Characterization of cellulases produced by trichoderma longibrachiatum DIBAF-10 using differential scanning calorimetry

Conversion of cellulose to soluble sugars by enzymatic hydrolysis is a key step for production of biofuels from lignocellulosic biomass. Efficient hydrolysis of cellulose requires the action of three different classes of enzymes: endoglucanase (1,4-β-d-glucan glucohydrolase [EC 3.2.1.4]), exoglucanase (1,4-β-d-glucan cellobiohydrolase [EC 3.2.1.91]), and β-glucosidase (β-d-glucoside glucohydrolase, [EC3.2.1.21]) [1]. Trichoderma longibrachiatum DIBAF-10 produced a cellulase cocktail tailored for the saccharification of milk thistle (Silybum marianum) with the potential to compete with commercial enzymes. In this study, crude cellulase preparations from T. longibrachiatum DIBAF-10 were thermally studied by differential scanning calorimetry (DSC), to assess the conformational transitions between the folded and unfolded structure of the proteins and the relationship between them and the energetics of their stability. Generally, the aggregation can take place concurrently with the irreversible thermal denaturation and the conformational unfolding or accompanies an exothermal effect that results in formation of precipitation [2]. The study of thermal properties is essential to better characterize and understand the interactions between cellulase and its substrates and their dependence on temperature. In fact, enzyme thermo-stability is essential during saccharification reaction because steam is always used to make the substrates more suitable for the enzymatic hydrolysis [3]. The present work shows that DSC profiles of crude enzyme samples from T. longibrachiatum DIBAF-10 provide important thermodynamic information, about the thermostability of the included proteins. The thermograms of crude enzyme coktails, and of the commercial ones, show similar exothermic peaks at 52,45±0,90°C and 49,5±0,95°C, respectively, and comparable H values. This is probably due to the same conformational change leading to aggregation of proteins. DSC, moreover, is cost-effective tool to obtain “conformational fingerprinting” of the crude enzyme cellulase preparations.