β-Galactosidase from Bacillus stearothermophilus

Abstract

Several strains of thermophilic aerobic spore-forming bacilli synthesize b-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The .beta.-galactosidase from 1 strain, investigated in cell-free extracts, has a pH optimum between 6.0-6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65.degree. C and the Arrhenius activation energy is about 24 kcal/mol below 47.degree. C and 16 kcal/mol above that temperature. At 55.degree. C the Km is 0.11 M for lactose and 9.8 .times. 10-3 M for o-nitrophenyl-.beta.-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki = 2.5 .times. 10-3 M). It is relatively stable at 50.degree. C, losing only half of its activity after 20 days at this temperature. At 60.degree. C more than 60% of the activity is lost in 10 min. The enzyme is protected somewhat against thermal inactivation by protein and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60.degree. C. Its MW, estimated by disc gel electrophoresis, is 215,000.