Domain engineering of Saccharomyces cerevisiae exoglucanases.
To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg(-1) respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.
Moses
S B Gundllapalli
SB
Institute for Wine Biotechnology and Department of Viticulture & Oenology, Stellenbosch University, Victoria Street, Stellenbosch, ZA 7602, South Africa.
Otero
R R Cordero
RR
Pretorius
I S
IS
D000327:Adsorption; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014242:Trichoderma / Q000201:enzymology
D017931:DNA Primers; D010455:Peptides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C468487:EXG1 protein, S cerevisiae; D043326:Glucan 1,3-beta-Glucosidase; C117648:beta-1,3-exoglucanase