Structure-function studies of chitinases and engineering of variants with improved properties

SAMMENDRAG: Chitinases degrade chitin, an abundant polymer of N-acetylglucosamine occurring in, for example, shrimp shells, insect cuticles and fungal cell walls. Chitinases are of biotechnological interest because (1) they can convert c hitin-containing biomass into useful products, (2) they have anti-fungal activity, and (3) chitinase inhibitors inhibit growth of chitin-containing organisms. This project is focussed on two chitinases from Serratia marcescens, ChiA and ChiB. The catalytic mechanism and the structural basis of substrate- and product specificity will be studied. Structural determinants of enzymological differences between ChiA and ChiB will be identified. Improved variants of ChiA and ChiB wil l be constructed, using both site-directed mutagenesis and random approaches (e.g. gene shuffling) coupled to high-throughput screening of mutants. One part of the project focuses on engineering stability at low pH and high temperatures. Anoth er part aims at manipulating substrate- and product specificities by careful analysis and subsequent manipulation of the architecture of substrate binding sites. Optimisation of both these parameters should result in robust biocatalysts that can be used t o convert chitin or partially deacetylated chitin into oligomeric fragments, preferably of defined lengths. The structures of interesting chitinase variants, as well as chitinases complexed with substrates or inhibitors will be studied using X-ray crystal lography.