Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose

Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages, To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M)Delta Y13 (1-3,1-4)-beta-glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE, However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris, CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme, HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase, Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers, Compared with the parental enzymes, CELGLU exhibits substantially higher V-max for degradation of both soluble cellulose and barley (1-3, 1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.