Degradation of insoluble carbohydrate polymers: how do microbial chitinolytic machineries work?

Enzymatic degradation of abundant insoluble carbohydrate polymers such as cellulose and chitin is of tremendous biological and biotechnological importance. Microbes produce numerous enzymes to hydrolyze these polymers, but the question of how they manage to disrupt the crystalline structure of the substrate, thus increasing enzymatic turnover rates, remains largely unanswered. This limits the application of enzyme technology, e.g. in biofuel production. We will address the issue of substrate disruption an d accessibility by studying enzyme domains and non-hydrolytic proteins that are potentially involved in these processes. We will use chitin degradation by the Gram-negative soil bacterium Serratia marcescens as a model system. The work will be partly base d on recent discoveries in our own laboratory, which constitute a new concept in the field (G. Vaaje-Kolstad et al., ?The non-catalytic chitin-binding protein CBP21 from Serratia marcescens, is essential for chitin degradation?; Journal of Biological Chem istry, 2005). We will study the functionality of S. marcescens chitinases (with or without substrate-binding domains), isolated substrate-binding domains, and non-hydrolytic substrate disrupting proteins such as CBP21, by analyzing (1) their contributions to the enzymatic turnover of chitin and (2) their abilities to bind, invade and disrupt chitin particles. These studies will be complemented by in vivo studies with S. marcescens strains in which one or more of the genes involved in chitin turnover have been deleted or mutated. We will also make a limited effort in proteomics, aimed at identifying other secreted proteins that may play hitherto unknown roles in turnover of natural chitinous materials by the bacterium. Finally, similar studies on cellulose degradation will be performed during a one-year stay of the project post-doc in a foreign laboratory.