title: Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases.
abstract: 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 without CBM throughout 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.
authors.affiliation: From Roskilde University, Nature, Systems, and Models, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and.
authors.forename: Trine Holst
authors.initials: TH
authors.lastname: Sørensen
authors.affiliation: From Roskilde University, Nature, Systems, and Models, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: From Roskilde University, Nature, Systems, and Models, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Michael Skovbo
authors.initials: MS
authors.lastname: Windahl
authors.affiliation: From Roskilde University, Nature, Systems, and Models, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Silke Flindt
authors.initials: SF
authors.lastname: Badino
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: From Roskilde University, Nature, Systems, and Models, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000465:Algorithms; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics; D001667:Binding, Competitive; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D002475:Cellobiose; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Hypocrea jecorina; Rasamsonia emersonii; carbohydrate-binding protein; cellulase; enzyme kinetics; thermoactivation; thermophile