Effect of the cellulose-binding domain on the catalytic activity of a beta-glucosidase from Saccharomycopsis fibuligera.
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.
Gundllapalli
Sarath B
SB
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Victoria Street, Stellenbosch, ZA, 7602, South Africa.
Pretorius
Isak S
IS
Cordero Otero
Ricardo R
RR
D002384:Catalysis; D020134:Catalytic Domain; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism* / Q000648:ultrastructure; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012442:Saccharomycopsis / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases; D043366:Cellulose 1,4-beta-Cellobiosidase