Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungusHumicola griseavar.thermoidea

Abstract

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, includingHypocrea jecorinaCel7A as a reference. The most stable of the homologues,Humicola griseavar.thermoideaCel7A, exhibits a 10°C higher melting temperature (Tmof 72.5°C) and showed a 4–5 times higher initial hydrolysis rate thanH. jecorinaCel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity withH. jecorinaCel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of theH. griseavar.thermoideaCel7A catalytic module (1.8 Å resolution;RworkandRfreeof 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostableH. jecorinaCel7A.