Purification, properties and characterization of a high-molecular-mass beta-galactosidase isoenzyme from Thermus aquaticus YT-I

An unusual high-molecular-mass beta-galactosidase isoenzyme from Thermus aquaticus YT-1 was purified to homogeneity by chromatography using gel filtration (Ultrogel AcA 34), anion-exchange (Mono Q) and gel filtration (Superose-12), The molecular mass of the purified enzyme was > 700 kDa as estimated by gel filtration, and its subunit molecular mass was 59 +/- 1 kDa as determined by SDS/PAGE, A single protein band of pl 4,9 was obtained by isoelectric-focusing gel electrophoresis, The optimum temperature and pH for enzyme activity were 80 degrees C and pH 5.5 respectively, The enzyme was stable over a wide pH range (pH 3-12), and the thermostability of the enzyme was enhanced by CaCl2, The enzyme was significantly activated by alkali and alkaline-earth-metal salts, Whereas reducing agents enhanced beta-galactosidase activity, thiol-binding agents drastically decreased the enzyme activity, The enzyme was strongly inhibited by the end products of the lactose hydrolysis reaction, The beta-galactosidase was specific for beta-D anomeric linkages, and the identity of the aglycone moiety also influenced enzyme activity dramatically, Oligosaccharide (OS) formation rates were directly related to lactose concentration, At a concentration of 19.4% (w/v) lactose, OS accounted for approx, 20% of the total carbohydrates, and only two types of OS were observed, Moreover, once formed, OS were not rehydrolysed to monosaccharides, even during long residence times, OS synthesis was also observed with cellobiose and lactulose, Fructose, xylose, lyxose, rhamnose and arabinose were active as galactosyl accepters, in contrast with glucose, ribose, fucose, tagatose and N-acetyl-glucosamine.