EMP171 “Role of enzymes processivity in degradation of recalcitrant polysaccharides” Priit Väljamäe – docent Silja Kuusk – research scientist Riin Kont – doctoral student Mihhail Kurashin – doctoral student Jürgen Jalak – doctoral student Morten Sørlie – professor Vincent Eijsink – professor Daniel Schaupp – doctoral student

Why? – For better use of biomass Recalcitrant polysaccharides, cellulose and chitin, are abundant – potential resource of renewable energy and chemicals. Second generation bioethanol – bioethanol from cellulose (used to replace oil based motor fuels) First production scale plant was opened in in Italy Cellulose to ethanol process includes three major steps: Pre-treatment of biomass Enzymatic hydrolysis to glucose - increasing the efficiency of enzymatic hydrolysis will increase the cost efficiency of the process Fermentation of glucose to ethanol Cellulose is the major component of cell walls of plants and algae Chitin is the major component of exosceletons of crustaceans and arthropodes and cell walls of fungy

Objective 1 – what are the costs and benefits of enzymes processivity? Cellulose – homopolymer of β-1,4-linked glucose units Chitin – homopolymer of β-1,4-linked N-acetylglucosamine units Both polymers have similar crystalline structure, which makes them recalcitrant towards enzymatic degradation. Degradation is mediated by the set of hydrolytic and oxidative enzymes – the key enzymes are processive hydrolases, which once bound will release a number of glucose units (processivity). Processive cellulases and chitinases are slow enzymes – why? Processive cellulase acting on cellulose (courtesy to Christina Divne)Processive chitinase acting on chitin (From Uchiyama et al 2001, J. Biol. Chem.) Objective 2 – Optimization of pre-treatment regimes of cellulosic biomass to minimize the formation of cellulase inhibitors

Expected achievements Aromatic amino acids (W, F, and Y) are thought to play central role in determining the processivity of chitinases (From Zakariassen et al 2009, J. Biol. Chem.) 1.Methods for measuring chitinase processivity Currently there are no methods for quantification of chitinase processivity (Horn, S.J., Sørlie, M., Vårum, K.M., Väljamäe, P., and Eijsink, V.G.H. (2012) Measuring processivity. Methods Enzymol. 510, ) 2. Elucidation of the role of processivity in degradation efficiency and identification of structural determinants of processivity 3. Optimized regimes for pre-treatment of cellulosic biomass

Role of the partners Project promoter – University of Tartu Development of the methods for measuring chitinase processivity. Measuring the processivity and other kinetic parameters of chitinases and their variants. Characterization of the liquid fractions from pre-treatment of cellulosic biomass in terms of cellulase inhibitors. Donor project partner – Norwegian University of Life Sciences Production of chitinases and their variants. Pre-treatment of cellulosic biomass. In the project we combine the large and unique catalogue of chitinases available at the Norwegian University of Life Sciences, with the equally unique research methodologies available at the University of Tartu. By pursuing the goals of the project we will establish a solid basis for a long-term scientific collaboration.