Glucose-cellobiose tolerant thermostable cellulases from Scytalidium thermophilum: heterologous expression, characterization and application in lignocellulosic biomass saccharification

Abstract

There is currently a great interest in the economically viable production of ethanol from lignocellulosic biomass. Intense research is being conducted aiming to the development of efficient and inexpensive processes for the enzymatic hydrolysis of lignocellulose in agroindustrial residues or byproducts, as well as in urban garbage. Enzymes are responsible for a great share of the hydrolysis total costs, since high enzymatic loads must be used to attain acceptable yields. This is mainly attributed to product inhibition of cellulases and hemicellulases. Among the strategies proposed to enhance the efficiency and lower the costs of the enzymatic hydrolysis, the identification of more efficient enzymes (particularly those that are stimulated or tolerant to the products), the heterologous expression of enzymes with attractive properties and the formulation of more efficient enzymatic cocktails must be stressed. In the laboratory of the supervisor it was identified, purified and characterized a ß-glucosidase from Humicola insolens RP86 which is stimulated by glucose and xylose. The enzyme was cloned and expressed in E. coli and P. pastoris, and maintained the property of xylose and glucose stimulation. Recently, an endoglucanase and a cellobiohydrolase from Scytalidium thermophilum CBS 619.91 were also purified and characterized, and revealed to be thermostable and tolerant to celobiose and glucose, but produced at low levels. In parallel, efficient cocktails were developed for the hydrolysis of raw sugarcane trash and bagasse, as well as untreated waste papers of different kinds, without the inclusion of product stimulated/tolerant ß-glucosidases or cellulases. In this project we intend to clone and express in P. pastoris, in active form, the cellobiose/glucose tolerant cellulases from S. thermophilum. The heterologous enzymes will be purified and submitted to biochemical, biophysical and kinetic characterization, with special emphasis in their properties of product tolerance. The recombinant forms of the S. thermophilum cellulases and the H. insolens ß-glucosidase, tolerant/stimulated by the products, will be also evaluated for their potential to compose enzymatic cocktails, particularly when added to previously standardized cocktails. The intended results may contribute to the understanding of the molecular mechanisms of celobiose and glucose stimulation of the S. thermophilum cellulases, as well as for the formulation of more efficient cocktails for the hydrolysis of lignocellulosic biomass, thus contributing to develop viable processes for the production of second generation ethanol, a renewable fuel strategically important for the development of our country. (AU)