester
Can be found out by the result in table 3, the Km value that esterase B SE01701 is hydrolyzed p-NP butyric ester is minimum, and Kcat is maximum, illustrate to the avidity of butyric ester and hydrolysis efficiency the highest.
5.5 metal ions, tensio-active agent are on the impact of esterase B SE01701 activity
According to the metal ion in Fig. 7 and the kinds of surfactants in Fig. 8 and corresponding final concentration, metal ion or tensio-active agent process esterase B SE01701 is added in reaction system Tris-HCl (pH8.5) buffered soln, reaction conditions is 35 DEG C, hatch 1h, live in contrast (100%) with the enzyme not adding any ion or tensio-active agent, then it is alive to measure relative enzyme by 4.3 measuring methods (using p-NP butyric ester as substrate).Fig. 7 is shown in the impact of metal ion on esterase B SE01701 enzymic activity.Compared with the control, under the Action of Metal Ions of 1mM concentration, Ca
2+, Mn
2+have activation to the vigor of esterase B SE01701 catalysis p-NP butyric ester, relative enzyme is lived and is respectively 117.05% and 115.13%; Mg
2+, K
+, Fe
2+live without impact on esterase B SE01701 enzyme, Cu
2+there is weak restraining effect, Ni
2+, Zn
2+there is strong restraining effect.Under the Action of Metal Ions of 5mM concentration, Ca
2+still there is promoter action, but promote that intensity is obvious not as 1mM concentration; Other ions enzymes have restraining effect.Fig. 8 is shown in the impact of tensio-active agent on esterase B SE01701 enzymic activity.Under 0.05% concentration, the activation that Tween-20, Tween-80, TritonX-100 and TPP live to enzyme is obvious, and relative enzyme is lived and is respectively 119.53%, 147.72,145.96 and 123.84%; SDS and SDBS has strong restraining effect.Under 0.1% concentration, Tween-80 increases along with concentration the promoter action that enzyme is lived and strengthens.Compared with lower concentration, the promoter action change of Tween-20, TritonX-100 and TPP of 0.1% concentration is little.
5.6 organic solvents are on the impact of esterase B SE01701 activity
According to the kind of organic solvent shown in Fig. 9 and final concentration (v/v), organic solvent process esterase B SE01701 enzyme liquid is added in reaction system Tris-HCl (pH8.5) buffered soln, reaction conditions is 35 DEG C, be placed in shaking table and hatch 1h, not add the esterase B SE01701 activity of organic solvent process in contrast (100%), measure the work of relative enzyme according to the measuring method (using p-NP butyric ester as substrate) of 4.3 again, result as shown in Figure 9.Under the concentration of 5%, normal hexane has activation to esterase B SE01701; Normal heptane, methyl alcohol, n-propyl alcohol, dimethyl sulfoxide (DMSO) and acetone are lived to enzyme and are had no significant effect; And n-decane, n-Octanol, tetrahydrofuran (THF), toluene, dimethylbenzene, Isosorbide-5-Nitrae dioxane and methylene dichloride have restraining effect to esterase B SE01701, and more strengthen along with concentration increases restraining effect; Butanic acid and the complete inhibitory enzyme of n-caproic acid are lived.Under 10% concentration, normal hexane and normal heptane strengthen the promoter action that enzyme is lived, and relative enzyme is lived and is respectively 122.33% and 110.39%, and dimethyl sulfoxide (DMSO) is lived on enzyme does not have impact, and other solvents make enzymic activity reduce.
Embodiment 6: esterase B SE01701 prepares D-ALPHA-Hydroxypropionic acid methyl esters
6.1pH and temperature of reaction prepare the impact of D-ALPHA-Hydroxypropionic acid methyl esters to esterase B SE01701
In the reaction system of the different pH (PBS, Tris-HCl, Glycine-NaOH) of 500 μ L, add 25 μ L (2.14mg/mL) esterase B SE01701,50mMDL-methyl lactates, be placed in 37 DEG C, 200rpm shaking table reaction 40min.Add isopyknic extraction into ethyl acetate twice subsequently, adding final concentration is mark in 20mM2-octanol is done, and detects, according to calculated by peak area substrate enantiomer excessive value (ee with gas chromatography chiral post
s), transformation efficiency (C) and enantioselectivity (E), formula is as follows.
Formula 1:
formula 3:
formula 3:
In formula: A
dand A
lrepresent the peak area of D-ALPHA-Hydroxypropionic acid methyl esters and Pfansteihl methyl esters respectively, A
0with the peak area reacting rear methyl lactate before representing reaction respectively with A.
As can be seen from Table 4, substrate enantiomer excessive value (ee
s) raise along with pH and raise rear reduction, reach maximum value (about 81%) at about pH8.8, between pH7.0 – 9.5, ee
smaintain more than 60%, pH higher than 9.5 time, ee
srapid decline.Between pH7.0 – 9.0, transformation efficiency is comparatively steady, and during more than 9.5, transformation efficiency rises.Enantioselectivity also has the rear downward trend of first rising.Illustrate that high pH affects the selectivity of enzyme.Therefore, optimum fractionation pH is about 8.8.
25 μ L (2.14mg/mL) esterase B SE01701 are added in 500 μ LpH8.8Tris-HCl reaction systems, 50mMDL-methyl lactate, is placed in 20-45 DEG C, 200rpm shaking table reaction 40min, measure the stereoselectivity that esterase B SE01701 splits DL-LACTIC ACID methyl esters, the results are shown in Table 4.Ee
sreduce afterwards along with temperature raises first to increase with C, all reach maximum at 35 DEG C, be respectively 75.16%, 59.32%, during higher than 45 DEG C, both all decline, may be active low relevant at 45 DEG C with enzyme.E value is then along with temperature slowly increases.Pay the utmost attention to ee
s, therefore optimum fractionation temperature is 35 DEG C.
Table 4pH and temperature of reaction optionally affect esterase B SE01701
D-ALPHA-Hydroxypropionic acid methyl esters and Pfansteihl methyl esters standard specimen GC scheme as shown in Figure 10, Figure 11 is the GC figure that esterase B SE01701 splits DL-LACTIC ACID methyl esters under Tris-HCl (pH8.8), 35 DEG C of conditions, under the katalysis of esterase B SE01701, the methyl lactate overwhelming majority be split as D-ALPHA-Hydroxypropionic acid methyl esters, few part is Pfansteihl methyl esters.
6.2 organic solvents prepare the impact of D-ALPHA-Hydroxypropionic acid methyl esters to esterase B SE01701
The pure enzyme of 15 μ L (2.14mg/mL) esterase B SE01701 is added in reaction system Tris-HCl (pH8.8) buffered soln, 50mMDL-methyl lactate, organic solvent in the table 5 of 5% (v/v) volume, not add organic solvent in contrast, be placed in 35 DEG C, 200rpm shaking table reaction 30min.Add isopyknic extraction into ethyl acetate twice subsequently, adding final concentration is mark in 20mM2-octanol is done, and detects with gas chromatography chiral post.Result is as shown in table 5.
Normal hexane, normal heptane and n-decane can improve the stereoselectivity of esterase B SE01701 as can be seen from Table 5, and wherein the promoter action of normal heptane is the strongest, ee
sreach 91.88%; The selectivity of DMSO on enzyme does not affect; N-propyl alcohol and acetone reduce the selectivity of enzyme on the contrary.
Table 5 organic solvent optionally affects esterase
Subsequently, take normal heptane as solvent, the normal heptane detecting different concns optionally affects esterase.As can be seen from Table 6, substrate enantiomer excessive value ee
salong with the increase of concentration first raises rear reduction, when concentration is 60%, the fractionation DL-LACTIC ACID methyl esters of esterase the highest, be 98.19%, stereoselectivity is also maximum, is 19.25.By these results suggest that esterase is comparatively strong to the normal heptane tolerance of high density, and the activation that the normal heptane of 60% concentration splits DL-LACTIC ACID methyl esters to esterase is the highest.The normal heptane of therefore follow-up experiment employing 60% is solvent.
Table 6 normal heptane is to esterase optionally concentration optimization
6.3 tensio-active agents prepare the impact of D-ALPHA-Hydroxypropionic acid methyl esters to esterase B SE01701
The pure enzyme of 25 μ L (2.14mg/mL) esterase B SE01701 is added in reaction system Tris-HCl (pH8.8) buffered soln, 50mMDL-methyl lactate, the tensio-active agent (Tween-20, Tween-80, tripoly phosphate sodium STPP (TPP) TritonX-100) of 0.05% (w/v), be placed in 35 DEG C, 200rpm shaking table reaction 30min.Add isopyknic extraction into ethyl acetate twice subsequently, adding final concentration is mark in 20mM2-octanol is done, and sample is used for GC and detects, and the results are shown in Table 7.Can find out from result, compared with the control, Tween-20 and Tween-80 reduces the selectivity of esterase B SE01701, the ee of methyl lactate
sreduce, TritonX-100 and TPP promotes the selectivity of esterase, and wherein the promoter action of TritonX-100 is the strongest, ee
sbe 91.15%, higher than control group by 21%.
Table 7 tensio-active agent optionally affects esterase
Subsequently, add the TritonX-100 of different concns as promotor in reaction system, the normal heptane detecting different concns optionally affects esterase.Compared with control group, containing the system of TritonX-100, the selectivity of esterase is higher, but difference little (table 8) between different concns.
Table 8TritonX-100 is to esterase optionally concentration optimization
Whether DL-LACTIC ACID methyl esters is split to esterase to check the system of mixing normal heptane and TritonX-100 and have promoter action, then carry out following experiment.The suitableeest normal heptane concentration (60%, v/v), the reaction result of the suitableeest TritonX-100 concentration (0.05%, w/v) and mixing two kinds of reagent is in table 9.The normal heptane of 60% (v/v) is than the ee of other two groups experiments
sall high, selectivity E value is 19.54.Therefore follow-up experiment adopts the normal heptane of 60% (v/v) to be solvent.
Table 9 mixes normal heptane and TritonX-100 prepares the impact of D-ALPHA-Hydroxypropionic acid methyl esters to esterase
The kinetics of 6.4 concentration of substrate and time prepares D-ALPHA-Hydroxypropionic acid methyl esters
(35 DEG C with optimal conditions, pH8.8Tris-HCl), 15 μ L (2.14mg/mL) esterase B SE01701 are added in 500 μ L reaction systems, 25-100mMDL-methyl lactate, 60% (v/v) normal heptane is solvent, be placed in 200rpm shaking table successive reaction 40min, be used for GC at interval of 10min sampling and detect, the results are shown in Table 10.As can be seen from the table, when concentration of substrate is 25mM and 50mM, ee
sincreasing gradually along with the prolongation of time, reach maximum after 40min, is 99.99%.When concentration is 75mM, ee
sincrease very fast before 20min, after 20min, speed is slow.When concentration is 100mM, ee
sdo not extend in time and raise, but maintaining about 55%.Can find out thus, the suitableeest concentration of substrate is 50mM, and the reaction times is 40min.When the suitableeest concentration of substrate and reaction 40min, transformation efficiency is about 61.79%, and stereoselectivity E is 37.82.
Table 10 concentration of substrate and time prepare the impact of D-ALPHA-Hydroxypropionic acid methyl esters to esterase
Claims (10)
1. an esterase B SE01701, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.2.
2. the esterase gene bse01701 of a coding esterase B SE01701 according to claim 1.
3. esterase gene bse01701 according to claim 2, is characterized in that, its nucleotide sequence is as shown in SEQIDNO.1.
4. the recombinant expression vector containing esterase gene bse01701 according to claim 2.
5. recombinant expression vector according to claim 4, is characterized in that, described expression vector is pET-28a (+) carrier.
6. the genetic engineering bacterium containing esterase gene bse01701 according to claim 2.
7. genetic engineering bacterium according to claim 6, is characterized in that, described genetic engineering bacterium is e. coli bl21 (DE3).
8. the application of esterase B SE01701 according to claim 1 in catalysis ester-type hydrolysis, esterification or transesterification.
9. application according to claim 8, is characterized in that, described esterase B SE01701 is obtained by reacting the application in D-ALPHA-Hydroxypropionic acid methyl esters at catalysis fractionation DL-LACTIC ACID methyl esters.
10. esterase B SE01701 according to claim 1 is at tolerance Ca
2+, Mn
2+, Tween-20, Tween-80, TritonX-100, TPP, carry out the application of catalysis under normal hexane and/or normal heptane environment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610101577.0A CN105543192B (en) | 2016-02-24 | 2016-02-24 | A kind of esterase B SE01701 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610101577.0A CN105543192B (en) | 2016-02-24 | 2016-02-24 | A kind of esterase B SE01701 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105543192A true CN105543192A (en) | 2016-05-04 |
| CN105543192B CN105543192B (en) | 2019-03-01 |
Family
ID=55822750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610101577.0A Expired - Fee Related CN105543192B (en) | 2016-02-24 | 2016-02-24 | A kind of esterase B SE01701 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105543192B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802935A (en) * | 2016-05-05 | 2016-07-27 | 中国科学院南海海洋研究所 | Esterase PHE14 as well as encoding gene and application thereof |
| CN107475218A (en) * | 2017-09-19 | 2017-12-15 | 中国科学院南海海洋研究所 | A kind of esterase B ae02030 and its encoding gene and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544992A (en) * | 2008-03-26 | 2009-09-30 | 苏州普瑞信生物技术有限公司 | Method for preparing high optical purity D-lactic acid by fermentation |
| CN104962533A (en) * | 2015-06-30 | 2015-10-07 | 中国科学院南海海洋研究所 | Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate |
| CN105296513A (en) * | 2015-12-09 | 2016-02-03 | 山东大学 | Marine esterase as well as coding gene E22 and application thereof |
-
2016
- 2016-02-24 CN CN201610101577.0A patent/CN105543192B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544992A (en) * | 2008-03-26 | 2009-09-30 | 苏州普瑞信生物技术有限公司 | Method for preparing high optical purity D-lactic acid by fermentation |
| CN104962533A (en) * | 2015-06-30 | 2015-10-07 | 中国科学院南海海洋研究所 | Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate |
| CN105296513A (en) * | 2015-12-09 | 2016-02-03 | 山东大学 | Marine esterase as well as coding gene E22 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| DUNLAP C.A. ET AL: "Bacillus subtilis subsp.subtilis str.OH131.1,complete genome", 《GENBANK登录号：CP007409 REGION:1133907..1134674》 * |
| LIANG J ET AL: "Enantioselective resolution of (±)-1-phenylethanol and (±)-1-phenylethyl acetate by a novel esterase from bacillus sp.SCSIO 15121", 《APPL BIOCHEM BIOTECHNOL》 * |
| 周丽 等: "微生物发酵产光学纯度D-乳酸研究进展", 《中国生物工程杂志》 * |
| 胡永红 等: "发酵法生产D-乳酸的研究进展", 《食品与发酵工业》 * |
| 高强: "海洋芽孢杆菌酯酶BSE-1的分离纯化和生化性质研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑 B018-14》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802935A (en) * | 2016-05-05 | 2016-07-27 | 中国科学院南海海洋研究所 | Esterase PHE14 as well as encoding gene and application thereof |
| CN105802935B (en) * | 2016-05-05 | 2019-06-25 | 中国科学院南海海洋研究所 | A kind of esterase PHE14 and its encoding gene and application |
| CN107475218A (en) * | 2017-09-19 | 2017-12-15 | 中国科学院南海海洋研究所 | A kind of esterase B ae02030 and its encoding gene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105543192B (en) | 2019-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105543190A (en) | Esterase BSE00077 and encoding gene and application thereof | |
| CN102533705B (en) | Nitrilase and gene and application thereof | |
| CN105543191A (en) | Esterase PHE21 and encoding gene and application thereof | |
| CN103881992A (en) | Lipase mutant and uses thereof | |
| CA2883141C (en) | Process for preparing sebacic acid | |
| CN104673810A (en) | Malic dehydrogenase gene MIMDH1 and recombinant expression vector thereof | |
| CN105420211A (en) | Thermophilic esterase AFEST mutant and screening method and application thereof | |
| CN108285895A (en) | A kind of esterase EstC11 and its encoding gene and application | |
| CN106244569B (en) | Esterase EstC10, and coding gene and application thereof | |
| CN102206686B (en) | Preparation method of methyl (R)-o-chloromandelate utilizing biocatalytic asymmetric reduction | |
| CN105543192A (en) | Esterase BSE01701 and encoding gene and application thereof | |
| CN105112385A (en) | Recombinant esterase, coding gene, carrier, engineering bacterium and application | |
| CN104212850B (en) | The method that 1 cyanocyclohexanoic guanidine-acetic acid is prepared using nitrilase engineering bacteria | |
| CN106119224B (en) | Esterase EstP00714 and coding gene and application thereof | |
| CN105296513A (en) | Marine esterase as well as coding gene E22 and application thereof | |
| CN105238769B (en) | A kind of lipase LIPASE7 and its encoding gene and application | |
| CN102174422B (en) | Lipase producing strain tolerant to organic solvent as well as genes and applications of lipase | |
| CN114350630A (en) | L-pantolactone dehydrogenase, mutant and application thereof | |
| CN107475218A (en) | A kind of esterase B ae02030 and its encoding gene and application | |
| CN103013949B (en) | Acetylation hydroxy acid hydrolase, gene and application thereof | |
| CN105132394A (en) | LIPASE 6 as well as encoding gene and application thereof | |
| CN101659960A (en) | Biological preparation method of chitin deacetylase | |
| CN105349506A (en) | Lipase LIPASEB5, and encoding gene and application thereof | |
| CN105368802A (en) | Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase | |
| CN101457217B (en) | Thermophilic lipase with alpha-phenyl ethyl alcohol disassemble activity, coding gene and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190301 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |