Separation and characterization of three beta-galactosidases from Bacillus circulans

Crude preparation of Bacillus circulans beta-galactosidase is known to have a good transglycolytic activity. Two isoforms of the enzyme have been described so far in the literature. Aiming at separating these two forms to assess their relative contribution to the regioselectivity of transglycosylation, we observed the presence of a third isoform never described before. This paper deals with the isolation procedures of the three enzymes and a re-consideration of their properties. The estimated molecular weight for the isoforms were 212 kDa (I), 145 kDa (II) and 86 kDa (III), respectively. Kinetic parameters were determined towards the hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) and lactose. For ONPG the following values of Km were found: 3.6, 5.0 and 3.3 mM for I, II and III, respectively, whereas for lactose the values were 3.7, 2.94 and 2.71 mM, respectively.