Studies on β-Galactosidase. II. Purification of β-Galactosidase from Macrophomina phaseoli and Its Enzymatic Properties

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Acid β-galactosidase (EC 3. 2. 1. 23) was purified from the culture filtrate of Macrophomina phaseoli by column chromatography using DEAE-cellulose and Sephadex G-200,and isoelectric focusing (pI, 3.6). The purified enzyme was homogeneous on disc electrophoresis. The enzyme is considered to be a glycoprotein, because the mobility of the protein band coincided with that of sugar one in disc electrophoresis, and because sugar content was not varied before and after isoelectric focusing of the purified enzyme. The sugar content of the enzyme was estimated to be 12% with phenol-sulfuric acid method. The enzyme was most active at pH 5.0 and 60°, and stable over a pH range from 4.0 to 8.0 and below 55°. The enzymatic activity was completely inactivated only by N-bromosuccinimide at 0.1 mM. K_m value was determined to be 0.45 mM for o-nitrophenyl β-D-galactopyranoside (ONPG) and 15 mM for lactose, and V_max was calculated to be 93.6 μmoles・min^<-1>・mg^<-1> for ONPG and 54.5 μmoles・min^<-1>・mg^<-1> for lactose.
1976-04-25