Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Abstract

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (DeltaT (m) = 2.5 degrees C). By adding the earlier found thermostability-increasing mutation S290T (DeltaT (m) = 1.5 degrees C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4 degrees C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75 degrees C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (DeltaT (m) = 2.5 degrees C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70 degrees C.

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

“…They also stabilize irreversibly unfolding proteins by decreasing the unfolding rate (15). Although a number of studies have reported that the introduction of disulfide bridges can increase enzyme thermostability (8,(10)(11)(12)(13), they have usually introduced only one beneficial disulfide bridge. Few reports describe the introduction of multiple disulfide bridges to improve the enzyme thermostability (9).…”

“…The engineering of disulfide bridges is a promising strategy for improving thermostability that has created stabilized enzymes such as cellobiohydrolases (8,9), lipases (10,11), and xylanases (12,13). Disulfide bridges stabilize proteins undergoing reversible unfolding by decreasing the main-chain entropy of their unfolded states (14).…”

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

“…Figure 6 shows a general shape of the spectra at 25°C, which is characteristic of β-class proteins, comparable to the secondary structure of other cellulases (Voutilainen et al 2009;Colussi et al 2012). The far UV CD spectra of both constructs did not display a negative band around 222 nm, indicating a relatively low content of α-helices (Fig.…”

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

“…While published impacts of CBM addition on enzyme thermostability vary, earlier studies demonstrate the potential of CBMs from thermophilic organisms to increase the thermostability of appended catalytic domains [40,41]. Accordingly, to evaluate the effect of the CtCBM3 module on AnAXE stability, the half-life of AnAXE at 60 • C was used to as a baseline to study impacts of the CtCBM3 fusion.…”