PMID: 2294974Jan 19, 1990

Comparison of the hydrolytic activity and fluorescence of native, guanidine hydrochloride-treated and renatured cellobiohydrolase I from Trichoderma reesei

Biochimica Et Biophysica Acta
J WoodwardJ M Wichert

Abstract

Guanidine hydrochloride (GdnHCl) is an effective agent for the elution of cellulase protein from unhydrolyzed cellulosic residues, but once eluted the enzyme is inactive. The studies described in this paper examine the effect of GdnHCl on the hydrolytic activity and tryptophan fluorescence of cellobiohydrolase I (CBH I) from Trichoderma reesei. CBH I was found to be completely inactivated by 0.25 M GdnHCl, but higher concentrations of GdnHCl were required to partially unfold this enzyme, as determined from the measurement of a decrease in its tryptophan fluorescence. Binding of CBH I to microcrystalline cellulose was prevented by 4 M GdnHCl, suggesting that a conformational change of CBH I resulted in the loss of substrate binding. Removal of the denaturant from CBH I by dialysis or gel filtration allowed the kinetics of the reactivation of CBH I, after 4 M GdnHCl treatment, to be studied. The fluorescence and specific hydrolytic activity of native and renatured CBH I were comparable. It is concluded, therefore, that GdnHCl may be used to elute cellulase components, such as CBH I, adsorbed on undigested cellulosic substrates since this component can easily be renatured and subsequently reused.

Related Concepts

Sulfite Cellulose
Hyphomycetes
Exoglycosidases
Guanidines
Hydrolysis
Protein Denaturation
Fluorescence Spectroscopy
Structure-Activity Relationship
Trichoderma
Cellobiohydrolase II

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