Modeling of enzyme kinetics on polymeric substrates by simulation : Enzymatic hydrolysis of cellulose

Sammanfattning: Biopolymers are an essential constituent of living matter. To develop new products and processes based on enzyme production and modification and hydrolysis of biopolymers, enzymes with well understood mechanism are required. It is impossible to derive a universal analytical model for every particular enzyme/polymer system. Therefore, we have developed an object-oriented tool for modeling of the dynamics of these systems. The tool is intended to be "soft", which means that system components and interactions between components are easy to construct and modify.In order to demonstrate our method in use, modeling is applied for design of experimental systems which allow us to discriminate between different mechanistic models of the cellulose/cellulase system:I Experimental system with reducing-end-labeled cellulose:Modeling shows that the hydrolysis pattern of reducing-end-labeled crystalline cellulose can be used to answer questions about 1) end-preference of enzymes 2) the role of processivity in enzyme actions 3) the existence of probability for endoaction 4) the role of non-productive binding of enzymes. Modeling shows also that hydrolysis of reducing-end-labeled cellulose by enzymes with a mixed exo-endoaction does not provide us with information about the mode of exo-action. The system has been applied efficiently for discussion of the mode of action of CBHI and CBHII from Trichoderma reesei and of CBH50 and CBH58 from Phanerochaete chrysosporium.II Experimental system with randomly substituted end-labeled cellulose:Modeling shows that the hydrolysis pattern of substituted reducing-end-labeled crystalline cellulose is very sensitive to changes in 1) end-preference of enzymes 2) processivity 3) exo-endoprobability 4) substitution degree of substrates. It implies that randomly substituted cellulose has a high potential for the determination of characteristic constants describing modes of enzyme action. Furthermore, modeling is used to introduce a new hypothesis about the reason for the dramaticretardation of mono-enzymatic hydrolysis of cellulose already at low degrees of conversion. The results of modeling demonstrate clearly that in this system ((strict exo-enzyme)/ (crystalline cellulose)), a clear retardation of the initial hydrolysis rate is observed. It allows us to propose that the change in the microstructure ("erosion") of fhe cellulose surface can be an adequate explanation for this phenomenon. The synergism of enzymes with different modes of action is thus considered to avoid the disadvantageous changes in cellulose microstructure.