Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

Abstract

We have measured hydrolytic rates of four purified cellulases in small increments of tem-perature (10-50 degree Celsius) and substrate loads (0-100 g/l), and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from respectively 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 while 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 hy-drolytic 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, it had a pronounced negative effect on the maximal rate, which was two-fold larger in variants without CBM throughout the investigated temperature range. We conclude that while 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. Forfattere: Trine Holst S?rensen, Nicolaj Cruys-Bagger, Michael Skovbo Windahl, Silke Flindt Badino, Kim Borch & Peter Westh