Effect of the cellulose-binding domain on the catalytic activity of a β-glucosidase from Saccharomycopsis fibuligera

Abstract

Enzyme engineering was performed to link the beta-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera beta-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 ( P )) and terminator (PGK1 ( T )) and yeast mating pheromone alpha-factor secretion signal (MFalpha1 ( S )). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2-4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…Cellulases (exoglucanases and endoglucanases) possess cellulose binding domains that are crucial for these enzymes to interact with their substrate cellulose (45). To improve the activity of single cellulolytic enzymes expressed and secreted by S. cerevisiae, domain engineering of cellulose binding domains has been a frequent target in the recent past (109,130,229).…”

Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovoranscellulose binding domain and its application in hydrolysis of microcrystalline cellulose

“…Further, Sarath reported the effect of a fungal CBD on the enzymatic characteristics of the β-glucosidase from Saccharomycopsis fibuligera . The fusion enzyme displayed a 2–4 fold increasing in their hydrolytic activity toward cellulosic substrates [11]. In the present study, we successfully over-expressed the β-glucosidase (BGL) gene from T. thermosaccharolyticum DSM 571 in E. coli .…”

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

“…Ni-NTA affinity purification of the soluble fraction resulted in a >90% purified enzyme as detected by SDS-PAGE and β-glucosidase activity assay. As fusion of a binding module has been shown to alter the activity and the thermal stability of the catalytic domain (28,29), the kinetic parameters (K m and k cat ), thermal stability and the optimal pH and temperature profiles of BglA-CohII were determined and compared to those of the wild-type enzyme (WT BglA). The thermal stability assay revealed that BglA-CohII retains 80% of its initial activity after 3 h at 60°C as compared to 91% retention of the activity shown by WT BglA thus indicating a decrease of 13%.…”