Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.
A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.
Onishi
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Central Research Laboratories of Ajinomoto Co. Inc., Kawasaki-shi, Japan.
Tanaka
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T
D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000096:biosynthesis / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D014312:Trisaccharides; C023872:mannotriose; D001616:beta-Galactosidase