JPH06339376A - New beta-galactosidase and its production - Google Patents
New beta-galactosidase and its productionInfo
- Publication number
- JPH06339376A JPH06339376A JP15426993A JP15426993A JPH06339376A JP H06339376 A JPH06339376 A JP H06339376A JP 15426993 A JP15426993 A JP 15426993A JP 15426993 A JP15426993 A JP 15426993A JP H06339376 A JPH06339376 A JP H06339376A
- Authority
- JP
- Japan
- Prior art keywords
- galactosidase
- novel
- range
- producing
- klebsiella
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010005774 beta-Galactosidase Proteins 0.000 title claims abstract description 50
- 102000005936 beta-Galactosidase Human genes 0.000 title claims abstract 15
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000008101 lactose Substances 0.000 claims abstract description 11
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 10
- 241000588746 Raoultella planticola Species 0.000 claims abstract description 10
- 229930182830 galactose Natural products 0.000 claims abstract description 10
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 9
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 6
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims abstract description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 5
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims abstract description 5
- 235000021255 galacto-oligosaccharides Nutrition 0.000 claims abstract description 5
- 150000003271 galactooligosaccharides Chemical class 0.000 claims abstract description 5
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims abstract description 4
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims abstract description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims abstract description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 3
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 claims abstract description 3
- SHZGCJCMOBCMKK-PQMKYFCFSA-N L-Fucose Natural products C[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O SHZGCJCMOBCMKK-PQMKYFCFSA-N 0.000 claims abstract description 3
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims abstract description 3
- SHZGCJCMOBCMKK-SVZMEOIVSA-N D-fucopyranose Chemical compound C[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O SHZGCJCMOBCMKK-SVZMEOIVSA-N 0.000 claims abstract 2
- SRBFZHDQGSBBOR-SOOFDHNKSA-N D-ribopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@@H]1O SRBFZHDQGSBBOR-SOOFDHNKSA-N 0.000 claims abstract 2
- 244000005700 microbiome Species 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- -1 D-galactosyl group Chemical group 0.000 claims description 4
- 241000588748 Klebsiella Species 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 125000001488 beta-D-galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 claims description 3
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 claims description 3
- 229920001542 oligosaccharide Polymers 0.000 claims description 3
- 150000002482 oligosaccharides Chemical class 0.000 claims description 3
- SRBFZHDQGSBBOR-OWMBCFKOSA-N L-ribopyranose Chemical compound O[C@H]1COC(O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-OWMBCFKOSA-N 0.000 claims description 2
- 229930182470 glycoside Natural products 0.000 claims description 2
- 150000002338 glycosides Chemical class 0.000 claims description 2
- 150000002772 monosaccharides Chemical class 0.000 claims description 2
- 150000005846 sugar alcohols Chemical class 0.000 claims description 2
- 239000000370 acceptor Substances 0.000 claims 1
- 238000000354 decomposition reaction Methods 0.000 claims 1
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 abstract description 10
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 abstract description 3
- 239000000600 sorbitol Substances 0.000 abstract description 3
- 235000010356 sorbitol Nutrition 0.000 abstract description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 abstract description 2
- 102000002464 Galactosidases Human genes 0.000 abstract description 2
- 108010093031 Galactosidases Proteins 0.000 abstract description 2
- 229960005070 ascorbic acid Drugs 0.000 abstract description 2
- 235000010323 ascorbic acid Nutrition 0.000 abstract description 2
- 239000011668 ascorbic acid Substances 0.000 abstract description 2
- 235000011187 glycerol Nutrition 0.000 abstract description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 34
- 230000000694 effects Effects 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- 150000001875 compounds Chemical class 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N benzopyrrole Natural products C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- 238000005918 transglycosylation reaction Methods 0.000 description 3
- 108010082340 Arginine deiminase Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102000051366 Glycosyltransferases Human genes 0.000 description 2
- 108700023372 Glycosyltransferases Proteins 0.000 description 2
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 2
- 108010048581 Lysine decarboxylase Proteins 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229910017464 nitrogen compound Inorganic materials 0.000 description 2
- 150000002830 nitrogen compounds Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XUCIJNAGGSZNQT-JHSLDZJXSA-N (R)-amygdalin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O[C@@H](C#N)C=2C=CC=CC=2)O1 XUCIJNAGGSZNQT-JHSLDZJXSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- DNUYOWCKBJFOGS-UHFFFAOYSA-N 2-[[10-(2,2-dicarboxyethyl)anthracen-9-yl]methyl]propanedioic acid Chemical compound C1=CC=C2C(CC(C(=O)O)C(O)=O)=C(C=CC=C3)C3=C(CC(C(O)=O)C(O)=O)C2=C1 DNUYOWCKBJFOGS-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 1
- 108050008072 Cytochrome c oxidase subunit IV Proteins 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 102000030902 Galactosyltransferase Human genes 0.000 description 1
- 108060003306 Galactosyltransferase Proteins 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-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
- 108010059881 Lactase Proteins 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 229920002472 Starch Polymers 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
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- PNNNRSAQSRJVSB-DPYQTVNSSA-N aldehydo-D-fucose Chemical compound C[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-DPYQTVNSSA-N 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229940089837 amygdalin Drugs 0.000 description 1
- YZLOSXFCSIDECK-UHFFFAOYSA-N amygdalin Natural products OCC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC(C#N)c3ccccc3 YZLOSXFCSIDECK-UHFFFAOYSA-N 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- UZUODNWWWUQRIR-UHFFFAOYSA-L disodium;3-aminonaphthalene-1,5-disulfonate Chemical compound [Na+].[Na+].C1=CC=C(S([O-])(=O)=O)C2=CC(N)=CC(S([O-])(=O)=O)=C21 UZUODNWWWUQRIR-UHFFFAOYSA-L 0.000 description 1
- YGHHWSRCTPQFFC-UHFFFAOYSA-N eucalyptosin A Natural products OC1C(O)C(O)C(CO)OC1OC1C(OC(C#N)C=2C=CC=CC=2)OC(CO)C(O)C1O YGHHWSRCTPQFFC-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229940116108 lactase Drugs 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- IJFXRHURBJZNAO-UHFFFAOYSA-N meta--hydroxybenzoic acid Natural products OC(=O)C1=CC=CC(O)=C1 IJFXRHURBJZNAO-UHFFFAOYSA-N 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 230000006098 transglycosylation Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、新規な糖転移活性の強
いβ−ガラクトシダーゼ自体およびその製造方法に関
し、更に詳しくは、クレビジーラ（Klebsiella）属に属
する微生物によって生産されるβ−ガラクトシダーゼ自
体およびその製造方法に関する。BACKGROUND OF THE INVENTION The present invention relates to a strong beta-galactosidase itself, and a method of manufacturing the same novel glycosyltransferase activity, more particularly, beta-galactosidase itself and its produced by the microorganisms belonging to Kurebijira (Klebsiella) genus It relates to a manufacturing method.
【０００２】[0002]
【従来の技術】β−ガラクトシダーゼは、β−Ｄ−ガラ
クトシド結合を加水分解してＤ−ガラクトースを遊離す
る酵素であり、一般に微生物および動植物中に広く存在
している。β−ガラクトシダーゼは、別名、ラクターゼ
とも呼ばれ、乳糖不耐症用の低乳糖牛乳の製造、あるい
はチーズ製造時に副産する乳清からのホエーシロップの
製造などに用いられている。BACKGROUND OF THE INVENTION β-Galactosidase is an enzyme that hydrolyzes β-D-galactoside bonds to release D-galactose, and is generally widely present in microorganisms and animals and plants. β-Galactosidase is also known as lactase, and is used for producing low-lactose milk for lactose intolerance, or for producing whey syrup from whey produced as a by-product during cheese production.
【０００３】また、β−ガラクトシダーゼは、ガラクト
シド結合を転移させる能力をも有しており、この能力を
利用してガラクトオリゴ糖（ガラクトース残基を有する
オリゴ糖）を製造する方法が知られている。β−ガラク
トシダーゼを生産する微生物の例には、アスペルギルス
・オリーゼ（Aspergillus oryzae）やバシルス・サーキ
ュランス（Bacillus circulans）などが報告されてい
る。Further, β-galactosidase also has an ability to transfer a galactoside bond, and a method for producing a galactooligosaccharide (oligosaccharide having a galactose residue) is known utilizing this ability. Examples of β-galactosidase producing microorganisms include Aspergillus.
・ Olize ( Aspergillus oryzae ) and Bacillus sark
And the like ( Bacillus circulans ) have been reported.
【０００４】[0004]
【発明が解決しようとする課題】しかしながら、従来、
β−ガラクトシダーゼの糖転移反応に関する研究におい
て、受容体としての乳糖への転移反応についてよく研究
されているが、受容体特異性について検討されている例
は少なく、大腸菌（E.coli ML309）に関する報告がある
のみである。Advances in Carbohydrate Chemistry 16.
263 (1961) の報告によれば、Ｄ−ガラクトース、Ｄ−
グルコース、Ｄ−及びＬ−アラビノース、Ｄ−フラクト
ース及びサッカロースについては転移すると報告されて
いる。その他の糖類については検討されていない。この
ように受容体特異性の範囲についてはあまり報告がなさ
れていない。したがって、従来のβ−ガラクトシダーゼ
においては、広い範囲の受容体特異性を持つβ−ガラク
トシダーゼの存在は知られていなかった。[Problems to be Solved by the Invention] However, in the past,
In the study on the transglycosylation reaction of β-galactosidase, the transglycosylation reaction to lactose as a receptor has been well studied, but the specificity of the receptor has not been examined, and E. coli ML309 is reported. There is only. Advances in Carbohydrate Chemistry 16.
263 (1961) reported that D-galactose, D-
Glucose, D- and L-arabinose, D-fructose and sucrose are reported to transpose. Other sugars have not been investigated. Thus, there have been few reports on the range of receptor specificity. Therefore, in the conventional β-galactosidase, the existence of β-galactosidase having a wide range of receptor specificity was not known.
【０００５】本発明者は、β−ガラクトシダーゼが受容
体化合物にガラクトースを転移させることにより、その
化合物がそれまでに持っていなかった新しい機能を有す
る糖質を合成できる可能性に着目し、広い範囲の受容体
特異性を持つβ−ガラクトシダーゼを提供すること、及
びそのβ−ガラクトシダーゼの製造方法を提供すること
を目的とする。The present inventor has focused on the possibility that β-galactosidase can transfer galactose to an acceptor compound to synthesize a sugar having a new function which the compound does not have until then, and has a wide range. It is an object of the present invention to provide β-galactosidase having the receptor specificity of, and a method for producing the β-galactosidase.
【０００６】[0006]
【課題を解決するための手段】上記した問題点を解決す
るために、本発明者らは、乳糖から付加価値の高い糖質
を合成し得る糖転移活性の高いガラクトシル基転移酵素
を菌体外に生産する微生物を検索してきた。その結果、
徳島県板野郡の土壌中より分離されたクレビジーラ属に
属する微生物を栄養培地で培養することにより、目的と
する広い範囲の受容体特異性を持つ、糖転移活性の強い
β−ガラクトシダーゼを菌体外に生産することを見いだ
し、本発明を完成するに至った。In order to solve the above-mentioned problems, the inventors of the present invention used a galactosyltransferase having high transglycosylation activity capable of synthesizing a sugar having high added value from lactose to the outside of the cell. Have searched for microorganisms that produce. as a result,
By culturing microorganisms belonging to the genus Klebijira isolated from the soil of Itano-gun, Tokushima Prefecture in a nutrient medium, β-galactosidase with a wide range of target receptor specificity and strong glycosyltransferase activity was extracellularly extracellular. The present invention has been completed and the present invention has been completed.
【０００７】本発明は以下に示す性質を有する新規β−
ガラクトシダーゼに関する。The present invention is a novel β- having the following properties:
Regarding galactosidase.
【０００８】新規β−ガラクトシダーゼの生産能を有す
る微生物、例えば、クレビジーラ・プランティコラＧ−
１７（Klebsiella planticola Ｇ−１７）（ＦＥＲＭ
Ｐ−１３５７１号）の生産するβ−ガラクトシダーゼは
幅広い受容体特異性を有しており、種々の化合物にガラ
クトースを転移させることにより、その化合物がそれま
でに持っていなかった新しい機能を有した糖質を合成す
ることができる。A microorganism having the ability to produce a novel β-galactosidase, for example, Klebijira planticola G-
17 ( Klebsiella planticola G-17) (FERM
Β-galactosidase produced by P-13571) has a wide range of receptor specificities, and by transferring galactose to various compounds, a sugar having a new function that the compound did not have up to that point. Quality can be combined.
【０００９】本発明の新規β−ガラクトシダーゼは後述
する新規な理化学的性質を有する。このβ−ガラクトシ
ダーゼは、クレビジーラ（Klebsiella）属に属する新規
β−ガラクトシダーゼの生産能を有する微生物、例え
ば、クレビジーラ・プランティコラＧ−１７（Klebsiel
la planticola Ｇ−１７）（ＦＥＲＭ Ｐ−１３５７１
号）を培養し、該培養物からβ−ガラクトシダーゼを分
離、採取することにより製造することができる。The novel β-galactosidase of the present invention has novel physicochemical properties described below. The β- galactosidase, microorganisms having an ability to produce novel β- galactosidase belonging to Kurebijira (Klebsiella) genus, for example, Kurebijira plan tee Kola G-17 (Klebsiel
la planticola G-17) (FERM P-13571
No.) is cultured, and β-galactosidase is separated and collected from the culture.
【００１０】以下に本発明について詳述する。The present invention will be described in detail below.
【００１１】（１）本発明に使用される微生物：本発明
に使用される微生物は、新規β−ガラクトシダーゼの生
成能を有するものであり、クレビジーラ属に属する菌種
である。クレビジーラ属に属する新規β−ガラクトシダ
ーゼの生産能を有する微生物としては、例えば、クレビ
ジーラ・プランティコラＧ−１７（Klebsiella plantic
ola Ｇ−１７）（ＦＥＲＭ Ｐ−１３５７１）である。(1) Microorganism used in the present invention: The microorganism used in the present invention has the ability to produce a novel β-galactosidase, and is a species belonging to the genus Klebijira. The microorganism capable of producing novel β- galactosidase belonging to Kurebijira genus, for example, Kurebi
Gira Planticola G-17 ( Klebsiella plantic
ola G-17) (FERM P-13571).
【００１２】前記クレビジーラ・プランティコラＧ−１
７（Klebsiella planticola Ｇ−１７）（ＦＥＲＭ Ｐ
−１３５７１）は次の菌学的性質を有する。なお、以下
に記載の菌学的諸性質の試験は、The National Collect
ions of Industrial and Marine Bacteria Limitedに依
頼して作成したものである。The Klebijira Planticola G-1
7 ( Klebsiella planticola G-17) (FERM P
-13571) has the following mycological properties. The tests for the mycological properties described below are based on The National Collect
It was created at the request of ions of Industrial and Marine Bacteria Limited.
【００１３】 試験条件：４８時間、３０℃〔Ｒａｐｉｄ Ｔｅｓｔ（ＡＰＩ）〕 β−ｇａｌａｃｔｏｓｉｄａｓｅ ＋ Ａｒｇｉｎｉｎｅ ｄｉｈｙｄｒｏｌａｓｅ − Ｌｙｓｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ ＋ Ｏｒｎｉｔｈｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ − Ｃｉｔｒａｔｅ ｕｔｉｌｉｓａｔｉｏｎ ＋ Ｈ2 Ｓ ｐｒｏｄｕｃｔｉｏｎ − Ｕｒｅａｓｅ ＋ Ｔｒｙｐｔｏｐｈａｎ ｄｅａｍｉｎａｓｅ − Ｉｎｄｏｌｅ ｐｒｏｄｕｃｔｉｏｎ − Ｖｏｇｅｓ ｐｒｏｓｋａｕｅｒ ＋ Ｇｅｌａｔｉｎａｓｅ − Ａｃｉｄ ｐｒｏｄｕｃｔｉｏｎ ｆｒｏｍ： Ｇｌｕｃｏｓｅ ＋ Ｍａｎｎｉｔｏｌ ＋ Ｉｎｏｓｉｔｏｌ ＋ Ｓｏｒｂｉｔｏｌ ＋ Ｒｈａｍｎｏｓｅ ＋ Ｓｕｃｒｏｓｅ ＋ Ｍｅｌｉｂｉｏｓｅ ＋ Ａｍｙｇｄａｌｉｎ ＋ Ｌ−（＋）−Ａｒａｂｉｎｏｓｅ ＋ Ｃｙｔｏｃｈｒｏｍｅ ｏｘｉｄａｓｅ − 試験条件：７日間、３０℃ Ｉｎｄｏｌｅ ｐｒｏｄｕｃｔｉｏｎ − Ｈ2 Ｓ ｐｒｏｄｕｃｔｉｏｎ − Ｌｙｓｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ ＋ Ａｒｇｉｎｉｎｅ ｄｉｈｙｄｒｏｌａｓｅ − Ｏｒｎｉｔｈｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ ＋ Ｍｅｔｈｙｌ ｒｅｄ ＋ Ｖｏｇｅｓ ｐｒｏｓｋａｕｅｒ ＋ Ｇｒｏｗｔｈ ａｔ １０℃ （＋） Ｕｔｉｌｉｓａｔｉｏｎ ｏｆ ｍ−ｈｙｄｒｏｘｙｂｅｎｚｏｉｃ ａｃｉｄ − Ａｃｉｄ ｆｒｏｍ ｍｅｌｉｚｉｔｏｓｅ ＰＷＳ − Ｇａｓ ｆｒｏｍ ｍｅｌｉｚｉｔｏｓｅ ＰＷＳ − 試験条件：６日間，４４．５℃ Ａｃｉｄ ｆｒｏｍ ｌａｃｔｏｓｅ ＰＷＳ ＋ Ｇａｓ ｆｒｏｍ ｌａｃｔｏｓｅ ＰＷＳ − ＰＷＳ：Ｐｅｐｔｏｎｅ ｗａｔｅｒ ｓｕｇａｒ （＋）：ｗｅａｋ ｐｏｓｉｔｉｖｅ 以上の菌学的性質により、上記菌株はクレビジーラ・プ
ランティコラ（Klebsiella planticola ）に属するもの
と同定された。[0013] Test Conditions: 48 hours, 30 ° C. [Rapid Test (API)] β-galactosidase + Arginine dihydrolase - Lysine decarboxylase + Ornithine decarboxylase - Citrate utilisation + H 2 S production - Urease + Tryptophan deaminase - Indole production - Voges proskauer + Gelatinase-Acid production from: Glucose + Mannitol + Inositol + Sorbitol + Rhamnose + Sucrose + Melibose + Amygdalin + Ar- (L- (L-)). binose + Cytochrome oxidase - Test conditions: 7 days, 30 ℃ Indole production - H 2 S production - Lysine decarboxylase + Arginine dihydrolase - Ornithine decarboxylase + Methyl red + Voges proskauer + Growth at 10 ℃ (+) Utilisation of m-hydroxybenzoic acid - Acid from melizetose PWS-Gas from melizetose PWS-Test condition: 6 days, 44.5 ° C Acid from lactose PWS + Gas from lactose PWS-PWS: Peptone sugar (+): The mycological properties of more than weak positive, the strain is Kurebijira Preferences
It was identified as belonging to the Lantikola ( Klebsiella planticola ).
【００１４】本発明における使用可能な微生物には、ク
レビジーラ・プランティコラＧ−１７（Klebsiella pla
nticola Ｇ−１７）（ＦＥＲＭ Ｐ−１３５７１）が挙
げられるが、本菌株はその一例であり、その自然的およ
び人工的変異株は勿論、クレビジーラ属に属する菌種で
新規β−ガラクトシダーゼ生成能を有する微生物はすべ
て本発明において使用することができる。The microorganisms that can be used in the present invention include :
Revisirah Planticola G-17 ( Klebsiella pla
nticola G-17) (FERM P-13571), but this strain is one example, and naturally occurring and artificial mutants thereof, as well as strains belonging to the genus Klebijira, have a novel β-galactosidase-producing ability. All microorganisms can be used in the present invention.
【００１５】培養方法：上記微生物の培養方法は、通常
用いられる固体培地、液体培地のどちらを用いてもよい
が、この微生物の生産するβ−ガラクトシダーゼは菌体
外に分泌するので、酵素を採取する観点からは、液体培
地の方が効率的に採取でき好ましい。培養に用いられる
培地は、炭素源としては微生物が同化し得る炭素源を用
いることが可能であるが、好ましくはガラクトース、乳
糖、ガラクトオリゴ糖などである。また、窒素源として
は、酵母エキス、コーンスティープリカー、ペプトン、
肉エキスなどの窒素化合物や、ＮａＮＯ3 ・（ＮＨ4 ）
2 ＳＯ4 などの無機窒素化合物を用いることができ、無
機塩類としては、ナトリウム塩、カリウム塩、マグネシ
ウム塩、リン酸塩などを適宜に用いることができる。さ
らに、ビタミン類や微量金属塩を培地に追加して使用菌
の生育を良好ならしめることができる。Culturing method: For the above-mentioned microorganism culturing method, either a commonly used solid medium or liquid medium may be used. Since β-galactosidase produced by this microorganism is secreted outside the cells, the enzyme is collected. From the viewpoint of doing so, the liquid medium is preferable because it can be collected efficiently. The medium used for culturing can use a carbon source that can be assimilated by microorganisms, but is preferably galactose, lactose, galactooligosaccharide or the like. Further, as the nitrogen source, yeast extract, corn steep liquor, peptone,
Nitrogen compounds such as meat extract, NaNO 3 · (NH 4 )
An inorganic nitrogen compound such as 2 SO 4 can be used, and as the inorganic salt, a sodium salt, a potassium salt, a magnesium salt, a phosphate or the like can be appropriately used. Furthermore, vitamins and trace metal salts can be added to the medium to improve the growth of the bacteria used.
【００１６】炭素源の濃度は０．５〜２０重量％の範
囲、培養温度は２０〜４５℃、培養液のｐＨは４〜９の
範囲内、培養時間は１〜４日間程度である。培養方法は
静置および振とう培養のいずれの方法でも行なうことが
できる。The concentration of the carbon source is in the range of 0.5 to 20% by weight, the culture temperature is in the range of 20 to 45 ° C., the pH of the culture solution is in the range of 4 to 9, and the culture time is about 1 to 4 days. The culture method may be either static or shake culture.
【００１７】[0017]
【実施例】以下に、クレビジーラ・プランティコラＧ−
１７（Klebsiella planticola Ｇ−１７）（ＦＥＲＭ
Ｐ−１３５７１）の培養の一例を示す。[Embodiment] Below, Klebijira Planticola G-
17 ( Klebsiella planticola G-17) (FERM
An example of culture of P-13571) is shown.
【００１８】培地組成： 無機塩類 ＮａＮＯ3 ０．３％ Ｋ2 ＨＰＯ4 ０．１％ ＭｇＳＯ4 ．７Ｈ2 Ｏ ０．０５％ ＫＣｌ ０．０５％ ＦｅＳＯ4 ．７Ｈ2 Ｏ ０．００１％ 窒素源 ペプトン ０．５％ 肉エキス ０．５％ 炭素源 ガラクトオリゴ糖 １％ 培地のｐＨ ７．０ 上記の組成の培地を滅菌後、クレビジーラ・プランティ
コラＧ−１７（Klebsiella planticola Ｇ−１７）（Ｆ
ＥＲＭ Ｐ−１３５７１）を植菌し、３０℃で３日間振
とう培養した。遠心分離により得た上清に０．８飽和に
なるように硫安を加え、一晩放置した。沈殿物を集め、
ｐＨ７．０のリン酸バッファーに溶解し、同バッファー
に対して透析したものをβ−ガラクトシダーゼの粗酵素
品とした。Medium composition: Inorganic salts NaNO 3 0.3% K 2 HPO 4 0.1% MgSO 4 . 7H 2 O 0.05% KCl 0.05% FeSO 4 . 7H 2 O 0.001% Nitrogen source Peptone 0.5% Meat extract 0.5% Carbon source Galactooligosaccharides 1% pH of medium 7.0 After sterilizing the medium of the above composition, Klebijira planty
Kola G-17 (Klebsiella planticola G- 17) (F
ERM P-13571) was inoculated and shake-cultured at 30 ° C. for 3 days. Ammonium sulfate was added to the supernatant obtained by centrifugation to give a saturation of 0.8, and the mixture was allowed to stand overnight. Collect the precipitate,
The product was dissolved in a phosphate buffer of pH 7.0 and dialyzed against the same buffer to give a crude β-galactosidase enzyme product.
【００１９】β−ガラクトシダーゼ：β−ガラクトシダ
ーゼは上記微生物の培養物から分離、採取される。分離
手段としては、公知の酵素精製方法を用いることができ
る。また、微生物培養物を濾過または遠心分離した上清
液を粗酵素とすることができ、塩析、透析、ゲル濾過、
イオン交換クロマトグラフィー、疎水クロマトグラフィ
ーなどの精製手段を用いて精製酵素とすることができ
る。Β-galactosidase: β-galactosidase is isolated and collected from the culture of the above microorganism. As a separation means, a known enzyme purification method can be used. Further, the supernatant liquid obtained by filtering or centrifuging the microbial culture can be used as a crude enzyme, and salting out, dialysis, gel filtration,
A purified enzyme can be obtained by using a purification means such as ion exchange chromatography or hydrophobic chromatography.
【００２０】β−ガラクトシダーゼは以下の理科学的性
質を有する新規な酵素である。Β-galactosidase is a novel enzyme having the following physical and scientific properties.
【００２１】〔理科学的性質〕 （１）作用および基質特異性 β−ガラクトシド結合を有する乳糖、ο−ニトロフェニ
ル−β−Ｄ−ガラクトピラノサイド（以下、ＯＮＰＧと
いう）等のβ−Ｄ−ガラクトピラノサイド誘導体に作用
し、そのβ−Ｄ−ガラクトシド結合を分解して、ガラク
トースとグルコース、ガラクトースとο−ニトロフェノ
ールなどを生成すると同時に、生成したＤ−ガラクトシ
ル基を転移する。[Scientific Properties] (1) Action and Substrate Specificity Lactose having a β-galactoside bond, β-D- such as o-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as ONPG) It acts on a galactopyranoside derivative and decomposes its β-D-galactoside bond to produce galactose and glucose, galactose and o-nitrophenol, and at the same time, transfers the produced D-galactosyl group.
【００２２】（２）受容体特異性 受容体として、各種単糖、糖アルコール、配糖体、オリ
ゴ糖等の存在下、β−Ｄ−ガラクトピラノシル誘導体を
基質とした場合に、分解生成したガラクトシル基を受容
体分子に転移させ、その受容体特異性は極めて広い。例
えば、ＯＮＰＧを基質として、各種糖類を受容体とした
ときの薄層クロマトグラフの例を図１に示した。図１に
示すように、Ｌ−アラビノース、Ｄ−アラビノース、Ｄ
−リボース、Ｌ−ソルボース、Ｄ−フラクトース、Ｄ−
フコース、Ｌ−フコース、マンノース、ラムノース、Ｄ
−グルコサミン塩酸塩、Ｄ−キシロース、ラクトース、
ソルビトール、グリセリン、アスコルビン酸、ＯＮＰＧ
についてＤ−ガラクトシル基の転移が認められる。(2) Receptor specificity When the β-D-galactopyranosyl derivative is used as a substrate in the presence of various monosaccharides, sugar alcohols, glycosides, oligosaccharides, etc. The transferred galactosyl group is transferred to a receptor molecule, and its receptor specificity is extremely wide. For example, an example of a thin layer chromatograph using ONPG as a substrate and various saccharides as an acceptor is shown in FIG. As shown in FIG. 1, L-arabinose, D-arabinose, D
-Ribose, L-sorbose, D-fructose, D-
Fucose, L-Fucose, Mannose, Rhamnose, D
-Glucosamine hydrochloride, D-xylose, lactose,
Sorbitol, glycerin, ascorbic acid, ONPG
The transfer of the D-galactosyl group is observed for.
【００２３】（３）力価の測定法（ＯＮＰＧに対する力
価） １０ｍＭのＯＮＰＧ０．５ｍｌ（ｐＨ７．０の５０ｍＭ
リン酸緩衝液）と希釈（同緩衝液）した粗酵素液０．５
ｍｌを混合し、４０℃で１０分間反応させた後、０．２
ＭＮａ2 ＣＯ3 を添加して反応を停止させた。生成した
ο−ニトロフェノールを４１０ｎｍにおける吸収を測定
することにより定量した。この条件で、１分間に１μＭ
のο−ニトロフェノールを生成する能力を１ユニット
（Ｕ）とした。(3) Method for measuring titer (titer against ONPG) 0.5 ml of 10 mM ONPG (50 mM at pH 7.0)
0.5 crude enzyme solution diluted with phosphate buffer)
After mixing ml and reacting at 40 ° C. for 10 minutes, 0.2
The reaction was stopped by adding MNa 2 CO 3 . The o-nitrophenol formed was quantified by measuring the absorption at 410 nm. Under this condition, 1 μM for 1 minute
The ability to produce o-nitrophenol was defined as 1 unit (U).
【００２４】（４）至適ｐＨおよび安定ｐＨ範囲 ＯＮＰＧを基質として各ｐＨにおいて４０℃で１０分反
応させた活性を測定した結果をｐＨ活性曲線として図２
に示す。図２に示すように、至適ｐＨは、７．０であっ
た。また、各ｐＨにおいて４０℃、１２０分間インキュ
ベートした後の残存活性を測定した結果をｐＨ安定曲線
として図３に示す。図３に示すようにｐＨ５〜７．５で
安定であった。(4) Optimum pH and stable pH range The activity was measured by reacting ONPG as a substrate at 40 ° C. for 10 minutes at each pH.
Shown in. As shown in FIG. 2, the optimum pH was 7.0. Moreover, the result of measuring the residual activity after incubation at 40 ° C. for 120 minutes at each pH is shown in FIG. 3 as a pH stability curve. As shown in FIG. 3, it was stable at pH 5 to 7.5.
【００２５】（５）作用適温の範囲 ＯＮＰＧを基質として、各温度においてｐＨ７．０で１
０分間反応させた活性を測定した結果を温度活性曲線と
して図４に示す。図４に示すように、作用適温は４０〜
５０℃の範囲であった。(5) Optimum temperature range of action Using ONPG as a substrate, 1 at pH 7.0 at each temperature.
The result of measuring the activity after reacting for 0 minutes is shown in FIG. 4 as a temperature activity curve. As shown in FIG. 4, the optimum working temperature is 40-
It was in the range of 50 ° C.
【００２６】（６）熱安定性 粗酵素液を各温度において、１５分間インキュベートし
た後、ＯＮＰＧを基質として残存活性を測定した。その
結果を温度安定曲線として図５に示す。図５に示すよう
に、４５℃まで安定であり、５０℃においては約２０％
程度にまで低下した。(6) Thermostability The crude enzyme solution was incubated at each temperature for 15 minutes, and the residual activity was measured using ONPG as a substrate. The result is shown in FIG. 5 as a temperature stability curve. As shown in Fig. 5, it is stable up to 45 ° C and is about 20% at 50 ° C.
Fell to a degree.
【００２７】（７）粗酵素液中の他酵素活性 粗酵素液中に存在する他の酵素の活性（α−ガラクトシ
ダーゼ、β−フラクトシダーゼ、α−グルコシダーゼ、
β−グルコシダーゼ、α−アミラーゼ）を各々測定し
た。基質は各々ラフィノース、サッカロース、マルトー
ス、セロビオース、可溶性デンプンを用い、生成した各
々ガラクトース、フラクトース、グルコース及び還元糖
量を各々Ｆキット及びソモジ・ネルソン法で測定した。
その結果を、β−ガラクトシダーゼ活性を１００とした
時の各活性の値で次の表１に示した。(7) Other enzyme activities in crude enzyme solution Activities of other enzymes present in the crude enzyme solution (α-galactosidase, β-fructosidase, α-glucosidase,
β-glucosidase and α-amylase) were measured. Raffinose, saccharose, maltose, cellobiose, and soluble starch were used as substrates, and the amounts of galactose, fructose, glucose, and reducing sugars produced were measured by the F kit and the Somogy Nelson method.
The results are shown in Table 1 below as the value of each activity when the β-galactosidase activity was set to 100.
【００２８】[0028]
【表１】 [Table 1]
【００２９】[0029]
【発明の効果】新規β−ガラクトシダーゼ生産能を有す
る微生物クレビジーラ・プランティコラＧ−１７（Kleb
siella planticola Ｇ−１７）（ＦＥＲＭ Ｐ−１３５
７１）は、幅広い受容体特異性を有しており、種々の化
合物にガラクトースを転移させることにより、その化合
物がそれまでに持っていなかった新しい機能を付与する
ことができる。EFFECTS OF THE INVENTION A microorganism having a novel β-galactosidase-producing ability, Klebijira planticola G-17 ( Kleb
siella planticola G-17) (FERM P-135
71) has a wide range of receptor specificities, and by transferring galactose to various compounds, it is possible to impart new functions that the compounds did not have until then.
【図面の簡単な説明】[Brief description of drawings]
【図１】各種糖類の受容体特異性を示す薄層クロマトグ
ラフ。FIG. 1 is a thin layer chromatograph showing the receptor specificity of various saccharides.
【図２】新規β−ガラクトシダーゼの至適ｐＨを示す。FIG. 2 shows the optimum pH of a novel β-galactosidase.
【図３】新規β−ガラクトシダーゼの安定ｐＨ範囲を示
す。FIG. 3 shows the stable pH range of the novel β-galactosidase.
【図４】新規β−ガラクトシダーゼの作用適温の範囲を
示す。FIG. 4 shows the optimum temperature range for action of the novel β-galactosidase.
【図５】新規β−ガラクトシダーゼの熱安定性を示す。FIG. 5 shows the thermostability of the novel β-galactosidase.
Claims (4)
ダーゼ。 作用および基質特異性：β−Ｄ−ガラクトピラノシル
誘導体に作用して、そのβ−ガラクトシド結合を分解す
るとともに、分解して生成したガラクトシル基を転移す
る。受容体としての各種単糖、糖アルコール、配糖体及
びオリゴ糖の存在下でβ−Ｄ−ガラクトピラノシル誘導
体を基質として上記新規β−ガラクトシダーゼを作用さ
せた場合に、分解生成したガラクトシル基を受容体分子
に転移させる。 受容体特異性：少なくとも次の受容体に対してＤ−ガ
ラクトシル基を転移する。Ｌ−アラビノース、Ｄ−アラ
ビノース、Ｄ−リボース、Ｌ−ソルボース、Ｄ−フラク
トース、Ｄ−フコース、Ｌ−フコース、マンノース、ラ
ムノース、Ｄ−グルコサミン塩酸塩、Ｄ−キシロース。 至適ｐＨ及びｐＨ安定性：至適ｐＨはｐＨ７付近で、
安定ｐＨ範囲はｐＨ５〜７．５（４０℃、１２０分間処
理）である。 作用温度の範囲：４０〜５０℃に最適作用温度を有す
る。 温度安定性：１５分間処理の場合、４５℃まで安定で
ある。1. A novel β-galactosidase having the following properties. Action and substrate specificity: acts on the β-D-galactopyranosyl derivative to decompose its β-galactoside bond and transfer the galactosyl group produced by decomposition. A galactosyl group decomposed and produced when the above novel β-galactosidase is allowed to act on a β-D-galactopyranosyl derivative as a substrate in the presence of various monosaccharides, sugar alcohols, glycosides and oligosaccharides as acceptors. Is transferred to the receptor molecule. Receptor specificity: Transfers the D-galactosyl group to at least the next receptor. L-arabinose, D-arabinose, D-ribose, L-sorbose, D-fructose, D-fucose, L-fucose, mannose, rhamnose, D-glucosamine hydrochloride, D-xylose. Optimum pH and pH stability: The optimum pH is around pH 7,
The stable pH range is pH 5 to 7.5 (treatment at 40 ° C. for 120 minutes). Range of working temperature: It has an optimum working temperature in the range of 40 to 50 ° C. Temperature stability: Stable up to 45 ° C when treated for 15 minutes.
請求項１記載の性質を有する新規β−ガラクトシダーゼ
の生成能を有する微生物を培養し、該培養液から、新規
β−ガラクトシダーゼを分離、採取することを特徴とす
る新規β−ガラクトシダーゼの製造方法。It belongs to the [claim 2] Kurebijira (Klebsiella) genus,
A method for producing a novel β-galactosidase, which comprises culturing a microorganism having the ability to produce a novel β-galactosidase having the property according to claim 1, and separating and collecting the novel β-galactosidase from the culture solution.
請求項１記載の性質を有する新規β−ガラクトシダーゼ
の生成能を有する微生物を、炭素源としてガラクトー
ス、乳糖およびガラクトオリゴ糖の一種以上を含む培地
に培養し、該培養液から、新規β−ガラクトシダーゼを
分離、採取することを特徴とする新規β−ガラクトシダ
ーゼの製造方法。It belongs to the [claim 3] Kurebijira (Klebsiella) genus,
A microorganism capable of producing a novel β-galactosidase having the property according to claim 1 is cultured in a medium containing at least one of galactose, lactose and galactooligosaccharide as a carbon source, and the novel β-galactosidase is separated from the culture solution. And a method for producing a novel β-galactosidase, which comprises collecting the β-galactosidase.
を有する微生物がクレビジーラ・プランティコラＧ−１
７（Klebsiella planticola Ｇ−１７）（ＦＥＲＭ Ｐ
−１３５７１）である請求項２又は３記載の新規β−ガ
ラクトシダーゼの製造方法。4. The microorganism having the ability to produce the novel β-galactosidase is Klebijira planticola G-1.
7 ( Klebsiella planticola G-17) (FERM P
-13571), The method for producing the novel β-galactosidase according to claim 2 or 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15426993A JPH06339376A (en) | 1993-05-31 | 1993-05-31 | New beta-galactosidase and its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15426993A JPH06339376A (en) | 1993-05-31 | 1993-05-31 | New beta-galactosidase and its production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH06339376A true JPH06339376A (en) | 1994-12-13 |
Family
ID=15580480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15426993A Pending JPH06339376A (en) | 1993-05-31 | 1993-05-31 | New beta-galactosidase and its production |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06339376A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017018135A (en) * | 2010-07-19 | 2017-01-26 | アルラ フーズ アムバＡｒｌａ Ｆｏｏｄｓ Ａｍｂａ | Galacto-oligosaccharide-containing composition and method of producing the same |
| CN107904189A (en) * | 2017-11-13 | 2018-04-13 | 浙江工业大学 | acid-producing Klebsiella and application thereof |
-
1993
- 1993-05-31 JP JP15426993A patent/JPH06339376A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017018135A (en) * | 2010-07-19 | 2017-01-26 | アルラ フーズ アムバＡｒｌａ Ｆｏｏｄｓ Ａｍｂａ | Galacto-oligosaccharide-containing composition and method of producing the same |
| CN107904189A (en) * | 2017-11-13 | 2018-04-13 | 浙江工业大学 | acid-producing Klebsiella and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5888776A (en) | Bacteria and enzymes for production of alternant fragments | |
| US7491518B2 (en) | Method of elevating yield of oligosaccharides containing α-galactosyl and anti-candida compositions | |
| Onishi et al. | Gluco-oligosaccharide and galacto-oligosaccharide production by Rhodotorula minuta IFO879 | |
| JPH0224519B2 (en) | ||
| JPH06339376A (en) | New beta-galactosidase and its production | |
| Akiba et al. | Properties of α-galactosidases of alkalophilic bacteria | |
| JPH0327285A (en) | Beta-fructofrasinodase having strong sugar-transferring activity, production thereof and production of aldosyl fructoside using same enzyme | |
| Yebra et al. | Characterization of novel neopullulanase from Bacillus polymyxa | |
| JP3028258B2 (en) | α-galacto-oligosaccharide composition | |
| EP0771868B1 (en) | Novel deaminoneuraminidase and process for producing the same | |
| JP2970932B2 (en) | Novel heat-stable β-galactosyltransferase, its production method and its use | |
| JP2688854B2 (en) | Method for producing α-galactosidase with strong transglycosylation activity | |
| US5110734A (en) | Purified cyclodextrinase | |
| JPH0523183A (en) | New alpha-galactosidase and production of sugar using the same | |
| JP2688782B2 (en) | Process for producing thermostable α-galactosidase with strong transglycosylation activity and its complex | |
| JPH0119877B2 (en) | ||
| JP2000041693A (en) | Production of galactooligosaccharide | |
| JPH07107988A (en) | Production of lysoglycolipid | |
| JPH062057B2 (en) | Novel β-galactosidase A and method for producing the same | |
| JP3617688B2 (en) | β-N-acetylglucosaminidase | |
| JP2866460B2 (en) | Saccharification method of polysaccharide | |
| JPH05252946A (en) | Novel saccharide hydrolase | |
| JP3812954B2 (en) | Method for producing isomaltosyl fructoside | |
| JPH0761260B2 (en) | New Bacillus microorganism | |
| JPH0284191A (en) | Production of oligosaccharide or glycoside containing alpha-galactosyl group |