JP6315978B2 - Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase - Google Patents
Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase Download PDFInfo
- Publication number
- JP6315978B2 JP6315978B2 JP2013265666A JP2013265666A JP6315978B2 JP 6315978 B2 JP6315978 B2 JP 6315978B2 JP 2013265666 A JP2013265666 A JP 2013265666A JP 2013265666 A JP2013265666 A JP 2013265666A JP 6315978 B2 JP6315978 B2 JP 6315978B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- amino acid
- acid sequence
- cutinase
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 81
- 108010005400 cutinase Proteins 0.000 title claims description 77
- 229920000728 polyester Polymers 0.000 title claims description 39
- -1 ester compound Chemical class 0.000 title claims description 31
- 238000000034 method Methods 0.000 title claims description 22
- 230000015556 catabolic process Effects 0.000 title description 26
- 238000006731 degradation reaction Methods 0.000 title description 26
- 230000000694 effects Effects 0.000 claims description 102
- 150000001413 amino acids Chemical group 0.000 claims description 83
- 102000004169 proteins and genes Human genes 0.000 claims description 61
- 229920009537 polybutylene succinate adipate Polymers 0.000 claims description 33
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 23
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 23
- 108010021466 Mutant Proteins Proteins 0.000 claims description 19
- 102000008300 Mutant Proteins Human genes 0.000 claims description 19
- 238000006467 substitution reaction Methods 0.000 claims description 19
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 17
- 229920000118 poly(D-lactic acid) Polymers 0.000 claims description 15
- 229920001610 polycaprolactone Polymers 0.000 claims description 15
- 239000011541 reaction mixture Substances 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 12
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 10
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 9
- 239000004473 Threonine Substances 0.000 claims description 9
- 229920001432 poly(L-lactide) Polymers 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 8
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 8
- 239000004472 Lysine Substances 0.000 claims description 7
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 7
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims description 6
- 229920006267 polyester film Polymers 0.000 claims description 6
- 229920001634 Copolyester Polymers 0.000 claims description 4
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 4
- ZMKVBUOZONDYBW-UHFFFAOYSA-N 1,6-dioxecane-2,5-dione Chemical compound O=C1CCC(=O)OCCCCO1 ZMKVBUOZONDYBW-UHFFFAOYSA-N 0.000 claims description 3
- 229920003232 aliphatic polyester Polymers 0.000 claims description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 2
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 description 64
- 108090000790 Enzymes Proteins 0.000 description 64
- 235000018102 proteins Nutrition 0.000 description 46
- 239000011575 calcium Substances 0.000 description 35
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 34
- 108091033319 polynucleotide Proteins 0.000 description 28
- 239000002157 polynucleotide Substances 0.000 description 28
- 102000040430 polynucleotide Human genes 0.000 description 28
- 235000001014 amino acid Nutrition 0.000 description 24
- 229940024606 amino acid Drugs 0.000 description 24
- UXDAWVUDZLBBAM-UHFFFAOYSA-N n,n-diethylbenzeneacetamide Chemical compound CCN(CC)C(=O)CC1=CC=CC=C1 UXDAWVUDZLBBAM-UHFFFAOYSA-N 0.000 description 24
- 239000002773 nucleotide Substances 0.000 description 23
- 125000003729 nucleotide group Chemical group 0.000 description 23
- 239000000872 buffer Substances 0.000 description 21
- 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 16
- 235000011187 glycerol Nutrition 0.000 description 16
- 239000000835 fiber Substances 0.000 description 13
- DVDUMIQZEUTAGK-UHFFFAOYSA-N p-nitrophenyl butyrate Chemical compound CCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 DVDUMIQZEUTAGK-UHFFFAOYSA-N 0.000 description 13
- 229920005839 ecoflex® Polymers 0.000 description 12
- 238000011534 incubation Methods 0.000 description 12
- 238000005259 measurement Methods 0.000 description 12
- 229920002799 BoPET Polymers 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 238000012289 standard assay Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 8
- 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 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000187810 Saccharomonospora viridis Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002983 circular dichroism Methods 0.000 description 6
- 239000002361 compost Substances 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 239000006391 Luria-Bertani Medium Substances 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 241000521303 Thermobifida alba Species 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 150000003355 serines Chemical group 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 108090001060 Lipase Proteins 0.000 description 4
- 102000004882 Lipase Human genes 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 4
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000004744 fabric Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000000465 moulding Methods 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- KKEYFWRCBNTPAC-UHFFFAOYSA-N terephthalic acid group Chemical group C(C1=CC=C(C(=O)O)C=C1)(=O)O KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 4
- PVNIQBQSYATKKL-UHFFFAOYSA-N tripalmitin Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PVNIQBQSYATKKL-UHFFFAOYSA-N 0.000 description 4
- 230000004580 weight loss Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 241001647802 Thermobifida Species 0.000 description 3
- 241000203780 Thermobifida fusca Species 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000010432 diamond Substances 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 229960000310 isoleucine Drugs 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
- 235000019421 lipase Nutrition 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 3
- 239000004014 plasticizer Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JDIIGWSSTNUWGK-UHFFFAOYSA-N 1h-imidazol-3-ium;chloride Chemical compound [Cl-].[NH2+]1C=CN=C1 JDIIGWSSTNUWGK-UHFFFAOYSA-N 0.000 description 2
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229920000832 Cutin Polymers 0.000 description 2
- 101710201394 Cutinase 1 Proteins 0.000 description 2
- MQIUGAXCHLFZKX-UHFFFAOYSA-N Di-n-octyl phthalate Chemical compound CCCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC MQIUGAXCHLFZKX-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 101710088336 Putative cutinase Proteins 0.000 description 2
- 241001496696 Saccharomonospora viridis DSM 43017 Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 229930183415 Suberin Natural products 0.000 description 2
- 241001295025 Thermobifida cellulosilytica Species 0.000 description 2
- ZFOZVQLOBQUTQQ-UHFFFAOYSA-N Tributyl citrate Chemical compound CCCCOC(=O)CC(O)(C(=O)OCCCC)CC(=O)OCCCC ZFOZVQLOBQUTQQ-UHFFFAOYSA-N 0.000 description 2
- BAECOWNUKCLBPZ-HIUWNOOHSA-N Triolein Natural products O([C@H](OCC(=O)CCCCCCC/C=C\CCCCCCCC)COC(=O)CCCCCCC/C=C\CCCCCCCC)C(=O)CCCCCCC/C=C\CCCCCCCC BAECOWNUKCLBPZ-HIUWNOOHSA-N 0.000 description 2
- PHYFQTYBJUILEZ-UHFFFAOYSA-N Trioleoylglycerol Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCCCCCCCC)COC(=O)CCCCCCCC=CCCCCCCCC PHYFQTYBJUILEZ-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 239000001361 adipic acid Substances 0.000 description 2
- 235000011037 adipic acid Nutrition 0.000 description 2
- 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 2
- 229960000723 ampicillin Drugs 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 229920001225 polyester resin Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 2
- 229940117972 triolein Drugs 0.000 description 2
- 229960001947 tripalmitin Drugs 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 1
- XVARCVCWNFACQC-MBJXGIAVSA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-(1h-indol-3-yloxy)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=CC=C12 XVARCVCWNFACQC-MBJXGIAVSA-N 0.000 description 1
- VOOGILFUHUXBEV-ZBRNBAAYSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-pyrrolidine-2-carboxylic acid Chemical group OC[C@H](N)C(O)=O.OC(=O)[C@@H]1CCCN1 VOOGILFUHUXBEV-ZBRNBAAYSA-N 0.000 description 1
- WPMUZECMAFLDQO-UHFFFAOYSA-N 2-[2-(2-hexanoyloxyethoxy)ethoxy]ethyl hexanoate Chemical compound CCCCCC(=O)OCCOCCOCCOC(=O)CCCCC WPMUZECMAFLDQO-UHFFFAOYSA-N 0.000 description 1
- SSKNCQWPZQCABD-UHFFFAOYSA-N 2-[2-[2-(2-heptanoyloxyethoxy)ethoxy]ethoxy]ethyl heptanoate Chemical compound CCCCCCC(=O)OCCOCCOCCOCCOC(=O)CCCCCC SSKNCQWPZQCABD-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241001480714 Humicola insolens Species 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-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
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000135044 Thermobifida fusca YX Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 241000498583 Ureibacillus thermosphaericus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000012773 agricultural material Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 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
- 239000011942 biocatalyst Substances 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000004566 building material Substances 0.000 description 1
- 238000007707 calorimetry Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000000748 compression moulding Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 150000002085 enols Chemical class 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000010097 foam moulding Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229940094991 herring sperm dna Drugs 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000012770 industrial material Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000002366 lipolytic effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 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
- 239000003209 petroleum derivative Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- JQCXWCOOWVGKMT-UHFFFAOYSA-N phthalic acid diheptyl ester Natural products CCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCC JQCXWCOOWVGKMT-UHFFFAOYSA-N 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000004645 polyester resin Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000001175 rotational moulding Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 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
- APSBXTVYXVQYAB-UHFFFAOYSA-M sodium docusate Chemical compound [Na+].CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC APSBXTVYXVQYAB-UHFFFAOYSA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000007725 thermal activation Methods 0.000 description 1
- 238000003856 thermoforming Methods 0.000 description 1
- 229920005992 thermoplastic resin Polymers 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000002759 woven fabric Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Separation, Recovery Or Treatment Of Waste Materials Containing Plastics (AREA)
Description
本発明は、新規なクチナーゼ、クチナーゼをコードする遺伝子、及びクチナーゼを用いたポリエステル又はエステル化合物の分解方法に関する。 The present invention relates to a novel cutinase, a gene encoding cutinase, and a method for decomposing a polyester or ester compound using the cutinase.
生分解性プラスチックの一般的な形式であるポリエステルはクチナーゼ型のポリエステラーゼにより加水分解される。クチナーゼは、リパーゼ、クチナーゼ、及びエステラーゼの３つの主要なグループからなるリパーゼファミリーに属し、リパーゼとエステラーゼの間の中間的位置を占め、親水性基質及び疎水性基質に対する種々の活性を示し、バイオディーゼル、石油製品、日用品、フレーバー化合物、フェノール化合物、及びキラル化合物等の化合物の合成及び修飾のための生体触媒として産業上の利用可能性を有している。また、クチナーゼは繊維工業、クリーニング産業、皮革工業及び食品産業における脂質分解酵素として利用可能である。植物表面の柔軟化加工への応用はバイオマス利用の前処理として有用である。さらに近年はクチナーゼの種々のポリエステルを分解する能力に対する関心が高まっている。 Polyester, a common form of biodegradable plastic, is hydrolyzed by cutinase type polyesterases. Cutinase belongs to the lipase family consisting of three main groups, lipase, cutinase, and esterase, occupies an intermediate position between lipase and esterase, and exhibits various activities against hydrophilic and hydrophobic substrates. It has industrial applicability as a biocatalyst for the synthesis and modification of compounds such as petroleum products, daily necessities, flavor compounds, phenolic compounds, and chiral compounds. Cutinase can also be used as a lipolytic enzyme in the textile industry, cleaning industry, leather industry and food industry. Application to plant surface softening is useful as a pretreatment for biomass utilization. In recent years, there has been an increasing interest in the ability of cutinase to degrade various polyesters.
好熱性菌であるThermobifida fusca及びThermobifida albaが堆肥中から分離され、これらが脂肪族芳香族コポリエステル等のポリエステルの有力な分解菌であり、これらのポリエステラーゼがポリエチレンテレフタレート（PET）その他のポリエステル繊維製品の質感（texture）を高め、ピリングを防止し、染色性及び仕上げを改善するために利用可能であることが報告されている（非特許文献1-3）。 Thermobifida fusca and Thermobifida alba, which are thermophilic bacteria, have been isolated from compost, and these are the major degradation bacteria of polyesters such as aliphatic aromatic copolyesters. These polyesterases are polyethylene terephthalate (PET) and other polyester fibers. It has been reported that it can be used to enhance the texture of products, prevent pilling, and improve dyeability and finishing (Non-Patent Documents 1-3).
PETを初めとするすべてのポリエステルの生化学的再生及び分解のための、有用なポリエステラーゼの単離及び開発のための新規な微生物資源も関心を集めており、これまでにPET分解性のクチナーゼとしてHumicola insolens（非特許文献4）及びThermobifida fusca （非特許文献5,6）由来の２つが知られていた。しかしながら、PET分解に関する情報は限られており、PETの酵素分解の詳細な生化学機構は不明であった。 There is also interest in new microbial resources for the isolation and development of useful polyesterases for biochemical regeneration and degradation of all polyesters, including PET, to date PET-degradable cutinase Two of them were derived from Humicola insolens (Non-patent Document 4) and Thermobifida fusca (Non-Patent Documents 5 and 6). However, information on PET degradation is limited, and the detailed biochemical mechanism of enzymatic degradation of PET is unknown.
本願発明者らは以前に、堆肥中からUreibacillus thermosphaericus、Streptmyces sp、Thermobifida alba及びSaccharomonospora viridisに属する４つの新規な芳香族含有ポリエステル分解菌を同定し、受託番号ＦＥＲＭ ＢＰ−１０８２７，ＦＥＲＭ ＢＰ−１０８２８，ＦＥＲＭ ＢＰ−１０８２９，及びＦＥＲＭ Ｐ−２１５０７として寄託した（特許文献１）。しかしながら、当該文献では芳香族含有ポリエステル分解作用を有する酵素が同定されておらず、酵素処理の分子機構は不明であった。 The present inventors previously identified four novel aromatic-containing polyester-degrading bacteria belonging to Ureibacillus thermosphaericus , Streptmyces sp, Thermobifida alba and Saccharomonospora viridis from compost, and have accession numbers FERM BP-10828, FERM BP-10828, Deposited as FERM BP-10829 and FERM P-21507 (Patent Document 1). However, in this document, an enzyme having an aromatic-containing polyester decomposing action has not been identified, and the molecular mechanism of the enzyme treatment has been unknown.
本発明の目的は、新規なクチナーゼ、クチナーゼ遺伝子、及びクチナーゼを用いたポリエステル又はエステル化合物の分解方法を提供することにある。 An object of the present invention is to provide a novel cutinase, a cutinase gene, and a method for decomposing a polyester or ester compound using the cutinase.
本願発明者らは、PET分解酵素を得、かつPET分解の生化学的機構を解明すべく、堆肥から単離した好熱性放線菌であるShaccharomonospora viridis AHK190から新規なクチナーゼ型ポリエステラーゼ(Cut190)をクローニングし、本発明を完成するに至った。また、活性及び熱安定性を高めたCut190の変異型も作製した。 The present inventors obtained a novel cutinase-type polyesterase (Cut190) from Shaccharomonospora viridis AHK190, a thermophilic actinomycete isolated from compost, in order to obtain a PET-degrading enzyme and elucidate the biochemical mechanism of PET degradation. Cloning has led to the completion of the present invention. A mutant of Cut190 with enhanced activity and thermal stability was also produced.
すなわち、本発明は以下の通りである。
［１］下記（ａ）〜（ｃ）のいずれかのタンパク質。
（ａ）配列番号４で表されるアミノ酸配列からなるタンパク質
（ｂ）配列番号４で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質
（ｃ）配列番号４で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質
［２］配列番号４で表されるアミノ酸配列に対し、
１８２位のセリンのプロリンによる置換、
１８４位のアルギニンのセリンによる置換、及び
２１８位のスレオニンのリジンによる置換
のうちの少なくともいずれか一つを有する変異型タンパク質である項１に記載のタンパク質。
That is, the present invention is as follows.
[1] A protein according to any one of (a) to (c) below.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having a cutinase activity (c) sequence A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by No. 4, and having cutinase activity [2] The amino acid sequence represented by SEQ ID No. 4 Whereas
Substitution of serine at position 182 with proline,
Substitution of arginine at position 184 with serine, and
Item 2. The protein according to Item 1, which is a mutant protein having at least one of substitution of threonine at position 218 with lysine.
［３］下記（ｄ）〜（ｆ）のいずれかのタンパク質。
（ｄ）配列番号２で表されるアミノ酸配列からなるタンパク質
（ｅ）配列番号２で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質
（ｆ）配列番号２で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質
［４］配列番号４で表されるアミノ酸配列からなる野生型クチナーゼに対して耐熱性が改善された項１に記載の（ｂ）若しくは（ｃ）又は項３に記載の（ｅ）若しくは（ｆ）のタンパク質。
［５］項１〜４のいずれか一項に記載のタンパク質をコードする遺伝子。
［６］下記（ｉ）〜（ｖｉ）のいずれかのポリヌクレオチドからなる遺伝子。
（ｉ）配列番号３で表されるヌクレオチド配列からなるポリヌクレオチド
（ｉｉ）配列番号３で表されるヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｉｉｉ）配列番号３で表されるヌクレオチド配列と９０％以上の同一性を有するポリヌクレオチドからなり、クチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉ）配列番号３で表されるヌクレオチド配列において、１もしくは数個の塩基が欠失、置換、挿入もしくは付加されたヌクレオチド配列からなり、かつクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
［７］下記（ｖ）〜（ｖｉｉｉ）のいずれかのポリヌクレオチドからなる遺伝子。
（ｖ）配列番号１で表されるヌクレオチド配列からなるポリヌクレオチド
（ｖｉ）配列番号１で表されるヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉｉ）配列番号１で表されるヌクレオチド配列と９０％以上の同一性を有するポリヌクレオチドからなり、クチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉｉｉ）配列番号１で表されるヌクレオチド配列において、１もしくは数個の塩基が欠失、置換、挿入もしくは付加されたヌクレオチド配列からなり、かつクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
［８］項５〜７のいずれか一項に記載の遺伝子を含有するベクター。
［９］項８に記載のベクターを含む形質転換体。
［１０］項１〜４のいずれか一項に記載のタンパク質又は請求項９に記載の形質転換体を、ポリエステル又はエステル化合物に接触させることを含む、ポリエステル又はエステル化合物の分解方法。
[3] A protein of any one of (d) to (f) below.
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (e) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having cutinase activity (f) sequence A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by No. 2 and having cutinase activity [4] The amino acid sequence represented by SEQ ID No. 4 Item (b) or (c) or Item (e) or (f), wherein the heat resistance of the wild-type cutinase is improved.
[5] A gene encoding the protein according to any one of Items 1 to 4.
[6] A gene comprising a polynucleotide according to any one of (i) to (vi) below.
(I) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 (ii) a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 3 under stringent conditions and encodes a protein having a cutinase activity (Iii) a polynucleotide comprising a polynucleotide having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 3 and encoding a protein having cutinase activity (vi) in the nucleotide sequence represented by SEQ ID NO: 3, A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted, inserted or added, and encoding a protein having cutinase activity. [7] A polynucleotide according to any one of (v) to (viii) below: A gene consisting of
(V) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (vi) a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having a cutinase activity (Vii) a polynucleotide consisting of a polynucleotide having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and encoding a protein having cutinase activity (viii) in the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide according to any one of [8] to [5], wherein the polynucleotide comprises a nucleotide sequence in which one or several bases are deleted, substituted, inserted or added, and which encodes a protein having cutinase activity. Contains vector.
[9] A transformant comprising the vector according to item 8.
[10] A method for decomposing a polyester or ester compound, comprising bringing the protein according to any one of items 1 to 4 or the transformant according to claim 9 into contact with the polyester or ester compound.
本発明によれば、遺伝子工学的手法等により新規なクチナーゼを生産することが可能である。かかるクチナーゼを用いて種々のポリエステル又はエステル化合物を分解することが可能である。 According to the present invention, a novel cutinase can be produced by a genetic engineering technique or the like. It is possible to decompose various polyester or ester compounds using such cutinase.
本発明において、「ポリエステル」とは、脂肪族ポリエステル、脂肪族芳香族コポリエステル、及び芳香族ポリエステルを含む。これらのうち、脂肪族ポリエステルとしてはポリ(カプロラクトン)(PCL)、ポリ(ブチレンスクシナート-co-アジペート) (PBSA)、ポリ(ブチレンスクシナート)(PBS)、ポリ(L-乳酸) (PLLA)、ポリ(D-乳酸)（PDLA）、及びポリ−3−ヒドロキシ酪酸(PHB)が挙げられるがこれらに限定されない。脂肪族芳香族コポリエステルとして好ましい一例は、芳香族ジカルボン酸として特にテレフタル酸を用い，ジオールとしてＣ２〜Ｃ４のグリコールモノマー及び／又はオリゴマーを用い，更に脂肪族ジカルボン酸としてＨＯＣ(Ｏ)(ＣＨ2)nＣ(Ｏ)ＯＨ（ｎは０〜１０の整数を表す。）を含む原料を重合させることにより製造したポリエステルである。特に好ましい例としては市販のアペクサ〔Apexa（登録商標），デュポン株式会社製(日本国、東京)〕及びエコフレックス〔Ecoflex（登録商標）：ＢＡＳＦジャパン株式会社（日本国、東京）製〕である。芳香族ポリエステルとしてはポリエチレンテレフタレート(PET)が挙げられるがこれらに限定されない。 In the present invention, “polyester” includes aliphatic polyester, aliphatic aromatic copolyester, and aromatic polyester. Of these, poly (caprolactone) (PCL), poly (butylene succinate-co-adipate) (PBSA), poly (butylene succinate) (PBS), poly (L-lactic acid) ( PLLA), poly (D-lactic acid) (PDLA), and poly-3-hydroxybutyric acid (PHB), but are not limited to these. One preferred example of the aliphatic aromatic copolyester is terephthalic acid as the aromatic dicarboxylic acid, C2 to C4 glycol monomers and / or oligomers as the diol, and HOC (O) (CH 2 as the aliphatic dicarboxylic acid. ) n Polyester produced by polymerizing a raw material containing C (O) OH (n represents an integer of 0 to 10). Particularly preferable examples are commercially available apexa (Apexa (registered trademark), manufactured by DuPont (Tokyo, Japan)) and Ecoflex (Ecoflex (registered trademark): manufactured by BASF Japan (Tokyo, Japan)). . Aromatic polyesters include, but are not limited to, polyethylene terephthalate (PET).
本発明のクチナーゼは、好熱性Saccharomonospora viridis AHK190（受託番号ＦＥＲＭ Ｐ−２１５０７）から単離及び同定された推定クチナーゼ(cutinase)、その相同タンパク質、又はその変異型タンパク質である。 The cutinase of the present invention is a putative cutinase, a homologous protein thereof, or a mutant protein thereof isolated and identified from the thermophilic Saccharomonospora viridis AHK190 (Accession No. FERM P-21507).
すなわち本発明は、下記（ａ）〜（ｃ）のいずれかのタンパク質を提供するものである。
（ａ）配列番号４で表されるアミノ酸配列からなるタンパク質
（ｂ）配列番号４で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質
（ｃ）配列番号４で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質
ここで、配列番号４で表されるアミノ酸配列において、「１若しくは数個のアミノ酸が欠失、置換、若しくは付加されたアミノ酸配列」とは、配列番号４のアミノ酸配列とそれぞれ等価のアミノ酸配列を意味し、１若しくは数個、好ましくは１〜１０個、より好ましくは１〜５個、さらにより好ましくは１〜３個のアミノ酸が欠失、置換、若しくは付加されたアミノ酸配列であって、依然としてクチナーゼ活性を保持する配列をいう。
That is, the present invention provides any one of the following proteins (a) to (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having a cutinase activity (c) sequence A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by No. 4 and having cutinase activity. Here, the amino acid sequence represented by SEQ ID No. 4 In the above, “amino acid sequence in which one or several amino acids are deleted, substituted, or added” means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 4, one or several, preferably 1 to An amino acid in which 10 amino acids, more preferably 1 to 5, even more preferably 1 to 3 amino acids have been deleted, substituted or added; A amino acid sequence that still retains cutinase activity.
配列番号４で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質である変異型タンパク質としては、（ｉ）配列番号４で表されるアミノ酸配列の２８位のグルタミン酸から４１位のスレオニンまでの間、（ｉｉ）５８位のバリンから６９位のメチオニンまでの間、（ｉｉｉ）８４位のフェニルアラニンから９５位のプロリンまでの間、（ｉｖ）１５２位のイソロイシンから１７０位のスレオニンまでの間、（ｖ）１７３位のイソロイシンから１８６位のヒスチジンまでの間、（ｖｉ）２０２位のチロシンから２１３位のプロリンまでの間、及び（ｖｉｉ）２３９位のセリンから２５２位のグルタミン酸までの間の少なくともいずれか一つの箇所で、１若しくは数個、好ましくは１〜１０個、より好ましくは１〜５個、さらにより好ましくは１〜３個のアミノ酸が欠失、置換、挿入若しくは付加されたアミノ酸配列を包含する。 In the amino acid sequence represented by SEQ ID NO: 4, a mutant protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, and having a cutinase activity includes (i) Between glutamic acid at position 28 to threonine at position 41 of the amino acid sequence represented by SEQ ID NO: 4, (ii) between valine at position 58 to methionine at position 69, (iii) phenylalanine at position 84 to position 95 (Iv) between isoleucine at position 152 to threonine at position 170, (v) between isoleucine at position 173 to histidine at position 186, (vi) from tyrosine at position 202 to proline at position 213 And (vii) at least any between serine at position 239 to glutamic acid at position 252 An amino acid sequence in which one or several, preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3 amino acids are deleted, substituted, inserted or added at one place. Include.
置換されるアミノ酸としては野生型のグルタミン酸（E）、グルタミン（Q）、アスパラギン酸（D）、アスパラギン（N）、アルギニン（R）、リジン（K）、ヒスチジン（H）、セリン（S）、トレオニン（T）、プロリン（P）等であり、代わって挿入又は付加されるアミノ酸としてはプロリン（P）、ロイシン（L）、イソロイシン（I）、バリン（V）、フェニルアラニン（F）、トリプトファン（W）、チロシン（Y）等である。 As amino acids to be substituted wild type glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), arginine (R), lysine (K), histidine (H), serine (S), Threonine (T), proline (P), and the like. Instead, amino acids inserted or added include proline (P), leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan ( W), tyrosine (Y) and the like.
本発明は、配列番号４で表されるアミノ酸配列からなる野生型クチナーゼに対して耐熱性が改善された上記のクチナーゼの相同タンパク質及び上記のクチナーゼの変異型タンパク質を提供する。 The present invention provides a homologous protein of the above cutinase and a mutant protein of the above cutinase, which have improved heat resistance with respect to the wild type cutinase consisting of the amino acid sequence represented by SEQ ID NO: 4.
さらに本発明は、配列番号４で表されるアミノ酸配列に対し、
１８２位のセリンのプロリンによる置換、
１８４位のアルギニンのセリンによる置換、及び
２１８位のスレオニンのリジンによる置換
のうちの少なくともいずれか一つを有する変異型タンパク質を提供するものである。
Furthermore, the present invention relates to the amino acid sequence represented by SEQ ID NO: 4,
Substitution of serine at position 182 with proline,
Substitution of arginine at position 184 with serine, and
The present invention provides a mutant protein having at least one of substitution of threonine at position 218 with lysine.
配列番号４で表されるアミノ酸配列を有するクチナーゼの上記相同タンパク質又は上記変異型タンパク質は、好ましくは野生型クチナーゼに対し高い耐熱性すなわち熱安定性を示す。例えば、１８２位のセリンのプロリンによる置換、１８４位のアルギニンのセリンによる置換、及び２１８位のスレオニンのリジンによる置換の少なくともいずれか一つにより、変異型タンパク質の高い活性と耐熱性がもたらされる。かかる変異型タンパク質の至適ｐＨは通常6.5-7.0であり、基質がPBSAの場合の至適ｐＨは8.0である。至適温度は65℃であり、20%のグリセリンの存在下では至適温度は65-75℃に増大する。また、かかる変異型タンパク質は50-65℃で24時間安定であり、高い耐熱性すなわち熱安定性を示す。 The homologous protein or the mutant protein of the cutinase having the amino acid sequence represented by SEQ ID NO: 4 preferably exhibits high heat resistance, that is, heat stability to the wild type cutinase. For example, at least one of substitution of serine at position 182 with proline, substitution of arginine at position 184 with serine, and substitution of threonine at position 218 with lysine results in high activity and heat resistance of the mutant protein. The optimum pH of such a mutant protein is usually 6.5-7.0, and the optimum pH when the substrate is PBSA is 8.0. The optimum temperature is 65 ° C, and in the presence of 20% glycerin, the optimum temperature increases to 65-75 ° C. Further, such a mutant protein is stable at 50-65 ° C. for 24 hours, and exhibits high heat resistance, that is, heat stability.
好ましくは、かかる変異型タンパク質は、１８２位のセリンのプロリンによる置換を有する変異型タンパク質、１８２位のセリンのプロリンによる置換及び１８４位のアルギニンのセリンによる置換を有する変異型タンパク質、又は１８２位のセリンのプロリンによる置換、１８４位のアルギニンのセリンによる置換、及び２１８位のスレオニンのリジンによる置換を有する変異型タンパク質である。 Preferably, such mutant proteins, 182 of the mutant protein having a substitution with proline serine, mutant proteins with substitution of serine for arginine substitution and 184 of by 182 serine proline, or position 182 of the A mutant protein having substitution of serine with proline, substitution of arginine at position 184 with serine, and substitution of threonine at position 218 with lysine.
また本発明は、配列番号２で表されるアミノ酸配列からなるタンパク質を提供するものである。 The present invention also provides a protein comprising the amino acid sequence represented by SEQ ID NO: 2.
さらに本発明は、配列番号２で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質、並びに配列番号２で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質も包含する。配列番号２で表されるアミノ酸配列の１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加された変異型タンパク質の場合、好ましい変異部位は配列番号４の変異型タンパク質について上述したのと同様である。 Furthermore, the present invention comprises an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and a protein having cutinase activity and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2. It also includes a protein having an amino acid sequence in which the amino acid is deleted, substituted, inserted or added and having cutinase activity. In the case of a mutant protein in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 2 are deleted, substituted, inserted or added, preferred mutation sites are the same as those described above for the mutant protein of SEQ ID NO: 4. It is.
本発明には、上記のいずれかのタンパク質をコードする遺伝子も含まれる。 The present invention also includes genes encoding any of the above proteins.
本発明はまた、下記（ｉ）〜（ｉｖ）のいずれかのポリヌクレオチドからなる遺伝子を提供するものである。
（ｉ）配列番号３で表されるヌクレオチド配列からなるポリヌクレオチド
（ｉｉ）配列番号３で表されるヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｉｉｉ）配列番号３で表されるヌクレオチド配列と９０％以上の同一性を有するポリヌクレオチドからなり、クチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉ）配列番号３で表されるヌクレオチド配列において、１もしくは数個の塩基が欠失、置換、挿入もしくは付加されたヌクレオチド配列からなり、かつクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
本発明はさらに、下記（ｖ）〜（ｖｉｉｉ）のいずれかのポリヌクレオチドからなる遺伝子を提供するものである。
（ｖ）配列番号１で表されるヌクレオチド配列からなるポリヌクレオチド
（ｖｉ）配列番号１で表されるヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉｉ）配列番号１で表されるヌクレオチド配列と９０％以上の同一性を有するポリヌクレオチドからなり、クチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
（ｖｉｉｉ）配列番号１で表されるヌクレオチド配列において、１もしくは数個の塩基が欠失、置換、挿入もしくは付加されたヌクレオチド配列からなり、かつクチナーゼ活性を有するタンパク質をコードするポリヌクレオチド
本発明の遺伝子には、配列番号１又は配列番号３のヌクレオチド（塩基）配列で示されるポリヌクレオチドにおいて、変異剤処理、ランダム変異、特定部位突然変異、欠損あるいは挿入等によって部分的に塩基配列が変化したものであっても、クチナーゼ活性を有するタンパク質をコードするポリヌクレトチドからなる遺伝子が包含される。
The present invention also provides a gene comprising a polynucleotide of any one of (i) to (iv) below.
(I) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 (ii) a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 3 under stringent conditions and encodes a protein having a cutinase activity (Iii) a polynucleotide comprising a polynucleotide having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 3 and encoding a protein having cutinase activity (vi) in the nucleotide sequence represented by SEQ ID NO: 3, A polynucleotide comprising a nucleotide sequence in which one or several bases have been deleted, substituted, inserted or added, and encoding a protein having cutinase activity. The present invention further includes any one of the following (v) to (viii) Provide genes consisting of polynucleotides It is something to offer.
(V) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (vi) a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having a cutinase activity (Vii) a polynucleotide consisting of a polynucleotide having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and encoding a protein having cutinase activity (viii) in the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide comprising a nucleotide sequence in which one or several bases have been deleted, substituted, inserted or added, and encoding a protein having cutinase activity. The gene of the present invention includes nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 ( Base) sequence indicated by the sequence Renucleotides include genes consisting of polynucleotides that encode proteins with cutinase activity, even if the nucleotide sequence is partially altered by mutagen treatment, random mutation, specific site mutation, deletion or insertion, etc. The
ここで「ストリンジエントな条件」とは、例えばMolecular cloning-a laboratory manual,2 nd edition（Sambrookら、1989）に記載の条件等が挙げられる。即ち、６ＸＳＳＣ（１ＸＳＳＣの組成：０．１５Ｍ塩化ナトリウム、０．０１５Ｍクエン酸ナトリウム、ｐＨ７．０）、０．５％ＳＤＳ、５Ｘデンハルト液及び１００ｍｇ／ｍＬニシン精子ＤＮＡを含む溶液にプローブとともに６５℃で８〜１６時間インキュベートし、ハイブリダイズさせる条件等が挙げられる。 Here, "stringent conditions" are, for example, Molecular cloning-a laboratory manual, 2 nd edition (Sambrook et al., 1989), and the conditions described. That is, 65 ° C. with a probe in a solution containing 6XSSC (composition of 1XSSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhardt's solution and 100 mg / mL herring sperm DNA And the like, and the like.
なお、アミノ酸配列及び塩基配列の同一性は、例えば、Lipman-Pearson法（Science, 227,1435, 1985）等の公知のアルゴリズムを用いてアミノ酸配列を比較することにより行うことができる。 The identity between the amino acid sequence and the base sequence can be determined by comparing the amino acid sequences using a known algorithm such as the Lipman-Pearson method (Science, 227, 1435, 1985).
また「クチナーゼ活性」には、エステル結合の分解活性（例えばpNP-acyl esters又はグリセリン脂肪酸エステル分解活性から推定されるエステル化合物又は脂質の分解活性）、エステル化反応、クチン及びスベリン等の木質層分解活性、及びポリエステル分解活性が含まれ、「クチナーゼ活性を有する」とは、上記のクチナーゼ活性の少なくともいずれか一つを有することを指す。好ましくは、本発明のクチナーゼの相同タンパク質又はその変異タンパク質は、野生型のクチナーゼよりも優れたクチナーゼ活性を有する。 In addition, “cutinase activity” includes ester bond degradation activity (eg, ester compound or lipid degradation activity estimated from pNP-acyl esters or glycerin fatty acid ester degradation activity), esterification reaction, wood layer degradation such as cutin and suberin. Activity and polyester degradation activity are included, and “having cutinase activity” means having at least one of the above cutinase activities. Preferably, the cutinase homologous protein of the present invention or a mutant protein thereof has a cutinase activity superior to that of the wild type cutinase.
本発明のクチナーゼは、好熱性Saccharomonospora viridis AHK190の培養液から得ることが可能であるが、その遺伝子を遺伝子工学的にクローニングし、生産、取得することも可能である。 The cutinase of the present invention can be obtained from a culture solution of thermophilic Saccharomonospora viridis AHK190, but the gene can also be cloned, produced and obtained by genetic engineering.
本発明のクチナーゼ遺伝子のクローニング方法としては、例えば当該遺伝子を安定に増幅できるＤＮＡベクターに連結させる、あるいは当該遺伝子を安定に維持できる染色体ＤＮＡ上に導入させる等の方法で本発明のクチナーゼをコードするＤＮＡを安定に増幅し、さらに当該遺伝子を安定にかつ効率よく発現させることが可能である宿主に導入し、クチナーゼを生産させる方法が採用できる。 As a method for cloning the cutinase gene of the present invention, for example, the cutinase gene of the present invention is encoded by a method such as ligation to a DNA vector capable of stably amplifying the gene or introduction into a chromosomal DNA capable of stably maintaining the gene. A method can be employed in which DNA is stably amplified and introduced into a host capable of stably and efficiently expressing the gene to produce cutinase.
また、本発明のクチナーゼ遺伝子を含む組換えベクターを作製するには、宿主菌体内で複製維持が可能であり、クチナーゼを安定に発現させることができ、当該遺伝子を安定に保持できるベクターにクチナーゼ遺伝子を組込めばよい。係る発現用ベクターとしては、大腸菌ベクター又は枯草菌ベクター等が挙げられる。 Further, in order to produce a recombinant vector containing the cutinase gene of the present invention, it is possible to maintain replication in the host cell, stably express cutinase, and cutinase gene into a vector capable of stably holding the gene. Can be incorporated. Examples of such expression vectors include E. coli vectors and Bacillus subtilis vectors.
得られた組換えベクターを用いて宿主細胞を形質転換するには、塩化カルシウム法、プロトプラスト法、コンピテントセル法やエレクトロポレーション法等を用いることができる。宿主細胞としては、例えば大腸菌、枯草菌、カビ、酵母、放線菌等が挙げられ、好ましくは大腸菌である。 In order to transform host cells using the obtained recombinant vector, a calcium chloride method, a protoplast method, a competent cell method, an electroporation method, or the like can be used. Examples of host cells include Escherichia coli, Bacillus subtilis, mold, yeast, actinomycetes, and the like, preferably Escherichia coli.
形質転換体は、該生物が資化し得る炭素源、窒素源、無機金属塩、ビタミン等を含む培地を用いて適度な条件下で培養すればよい。培養液から、一般的な方法により酵素の採取、精製を行い、凍結乾燥、噴霧乾燥、結晶化により必要な酵素形態を得ることができる。 The transformant may be cultured under appropriate conditions using a medium containing a carbon source, a nitrogen source, an inorganic metal salt, a vitamin and the like that can be assimilated by the organism. From the culture solution, the enzyme is collected and purified by a general method, and a necessary enzyme form can be obtained by lyophilization, spray drying, and crystallization.
本発明はまた、本発明のタンパク質又は形質転換体を、ポリエステル又はエステル化合物に接触させることを含む、ポリエステル又はエステル化合物の分解方法を提供するものである。ここで、「エステル化合物」とは、ポリマーではない、エステル結合を有する化合物を指す。エステル化合物としてはクチン、スベリン等の植物成分、グリセリン脂肪酸エステル等の脂質類、水溶性および水不溶性合成エステル化合物等が挙げられる。 The present invention also provides a method for decomposing a polyester or ester compound, which comprises contacting the protein or transformant of the present invention with the polyester or ester compound. Here, the “ester compound” refers to a compound having an ester bond that is not a polymer. Examples of the ester compound include plant components such as cutin and suberin, lipids such as glycerin fatty acid ester, and water-soluble and water-insoluble synthetic ester compounds.
分解されるポリエステル又はエステル化合物の試料は、液体又は固体の試料であってよい。分解されるポリエステル又はエステル化合物の試料が固体の場合、例えば、樹脂成形体、フィルム状、シート状、繊維状、トレイ状、ボトル状、パイプ状、その他特定形状等を有する、例えば、包装用資材、農業用資材、土木用資材、建築用資材、漁業用資材、衣料用資材、自動車部品、家電部品、その他工業用資材等の各種のものが挙げられ、これらは、熱可塑性樹脂の通常の溶融成形法、例えば、押出成形、圧縮成形、射出成形、中空成形、回転成形等、並びに、更にそれらに熱成形、延伸成形、発泡成形等の二次成形法を適用して成形される。 The sample of polyester or ester compound to be degraded may be a liquid or solid sample. When the sample of the polyester or ester compound to be decomposed is solid, it has, for example, a resin molded body, a film shape, a sheet shape, a fiber shape, a tray shape, a bottle shape, a pipe shape, and other specific shapes. , Agricultural materials, civil engineering materials, building materials, fishery materials, clothing materials, automobile parts, home appliance parts, other industrial materials, etc., these are the normal melting of thermoplastic resin The molding is performed by, for example, extrusion molding, compression molding, injection molding, hollow molding, rotational molding, and the like, and further applying a secondary molding method such as thermoforming, stretch molding, foam molding or the like.
本発明のポリエステル又はエステル化合物の分解方法において、本発明のタンパク質又は形質転換体をポリエステル、特にはポリエステル系樹脂成形体に接触させるには、本発明の酵素を含有する反応混合液を、ポリエステル系樹脂成形体の表面に噴霧、散布、或いは塗布するか、又はポリエステル系樹脂成形体を該反応混合液に浸漬させることにより、成形体に酵素を接触させるのが好ましい。この場合、反応混合液はカルシウムイオン（Ca2+）を含むことが好ましく、Ca2+の濃度は特に限定されないが通常25mMから50Mの範囲である。また、反応混合液は、ポリエステルフィルムの分解を促進する、DEPA、triethylene glycol dihexanoate, tetraethylene glycol diheptanoate, bis(2-ethylhexyl)sodium sulfosuccinate, bis(2-ethy hexyl)phthalate, di-n-octyl phthalate, 及びtributyl citrate等の可塑剤をさらに含んでもよい。 In the method for decomposing a polyester or ester compound of the present invention, in order to bring the protein or transformant of the present invention into contact with a polyester, particularly a polyester resin molded product, a reaction mixture containing the enzyme of the present invention is used. It is preferable that the enzyme is brought into contact with the molded body by spraying, spraying, or coating the surface of the resin molded body, or immersing the polyester-based resin molded body in the reaction mixture. In this case, the reaction mixture preferably contains calcium ions (Ca 2+ ), and the concentration of Ca 2+ is not particularly limited, but is usually in the range of 25 mM to 50 M. The reaction mixture also promotes the degradation of the polyester film, DEPA, triethylene glycol dihexanoate, tetraethylene glycol diheptanoate, bis (2-ethylhexyl) sodium sulfosuccinate, bis (2-ethy hexyl) phthalate, di-n-octyl phthalate, And a plasticizer such as tributyl citrate.
反応混合液のｐＨは通常5-9、好ましくは6.5-8.0とする。 The pH of the reaction mixture is usually 5-9, preferably 6.5-8.0.
反応の温度は通常20-70℃、好ましくは50-65℃、より好ましくは65℃前後とし、20%のグリセリンの存在下では好ましくは65-75℃とする。 The reaction temperature is usually 20-70 ° C., preferably 50-65 ° C., more preferably around 65 ° C., and preferably 65-75 ° C. in the presence of 20% glycerin.
反応時間は通常数時間〜数日、例えば１時間〜2日間（48時間）とする。 The reaction time is usually several hours to several days, for example, 1 hour to 2 days (48 hours).
一つの実施形態では、ポリエステル糸が用いられた布等に、本発明のクチナーゼ（相同タンパク質又は変異型タンパク質を含む）又は本発明のクチナーゼ遺伝子（相同ポリヌクレオチド又は変異型ポリヌクレオチドを含む）を含有するベクターを含む形質転換体を含む液を接触させることによって表面加工を行い、例えば風合いを改良したりすることができる。 In one embodiment, the cloth or the like using polyester yarn contains the cutinase (including homologous protein or mutant protein) of the present invention or the cutinase gene (including homologous polynucleotide or mutant polynucleotide) of the present invention. Surface treatment can be carried out by bringing a solution containing a transformant containing the vector to be brought into contact, for example, to improve the texture.
また、本願発明のクチナーゼは、バイオディーゼル、エステル化合物の合成にも応用できる。 The cutinase of the present invention can also be applied to the synthesis of biodiesel and ester compounds.
本明細書中に引用されているすべての特許出願および文献の開示は、それらの全体が参照により本明細書に組み込まれるものとする。 The disclosures of all patent applications and documents cited herein are hereby incorporated by reference in their entirety.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明がこれらに限定されないことは言うまでもない。 Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.
（材料および方法）
１．細菌株と化学薬品
S.viridis AHK190は堆肥(日本国、岡山)から分離し、寄託番号FERM P-21507の下、国際特許生物寄託機関（IPOD）である製品評価技術基盤機構 (NITE;日本国、千葉)に寄託した（特開2009-39095）。AHK190菌株は振とう下50℃でLuria-Bertani培地 (LB)にて培養した。クローニングのため、大腸菌DH5αおよびRosetta-gami B(DE3)を宿主として使用した。形質転換体は50μg/mlアンピシリンを補充したLB培地で、必要な場合には、0.1mMイソプロピルβ-D-1-チオガラクトピラノシド (IPTG)または1mM 5-ブロモ-4-クロロ-3-インドキシル-β-D-ガラクトピラノシドを補充したLB培地で増殖させた。ポリマーペレットを使用してポリマー寒天平板を調製した：ポリ(カプロラクトン)(PCL; 平均分子量(Mw)=40,000)は和光純薬工業株式会社(日本国、大阪)から購入した。ポリ(ブチレンスクシナート-co-アジペート) (PBSA) (Bionolle(登録商標)EM-301; 重量平均分子量=1.0×105)およびポリ(ブチレンスクシナート)(PBS)(Bionolle（登録商標）#1020;重量平均分子量=1.0×105)は、昭和電工株式会社 (日本国、東京)の製品である。Ecoflex（登録商標）はテレフタル酸、1，4−ブタンジオール、アジピン酸および未知の化合物からなる脂肪族芳香族ポリエステルである（Kleeberg I, et al. Biomacromolecules 6:262-270, 2005.）。ポリ(L-乳酸) (PLLA;重量平均分子量=1.69×105)およびポリ(D-乳酸)（PDLA;重量平均分子量=1.63×105）は、以前に記述された通りに合成した(Kawai F, et al., Polym. Degrad. Stab. 96:1342-1348, 2011)。ポリ−3−ヒドロキシ酪酸(PHB)は三菱ガス化学株式会社（日本国、東京）より提供された。ポリマー寒天平板は以前に記述された通りに調製した(Thumarat U, et al., Appl. Microbiol. Biotechnol. 95:419-430, 2012)。
(Materials and methods)
1. Bacterial strains and chemicals
S.viridis AHK190 was separated from compost (Okayama, Japan) and deposited with the National Institute of Technology and Evaluation (NITE; Chiba, Japan) under the deposit number FERM P-21507 (JP 2009-39095). The AHK190 strain was cultured in Luria-Bertani medium (LB) at 50 ° C. with shaking. For cloning, E. coli DH5α and Rosetta-gami B (DE3) were used as hosts. Transformants are LB medium supplemented with 50 μg / ml ampicillin, if necessary, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) or 1 mM 5-bromo-4-chloro-3- Growing in LB medium supplemented with indoxyl-β-D-galactopyranoside. Polymer agar plates were prepared using polymer pellets: Poly (caprolactone) (PCL; average molecular weight (Mw) = 40,000) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Poly (butylene succinate-co-adipate) (PBSA) (Bionolle® EM-301; weight average molecular weight = 1.0 × 10 5 ) and poly (butylene succinate) (PBS) (Bionolle®) # 1020; weight average molecular weight = 1.0 × 10 5 ) is a product of Showa Denko KK (Tokyo, Japan). Ecoflex® is an aliphatic aromatic polyester consisting of terephthalic acid, 1,4-butanediol, adipic acid and an unknown compound (Kleeberg I, et al. Biomacromolecules 6: 262-270, 2005.). Poly (L-lactic acid) (PLLA; weight average molecular weight = 1.69 × 10 5 ) and poly (D-lactic acid) (PDLA; weight average molecular weight = 1.63 × 10 5 ) were synthesized as previously described (Kawai F, et al., Polym. Degrad. Stab. 96: 1342-1348, 2011). Poly-3-hydroxybutyric acid (PHB) was provided by Mitsubishi Gas Chemical Co., Inc. (Tokyo, Japan). Polymer agar plates were prepared as previously described (Thumarat U, et al., Appl. Microbiol. Biotechnol. 95: 419-430, 2012).
PBSA(Bionolle(登録商標) 3001G、Mｗ=2.0-2.5×105、20μm)およびPBS(Bionolle (登録商標) 1001G、Mw=2.0-2.5×105、20μm)フィルムは、昭和電工株式会社により提供された。0.25mm厚非晶形PETフィルムをGoodfellows Cambridge株式会社(日本国、東京)から得た。PCL、Ecoflex(登録商標)およびPDLAのキャストフィルムを以前に記述された通りに調製した(Kawai F, et al., Polym. Degrad. Stab. 96:1342-1348, 2011)。他のすべての化学薬品は、利用可能な最高グレードのものとした。 PBSA (Bionolle (registered trademark) 3001G, Mw = 2.0-2.5 × 10 5 , 20 μm) and PBS (Bionolle (registered trademark) 1001G, Mw = 2.0-2.5 × 10 5 , 20 μm) films are provided by Showa Denko KK It was done. A 0.25 mm thick amorphous PET film was obtained from Goodfellows Cambridge, Inc. (Tokyo, Japan). PCL, Ecoflex® and PDLA cast films were prepared as previously described (Kawai F, et al., Polym. Degrad. Stab. 96: 1342-1348, 2011). All other chemicals were of the highest grade available.
２．S.viridis AHK190からの推定クチナーゼコード遺伝子およびその周辺領域の配列決定
全DNAをGentra Puregene Yeast/Bacteria Kit(Qiagen K.K.、日本国、東京)を使用して、S.viridis AHK190から抽出した。DNAの精製、形質転換および電気泳動をSambrookおよびRussellのプロトコル（Sambrook J, et al. 2001. Molecular cloning: a laboratory manual, 3rd ed.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY）に従って行った。制限酵素およびその他のDNA修飾酵素はTOYOBO(日本国、大阪)またはTakara Bio社(日本国、滋賀、大津市)から購入し、製造業者により指定された通りに使用した。KOD DNAポリメラーゼを製造業者(TOYOBO)の指示に従いPCRのために使用した。S.viridis DSM 43017(CP001683)の配列に基づいて、遺伝子(cut190)およびその上流及び下流領域を増幅するための適切なプライマーを設計した (表１)。PCR混合物(25μl)は1μｇの全DNA、0.3 pmol/μlの各プライマー、1mM MgCl2、0.2mMの各デオキシリボヌクレオチド、1×KOD DNAポリメラーゼ緩衝液、4%(v/v)ジメチルスルホキシドおよび0.5U KOD DNAポリメラーゼを含んでいた。増幅されたフラグメントを精製し、SolGent社(Daejeon、韓国)により配列決定し、GENETYXソフトウェアバージョン5.1(GENETYX社、日本国、東京)を使用してアセンブルした。計5,811bpのフラグメントが配列決定され、S.viridis DSM 43017の同じ領域と正確に一致することが見出され、cut190のORFは915 bp(304個のアミノ酸に相当)を含んでいた。ORF中の44個のアミノ酸を含むシグナルペプチド配列の存在を予測するためにSignalP 4.1 Server(http://www.cbs.dtu.dk/services/SignalP/)を使用した。
2. Sequencing of the putative cutinase-encoding gene from S. viridis AHK190 and its surrounding region Total DNA was extracted from S. viridis AHK190 using the Gentra Puregene Yeast / Bacteria Kit (Qiagen KK, Tokyo, Japan). DNA purification, transformation and electrophoresis were performed according to the Sambrook and Russell protocol (Sambrook J, et al. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Restriction enzymes and other DNA modifying enzymes were purchased from TOYOBO (Osaka, Japan) or Takara Bio (Shiga, Otsu, Japan) and used as specified by the manufacturer. KOD DNA polymerase was used for PCR according to the manufacturer's instructions (TOYOBO). Based on the sequence of S. viridis DSM 43017 (CP001683), appropriate primers were designed to amplify the gene (cut190) and its upstream and downstream regions (Table 1). The PCR mixture (25 μl) contains 1 μg of total DNA, 0.3 pmol / μl of each primer, 1 mM MgCl 2 , 0.2 mM of each deoxyribonucleotide, 1 × KOD DNA polymerase buffer, 4% (v / v) dimethyl sulfoxide and 0.5 U. KOD DNA polymerase was included. Amplified fragments were purified, sequenced by SolGent (Daejeon, Korea) and assembled using GENETYX software version 5.1 (GENETYX, Tokyo, Japan). A total of 5,811 bp fragments were sequenced and found to match exactly the same region of S. viridis DSM 43017, and the cut190 ORF contained 915 bp (corresponding to 304 amino acids). SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the presence of a signal peptide sequence containing 44 amino acids in the ORF.
３．推定クチナーゼ遺伝子(Cut190)のクローニング
順方向プライマーのCut190F(5′-GACCAGTTGCCTGGACTCTC-3′)および逆方向プライマーのCut190R(5′-GCGGACTCCGATATTCAGC-3′)を使用してcut190フラグメント(44個のシグナルペプチドアミノ酸に対応するヌクレオチドを除く)（配列番号３）を増幅し、これを精製し、pGEM-T easy vector（Promega、アメリカ合衆国ワィスコンシン州Madison）にライゲートした。プラスミド(pGEM-cut190)を従来の青白コロニースクリーニングのため大腸菌DH5αに形質転換した。プラスミドを白いコロニーから抽出し、単離したプラスミドの両方の鎖をSolGent社により配列決定した。pGEM-cut190中の成熟Cut190(260個のアミノ酸)に相当する正しいインサートを、順方向プライマーCut190F2(5′-GCCGGATCCCAGGACAACCCTTACGAACG-3′)および逆方向プライマーCut190R2(5′-GCGAAGCTTGTACGGGCAGGTCGAGCGG-3′)を用いて増幅し (BamHIとHindIIIの制限部位にそれぞれ下線)、BamH1およびHindIII で消化し、精製してpQE80LのBamHIおよびHindIII部位にライゲートし、6×Hisでタグ付けされた融合タンパク質としてCut190をクローニングした。その後、プラスミド(pQE80L-cut190)を熱ショック法(Sambrook J, et al. 2001. Molecular cloning: a laboratory manual, 3rd ed. 561 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)により、大腸菌Rosetta-gami B(DE3)に導入し、形質転換した。
3. Cloning of Putative Cutinase Gene (Cut190) Cut190 fragment (44 signal peptide amino acids) using the forward primer Cut190F (5′-GACCAGTTGCCTGGACTCTC-3 ′) and the reverse primer Cut190R (5′-GCGGACTCCGATATTCAGC-3 ′) (SEQ ID NO: 3) was amplified, purified and ligated into the pGEM-T easy vector (Promega, Madison, Wis., USA). The plasmid (pGEM-cut190) was transformed into E. coli DH5α for conventional blue-white colony screening. The plasmid was extracted from white colonies and both strands of the isolated plasmid were sequenced by SolGent. Correct inserts corresponding to mature Cut190 (260 amino acids) in pGEM-cut190 are identified as forward primer Cut190F2 (5′-GCC GGATCC CAGGACAACCCTTACGAACG-3 ′) and reverse primer Cut190R2 (5′-GCG AAGCTT GTACGGGCAGGTCGAGCGG-3 ′ ) (Underlined restriction sites for BamHI and HindIII, respectively), digested with BamH1 and HindIII, purified, ligated to the BamHI and HindIII sites of pQE80L, and Cut190 as a fusion protein tagged with 6 × His. Was cloned. Thereafter, the plasmid (pQE80L-cut190) was obtained by the heat shock method (Sambrook J, et al. 2001. Molecular cloning: a laboratory manual, 3rd ed. 561 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). It was introduced into (DE3) and transformed.
４．酵素の発現、精製および活性の分析
pQE80L-cut190プラスミドを導入した大腸菌Rosetta-gami B(DE3)を50μg/mlアンピシリンを含むLB培地で37℃にてOD600が0.4-0.5に達するまで生育させた。その後、IPTGを終濃度0.1 mMとなるよう培地へ添加し、細胞を12-15時間37℃で培養した。細胞ペレットを8,000rpm 15分、4℃で遠心分離により収集し、0.85%(w/v)NaClで2回洗浄し、溶解緩衝液(20mM NaH2PO4、500mM NaClおよび20mMイミダゾールHCl、pH7.4)に再懸濁し、INSONATOR 201M(株式会社クボタ、日本、大阪)を使用して、200Wで5分間、氷上の超音波処理により破砕した。破砕した細胞は、10,000rpm、4℃および20分間遠心分離し、上澄みを無細胞抽出液として使用した。Ni-Sepharose 6 Fast Flowカラム(GE Healthcare、スウェーデン)を使用して、無細胞抽出液から組換え酵素を生成した。組換え酵素は、300mM イミダゾールHClを含む20mMTris-HCl緩衝液(pH 7.0)で溶出した。溶出された酵素は、PD-10カラム(GE Healthcare)に通過させることにより脱塩した。有効画分を収集し、精製された酵素として使用した。
4). Analysis of enzyme expression, purification and activity
Escherichia coli Rosetta-gami B (DE3) introduced with the pQE80L-cut190 plasmid was grown in LB medium containing 50 μg / ml ampicillin at 37 ° C. until the OD 600 reached 0.4-0.5. IPTG was then added to the medium to a final concentration of 0.1 mM and the cells were cultured for 12-15 hours at 37 ° C. Cell pellets were collected by centrifugation at 8,000 rpm for 15 minutes at 4 ° C., washed twice with 0.85% (w / v) NaCl, and lysis buffer (20 mM NaH 2 PO 4 , 500 mM NaCl and 20 mM imidazole HCl, pH 7. The suspension was resuspended in 4) and disrupted by sonication on ice for 5 minutes at 200 W using INSONATOR 201M (Kubota Corporation, Osaka, Japan). The disrupted cells were centrifuged at 10,000 rpm, 4 ° C. and 20 minutes, and the supernatant was used as a cell-free extract. Recombinant enzymes were produced from cell-free extracts using Ni-Sepharose 6 Fast Flow columns (GE Healthcare, Sweden). The recombinant enzyme was eluted with 20 mM Tris-HCl buffer (pH 7.0) containing 300 mM imidazole HCl. The eluted enzyme was desalted by passing through a PD-10 column (GE Healthcare). Effective fractions were collected and used as purified enzyme.
クーマシーブリリアントブルー染色でSDS-PAGE(Laemmli, UK. Nature 227:680-685, 1970)によりCut190の純度を確認し、Bio-Radタンパク質アッセイキット(Bio-Rad Laboratories、アメリカ合衆国カリフォルニア州Hercules)を使用してタンパク濃度を決定した。なお、標準としてウシ血清アルブミンを用いた。酵素活性は以前に記述されたように(Thumarat U, et al., Appl. Microbiol. Biotechnol. 95:419-430., 2012) pNPBを基質として300mM CaCl2存在下で生成されるp-ニトロプェノール(pNP)を346nmでの分光測光法により測定した(ε=5.48 mmol-1cm-1)。酵素活性の1カタールユニットは、標準アッセイ条件下で１秒あたり1molのpNPを生産できる酵素の量として定義した。比活性は1mgタンパク質当たりの酵素活性として定義した。ポリエステルその他に対するCut190の活性は、以前に(Hu X, et al, Appl. Microbiol. Biotechnol. 87:71-779, 2010)記述した通りに、0.1%ポリエステル(Ecoflex（登録商標）、PCL、PBSA、PBS、PDLA、PLLA及びPHB) または炭素鎖の異なるグリセリン脂肪酸エステル(トリブチリン、トリパルミチン、トリオレイン)を含むLB寒天平板上のハロー形成により確認した。また、Cut190活性は600nmでPBSA懸濁液 (Bionolle（登録商標） EM-301)の濁度の減少をモニタすることによっても測定された。PBSA分析の場合、反応混合物は全量2mlにPBSA(約1.0-1.5の最終OD600)、50mMTris-HCl緩衝液(pH 8.2)、25mM Ca2+およびCut190の適量を含んでいた。反応混合物は水浴中で温和に振動させて30分間、37℃でインキュベートした。測定は二連または三連にて行い、標準偏差は5%以内とした。 Purity of Cut190 is confirmed by SDS-PAGE (Laemmli, UK. Nature 227: 680-685, 1970) with Coomassie brilliant blue staining and using Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) The protein concentration was determined. In addition, bovine serum albumin was used as a standard. Enzymatic activity was determined as previously described (Thumarat U, et al., Appl. Microbiol. Biotechnol. 95: 419-430., 2012) p-nitrop produced in the presence of 300 mM CaCl 2 using pNPB as a substrate. Enol (pNP) was measured spectrophotometrically at 346 nm (ε = 5.48 mmol −1 cm −1 ). One Qatar unit of enzyme activity was defined as the amount of enzyme capable of producing 1 mol of pNP per second under standard assay conditions. Specific activity was defined as enzyme activity per mg protein. The activity of Cut190 against polyesters and others is as described previously (Hu X, et al, Appl. Microbiol. Biotechnol. 87: 71-779, 2010), as described in 0.1% polyester (Ecoflex®, PCL, PBSA, PBS, PDLA, PLLA and PHB) or halo formation on LB agar plates containing glycerin fatty acid esters (tributyrin, tripalmitin, triolein) with different carbon chains. Cut190 activity was also measured by monitoring the decrease in turbidity of PBSA suspension (Bionolle® EM-301) at 600 nm. For PBSA analysis, the reaction mixture contained appropriate amounts of PBSA (final OD 600 of about 1.0-1.5), 50 mM Tris-HCl buffer (pH 8.2), 25 mM Ca 2+ and Cut190 in a total volume of 2 ml. The reaction mixture was incubated at 37 ° C. for 30 minutes with gentle rocking in a water bath. Measurement was performed in duplicate or triplicate, and the standard deviation was within 5%.
５．部位特異的突然変異
部位特異的突然変異を、KOD-plus-mutagenesis kit (TOYOBO)を使用して製造業者の指示に従って行い、特定の部位に突然変異を導入した。部位特異的突然変異のためのDNAテンプレートとしてpQE80L-est119を使用して変異導入に適切なprimersを使用してPCR産物を得た。このテンプレートのpQE80L-est119プラスミドはDpnIでの消化により除去し、PCR産物は大腸菌Rosetta-gami B(DE3)細胞内へ直接形質転換した。形質転換菌からプラスミドを精製し、そのDNA配列をSolGent社により分析した。変異型酵素はすべて、野生型Cut190と同じ条件下で発現および精製された。野生型と変異型Cut190の発現および生成量に違いは示されなかった。
5. Site-specific mutations Site-specific mutations were performed using the KOD-plus-mutagenesis kit (TOYOBO) according to the manufacturer's instructions to introduce mutations at specific sites. PCR products were obtained using primers appropriate for mutagenesis using pQE80L-est119 as a DNA template for site-directed mutagenesis. The pQE80L-est119 plasmid of this template was removed by digestion with DpnI, and the PCR product was directly transformed into Escherichia coli Rosetta-gami B (DE3) cells. The plasmid was purified from the transformant and the DNA sequence was analyzed by SolGent. All mutant enzymes were expressed and purified under the same conditions as wild-type Cut190. There was no difference in the expression and production of wild-type and mutant Cut190.
６．野生型と変異型Cut190の特徴付け
pH範囲4.0-9.0(酢酸緩衝液はpH4.0-5.5、MES緩衝液はpH6.0-7.0、Tris-HCl緩衝液はpH 7.0-9.0)の終濃度100 mMの異なる緩衝液を使用して、標準アッセイ条件で至適pHを測定した。至適温度は、種々の温度(30-80℃)で20%のグリセリンの有無の下、標準アッセイ条件で決定した。pH安定性は、5℃で18時間 pH の異なる100mM緩衝液中で酵素をインキュベートした後の残存活性を測定することにより決定した。熱安定性は精製された酵素を、20%のグリセリン有無の下、300mM Ca2+を含む100mM Tris-HCl緩衝液 (pH 7.0)中で50-70℃の異なる温度で１時間インキュベートした後の残存活性を測定することにより決定した。活性に対するEDTAおよびフェニルメチルスルホニルフルオリド(PMSF)の影響は、PMSFとEDTAを含む100mM Tris-HCl緩衝液 (pH 7.0)中で１時間氷上で酵素をインキュベートした後の残存活性を測定することにより決定した。残存活性は標準アッセイ条件で測定した。測定はすべて二連で行なった。
6). Characterization of wild-type and mutant Cut190
Use different buffers with a final concentration of 100 mM in the pH range 4.0-9.0 (acetate buffer pH 4.0-5.5, MES buffer pH 6.0-7.0, Tris-HCl buffer pH 7.0-9.0). The optimum pH was measured under standard assay conditions. The optimum temperature was determined under standard assay conditions with and without 20% glycerin at various temperatures (30-80 ° C). The pH stability was determined by measuring the residual activity after incubating the enzyme in 100 mM buffer at different pH for 18 hours at 5 ° C. Thermostability was achieved after incubation of the purified enzyme in 100 mM Tris-HCl buffer (pH 7.0) containing 300 mM Ca 2+ in the presence or absence of 20% glycerin at 50-70 ° C. for 1 hour. Determined by measuring residual activity. The effect of EDTA and phenylmethylsulfonyl fluoride (PMSF) on activity was determined by measuring the residual activity after incubation of the enzyme on ice for 1 hour in 100 mM Tris-HCl buffer (pH 7.0) containing PMSF and EDTA. Were determined. Residual activity was measured under standard assay conditions. All measurements were done in duplicate.
７．円偏光二色性 (CD)測定および融点 (Tm)測定
CD測定値は、室温でモデルJ-720 Jasco分光偏光計(JASCO社、日本国、東京)を使用して得た。遠紫外線(200-250nm)および近紫外線(250-320nm)CDスペクトルを、2mmおよび5mmの光路長を有する石英セル中の50mM Tris-HCl緩衝液 (pH 7.0)における0.2mg/mlおよび1.0mg/mlのタンパク濃度のそれぞれに対して得た。Tmは、ナノ示差走査熱量計(nano-DSC)(モデル6100 NanoII DSC、Calorimetry Sciences社、アメリカ合衆国ユタ州Lindon)を用いて測定した。DSC測定のためのサンプルは、測定に先立って15分間真空中で脱気した。測定は、サンプルの泡立ちを防ぐために1℃/分の加熱速度で、3気圧の圧力下で行なった。Tm測定は50mM MES緩衝液 (pH 7.0)中で行った。
7). Circular dichroism (CD) measurement and melting point (Tm) measurement
CD measurements were obtained using a model J-720 Jasco spectropolarimeter (JASCO, Tokyo, Japan) at room temperature. Far ultraviolet (200-250 nm) and near ultraviolet (250-320 nm) CD spectra were obtained at 0.2 mg / ml and 1.0 mg / ml in 50 mM Tris-HCl buffer (pH 7.0) in a quartz cell with 2 mm and 5 mm path lengths. Obtained for each protein concentration in ml. Tm was measured using a nano differential scanning calorimeter (nano-DSC) (model 6100 NanoII DSC, Calorimetry Sciences, Lindn, Utah, USA). Samples for DSC measurement were degassed in vacuum for 15 minutes prior to measurement. The measurement was performed at a pressure of 3 atm at a heating rate of 1 ° C./min to prevent sample bubbling. Tm measurement was performed in 50 mM MES buffer (pH 7.0).
８．ポリエステルフィルムの分解試験
ポリエステルフィルムを、50mM Tris-HCl緩衝液(pH8.2)、30mM CaCl2および1.1 μM Cut190(S226P/R228S) を含む5mlの反応混合液中で振とう下、48℃でインキュベートした。各々約30mgのPCL、Ecoflex(登録商標)およびPDLAフィルムを、PBSAおよびPBSフィルム(各々約1×1cm)と共に試験に使用した。PETフィルムは50-100mM Tris-HCl緩衝液(pH8.2)、50mM CaCl2、20%グリセリンおよび約10μM Cut190(S226P/R228S)を含む反応混合物 (1.5ml)中で1% N,N-ジエチル-2-フェニルアセトアミド(DEPA)有無の条件下で、50、60および65℃で2日間インキュベートした。反応後、これらのフィルムを水で繰り返し洗浄し、最後は50%のエタノールで洗浄した。フィルムを、重量減少及び走査型電子顕微鏡測定(SEM)に先立って室温(PCL、Ecoflex(登録商標)、及びPDLA)および60℃(PET)で2日間乾燥させた。DEPAを含むPETフィルムは、同じ条件下で繰り返し第2および第3の反応を行った。重量減少は2連反応の平均値として表した。SEMはS-300N走査電子顕微鏡(日立株式会社、日本、東京)を使用した。
8). Polyester film degradation test Polyester film was incubated at 48 ° C under shaking in a 5 ml reaction mixture containing 50 mM Tris-HCl buffer (pH 8.2), 30 mM CaCl 2 and 1.1 μM Cut190 (S226P / R228S). did. About 30 mg each of PCL, Ecoflex® and PDLA films were used in the test with PBSA and PBS films (about 1 × 1 cm each). PET film is 1% N, N-diethyl in a reaction mixture (1.5 ml) containing 50-100 mM Tris-HCl buffer (pH 8.2), 50 mM CaCl 2 , 20% glycerin and about 10 μM Cut190 (S226P / R228S). Incubated for 2 days at 50, 60 and 65 ° C. with or without -2-phenylacetamide (DEPA). After the reaction, these films were repeatedly washed with water and finally washed with 50% ethanol. The film was dried at room temperature (PCL, Ecoflex®, and PDLA) and 60 ° C. (PET) for 2 days prior to weight loss and scanning electron microscopy (SEM). The PET film containing DEPA was repeatedly subjected to the second and third reactions under the same conditions. Weight loss was expressed as an average of duplicate reactions. SEM used an S-300N scanning electron microscope (Hitachi, Tokyo, Japan).
９．ポリエステル繊維分解試験
Apexa（登録商標）（デュポン株式会社製(日本国、東京)）又はポリエチレンテレフタレート(PET)の糸(75dt-36f (DTY))約30cm (約0.1mg)を、25 mM Tris-HCl緩衝液(pH8.15)、25mM CaCl2、12%グリセリン、14μM Cut190(S226P/R228S) 、及び1% N,N-ジエチル-2-フェニルアセトアミド(DEPA)を含む1mlの反応混合液中で、振とう下、60℃で5日間インキュベートした。また上記の条件で、分解酵素Cut190(S226P/R228S)を含まない試験（対照）も行なった。他方、PET繊維の織物(約2(2cm、緯糸及び経糸ともに84dt-36f (DTY))を1%DEPAの有無下で、50 mM Tris-HCl緩衝液(pH8.15)、50 mM CaCl2、20 %グリセリン、20μM Cut190(S226P/R228S)を含む1mlの反応混合液中で、振とう下、65℃で5日間インキュベートした。また上記の条件で、分解酵素Cut190(S226P/R228S)を含まない試験（対照）も行なった。dt: decitex, f: filament, DTY: draw textured yarn
反応後、これらの糸および織物を水で繰り返し洗浄し、最後は50%のエタノールで洗浄した。これらを60℃で2日間乾燥させ、SEMで観察した。SEMはS-300N走査電子顕微鏡(日立株式会社、日本、東京)を使用した。
（結果）
１．cut190の遺伝子クローニングおよび発現
915bpのクチナーゼ遺伝子(cut190遺伝子)（配列番号１）、3,020 bpの上流領域、および1,876 bpの下流領域を含む5,811bpフラグメントを配列決定した。クチナーゼであるCut190タンパク質（配列番号２）は、保存されたペンタペプチド配列モチーフ(GHSMG)を含むことが分かると共に、リパーゼスーパーファミリーの３つ組触媒基 (Ser176-Asp222-His254)が識別された。Cut190はT.fusca YX 由来のYP_288944.1およびYP_288943.1(それぞれT.cellulosilytica由来のADV92526.1およびADV92527.1に相当)（Lykidis A, et al., J. Bacteriol. 189:2477-2486, 2007）とそれぞれ65%および66%のアミノ酸配列同一性を有し、T.alba AHK119由来のEst119と64%、Est1と62%のアミノ酸配列同一性を有し、堆肥メタゲノム由来の配列HQ704839（Sulaiman S, et al, Microbiol. 78:1556-1562, 2012）と55％のアミノ酸配列同一性を有する。すべての配列はシグナルペプチドのない成熟酵素に相当する(図１)。
9. Polyester fiber degradation test
Apexa (registered trademark) (manufactured by DuPont Co., Ltd. (Tokyo, Japan)) or polyethylene terephthalate (PET) thread (75dt-36f (DTY)) of about 30 cm (about 0.1 mg) was added to 25 mM Tris-HCl buffer ( pH8.15), 25mM CaCl 2, 12 % glycerol, 14μM Cut190 (S226P / R228S) , and 1% N, in the reaction mixture of 1ml containing N- diethyl-2-phenyl acetamide (DEPA), with shaking Incubated at 60 ° C. for 5 days. Under the above conditions, a test (control) not containing the degrading enzyme Cut190 (S226P / R228S) was also conducted. On the other hand, PET fiber fabric (about 2 (2 cm, both weft and warp 84dt-36f (DTY)) with or without 1% DEPA, 50 mM Tris-HCl buffer (pH8.15), 50 mM CaCl2, 20 mM Incubated for 5 days at 65 ° C with shaking in a 1 ml reaction mixture containing% glycerin and 20 μM Cut190 (S226P / R228S), and a test without the degrading enzyme Cut190 (S226P / R228S) under the above conditions. (Control) was also performed: dt: decitex, f: filament, DTY: draw textured yarn
After the reaction, these yarns and fabrics were repeatedly washed with water and finally with 50% ethanol. These were dried at 60 ° C. for 2 days and observed by SEM. SEM used an S-300N scanning electron microscope (Hitachi, Tokyo, Japan).
(result)
1. gene cloning and expression of cut190
A 5,811 bp fragment containing a 915 bp cutinase gene (cut190 gene) (SEQ ID NO: 1), a 3,020 bp upstream region, and a 1,876 bp downstream region was sequenced. The cutinase Cut190 protein (SEQ ID NO: 2) was found to contain a conserved pentapeptide sequence motif (GHSMG) and a lipase superfamily triplet catalytic group (Ser176-Asp222-His254) was identified. Cut190 is YP_288944.1 and YP_288943.1 from T.fusca YX (corresponding to ADV92526.1 and ADV92527.1 from T. cellulosilytica, respectively) (Lykidis A, et al., J. Bacteriol. 189: 2477-2486, 2007) and amino acid sequence identity of 65% and 66%, respectively. Est119 and 64% from T.alba AHK119, 62% amino acid sequence identity from Est1 and compost metagenome sequence HQ704839 (Sulaiman S, et al, Microbiol. 78: 1556-1562, 2012) and 55% amino acid sequence identity. All sequences correspond to mature enzyme without signal peptide (FIG. 1).
また、Cut190の44個のシグナルペプチドアミノ酸がない成熟タンパク質をコードするcut190フラグメントのヌクレオチド配列を配列番号３で表し、Cut190の44個のシグナルペプチドアミノ酸がない成熟タンパク質であるCut190フラグメントのアミノ酸配列を配列番号４で表す。 In addition, the nucleotide sequence of the cut190 fragment encoding the mature protein that does not have 44 signal peptide amino acids of Cut190 is represented by SEQ ID NO: 3, and the amino acid sequence of the Cut190 fragment that is a mature protein that does not have 44 signal peptide amino acids of Cut190 This is represented by the number 4.
Thermobifida属クチナーゼの配列は高い同一性を共有していたが、Cut190とHQ704839は、Thermobifida属および相同性クチナーゼのテンプレートと考えられるEst119（Kitadokoro K, et al., Polym. Degrad. Stab. 97:771-775, 2012）のX線結晶解析に基づくとThermobifida属クチナーゼのいくつかの領域 (主に外側のα−ヘリックスおよびループ領域)の配列が異なっていた。組換えCut190を大腸菌で発現させ、Hisタグアフィニティクロマトグラフィーにより均一になるよう精製した(図２)。発現されたCut190の分子量は組換えCut190に対する予測値(30,339Da)と合致していた。Est119に対して以前に報告されたように(Thumarat U, et al., Appl. Microbiol. Biotechnol. 95:419-430, 2012)、基質としてpNPBを用いた酵素活性の予備的アッセイではCa2+が活性を増強することを示したため、「材料および方法」の節で説明されるように300mM Ca2+の存在下で標準酵素アッセイを行った。酵素はpNP-C4からC10までに活性を有し、pNP-C6に対して最も高い活性が観察されたが、pNP-C2ではほとんど活性が検出されなかった(図３)。Cut190はトリブチリン、トリパルミチン、トリオレインも活性を有することが判明した（データ省略）。 Although the sequence of Thermobifida cutinase shared high identity, Cut190 and HQ704839 were found to be templates for Thermobifida and homologous cutinases, Est119 (Kitadokoro K, et al., Polym. Degrad. Stab. 97: 771 -775, 2012), the sequence of several regions of Thermobifida cutinase (mainly outer α-helix and loop regions) differed. Recombinant Cut190 was expressed in E. coli and purified to homogeneity by His tag affinity chromatography (FIG. 2). The molecular weight of expressed Cut190 was consistent with the predicted value for recombinant Cut190 (30,339 Da). As previously reported for Est119 (Thumarat U, et al., Appl. Microbiol. Biotechnol. 95: 419-430, 2012), a preliminary assay for enzyme activity using pNPB as a substrate produced Ca 2+ Was shown to enhance activity, standard enzyme assays were performed in the presence of 300 mM Ca 2+ as described in the Materials and Methods section. The enzyme was active from pNP-C 4 to C 10 and the highest activity was observed for pNP-C 6 but almost no activity was detected with pNP-C 2 (FIG. 3). Cut190 was also found to have activity on tributyrin, tripalmitin, and triolein (data not shown).
２．Cut190の部位特異的突然変異
本願発明者らは以前に、ProによるSer219の置換がEst119の耐熱性を増強し(Thumarat U, et al., Appl. Microbiol. Biotechnol. 95:419-430. 2012)、プロリンが相同配列のこの位置で保存されていることを実証した。野生型Cut190は226位(Est119の219位に相当)にセリンを有するため、本願発明者らは対応する変異型(S226P)を構築した。Cut190(S226P)のTm値は野生型Cut190のそれに対して5.9℃増加し、Cut190(S226P)はCut190よりも高い活性を示した (表２および図４Ａ)。Pro226の下流の正に荷電したArg228は、同じAsp222ループ内の負に荷電したアミノ酸(Glu220)と塩架橋を形成し、活性に影響を及ぼしうるため、中性アミノ酸Serと置き換えた。予想通り、Cut190(S226P/R228S)の活性は1.5倍増加し（図４Ａ)、Tmはわずかに増加し、ΔＨはCut190(S226P)と比較して有意に増加した(表２)。262位の残基の正に荷電したLysは相同性クチナーゼで保存されており、この残基はHis254ループの最後に位置する。しかしながら、Cut190は262位の残基が中性のThrを有するため、本願発明者らは、しばしば塩架橋が熱安定性化に寄与するので(Vieille C, et al., Microbiol. Mol. Biol. Rev. 65:1-43, 2001)、ThrをLysにより置換した。Cut190(S226P)、Cut190(S226P/R228S)およびCut190(S226P/R228S/T262K)の活性および耐熱性の比較を図４に示す。226位のSerのProによる置換により、表２に示されるようにTm値が増加し、すべての変異型の耐熱性が増加した。Arg228をSerにより、およびThr262をLysにより置換することにより、活性および耐熱性が改善した。これらの変異型酵素は300mM Ca2+の存在下で50-65℃で１時間インキュベートすると、加熱により活性化された。Cut190(S226P)およびCut190(S226P/R228S/T262K)の活性は、50および60℃でCut190(S226P/R228S)よりも高かったが、65℃で１時間インキュベート後、3つの変異型はほぼ同じ活性のレベルを示した。また、Cut190(S226P/R228S)は70℃で最も高い残存活性を示した。したがって、本願発明者らはさらなる特徴付けのためにCut190(S226P/R228S)を選択した。Cut190(S226P/R228S)よりも20倍高い濃度でもCut190(S176A/S226P/R228S)の活性は観察されなかったので、Ser176(３つ組触媒基に位置する)は活性に必要である。
2. Site-directed mutagenesis of Cut190. We have previously replaced Ser219 with Pro to enhance the heat resistance of Est119 (Thumarat U, et al., Appl. Microbiol. Biotechnol. 95: 419-430. 2012). We have demonstrated that proline is conserved at this position in the homologous sequence. Since wild-type Cut190 has serine at position 226 (corresponding to position 219 of Est119), the present inventors constructed a corresponding mutant (S226P). The Tm value of Cut190 (S226P) increased by 5.9 ° C. relative to that of wild-type Cut190, and Cut190 (S226P) showed higher activity than Cut190 (Table 2 and FIG. 4A). The positively charged Arg228 downstream of Pro226 was replaced with the neutral amino acid Ser because it can form a salt bridge with the negatively charged amino acid (Glu220) in the same Asp222 loop and affect activity. As expected, the activity of Cut190 (S226P / R228S) increased 1.5-fold (FIG. 4A), Tm increased slightly, and ΔH significantly increased compared to Cut190 (S226P) (Table 2). The positively charged Lys at residue 262 is conserved in the homologous cutinase, which is located at the end of the His254 loop. However, since Cut190 has a neutral Thr residue at position 262, the present inventors often find that salt bridges contribute to thermal stabilization (Vieille C, et al., Microbiol. Mol. Biol. Rev. 65: 1-43, 2001), Thr was replaced by Lys. A comparison of the activity and heat resistance of Cut190 (S226P), Cut190 (S226P / R228S) and Cut190 (S226P / R228S / T262K) is shown in FIG. Substitution of Ser at position 226 increased the Tm value as shown in Table 2 and increased the heat resistance of all variants. Replacing Arg228 with Ser and Thr262 with Lys improved the activity and heat resistance. These mutant enzymes were activated by heating when incubated at 50-65 ° C. for 1 hour in the presence of 300 mM Ca 2+ . Cut190 (S226P) and Cut190 (S226P / R228S / T262K) were more active than Cut190 (S226P / R228S) at 50 and 60 ° C, but after incubation for 1 hour at 65 ° C, the three variants had almost the same activity Showed the level. Cut190 (S226P / R228S) showed the highest residual activity at 70 ° C. Therefore, we selected Cut190 (S226P / R228S) for further characterization. Since Cut190 (S176A / S226P / R228S) activity was not observed even at concentrations 20 times higher than Cut190 (S226P / R228S), Ser176 (located in the triplicate catalytic group) is required for activity.
３．Cut190(S226P/R228S)の生化学的特徴付け
i) 基質特異性と速度論的定数値。pNPBに対する活性は図５に示されるようにCa2+により増強された。以前に説明されたように(Thumarat U, et al., Appl. Microbiol. Biotechnol. 95:419-430, 2012)、pNPBase活性を300mM Ca2+の存在下、標準アッセイ条件下で測定した。pNPBに対するKmおよびVmaxの値は、Lineweave-Burkプロット (表３)に基づくとそれぞれ0.68 mMおよび237 nkat・mg-1と推定された。
3. Biochemical characterization of Cut190 (S226P / R228S)
i) Substrate specificity and kinetic constant values. The activity against pNPB was enhanced by Ca 2+ as shown in FIG. As previously described (Thumarat U, et al., Appl. Microbiol. Biotechnol. 95: 419-430, 2012), pNPBase activity was measured under standard assay conditions in the presence of 300 mM Ca 2+ . Km and Vmax values for pNPB were estimated to be 0.68 mM and 237 nkat · mg −1 , respectively, based on the Lineweave-Burk plot (Table 3).
pNPBを使用してクチナーゼ活性の定量的分析を行うことに加えて、本願発明者らはPBSA懸濁液を使用してポリエステラーゼ活性の定量的分析も行った。PBSAの分解は、600nmにおける濁度の減少をモニタすることにより定量的に測定した。600nmにおける１の吸光度は、0.25mg乾燥重量PBSA/mlに相当した。PBSAユニット構造が[Ｏ-（ＣＨ2）4-Ｏ-ＣＯ-（ＣＨ2）2-ＣＯ][ Ｏ-（ＣＨ2）4-Ｏ-ＣＯ-（ＣＨ2）4-ＣＯ]である場合、1molのPBSA(約1.0×105の重量平均分子量を有する)は、ｎユニット(n=269)を含み、酵素加水分解を受けるエステル結合の総数は方程式ｎ×4−1＝1,075によると1075である。したがって、PBSA懸濁液のOD600の１の減少は約1.5 nkatに相当する。pNPBase活性で観察されるように、PBSA加水分解活性はCa2+により増強され、PBSAの加水分解活性は2.5-5.0 mM Ca2+で100%、10-50 mM Ca2+で90%を超え、より高いCa2+濃度ではさらに減少した。25-50 mM Ca2+の存在下では、対照(加熱はなし)と比較して、60℃で1時間加熱後に100%よりも大きい残存活性が維持された。後続で説明するように、pNPBに対する至適酵素pHは6.5-7.0であったが、PBSAの分解速度はpH 7.0でよりもpH 8.2で高かった。Lineweave-Burkプロットを用いてOD600の減少を分析したところ、PBSAに対する推定Km値およびVmax値は0.04 mMおよび268 nkat・mg-1であることが明らかとなった(表３)。pNPBおよびPBSAに対する触媒速度定数(kcat値)はほぼ同一であるが、PBSAに対する触媒効率(kcat/Km値)はpNPBのそれより20倍高い。 In addition to performing quantitative analysis of cutinase activity using pNPB, the inventors also performed quantitative analysis of polyesterase activity using PBSA suspension. PBSA degradation was measured quantitatively by monitoring the decrease in turbidity at 600 nm. An absorbance of 1 at 600 nm corresponded to 0.25 mg dry weight PBSA / ml. When the PBSA unit structure is [O— (CH 2 ) 4 —O—CO— (CH 2 ) 2 —CO] [O— (CH 2 ) 4 —O—CO— (CH 2 ) 4 —CO], 1 mol of PBSA (having a weight average molecular weight of about 1.0 × 10 5 ) contains n units (n = 269) and the total number of ester bonds that undergo enzymatic hydrolysis is 1075 according to the equation n × 4-1 = 11,075. is there. Therefore, a decrease of 1 in OD 600 of PBSA suspension corresponds to about 1.5 nkat. As observed for pNPBase activity, PBSA hydrolysis activity is enhanced by Ca 2+ , with PBSA hydrolysis activity exceeding 100% at 2.5-5.0 mM Ca 2+ and over 90% at 10-50 mM Ca 2+ It was further reduced at higher Ca 2+ concentrations. In the presence of 25-50 mM Ca 2+ , greater than 100% residual activity was maintained after 1 hour heating at 60 ° C. compared to the control (no heating). As explained below, the optimal enzyme pH for pNPB was 6.5-7.0, but the degradation rate of PBSA was higher at pH 8.2 than at pH 7.0. Analysis of the decrease in OD 600 using Lineweave-Burk plots revealed that the estimated Km and Vmax values for PBSA were 0.04 mM and 268 nkat · mg −1 (Table 3). The catalytic rate constants (k cat value) for pNPB and PBSA are nearly identical, but the catalytic efficiency for PBSA (k cat / Km value) is 20 times higher than that of pNPB.
最終1-6 mMのエチレングリコール、ブチレングリコール、コハク酸およびアジピン酸が反応液に加えられた時に、これらの分解産物による阻害は観察されなかった。テレフタル酸-2Naは1-2 mMの終濃度で使用された時は検出できる阻害を示さなかったが、より高い濃度では反応液中の濁りを引き起こし、これにより酵素活性の測定が妨げられた。従って、Cut190(226/228)は反応産物により阻害を受けないか、せいぜいでも弱く阻害されるにすぎない。 When the final 1-6 mM ethylene glycol, butylene glycol, succinic acid and adipic acid were added to the reaction, no inhibition by these degradation products was observed. Terephthalic acid-2Na showed no detectable inhibition when used at a final concentration of 1-2 mM, but higher concentrations caused turbidity in the reaction, which hindered measurement of enzyme activity. Therefore, Cut190 (226/228) is not inhibited by the reaction product, or at best it is only weakly inhibited.
ii)pHおよび温度の最適条件および安定化。精製された組換えCut190(226/228)の至適pHおよび温度の値は、それぞれ約pH 6.5-7.0および65℃であった(図６Ａおよび６Ｂ)。70℃でさえ76.3%の活性が維持されたが、75℃で活性は急速に (10%未満まで)減少した。20%のグリセリンの存在下で、至適温度は増大し(65-75℃)、80℃で約90%の活性が観察された。Cut190(226/228)は弱酸から弱アルカリまでのpH値(5-9)で非常に安定であり、pH 7では100%の活性、pH 6および8-9では約90%の活性、およびpH 5では約80%の活性を有していた。しかしながら、pH 4.0では活性は一晩で約30%まで減少した(図６Ｃ)。酵素の耐熱性を、酵素を300mM Ca2+を含む50mM Tris-HCl緩衝液(pH 7.0)中で50-70℃、1時間でインキュベートした後の残存活性に基づいて決定した(図８)。酵素は50-65℃で熱活性化を示したが、70℃では活性が約40%まで減少した(図６Ｄ)。20%のグリセリン中では、図８に示されるように酵素活性は50-65℃で24時間安定だったが、70℃では1時間以内に50%未満に減少した。 ii) Optimum conditions and stabilization of pH and temperature. The optimum pH and temperature values of purified recombinant Cut190 (226/228) were about pH 6.5-7.0 and 65 ° C., respectively (FIGS. 6A and 6B). Even at 70 ° C, 76.3% activity was maintained, but at 75 ° C the activity decreased rapidly (to less than 10%). In the presence of 20% glycerin, the optimum temperature increased (65-75 ° C.) and about 90% activity was observed at 80 ° C. Cut190 (226/228) is very stable at pH values (5-9) from weak acids to weak alkalis, 100% activity at pH 7, about 90% activity at pH 6 and 8-9, and pH 5 had about 80% activity. However, at pH 4.0 the activity decreased to about 30% overnight (Figure 6C). The thermostability of the enzyme was determined based on the residual activity after incubation of the enzyme in 50 mM Tris-HCl buffer (pH 7.0) containing 300 mM Ca 2+ at 50-70 ° C. for 1 hour (FIG. 8). The enzyme showed thermal activation at 50-65 ° C., but at 70 ° C. the activity decreased to about 40% (FIG. 6D). In 20% glycerin, as shown in FIG. 8, the enzyme activity was stable at 50-65 ° C. for 24 hours, but at 70 ° C., it decreased to less than 50% within 1 hour.
iii) 阻害剤とカルシウムイオンの影響。Cut190(S226P/R228S)は、5 mMの最終PMSF濃度により完全に阻害された。これは、Cut190(S176A/S226P/R228S)の不活性と組み合わせると、Ser-176が触媒残基であることを示している。酵素は、Ca2+のない標準分析条件下では5 mMの最終EDTA濃度でも10 mMの最終EDTA濃度でも阻害されなかったが、これは、金属イオン(特にCa2+)が補欠分子族として必要ではないことを示している。PBSA分解の至適Ca2+濃度は2.5-5.0 mMであったが、上述したようにpNPB加水分解には高いCa2+濃度(300mMよりも高い濃度)が有効である。従って、Ca2+濃度の変化によってCut190(S226P/R228S)構造に対するその影響が変化し、pNPBおよびPBSAに対する活性が変化し得る。この推測を確認するために、図７に示されるようにCa2+有りおよび無しでCD測定を行なった。25mMおよび300 mMでは、Ca2+は遠紫外線スペクトルに影響を及ぼさず、二次構造に変化がないことが示唆された。しかしながら、Ca2+の存在下ではCut190(S226P/R228S)の近紫外線スペクトルにわずかな変化が観察され、Ca2+がタンパク質の三次構造を変化させることが示された。この発見はDSCによって観察される、Ca2+の添加時のCut190(S226P/R228S)の耐熱性(Tm値)の増加と相関する。 iii) Effects of inhibitors and calcium ions. Cut190 (S226P / R228S) was completely inhibited by a final PMSF concentration of 5 mM. This indicates that Ser-176 is a catalytic residue when combined with the inactivation of Cut190 (S176A / S226P / R228S). The enzyme was not inhibited at 5 mM final EDTA concentration or 10 mM final EDTA concentration under standard analytical conditions without Ca 2+ , which requires metal ions (especially Ca 2+ ) as a prosthetic group It is not. The optimal Ca 2+ concentration for PBSA degradation was 2.5-5.0 mM, but as described above, a high Ca 2+ concentration (a concentration higher than 300 mM) is effective for pNPB hydrolysis. Thus, changes in Ca 2+ concentration can change its effect on the Cut190 (S226P / R228S) structure and change its activity on pNPB and PBSA. In order to confirm this assumption, CD measurement was performed with and without Ca 2+ as shown in FIG. At 25 mM and 300 mM, Ca 2+ did not affect the deep ultraviolet spectrum, suggesting no change in secondary structure. However, in the presence of Ca 2+ , a slight change was observed in the near-ultraviolet spectrum of Cut190 (S226P / R228S), indicating that Ca 2+ changes the tertiary structure of the protein. This finding correlates with the increase in heat resistance (Tm value) of Cut190 (S226P / R228S) upon addition of Ca 2+ observed by DSC.
４．種々のポリエステルの分解
Cut190(S226P/R228S)は、PBSA、 PBS、PCL、Ecoflex(登録商標)、PHB、PDLAおよびPLLAを含むポリエステル寒天平板で明瞭なハローを示した（図９）。ポリエステルに対する見かけ上の活性は以下のように順位付けたPBSA>PBS，PCL>Ecoflex（登録商標）>PHB，PDLA>PLLA。また、酵素はPBSAフィルムおよびPBSフィルム(20μm厚の農業用マルチングフィルム)を図１０Ａに示されるように完全に破断した。PBSAは5時間以内に破断され、PBSの破断はよりゆっくりと進行した。PCL、Ecoflex（登録商標）、およびPDLAフィルム（100μm厚以上）では破断が検出されなかったが、酵素ありではポリエステルの分解による濁りが生じ（図１０Ａ）、分解処理後のフィルムの重量を測定したところ、重量は図１０Ｂに示されるように有意に減少した。野生型と変異型Cut190はPETフィルムを50℃で一晩で親水化し、インキュベートを開始したときに浮かんでいた疎水性PETは、インキュベート後にはインキュベーション混合物の底まで沈んだが、対照PETフィルム(酵素無し)は依然として疎水性を維持して浮かんでいた。SEM観察は、図１１Ａに示されるように、60℃および65℃でCut190(S226P/R228S)とインキュベートされたPETフィルム上に分解痕があることを示した。しかしながら、PETフィルムが50℃でインキュベートされると分解の痕跡は観察されなかった。質量の減少は65℃では約1-2%（50-55mgに対して約0.4-1.2mg）であったが、対照では質量の減少は観察されなかった。可塑剤がプラスチックのガラス転移温度(Tg)を減少させると予想されるため、PETフィルムをDEPA可塑剤の存在下でインキュベートした。本願発明者らはDSCでTgを測定することに失敗したが、これはおそらくフィルムの表面にDEPAの小さな小滴が不規則に付着していたためと考えられる(図１１Ｂ)。65℃で酵素とインキュベートすると、60℃での酵素とのインキュベーションよりも深い穴と裂け目が生じ、酵素が存在しないいずれの対照区でも穴と裂け目は示されなかった。酵素が存在しない場合は、フィルム表面に付着したDEPAのサイズは65℃で増大した。対照区ではDEPAではフィルム表面の艶は失われなかったが、酵素反応液のフィルムは艶が消失し、白色化した。PETフィルムは65℃で2日間(酵素活性が24時間よりも長く維持されたため)インキュベートすると、DEPAがない場合よりも大きな質量減少 (約7-8%)を示した。反応を3回繰り返すと約20%の質量減少が生じた。
4). Degradation of various polyesters
Cut190 (S226P / R228S) showed a clear halo on polyester agar plates containing PBSA, PBS, PCL, Ecoflex®, PHB, PDLA and PLLA (FIG. 9). Apparent activity with respect to polyester was ranked as follows: PBSA> PBS, PCL> Ecoflex (registered trademark)> PHB, PDLA> PLLA. In addition, the enzyme completely ruptured PBSA film and PBS film (20 μm thick agricultural mulching film) as shown in FIG. 10A. PBSA was ruptured within 5 hours and PBS rupture proceeded more slowly. No breakage was detected in PCL, Ecoflex (registered trademark), and PDLA film (100 μm thickness or more), but turbidity occurred due to degradation of the polyester with the enzyme (FIG. 10A), and the weight of the film after the degradation treatment was measured. However, the weight decreased significantly as shown in FIG. 10B. Wild-type and mutant Cut190 hydrophilized the PET film overnight at 50 ° C, and the hydrophobic PET that floated when the incubation started began to sink to the bottom of the incubation mixture after the incubation, but the control PET film (no enzyme) ) Still floated while maintaining hydrophobicity. SEM observation showed that there were degradation marks on the PET film incubated with Cut190 (S22 6 P / R228S) at 60 ° C. and 65 ° C. as shown in FIG. 11A. However, no traces of degradation were observed when the PET film was incubated at 50 ° C. The weight loss was about 1-2% at 65 ° C. (about 0.4-1.2 mg versus 50-55 mg), but no weight loss was observed in the controls. Because the plasticizer is expected to reduce the glass transition temperature (Tg) of the plastic, the PET film was incubated in the presence of the DEPA plasticizer. The inventors of the present application failed to measure Tg by DSC, probably because small droplets of DEPA adhered irregularly on the film surface (FIG. 11B). Incubation with the enzyme at 65 ° C resulted in deeper holes and tears than incubation with the enzyme at 60 ° C, and showed no holes and tears in any of the controls where no enzyme was present. In the absence of enzyme, the size of DEPA attached to the film surface increased at 65 ° C. In the control group, the gloss of the film surface was not lost by DEPA, but the gloss of the film of the enzyme reaction solution disappeared and became white. PET film incubated at 65 ° C. for 2 days (because enzyme activity was maintained longer than 24 hours) showed a greater mass loss (about 7-8%) than without DEPA. When the reaction was repeated three times, a mass loss of about 20% occurred.
さらに、Apexa（登録商標）及びPET繊維（糸）をDEPAの存在下、Cut190(S226P/R228S)の有無の下、インキュベートしたところ、図１２に示されるように、酵素無添加の場合、Apexa（登録商標）繊維の糸の表面に変化が見られないのに対し（図１２Ａ）、酵素を添加した場合、糸の分解が観察された（図１２Ｂ，Ｃ）。図１３に示されるように、PETでも酵素無添加の場合、繊維の糸の表面に変化が見られないのに対し（図１３Ａ）、酵素を添加した場合、糸の分解が観察された（図１３Ｂ，Ｃ）。また、PET繊維の織物に対する酵素反応では、反応混合液中にDEPAを含む方（図１４Ｂ）が、DEPAを含まない場合（図１４Ｄ）よりもPET繊維の分解の程度が大きく、DEPAがポリエステル繊維の分解を促進することが示された。このように、本発明の酵素は種々のポリエステル糸を分解できるため、ポリエステル糸が用いられた布に本発明の酵素を含む液を接触させることによって表面処理を行なうことも可能である。 Furthermore, when Apexa (registered trademark) and PET fiber (yarn) were incubated in the presence of DEPA in the presence or absence of Cut190 (S22 6 P / R228S), as shown in FIG. 12, when no enzyme was added, While no changes were observed on the surface of the Apexa® fiber yarn (FIG. 12A), degradation of the yarn was observed when the enzyme was added (FIGS. 12B, C). As shown in FIG. 13, when PET was not added with enzyme, no change was observed on the surface of the fiber yarn (FIG. 13A), whereas when enzyme was added, yarn degradation was observed (FIG. 13). 13B, C). In addition, in the enzymatic reaction of PET fiber to the fabric, the degree of degradation of PET fiber is greater when DEPA is contained in the reaction mixture (FIG. 14B) than when DEPA is not included (FIG. 14D), and DEPA is polyester fiber. Has been shown to promote the degradation of. Thus, since the enzyme of this invention can decompose | disassemble various polyester yarn, it is also possible to surface-treat by making the liquid in which the polyester yarn was used contact the liquid containing the enzyme of this invention.
Claims (11)
（ａ）配列番号４で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質
（ｂ）配列番号４で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質 The protein of any one of the following (a) and (b) , wherein the serine at position 182 of the amino acid sequence represented by SEQ ID NO: 4 is substituted with proline.
Consisting of an amino acid sequence having the amino acid sequence identity of 90% or more (a) represented by SEQ ID NO: 4, the amino acid sequence represented by (b) a protein SEQ ID NO: 4 having cutinase activity, one or several A protein having an amino acid sequence in which amino acids are deleted, substituted, inserted or added, and having cutinase activity
配列番号４で表されるアミノ酸配列の２１８位のスレオニンのリジンによる置換
をさらに有する変異型タンパク質である請求項１に記載のタンパク質。 Substitution of arginine at position 184 of the amino acid sequence represented by SEQ ID NO: 4 with serine, and / or
Replacement of threonine at position 218 of the amino acid sequence represented by SEQ ID NO: 4 with lysine
The protein according to claim 1, which is a mutant protein further comprising :
ポリ(ブチレンスクシナート-co-アジペート) （PBSA）フィルム、ポリ(ブチレンスクシナート)（PBS）フィルム、ポリ(カプロラクトン)（PCL）フィルム、ポリ(L-乳酸) (PLLA)、ポリ(D-乳酸)（PDLA）又はポリ（ヒドロキシ酪酸）(PBA)、である脂肪族ポリエステル、Poly (butylene succinate-co-adipate) (PBSA) film, poly (butylene succinate) (PBS) film, poly (caprolactone) (PCL) film, poly (L-lactic acid) (PLLA), poly (D -Lactic acid) (PDLA) or poly (hydroxybutyric acid) (PBA), an aliphatic polyester,
脂肪族芳香族コポリエステル、又はAn aliphatic aromatic copolyester, or
ポリエチレンテレフタレート（PET）である請求項７に記載の方法。The method according to claim 7, which is polyethylene terephthalate (PET).
（ａ）配列番号４で表されるアミノ酸配列からなるタンパク質(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4
（ｂ）配列番号４で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、クチナーゼ活性を有するタンパク質(B) a protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having cutinase activity
（ｃ）配列番号４で表されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列からなり、かつクチナーゼ活性を有するタンパク質(C) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4 and having cutinase activity
配列番号４で表されるアミノ酸配列の１８４位のアルギニンのセリンによる置換、及び／又は Substitution of arginine at position 184 of the amino acid sequence represented by SEQ ID NO: 4 with serine, and / or
配列番号４で表されるアミノ酸配列の２１８位のスレオニンのリジンによる置換 Replacement of threonine at position 218 of the amino acid sequence represented by SEQ ID NO: 4 with lysine
を有する変異型タンパク質である請求項１０に記載のタンパク質。The protein according to claim 10, which is a mutant protein having
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013265666A JP6315978B2 (en) | 2013-12-24 | 2013-12-24 | Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013265666A JP6315978B2 (en) | 2013-12-24 | 2013-12-24 | Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015119670A JP2015119670A (en) | 2015-07-02 |
| JP6315978B2 true JP6315978B2 (en) | 2018-04-25 |
Family
ID=53531990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013265666A Active JP6315978B2 (en) | 2013-12-24 | 2013-12-24 | Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6315978B2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2018014090A (en) | 2016-05-19 | 2019-04-01 | Carbios | A process for degrading plastic products. |
| JP7189866B2 (en) * | 2016-07-12 | 2022-12-14 | キャルビオス | Novel esterase and use thereof |
| MX2019000492A (en) * | 2016-07-12 | 2019-08-16 | Carbios | Novel esterases and uses thereof. |
| EP3517608A1 (en) * | 2018-01-30 | 2019-07-31 | Carbios | New polypeptides having a polyester degrading activity and uses thereof |
| JP2023546506A (en) * | 2020-10-27 | 2023-11-02 | キャルビオス | Novel esterases and their uses |
| CN113698742B (en) * | 2021-08-31 | 2022-11-22 | 江南大学 | Modification method of polyethylene terephthalate and application of polyethylene terephthalate in enzymolysis process |
| WO2024122636A1 (en) * | 2022-12-09 | 2024-06-13 | キリンホールディングス株式会社 | Protein having polyethylene terephthalate decomposing activity and method for decomposing polyethylene terephthalate |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR087745A1 (en) * | 2011-08-31 | 2014-04-16 | Danisco Us Inc | COMPOSITIONS AND METHODS THAT INCLUDE A VARIANT OF LIPOLITIC ENZYME |
-
2013
- 2013-12-24 JP JP2013265666A patent/JP6315978B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2015119670A (en) | 2015-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6315978B2 (en) | Novel cutinase, gene encoding cutinase, and degradation method of polyester or ester compound using cutinase | |
| Hu et al. | Enzymatic degradation of poly (butylene succinate) by cutinase cloned from Fusarium solani | |
| JP7179002B2 (en) | Improved plastic-degrading protease | |
| US10508269B2 (en) | Polypeptide having a polyester degrading activity and uses thereof | |
| CN106852154B (en) | Polypeptide with polyester degradation activity and application thereof | |
| Kawai et al. | A novel Ca 2+-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190 | |
| Jung et al. | Purification, characterization, and gene cloning of an Aspergillus fumigatus polyhydroxybutyrate depolymerase used for degradation of polyhydroxybutyrate, polyethylene succinate, and polybutylene succinate | |
| Akutsu-Shigeno et al. | Cloning and sequencing of a poly (DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli | |
| Thumarat et al. | Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119 | |
| JP6599767B2 (en) | Aromatic polyester degrading enzyme and method for decomposing aromatic polyester using the enzyme | |
| CN112673104A (en) | Novel esterases and their use | |
| KR20210043599A (en) | Novel esterases and uses thereof | |
| JP5850342B2 (en) | A novel esterase derived from twig leaf compost | |
| CN115768902A (en) | Enzymatic degradation of polyolefin polymer plastics with KATG enzymes | |
| AU2010302460A1 (en) | Bacterial strains and variants thereof that can degrade polylactic acid, and uses of same | |
| Li et al. | Purification and characterization of poly (L-lactic acid)-degrading enzymes from Amycolatopsis orientalis ssp. orientalis | |
| KR20220032091A (en) | Novel esterases and uses thereof | |
| US10227575B2 (en) | Multi-domain enzymes having cutinase activity, compositions comprising same and uses thereof | |
| Li et al. | Two Extracellular poly (ε-caprolactone)-degrading enzymes from Pseudomonas hydrolytica sp. DSWY01T: purification, characterization, and gene analysis | |
| WO2021148713A1 (en) | Enzymes, micro-organisms and uses thereof, and a method of degrading a polyester | |
| JP4831664B2 (en) | Cutinase variant with excellent thermostability | |
| Zhang et al. | Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene | |
| Silva et al. | Enzymatic hydrolysis and modification of core polymer fibres for textile and other applications | |
| JP2023546505A (en) | Novel esterases and their uses | |
| KR20230093468A (en) | Novel esterases and their uses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20161219 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20170223 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170223 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20170322 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170322 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20171011 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20171107 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20171225 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180216 |
|
| TRDD | Decision of grant or rejection written | ||
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20180216 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20180306 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20180327 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6315978 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |