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 Vmax 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.
Olsen
O
O
Department of Physiology, Gamle Carlsberg Vej 10, Copenhagen, Denmark. olsen@biobase.dk
Thomsen
K K
KK
Weber
J
J
Duus
J O
JO
Svendsen
I
I
Wegener
C
C
von Wettstein
D
D
D019677:Artificial Gene Fusion; D002384:Catalysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005815:Genetic Code; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D009097:Multienzyme Complexes; D010843:Pichia; D017434:Protein Structure, Tertiary
D009097:Multienzyme Complexes; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D002480:Cellulase; D005947:Glucose