Description
Structural polysaccharides like cellulose and chitin provide a promising green alternative for the production of commodity products and motor fuels. Industrial scale usage of cellulose and chitin is hampered by their crystalline structure. Processive chitinases and cellulases are the key enzymes in degradation of recalcitrant polysaccharides. They are also primary targets in protein engineering for improved performance. However, the molecular mechanisms of their mode of action are still not fully understood. Therefore, this project focuses on in-depth studies of processive enzymes involved in cellulose and chitin degradation. We combine the large catalogue of chitinases and cellulases available at Norwegian University of Life Sciences with the substrate-labeling technologies and other research methods developed at University of Tartu. This will generate new fundamental insights into processivity, one of the most important properties of enzymes used in chitin and cellulose biorefining.
Summary of project results
Recalcitrant polysaccharides, cellulose and chitin, are abundant reservoirs of renewable organic carbon. Biorefining of these materials to produce biofuels and commodity products contributes to the development of environment-friendly sustainable industry. To date the processive enzymes (enzymes that release a number of soluble sugar units before leaving the insoluble polymeric substrate) are main components of enzyme cocktails used in enzymatic hydrolysis of cellulose and chitin. Therefore, the aim of the project was to elucidate the role of enzymes processivity in hydrolysis of recalcitrant polysaccharides. The basis of achieving the project goals relies in combining the large and unique catalogue of enzymes available at Norwegian University of Life Sciences, with the equally unique substrate-labelling methodologies available at the University of Tartu. Substrate-labelling technologies originally developed for cellulase research were successfully adapted for chitinase research during the project. Measurements of the processivity and underlying rate constants of chitinase ChiA from bacterium Serratia marcescens and its genetically engineered variants with altered processivity revealed that high processivity was required for efficient degradation of the recalcitrant portion of chitin. On more amorphous forms of chitin the processivity became a penalty reducing the rate of enzyme recruitment. Since strong product binding was necessary for processivity, another cost of processivity can be seen in the strong product inhibition of the enzyme. Therefore the benefits of processive enzymes in degradation of recalcitrant polysaccharides depend on the method for biomass pre-treatment that alters the substrate crystallinity and presence of synergistic enzyme components that alleviate the product inhibition. Besides the scientific output, the project founded a solid base for long-lasting cooperation between the project partners.
Summary of bilateral results
The Norwegian Project partner had a significant role in the bilateral cooperation, since it was the provider of valuable enzymatic activities, as well as the corresponding mutants that were needed for the proposed investigation. The enzymes provided were chitinases from bacterium Serratia marcescens and a human chitotriosidase. In addition, three lytic polysaccharide monooxygenases were provided that gave additional value in the proposed investigation. The Norwegian partner is a well-known and experienced research group on chitinases and lytic polysaccharide monooxygenases that has the potential to provide extensive knowledge to the Estonian group on these enzymatic systems. Active cooperation between the project partners was a prerequisite for all the work carried out and the papers published. As stated in the final report, both partners participated in planning and performing experiments as well as writing papers. In my opinion, considering the work published, the project was the outcome of effective teamwork with the Norwegian Project partner, which finally resulted in strong and sustainable bilateral cooperation.