Development of new biocatalysts for the hydrolysis of lignocellulosic substrates

The industrial conversion of lignocellulosic biomasses into second-generation biofuels or other high-added-value products includes a saccharification step to hydrolyze the polysaccharides into fermentable sugars carried out by means of an enzymatic cocktail including cellulases. The production costs of these enzymes represent the main contributor to the expense of the overall process and a huge amount of enzymes is required for the hydrolysis. Therefore, the production of new cellulolytic enzymes more efficient in lignocellulose conversion represents one of the main routes to contribute to the cost reduction of biomass conversion. In this context the aim of this PhD project has been to develop biocatalysts for the enhanced hydrolysis of cellulose into monosaccharides, particularly for spent mushroom substrate supplemented with wheat straw. Two new GH5 family cellulases (Cel1 and Cel2) have been cloned, recombinantly expressed in Escherichia coli, characterized and tested in the enzymatic hydrolysis of three pretreated biomasses Populus nigra, Panicum virgatum and spent mushroom substrate (SMS) supplemented with wheat straw (WS). Based on these results, Cel2 was selected for the development of improved biocatalysts by directed evolution. A library of 30,000 random mutants was generated and screened for their activity. The diversity was introduced into the cel2 gene by error-prone Polymerase Chain Reaction and the screening on solid and liquid medium was set up and applied for all the mutants. The improved variants were cultured in a flask and were characterized. The thermoresistant mutants were then tested in the enzymatic hydrolysis of pretreated SMS/WS in conjunction with a commercial enzymatic hydrolysis performance booster mixture (MetZyme® SUNO™ BOOSTER 144), allowing us to obtain improved biocatalysts compared to the wild type Cel2. Moreover, the selection of a lytic polysaccharide monooxygenase was performed in order to set up a recombinant expression system, considering the capability of these enzymes to enhance the bioconversion of lignocellulosic substrates when added to the enzymatic cocktail