Adsorption of cellulase from Trichoderma reesei QM9414 on cellulose and ultraviolet difference spectroscopic study on the interactions of cellulase from T. reesei with cellodextrins

Wen-Juin Shieh, Purdue University

Abstract

Purification and fractionation of the cellulase complex is an important prerequisite to any fundamental kinetic analysis of enzymatic hydrolysis of cellulose. Crude cellulase from Trichoderma reesei QM9414 was separated into five fractions by ion-exchange chromatography. Fractions I, II and III consisted of mainly endoglucanase. Fraction IV and fraction V were mainly cellobiohydrolase. Enzymatic hydrolysis of cellulose is a heterogeneous system. Before the hydrolysis reaction can take place, it is necessary for cellulase enzymes to adsorb onto the surface of cellulose. The fractionated cellulases showed Langmuir adsorption pattern. Fraction III showed the strongest adsorption affinity between endoglucanase and cellulose. Cellobiohydrolase had stronger adsorption affinity to cellulose although endoglucanase initiated the attack on native cellulose and created more chain ends for cellobiohydrolase (C$\sb1$) to react. The formation of cellodextrin-cellobiohydrolase complex was studied by ultraviolet difference spectroscopy. Upon the binding of cellodextrins (G$\sb7$-G$\sb3$), cellobiohydrolase (EC 3.2.1.91) produced characteristic difference spectra with maxima at 289-293 nm and 283-286 nm. Such phenomena indicate that the microenvironment of tryptophan residue(s) located at or near the active site is changed by the interactions between cellodextrins and cellobiohydrolase. The difference spectra maximum were shifted at acidic or alkaline pHs and were accompanied by a marked decrease of binding ability of cellobiohydrolase. The standard free energy change for the association of cellodextrins to the cellobiohydrolase, or the standard affinity, ranged from 3.99 to 3.91 kcal/mol. The association constant of enzyme for substrate decreased as temperature increased from 20$\sp\circ$C to 48$\sp\circ$C. The dissociation constants (25$\sp\circ$C) for the enzyme with cellohexaose and cellotriose were estimated to be 1.19 mM and 1.37 mM, respectively. The decrease in dissociation constants as glucosyl unit increases suggests that there exist at least six subsites in the active center of cellobiohydrolase.

Degree

Ph.D.

Advisors

Tsao, Purdue University.

Subject Area

Agricultural engineering

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS