Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei.
Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei. The enzyme contains a discrete cellulose-binding domain (CBD), which increases its binding and activity on crystalline cellulose. We studied cellulase-cellulose interactions using site-directed mutagenesis on the basis of the three-dimensional structure of the CBD of CBHI. Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism. The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality. The data also suggest that the more hydrophobic surface is not directly involved in the CBD function. The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption. The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect. High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD.
D000327:Adsorption; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D006026:Glycoside Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008278:Magnesium Sulfate / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology*
D011994:Recombinant Proteins; D008278:Magnesium Sulfate; D002482:Cellulose; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase