Purification and Properties of a Galacto- and Gluco-Oligosaccharide-Producing β-Glycosidase from Rhodotorula minuta IFO879

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A β-glycosidase with strong transglycosylation activity was solubilized from the cell wall fraction of Rhodotorula minuta IFO879 by a cell wall lytic enzyme, Usukizyme, and was purified to homogeneity with a yield of 53% by DEAE-Toyopearl, Butyl-Toyopearl, p-aminobenzyl 1-thio-β-D-galactopyranoside agarose and Con A agarose column chromatography. The native enzyme is a glycoprotein with a molecular weight of 144,000 and is composed of two subunits with molecular weights of about 72,000. Its isoelectric point was estimated to be 4.8 by polyacrylamide gel electrofocusing. The optimal temperature for enzyme activity is 70°C. It is stable at temperatures up to 55°C for 1 h. The optimal pH range is 4.7 to 5.2,and stability was maintained between pH 3.0 and 7.0. The purified β-glycosidase was found to be active toward β-D-fucopyranoside and α-L-arabinopyranoside as well as β-D-galactopyranoside and β-D-glucopyranoside. The K_m values measured for lactose, cellobiose, o-nitrophenyl-β-D-galactopyranoside and p-nitrophenyl-β-D-glucopyranoside are 2.4,11.1,6.2 and 1.2 mM, respectively, and V_<max> values for these substrates are 63.6,270.6,34.4 and 20.7 μmol/min per mg protein, respectively.In addition, this enzyme exhibits strong transglycosylation activities, producing 78 mg/ml galacto-oligosaccharide or 68 mg/ml gluco-oligosaccharide from 200 mg/ml lactose or cellobiose, respectively.
公益社団法人日本生物工学会の論文
1996-11-25