Comprehensive Enzymatic Analysis of the Cellulolytic System in Digestive Fluid of the Sea Hare Aplysia kurodai. Efficient Glucose Release from Sea Lettuce by Synergistic Action of 45 kDa Endoglucanase and 210 kDa ß-Glucosidase

Abstract

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.