WO2008047819A1 - Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation - Google Patents
Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation Download PDFInfo
- Publication number
- WO2008047819A1 WO2008047819A1 PCT/JP2007/070225 JP2007070225W WO2008047819A1 WO 2008047819 A1 WO2008047819 A1 WO 2008047819A1 JP 2007070225 W JP2007070225 W JP 2007070225W WO 2008047819 A1 WO2008047819 A1 WO 2008047819A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ibuprofen
- optically active
- ester
- sequence
- polypeptide
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title description 11
- 230000000694 effects Effects 0.000 claims abstract description 31
- 229920001184 polypeptide Polymers 0.000 claims abstract description 28
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 28
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960001680 ibuprofen Drugs 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 4
- 108090000604 Hydrolases Proteins 0.000 claims description 46
- 102000004157 Hydrolases Human genes 0.000 claims description 41
- YNZYUHPFNYBBFF-UHFFFAOYSA-N methyl 2-[4-(2-methylpropyl)phenyl]propanoate Chemical group COC(=O)C(C)C1=CC=C(CC(C)C)C=C1 YNZYUHPFNYBBFF-UHFFFAOYSA-N 0.000 claims description 35
- -1 carboxylic acid compound Chemical class 0.000 claims description 27
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 20
- HEFNNWSXXWATRW-SNVBAGLBSA-N levibuprofen Chemical compound CC(C)CC1=CC=C([C@@H](C)C(O)=O)C=C1 HEFNNWSXXWATRW-SNVBAGLBSA-N 0.000 claims description 19
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 12
- 241000187562 Rhodococcus sp. Species 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- HEFNNWSXXWATRW-JTQLQIEISA-N dexibuprofen Chemical compound CC(C)CC1=CC=C([C@H](C)C(O)=O)C=C1 HEFNNWSXXWATRW-JTQLQIEISA-N 0.000 claims description 7
- 238000001962 electrophoresis Methods 0.000 claims description 7
- 230000003287 optical effect Effects 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000002523 gelfiltration Methods 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 5
- 229920002401 polyacrylamide Polymers 0.000 claims description 5
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 claims description 3
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 abstract description 10
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 239000008177 pharmaceutical agent Substances 0.000 abstract 2
- 150000001735 carboxylic acids Chemical class 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 44
- 102000004190 Enzymes Human genes 0.000 description 43
- 108020004414 DNA Proteins 0.000 description 38
- 238000000034 method Methods 0.000 description 36
- 210000004027 cell Anatomy 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 19
- 239000000243 solution Substances 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000008057 potassium phosphate buffer Substances 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 239000013611 chromosomal DNA Substances 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 241001131785 Escherichia coli HB101 Species 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- YBADLXQNJCMBKR-UHFFFAOYSA-M (4-nitrophenyl)acetate Chemical compound [O-]C(=O)CC1=CC=C([N+]([O-])=O)C=C1 YBADLXQNJCMBKR-UHFFFAOYSA-M 0.000 description 5
- 108090000371 Esterases Proteins 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000010931 ester hydrolysis Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000003505 heat denaturation Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 230000000707 stereoselective effect Effects 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 1
- SYTBZMRGLBWNTM-SNVBAGLBSA-N (R)-flurbiprofen Chemical compound FC1=CC([C@H](C(O)=O)C)=CC=C1C1=CC=CC=C1 SYTBZMRGLBWNTM-SNVBAGLBSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ASNHGEVAWNWCRQ-UHFFFAOYSA-N 4-(hydroxymethyl)oxolane-2,3,4-triol Chemical compound OCC1(O)COC(O)C1O ASNHGEVAWNWCRQ-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108700023418 Amidases Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010053229 Lysyl endopeptidase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001024304 Mino Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101150056072 TUFB gene Proteins 0.000 description 1
- 229910052770 Uranium Inorganic materials 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000001458 anti-acid effect Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000012024 dehydrating agents Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 1
- 229940093496 esculin Drugs 0.000 description 1
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- QVLTXCYWHPZMCA-UHFFFAOYSA-N po4-po4 Chemical compound OP(O)(O)=O.OP(O)(O)=O QVLTXCYWHPZMCA-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 108010058004 pyrazinamide deamidase Proteins 0.000 description 1
- 125000004929 pyrrolidonyl group Chemical group N1(C(CCC1)=O)* 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 101150079601 recA gene Proteins 0.000 description 1
- 238000011945 regioselective hydrolysis Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 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
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 101150099542 tuf gene Proteins 0.000 description 1
- 101150071165 tuf1 gene Proteins 0.000 description 1
- 101150010742 tuf2 gene Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration 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)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
-
- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- 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
Definitions
- Novel ester hydrolases genes encoding them, and methods for using them
- the present invention relates to an ester hydrolase having an activity of asymmetrically hydrolyzing an ester compound to produce an optically active carboxylic acid or to leave an optically active ester, DNA encoding the enzyme,
- the present invention relates to a vector having DNA and a transformed cell transformed with this vector.
- it is useful as a catalyst in the production of optically active ibuprofen, which is a compound useful as a synthetic material for pharmaceutical and agricultural chemicals.
- Porcine liver-derived esterases and the like are generally used for such purposes, but they are expensive and have a limited supply amount, which is disadvantageous for industrial use. .
- a method of using a microorganism-derived esterase instead of porcine liver esterase has also been tried.
- each has a characteristic substrate specificity, so the selectivity and reaction rate vary greatly depending on the compound applied.
- Patent Document 1 As part of the enzymatic synthesis method, a method has been proposed to reduce the catalyst cost by using genetically modified microorganisms (Patent Document 1), but the enzyme used in this method has an optimal reaction temperature of 25- 4 It is hard to say that it is a practical method as low as 5 ° C. Therefore, development of a more efficient method for synthesizing (S) -ibuprofen, which is an alternative to these methods, has been desired! /.
- Patent Document 1 JP-A-8-242853
- Non-Patent Document 1 S. Adams, et al., J. Pharm. Pharmac, 28, 256 (1976)
- Non-patent document 2 A. J. Hutt and J. Caldwell, Clinical Pharmacokinetics., 9, 371 (1984)
- Non-patent document 3 Williams, Tsuji, et al., Biochem. Pharmac, 35, 3403 (1986)
- Non-patent document 4 J. Caldwell and MV Marsh, Biochem. Pharmac, 32, 1667 (1983)
- Non-patent document 5 Duan, G. and Chen, JY, Biotechnol. Lett., 16, 1065 (1994)
- Non-patent document 6 Mustranta, A., J. Org. Chem., 59, 4410 (1994)
- Non-Patent Document 7 Lee, W. ⁇ ., Et al., J. Ferment. Bioeng., 80, 613 (1995)
- the inventors of the present application have released a novel ester hydrolase having a high optimal reaction temperature of 60 to 70 ° C from a fine cattle belonging to the genus Rhodococcus. Furthermore, it was found that by using this enzyme, (S) -ibuprofen can be efficiently produced from racemic ibuprofen methyl, and the present invention was completed.
- the present invention is an ester hydrolase which is a polypeptide according to any one of (1) to (3) below.
- (1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
- the present invention is an ester hydrolase having the following physicochemical properties (1) to (5).
- Inhibitor Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
- the present invention is a DNA encoding the ester hydrolase.
- the present invention is a DN sequence consisting of the base sequence described in any of (1) to (4) below.
- the present invention is a vector having the DNA.
- the present invention also relates to a transformed cell transformed with the vector.
- the present invention is an optically active carboxylate compound and / or remaining by reacting the ester hydrolase of the present invention and / or the transformed cell of the present invention with an ester compound.
- a novel ester hydrolase a DNA encoding the same, a vector having the DNA, a transformant transformed with the vector, an optically active carboxylic acid compound using them, and / or Alternatively, a method for producing an optically active ester compound is provided.
- the organism used as the origin of the ester hydrolase of the present invention is not particularly limited, and examples thereof include fine cattle belonging to the genus Rhodococcus, and in particular, Rhodococcus sp. KNK0401 preferable.
- Rhodococcus sp. KNK0401 The mycological properties of Rhodococcus sp. KNK0401 are described below.
- Oxidase Yinsei
- Rhodococcus sp (The original deposit date was transferred to the international deposit under the Budapest Treaty (September 21, 2007)).
- the medium used for culturing the microorganism producing the enzyme of the present invention is not particularly limited as long as the microorganism can grow.
- a normal liquid nutrient medium containing a carbon source, nitrogen source, inorganic salts, organic nutrients and the like can be used.
- purification of the enzyme from the microorganism producing the enzyme of the present invention can be carried out by a conventional method.
- the enzyme of the present invention can be isolated from Rhodococcus sp. KNK0401 as follows, for example. First, the strain is cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation. The cells are crushed with, for example, Dynomill (Dyno-Mill), and the cell residue is removed by centrifugation to obtain a cell-free extract. For this cell-free extract, columns such as salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase, etc.
- salting out ammonium sulfate precipitation, sodium phosphate precipitation, etc.
- solvent precipitation protein fraction precipitation with acetone or ethanol, etc.
- dialysis gel filtration, ion exchange, reverse phase, etc.
- the enzyme can be purified by performing treatments such as chromatography and ultrafiltration alone or in combination. Ester hydrolysis activity is performed by measuring the increase in absorbance at 405 nm at 30 ° C by adding the substrate p-nitrophenyl acetate ImM and enzyme to lOOmM phosphate phosphate buffer (pH 7.0). obtain. Under these reaction conditions, the activity of hydrolyzing l ⁇ mol of p-nitrotrophyl acetate per minute is defined as lu nit.
- an enzyme is "inhibited" by a specific compound means that when a specific compound is added to a reaction solution of the enzyme at a concentration of ImM or less, the enzyme is not added. The activity decreases to% or less!
- the molecular weight of the enzyme was measured using Superdex 200 HR 10/30 (lOmml. D. X 30cm) Performed by gel filtration analysis using a column (GE Healthcare Biosciences). As eluent, 10 mM potassium phosphate buffer ( ⁇ 7 ⁇ 0) containing 0.2 M NaCl is used. Subunit molecular weight is calculated from the relative mobility of standard proteins by electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% ( ⁇ / ⁇ ) 2-mercaptoethanol). To be determined.
- the ester hydrolase of the present invention has, for example, the following physicochemical properties (1) to (5).
- Inhibitor Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
- the enzyme having almost the same properties as the enzyme of the present invention may be a natural enzyme or a recombinant enzyme.
- the recombinant enzyme can be obtained by introducing a gene of an enzyme derived from the genus Rhodococcus determined by the following method into an appropriate host.
- a mutant enzyme in which one or several amino acids in the amino acid sequence of the enzyme are substituted, deleted, inserted or added is added. It is also possible to produce it.
- Mutant enzymes thus obtained are also included in the present invention as long as they have the activity of acting on ibuprofen methyl to produce (R) -ibuprofen and leave (S) -ibuprofenmethyl.
- the number of amino acids to be deleted, replaced, inserted or added is preferably 40 or less, more preferably 25 or less, still more preferably 10 or less, most preferably 5 or 4 , 3 or 2 or less.
- a polypeptide having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing acts on ibuprofen methyl to produce (R) -ibuprofen (S)-
- a polypeptide having the activity of leaving buprofen methyl is also included in the polypeptide of the present invention.
- a polypeptide having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 in the sequence listing is a force S included in the polypeptide of the present invention, and its sequence identity is preferably 90% or more, more preferably 95% or more. More than 98% is more preferable. More than 99% is more preferable.
- sequence identity of amino acid sequences is determined by comparing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with the evaluated amino acid sequence and the number of positions where the amino acid matches in both sequences. Divided by the number of mino acids and then multiplied by 100.
- the enzyme of the present invention purified as described above can be denatured under appropriate conditions and then digested with an appropriate endopeptidase to obtain a peptide fragment constituting the enzyme of the present invention.
- These peptide fragments are purified by reverse phase HPLC using, for example, a YMC-Pack SIL-06 (manufactured by YMC) column, and then subjected to a protein sequencer, whereby the partial amino acid sequence of the enzyme of the present invention is obtained.
- YMC-Pack SIL-06 manufactured by YMC
- a PCR (Polymerase Chain Reaction) primer can be synthesized based on the partial amino acid sequence information obtained.
- chromosomal DNA of the microorganism is prepared from the microorganism that is the origin of the ester hydrolase of the present invention by, for example, the method of Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)). obtain.
- PCR can be performed using the PCR primers described above to amplify a part of the DNA encoding the enzyme (core arrangement IJ) and determine the nucleotide sequence.
- the base sequence can be determined by a dideoxy chain termination method or the like, and can be performed using, for example, ABI 3130xlDNA Sequencer (Applied Biosystems).
- the chromosomal DNA of the microorganism is digested with a restriction enzyme whose recognition sequence does not exist in the core sequence, and the generated DNA fragment is A cage DNA for reverse PCR (Nucleic Acids Res., 16: 8186 (1988)) is prepared by self-cyclization with T4 ligase.
- a primer that acts as a starting point for DNA synthesis is synthesized outside the core sequence, and the peripheral region of the core sequence is amplified by inverse PCR.
- the base sequence of the entire coding region of the target enzyme can be read.
- DNA encoding the polypeptide of the present invention for example, DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, or a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing And DNA encoding a polypeptide that has the activity of hybridizing under stringent conditions and acting on ibuprofen phenmethyl to produce (R) -ibuprofen and (S) -ibuprofenmethyl to remain. The power to raise S.
- DNA encoding a polypeptide having the activity of leaving ibuprofen methyl refers to a colony under stringent conditions using a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the Sequence Listing as a probe. DNA obtained by using the 'noisy hybridization method, plaque' hybridization method, or Southern hybridization method, etc. and acting on ibuprofen methyl to produce (R) -ibuprofen.
- S refers to DNA encoding a polypeptide having the activity of leaving ibuprofen methyl.
- noisy Pre-Daisyong is Molecular Cloning, A laboratory manual, second edition
- DNA that hybridizes under stringent conditions means, for example, hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After dialysis, filter at 65 ° C using 2x SSC solution (1x SSC solution consists of 150mM sodium chloride, 15mM sodium citrate). It is possible to increase the DNA that can be obtained by washing. Preferably washed with 0.5 times SSC solution at 65 ° C, more preferably washed with 0.2 times SSC solution at 65 ° C, more preferably 0.1 times SSC at 65 ° C This DNA can be obtained by washing with a solution.
- the DNA that can be hybridized under the above conditions is 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98%, with the DNA represented by SEQ ID NO: 1.
- SEQ ID NO: 1 the DNA represented by SEQ ID NO: 1.
- sequence identity means that two DNAs to be compared are optimally aligned and both nucleic acid bases (eg, A, T, C, G, U, or I) are both aligned. The number of matching positions in this sequence is divided by the total number of comparison bases, and this result is expressed by multiplying by 100.
- Sequence identity can be calculated, for example, using the following sequence analysis tool: GCG Wise onsin Package (Program Manual ror he Wisconsin Package, Version 8, September 1994, enetics Computer Group, 575 Science Drive Medison , Wisconsin, USA 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 IRQ, England), and the ExPASy World Wide Web for molecular biology Geneva University Hospital and University or ueneva, en eva, Switzerland.
- GCG Wise onsin Package Program Manual ror he Wisconsin Package, Version 8, September 1994, enetics Computer Group, 575 Science Drive Medison , Wisconsin, USA 53711
- Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 IRQ, England), and the ExPASy World Wide Web for molecular biology Geneva University Hospital and University or ueneva, en eva, Switzerland.
- the ester hydrolase is expressed in an appropriate host microorganism.
- Any vector can be used as long as it is available.
- examples of such vector DNA include plasmid vector 1, phage vector, cosmid vector and the like.
- a shuttle vector that can exchange genes with other host strains can also be used.
- such vector DNA is operably linked to promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter motor, PL promoter, etc.), enhancer sequences, etc.
- pUCNT WO94 / 03613
- This plasmid pUCNT is 1 Since it has an insertion site such as Ndel or EcoRI site downstream of the ac promoter, it can be suitably used.
- the obtained recombinant plasmid having ester hydrolase can be introduced into a host cell by a conventional method.
- host cells bacteria, yeasts, filamentous fungi, plant cells, animal cells and the like can be used.
- the use of E. coli is particularly preferred.
- the introduction of the plasmid into the host can be performed by methods well known to those skilled in the art, for example, a method including a step of mixing a recombinant host cell with a competent host cell, a conjugative transfer using a helper plasmid.
- the plasmid introduced into the host can replicate autonomously as an episome, or all or part of it can be integrated into the chromosome and replicated together with the chromosome.
- optically active carboxylic acid (S) -ibuprofen is obtained, for example, as follows.
- ibuprofen methyl may be used as the substrate.
- the reaction can be carried out by adding the substrate ibuprofen methyl, a microorganism culture or a treated product thereof in an appropriate solvent, and stirring under pH adjustment.
- the reaction is carried out at a temperature of 10 ° C to 70 ° C, pH 4 to 10;
- the substrate concentration is 0.1% to 90% (W / V), but the substrate can be added continuously.
- the reaction can be carried out batchwise or continuously.
- the treated product of microorganisms is, for example, a crude extract, cultured cells, freeze-dried organisms, acetone-dried organisms, or a ground product of these cells, and the catalytic activity of ester hydrolase. Means the remaining item.
- ibuprofen methyl is subjected to treatment such as centrifugation and filtration as necessary when microorganisms are used, etc. to remove cell suspensions, and then extracted with an organic solvent such as ethyl acetate or toluene.
- Dehydrate with a dehydrating agent such as sodium sulfate remove the organic solvent under reduced pressure, and It can be purified by performing a treatment such as distillation or chromatography (for example, silica gel column chromatography).
- a treatment such as distillation or chromatography (for example, silica gel column chromatography).
- the obtained optically active ibuprofen methyl can be chemically hydrolyzed by a conventional method. For example, it can be hydrolyzed by stirring under conditions of a strong base in an appropriate solvent.
- Quantification of ibuprofen methyl can be performed by gas chromatography using TC-FFAP (manufactured by GL Sciences Inc.), chromatography at a column temperature of 80 ° C to 200 ° C, and detection by FID. .
- the culture was performed for 3 days under the condition of 0.5 L / min.
- the pH was adjusted to 7.0 with a 5N aqueous sodium hydroxide solution.
- the cells were collected from the culture solution by centrifugation. In this way, 470 g of wet cells of the strain was obtained.
- the wet cells were suspended in 2 L of 10 mM potassium phosphate buffer (pH 7.0) and heat-treated by stirring for 20 minutes in a 50 ° C constant temperature bath.
- the cells were crushed with Dynomill (Dyno-Mill).
- the cell residue was removed from the crushed cell by centrifugation to obtain 2100 ml of a cell-free extract.
- the resulting precipitate was removed by centrifugation.
- ester hydrolase activity is basically performed by measuring lOOmM potassium phosphate buffer.
- the substrate p-nitrophenyl acetate ImM and enzyme were added to (pH 7.0), and the increase in absorbance at a wavelength of 405 nm was measured at 30 ° C.
- the reaction product was identified by gas chromatography using ibuprofen instead of the substrate P-nitrophenyl acetate.
- the molecular weight of the enzyme is measured using a Superdex 200 HR 10/30 (lOmml. D. X 30cm) column (manufactured by GE Healthcare Biosciences Inc.) and contains 0.2M sodium chloride as the eluent. When 10 mM potassium phosphate buffer (pH 7.0) was used, it was about 57,000.
- the molecular weight of the subunit of the enzyme is calculated from the relative mobility of the standard protein after electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% (V / v) 2-mercaptoethanol). Was determined by As a result, the molecular weight of the subunit of this enzyme was about 37,000. [0054] [Table 1]
- Rhodococcus sp. KNK0401 Chromosomal DNA was extracted in accordance with the method of Nakuryoku et al., Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)).
- the purified ester hydrolase obtained as in Example 1 was denatured in the presence of 8M urea and then digested with acrymopacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The sequence of the fragment was determined by the Edman method. Considering the DNA sequence expected from this amino acid sequence, two kinds of PCR primers 5'—CCRTGR TTRTANCCYTCCCA-3 ′ (primer 1: SEQ ID NO: 3), 5′—GARGCNG TNAGYGTNGAYGG-3 ′ (primer 2: Sequence listing SEQ ID NO: 4) was synthesized.
- Primer 1 and Primer 2 2 types of primers 50 pmol each, chromosomal DNA 240 ng, d NTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1. Prepare ExTaq buffer solution 50 1 containing 3 U, and heat denaturation ( 97 ° C, 30 seconds), annealing (50 ° C, 1 minute), extension reaction (72 ° C, 1 minute) for 30 cycles. After cooling to 4 ° C, amplified DNA was confirmed by agarose genomic electrophoresis. .
- the amplified DNA was subcloned into pT7Blue Vector (Novagen), and its nucleotide sequence was determined. From this result and the result of the core sequence, the entire nucleotide sequence of DNA encoding the ester hydrolase was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are shown in SEQ ID NO: 2 in the sequence listing.
- primers (primer 5 and primer 6) 50 pmol each, Rhodococcus sp. KNK0401 chromosomal DNA 15 ng, dNTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1.
- ExTaq buffer containing 3 U Prepare 501, heat denaturation (97 ° C, 30 seconds), annealing (60 ° C, 1 minute), extension reaction (72 ° C, 5 minutes) for 30 cycles, and after cooling to 4 ° C The amplified DNA was confirmed by agarose gel electrophoresis.
- This amplified fragment was digested with Ndel and EcoRI and inserted into the Ndel and EcoRI sites downstream of the lac promoter of the plasmid pUCNT (WO94 / 03613) to obtain a recombinant vector pNTHR.
- the recombinant vector pNTHR obtained in Example 4 was mixed with DNA Ligation Kit Ver. 2.1 I solution (manufactured by Takara Bio Inc.) and incubated at 16 ° C. for 30 minutes. The resulting reaction solution was added to Escherichia coli HB101 combined cell (manufactured by Takara Bio Inc.), incubated on ice for 30 minutes, incubated at 42 ° C for 45 seconds, and then cooled on ice for 2 minutes, Recombinant E. coli HB101 (pNTHR) was obtained.
- Escherichia coli HB101 (pNTHR), a transformant obtained in this way, has the accession number FERM P-20271, dated October 22, 2004, Tsukuba Sakai Higashi 1-chome, 1-chome, Ibaraki, Japan 1 No. (Postal code: 305-8566) Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary.
- Recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was mixed with 2 XYT medium containing 120 ⁇ g / ml ampicillin (batato'tryptone 1 ⁇ 6% (w / v), butato'yeast extract 1 ⁇ 0% (w / v), NaClO. 5% (w / v), pH 7.0), and the resulting culture broth was sonicated to obtain a cell-free extract. The ester hydrolysis activity of this cell-free extract was measured by the method described in Example 2.
- E. coli HB101 (pNTHR) showed a clear increase in ester hydrolysis activity as compared to E. coli HB101 (pUCNT), which is a transformant of only the vector plasmid.
- the recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was inoculated into 50 ml of 2X YT medium sterilized in a 500 ml volumetric flask and cultured with shaking at 37 ° C for 48 hours. The resulting culture solution to give a P H7.
- Cell-free extract by adjusting to sonication at 0. 10 mg of racemic ibuprofen methyl was added to 1 ml of the cell-free extract and shaken at 30 ° C. for 12 hours. After the conversion reaction, the reaction solution is saturated with ammonium sulfate, extracted with ethyl acetate, and remains.
- CHIRALCEL ⁇ J—H manufactured by Daicel Chemical Industries, Ltd.
- flow rate 1. Oml / min.
- elution time R-form 30 minutes, S-form 25 minutes
- the cells were obtained from 1 L of the Rhodococcus sp. KNK0401 culture solution obtained by the method described in Example 1 by centrifugation, and racemic into 250 ml of OOmM potassium phosphate buffer (pH 8.0). 2.5g of ibuprofen methinore was added and stirred for 25 days at 40 ° C. After the conversion reaction, the reaction solution was saturated with ammonium sulfate, extracted with ethyl acetate, and the remaining ibuprofen methyl and the produced ibuprofen were analyzed by the method described in Example 7. As a result, 750 mg of ibuprofen was produced.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
L'invention concerne : un polypeptide présentant une activité d'hydrolyse des esters, ledit polypeptide étant isolé à partir d'un microorganisme appartenant au genre Rhodococcus ; de l'ADN codant pour ledit polypeptide ; un transformant capable de produire ledit polypeptide ; et un procédé permettant de produire un acide carboxylique optiquement actif ou un ester optiquement actif par réaction dudit polypeptide ou transformant avec un ester. Il est ainsi possible de produire avec un bon rendement un composé optiquement actif (ibuprofène optiquement actif, par exemple) utile en tant qu'agent pharmaceutique ou bien un intermédiaire d'agent pharmaceutique.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006-283309 | 2006-10-18 | ||
JP2006283309 | 2006-10-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2008047819A1 true WO2008047819A1 (fr) | 2008-04-24 |
Family
ID=39314041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2007/070225 WO2008047819A1 (fr) | 2006-10-18 | 2007-10-17 | Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2008047819A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110592049A (zh) * | 2019-09-29 | 2019-12-20 | 北京工商大学 | 一种黑曲霉酯水解酶AnCu3、编码基因及其在水解DEHP中的应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03224496A (ja) * | 1989-12-21 | 1991-10-03 | Asahi Chem Ind Co Ltd | 光学活性なα―置換有機酸を製造する方法 |
-
2007
- 2007-10-17 WO PCT/JP2007/070225 patent/WO2008047819A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03224496A (ja) * | 1989-12-21 | 1991-10-03 | Asahi Chem Ind Co Ltd | 光学活性なα―置換有機酸を製造する方法 |
Non-Patent Citations (7)
Title |
---|
CHUNG Y.M. ET AL.: "Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeon Sulfolobus solfataricus", BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, vol. 5, no. 1, 2000, pages 53 - 56, XP003022309 * |
DATABASE SWISSPROT [online] XU Y. ET AL.: "Carboxyeasterase", XP003022311, Database accession no. (Q5MAF6) * |
DROGE M.J. ET AL.: "Comparison and functional characterization of three homologous intracellular carboxyesterases of Bacillus subtilis", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 32, no. 5-6, 2005, pages 261 - 270, XP004752016 * |
LEE W.H. ET AL.: "Enzymatic resolution of racemic ibuprofen esters: effects of organic cosolvents and temperature", J. FERMENT. BIOENG., vol. 80, no. 6, 1995, pages 613 - 615, XP009022668 * |
MADHAV M.V. ET AL.: "Study on the enzymatic hydrolysis of racemic methyl ibuprofen ester", J. CHEM. TECHNOL. BIOTECHNOL., vol. 76, no. 9, 2001, pages 941 - 948, XP001092368 * |
RHODOCOCCUS SP. CDT3 * |
SNELL D. ET AL.: "Enantioselective hydrolysis of racemic ibuprofen amide to S-(plus)-ibuprofen by RHodococcus AJ270", ENZYME AND MICROBIAL TECHNOLOGY, vol. 24, no. 3-4, 1999, pages 160 - 163, XP003022310 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110592049A (zh) * | 2019-09-29 | 2019-12-20 | 北京工商大学 | 一种黑曲霉酯水解酶AnCu3、编码基因及其在水解DEHP中的应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4510351B2 (ja) | 新規カルボニル還元酵素、その遺伝子、およびその利用法 | |
JP4746548B2 (ja) | 新規カルボニル還元酵素、その遺伝子、およびその利用法 | |
JP6027173B2 (ja) | 新規なアミノ酸脱水素酵素、およびl−アミノ酸、2−オキソ酸、又はd−アミノ酸の製造方法 | |
WO2007139055A1 (fr) | Méthode de production d'un composé amine optiquement actif, d'un vecteur recombinant et d'un transformant portant ledit vecteur | |
JPWO2007094178A1 (ja) | 新規な(s,s)−ブタンジオール脱水素酵素、その遺伝子、及びその利用法 | |
JP5005672B2 (ja) | 新規カルボニル還元酵素、その遺伝子、およびそれらを利用した光学活性アルコールの製造方法 | |
JP4205496B2 (ja) | 新規カルボニル還元酵素及びこれをコードするdna、ならびにこれらを利用した光学活性アルコールの製造方法 | |
EP2128258B1 (fr) | Nouvelle amidase, gène pour celle-ci, vecteur, transformant et procédé de fabrication d'un amide d'acide carboxylique optiquement actif et d'un acide carboxylique optiquement actif à l'aide de l'un quelconque de ces éléments | |
WO2012043653A1 (fr) | Nouvelle transaminase présentant une activité élevée vis-à-vis de l'acide glutamique, gène codant ladite transaminase, et sa méthode d'utilisation | |
JPWO2010123062A1 (ja) | (r)−3−キヌクリジノールの製造方法 | |
WO2008047819A1 (fr) | Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation | |
JP4880859B2 (ja) | 新規カルボニル還元酵素、その遺伝子、およびその利用法 | |
WO2007099994A1 (fr) | carbonyle reductase, gene pour la reductase, vecteur, transformant et procede de production d'alcool optiquement actif au moyen de ces materiaux | |
JP4345425B2 (ja) | クロロヒドリン及びヒドロキシカルボン酸エステル不斉加水分解酵素遺伝子 | |
US20080305534A1 (en) | Novel Glycerol Dehydrogenase, Gene Therefor, and Method of Utilizing the Same | |
JP4627039B2 (ja) | アミダーゼ活性を有するポリペプチド及びその遺伝子 | |
JP4796323B2 (ja) | 新規カルボニル還元酵素、その遺伝子、およびその利用法 | |
JP2005027552A (ja) | 新規な光学活性2−ヒドロキシメチル−3−アリールプロピオン酸の製造方法 | |
JP2008194037A (ja) | 生体触媒による4−ハロ−3−ヒドロキシ酪酸エステルの光学分割法 | |
JP2010279272A (ja) | 新規カルボニル還元酵素、その遺伝子、ベクター、形質転換体、およびそれらを利用した光学活性アルコールの製造方法 | |
US9493762B2 (en) | Vector, a transformant and a method to produce a polypeptide having activity to selectively hydrolyze a (R)-tropic acid amide in a racemic mixture | |
WO2005044973A2 (fr) | Nouvelle acetoacetyle-coa reductase et procede pour produire un alcool actif optiquement |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07829959 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 07829959 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: JP |