Tuesday, April 20, 2010
10-11

Rate-limiting factors in high solid enzymatic hydrolysis: A calorimetric approach

Søren N. Olsen1, Kim Borch2, and Peter Westh1. (1) Nsm, Rosilde University, Universitetsparken 1, Roskilde, 4000, Denmark, (2) Novozymes A/S, Krogshoejvej 36, Bagsvaerd, Denmark

Enzymatic hydrolysis of highly concentrated cellulosic biomass (>20 % w/w) has become increasingly important as a key step in the production of second generation bio-ethanol.  To this end development of quantitative real-time assays are desirable, both for empirical optimization and for detailed kinetic analysis. In the current work we have tested the applications of isothermal calorimetry to measure enzymatic hydrolysis of different substrates at high solid concentrations (up to 40 %), ranging from pure cellulose to pretreated corn stover. In all cases the heat-flow is directly proportional to the hydrolysis rate, with a sensitivity of 300 pmol glucose s-1, and hence, calorimetry is shown to be a highly sensitive real-time method, independent of the concentration (1-40 %) and complexity of the substrate. The real-time nature of calorimetry, i.e. reaction rate over time, allows for a thorough analysis of cellulase kinetics. In particular, multidimensional analysis varying enzyme, solid or inhibitor (sugar) concentrations is demonstrated to be useful for both empirical optimization of the cellulase mixture/reaction conditions and for more fundamental studies identifying the rate limiting factors/steps in hydrolysis.  

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