Purification, Crystallization, and Properties of β-Galactosidase from Bacillus macerans

Abstract

β-Galactosidase (EC 3.2.1.23) from Bacillus macerans was produced in a medium containing lactose. The enzyme was purified through the following steps: precipitation with ammonium sulfate, gel filtration on Sephadex G-100, chromatography on DEAE-Sephadex A-50, a second chromatography on DEAE-Sephadex A-50, and a second gel filtration on Sephadex G-100. Hexagonal plate crystals were formed when acetone was dropped into the purified enzyme solution containing calcium ion. The purified enzyme was homogeneous on polyacrylamide disc electrophoresis, and the molecular weight was estimated to be about 320, 000 by gel filtration on Sephadex G-200 and about 78, 000 by sodium dodecyl sulfate gel electrophoresis. The purified enzyme migrated as a single protein band in both assays of molecular weight. Therefore, the enzyme consisted of four probably identical subunits. The isoelectric point of the enzyme was about pH 4.4. The enzyme had its optimal pH at 6.5 and was stable between pH 6 and 9. The enzyme was relatively stable below 37°C but completely lost its activity after heating 60°C for 10min. The Michaelis constants were 2.0mM for p-nitrophenyl-βgalactoside and 30mM for lactose. The enzymic activity was completely inhibited by Ag⁺, Hg²⁺, Cu²⁺, and p-chloromercuribenzoate, and it was considerably inhibited by monoiodoacetic acid, trisaminomethane, and galactose.