The kinetics of cellulose enzymatic hydrolysis : Implications of the synergism between enzymes

• Main Author: Väljamäe, Priit
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Institutionen för naturvetenskaplig biokemi 2002
• Subjects: Biochemistry; Acetobacter; Cellobiohydrolase; Cellobiose; Cellulase; Cellulose; Diffusion; Endoglucanase; Hydrolysis; Inhibition; Kinetics; Model; Product; Substrate; Surface; Synergism; Trichoderma reesei; Biokemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3120; http://nbn-resolving.de/urn:isbn:91-554-5479-8

The hydrolysis kinetics of bacterial cellulose and its derivatives by Trichoderma reesei cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.