TRANSPLANTING 2 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. Depolymeri zation of the latter substrate is mainly caused by (1-3,1-4)-beta-gluc anases, 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 doma in and interdomain linker of the cellulase from Erwinia carotovora sub species atroseptica was fused to that for the heat stable Bacillus hyb rid, H(A12-M)Delta Y13 (1-3,1-4)-beta-glucanase. The chimeric enzyme s ecreted from Escherichia coli cells did not remain covalently assemble d as judged by SDS-PAGE, However, the glycosylated and intact enzyme ( denoted CELGLU) is secreted from the yeast Pichia pastoris, CELGLU exh ibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities , and was accordingly classified a true multienzyme, HPLC and NMR anal yses revealed that among the products from CELGLU, di- and trimeric ol igosaccharides were identical to those produced by the parental cellul ase, Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-gluc anase activity of CELGLU, were further degraded by the cellulase moiet y to yield glucose and trimers, Compared with the parental enzymes, CE LGLU exhibits substantially higher V-max for degradation of both solub le cellulose and barley (1-3, 1-4)-beta-glucan. These findings point t o construction of multienzymes as an effective approach for engineerin g enzymes with novel characteristics.