Abstract
Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.
Highlights
Plant cellulose presents the most abundant renewable biomass on Earth and the conversion of lignocellulose to biofuel by cellulolytic enzymes is considered a promising approach for providing renewable energy
Screening of cellulose binding proteins in the digestive fluid of sea hare Here, we attempted to isolate proteins enhancing enzymatic cellulose digestion by screening for cellulose binding activity in the A. kurodai digestive fluid which was incubated with cellulose fiber (CF-11) or semi-crystalline starch used as control
No reaction products were detected when Bemcot alone was incubated with 110K BGL, but glucose was detected in high amounts in the reaction containing Bemcot-bound AkEG21 and 110K BGL and especially in that containing AkEG45. These results indicate that cellobiose released from cellulose by AkEG21 and AkEG45 can be further converted to glucose by the activity of 110K BGL
Summary
Plant cellulose presents the most abundant renewable biomass on Earth and the conversion of lignocellulose to biofuel by cellulolytic enzymes is considered a promising approach for providing renewable energy. Proccessive ß-1,4-endoglucanase of sea hare production of biofuels since it produces a variety of enzymes in large quantities, including ß1,4-endoglucanases, cellobiohydrolases (CBHs) and ß-glucosidases [1, 2]. T. reesei secretes different types of cellulases possessing distinct cleavage specificity toward cellulose and oligocellulose including cellobiohydrolases, Cel7A (CBH 1) and Cel6A (CBH 2), endoglucanases (e.g., Cel 6B and Cel7B), and ß-glucosidases. CBHs play a critical role in the saccharification of crystalline cellulose. T. reesei CBHs Cel7A (CBH 1) and Cel6A (CBH 2) are processive cellulases acting on crystalline cellulose from reducing and nonreducing ends, respectively [7,8,9]. A CBH-independent mechanism was reported for a marine bacterium (Saccharophagus degradans) [15]
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