A laminaribiose-hydrolyzing enzyme, AkLab, from the common sea hare Aplysia kurodai and its transglycosylation activity

Endo-β-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 °C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-β-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences.