Temperature effects on kinetic parameters and substrate affinity of Cel7A cellobiohydrolases

Trine Holst Sørensen, Nicolaj Cruys-Bagger, Michael Skovbo Windahl, Silke Flindt Badino, Peter Westh

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We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants
of these enzymes designed to elucidate the role of the carbohydrate
binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced
negative effect on the maximal rate, which was 2-fold larger in variants withoutCBMthroughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general
theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.
TidsskriftJournal of Biological Chemistry
Sider (fra-til)22193-22202
StatusUdgivet - 2015

Citer dette

Sørensen, T. H., Cruys-Bagger, N., Windahl, M. S., Badino, S. F., & Westh, P. (2015). Temperature effects on kinetic parameters and substrate affinity of Cel7A cellobiohydrolases. Journal of Biological Chemistry, 290, 22193-22202.