Hydrolysis of soluble cellooligosaccharides by trichoderma reesei cellobiohydrolase 7A

Abstract

Cellulose is the main component of plant cell wall and the most abundant biopolymer on the earth. The complexity of cellulose structure and multiple binding modes of cellulose restrict the ease of understanding the mode of action of cellulolytic enzymes. Hence, in this study, soluble cellooligosaccharides were used as substrates. In nature, microorganisms are capable of degrading cellulose by secreting a set of enzymes called cellulases. Most studies have been conducted on the cellulolytic system that is secreted by the soft-rot fungus, Trichoderma reesei (Tr). This study aims to analyse the hydrolysis pattern of cellooligosaccharides (including cellotriose, cellotetraose, cellopentaose and cellohexaose) when hydrolysed by Tr cellobiohydrolase 7A (TrCel7A) and evaluate the reaction kinetics such as bond cleavage frequencies and bond cleavage probabilities. This work involves purification of enzyme TrCel7A from enzyme mixture (Celluclast®), analysis of purified enzyme TrCel7A, preparation of substrate (i.e. cellooligosaccharides), hydrolysis of the cellooligosaccharides with degree of polymerisation (DP) from 3 to 6 by TrCel7A at 25 °C for 1 hour, and high-performance liquid chromatography analysis of the product concentration. Based on kinetic modelling of cellooligosaccharides, the frequencies of hydrolysis of the glycosidic bond in the enzyme-substrate complex and the probabilities that hydrolysis of glycosidic bond took place specifically in the enzyme-substrate complex were calculated. Based on the quantitative data on the bond cleavage frequencies of cellooligosaccharides with DP 4 to 6 showed that the bond cleaved more frequent at glucose linkage (cellopentaose = 0.102 ± 0.021 s-1, cellohexaose = 0.109 ± 0.011 s-1) followed by at cellobiose linkage (cellopentaose = 0.085 ± 0.003 s-1, cellohexaose = 0.053 ± 0.002 s-1) and cellotriose linkage (cellohexaose = 0.040 ± 0.004 s-1). A similar trend was observed for the result of bond cleavage probabilities for most substrates that shows the glucose linkage (cellopentaose = 0.542 ± 0.050 s-1, cellohexaose = 0.540 ± 0.036 s-1) of the substrate chain has the highest probabilities than cellobiose linkage (cellopentaose = 0.458 ± 0.050 s-1, cellohexaose = 0.263 ± 0.021 s-1) and cellotriose linkage (cellohexaose = 0.197 ± 0.022 s-1). Therefore, this research suggested that TrCel7A catalysed degradation of cellooligosaccharides from the reducing end of the chain. Also, it specified that the position of whole cellooligosaccharides chain is in the active site for the TrCel7A.