Intramolecular synergism in an engineered exo-endo-1,4-beta-glucanase fusion protein.
Exoglucanase CelY and endoglucanase CelZ from the cellulolytic thermophile Clostridium stercorarium act in synergism to hydrolyse cellulosic substrates. To increase the efficiency of the hydrolytic degradation process, an artificial multienzyme carrying both enzymatic activities on one polypeptide chain was constructed by gene fusion. A segment of CelZ, CelZdeltaBB'C (designated CelZC'), comprising the catalytic domain and the adjacent domain C' homologous to the cellulose-binding domain family IIIc, was fused to the C-terminus of CelY, yielding the fusion protein CelY-CelZC', designated CelYZ. The large fusion protein (170 kDa) could be isolated from a recombinant Escherichia coli strain in its intact form retaining the pronounced thermostability of the fusion partners. As a true multienzmye, CelYZ exhibited both exoglucanase and endoglucanase activities. The cellulolytic activity of the fusion protein was three- to fourfold higher than the sum of the individual activities. Dilution experiments showed that the enhanced cellulolytic activity of the multienzyme resulted from intramolecular synergism of the fusion partners. The product profiles and the kinetic constants of cellulose hydrolysis support a new mechanistic model proposed for explaining the co-operativity of the two catalytic domains within the multienzmye.
Riedel
K
K
Lehrstuhl für Mikrobiologie, Technische Universität München, Munich, Germany.
Bronnenmeier
K
K
D019677:Artificial Gene Fusion; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D004357:Drug Synergism; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase