Transcript Binding isotherm of cellobiohydrolase TrCel7A depends

EMP171 “Role of enzymes
processivity in degradation of
recalcitrant polysaccharides”
Priit Väljamäe – docent
Morten Sørlie – professor
Silja Kuusk – research scientist
Vincent Eijsink – professor
Riin Kont – doctoral student
Daniel Schaupp – doctoral student
Mihhail Kurashin – doctoral student
Jürgen Jalak – doctoral student
Why? – For better use of biomass
Recalcitrant polysaccharides, cellulose and chitin, are abundant – potential
resource of renewable energy and chemicals.
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
Second generation bioethanol – bioethanol from cellulose (used to replace oil based motor fuels)
First production scale plant was opened in 09.10.2013 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
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
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, 69-95.)
2. Elucidation of the role of processivity in degradation efficiency and
identification of structural determinants of processivity
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.)
3. Optimized regimes for pre-treatment of cellulosic biomass
Role of the partners
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.
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.
By pursuing the goals of the project we will establish a solid basis for a
long-term scientific collaboration.