Molecular cloning, expression, purification and structural characterization of endoglucanase from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol

Author(s)

Vanessa de Oliveira Arnoldi Pellegrini

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2016-03-09

Examining board members

Igor Polikarpov; Raghuvir Krishnaswamy Arni; Carlos Eduardo Driemeier; Mario Tyago Murakami; Ronaldo Alves Pinto Nagem

• Advisor: Igor Polikarpov; Cesar Moises Camilo

Abstract

The increase in global energy demand, shrinkage perspective of energy resources and global concern about environmental issues, aroused interest in finding alternative sources of energy. Lignocellulosic biomass is plentiful and inexpensive, and has the potential to complement the large-scale production of fuel. The degradation of molecules that constitute the cell wall to fermentable sugars and then to ethanol, occurs via the enzymatic hydrolysis of biomass. However, the use of enzymes for this purpose is in exploratory stage and represents a bottleneck in the implementation of 2G ethanol technologies in industrial scale, supporting studies about biochemically most active, stable and economically viable cellulases. This work aimed the characterization of endoglucanase I from fungus Trichoderma harzianum and for that, was performed expression and biochemical and biophysical assays of the catalytic domain (ThCel7B-CCD) and full protein (ThCel7B-full). The enzyme exhibited a acidophilic profile, with optimal activity at pH 3.0 at 55°C. The protein was also able to hydrolyze a variety of substrates, with higher hydrolytic activity in β-glucano (75 U mg-1). Analyzing the thermal stability as pH 5, residual activity remained intact for over 2 months. Another important feature was the high degree of synergy between ThCel7B and ThCel7A (DS 3). Electron microscopy analysis of oat samples subjected to hydrolysis with ThCel7B show the substrate degradation effects in relation to the control samples. All these results, as well as important for understanding the molecular mechanism of ThCel7B and other endoglucanases of GH7 family, also disclose an enzyme of biotechnological interest because the acid behavior and thermal stability are promises of industrial applications under extremely acidic conditions. (AU)

• FAPESP's process: 10/18773-8 - Molecular cloning, expression, purification and structural characterization of endoglucanase from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol
• Grantee: Vanessa de Oliveira Arnoldi Pellegrini
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)