β-galactosidase stability at high substrate concentrations

Abstract

Enzymatic synthesis of galacto-oligosaccharides is usually performed at high initial substrate concentrations since higher yields are obtained. We report here on the stability of β-galactosidase from Bacillus circulans at 25, 40, and 60°C in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. In buffer, the half-life time was 220 h and 13 h at 25 and 40°C, respectively, whereas the enzyme was completely inactivated after two hours at 60°C. In systems with 5.0 and 30% (w/w) lactose, a mechanistic model was used to correct the o NPG converting activity for the presence of lactose, glucose, galactose, and oligosaccharides in the activity assay. Without correction, the stability at 5.0% (w/w) lactose was overestimated, while the stability at 30% (w/w) lactose was underestimated. The inactivation constant kd was strongly dependent on temperature in buffer, whereas only a slight increase in kd was found with temperature at high substrate concentrations. The enzyme stability was found to increase strongly with the initial substrate concentrations. The inactivation energy Ea appeared to be lower at high initial substrate concentrations.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-402) contains supplementary material, which is available to authorized users.