NEW WILD-TYPE AND ENGINEERED BIOCATALYSTS AND BIOSYSTEMS FOR CONVERSION OF THE LIGNOCELLULOSIC BIOMASS ARUNDO DONAX INTO FUEL ETHANOL

The hydrolysis of lignocellulosic biomasses to obtain fermentable sugars is complex process which requires multiple and synergistic enzymes such as cellulases, hemicellulases (xylanases) and accessory enzymes (e.g. arabinofuranosidases and pectinases). The improvement of the hydrolysis yield and the reduction of the needed amounts of the enzymes involved in the process and thus their costs represent the main challenges of second generation ethanol production. To meet these necessities, different strategies can be adopted such as the isolation and identification of new microorganisms producing high levels of (hemi)cellulase activities and the development of new tailor-made enzymes with high specific activity and high resistance at the conditions used in the hydrolysis process. In this project, compost obtained from agro-industrial wastes was investigated as a source of new bacteria producing new accessory enzymes. The Paenibacillus xylanolyticus 2-6L was isolated from agro-industrial wastes based compost and its pectinolytic activity was characterized. A pectate lyase (Paenxyl Pel) was identified, purified and characterized. Paenxyl Pel exhibits an optimum temperature of 60-70°C and optimal pH value of 9.0 for its pectinase activity on pectin from citrus fruit. Paenxyl Pel shows high thermoresistance and pH resistance and it follows a Michaelis–Menten kinetic towards pectin from citrus fruit, pectin from sugar beet pulp, high ester pectin extracted from citrus peel (>50% esterified) and polygalacturonic acid (PLA). These results suggest that pectate lyase from P. xylanoliticus shows an unusual activity combining traits of pectate lyase and pectin lyase. The development of tailor made arabinofuranosidase was carried out by using directed evolution of PoAbf, the -L-arabinofuranosidase produced by fungus Pleurotus ostreatus. The best evolved variant of the first generation library, rPoAbf F435Y/Y446F, was recombinantly expressed in Pichia pastoris, purified and characterized. It exhibited a Kcat of 7.3*103 ± 0.3 min-1, around 3-fold higher than that of the wild-type and a KM (0.54 ± 0.06 mM), 30% lower than that of the wild-type towards towards p-nitrophenyl--L-arabinofuranoside. The mutant showed also improved catalytic properties towards pNP-β-D-glucopyranoside (Kcat of 50.85 ± 0.21 min−1 versus 11.0 ± 0.6 min−1) and it was shown able to hydrolyze larch arabinogalactan that is not recognized by the wild-type. The mutant was also more active than the wild-type towards arabinoxylan and was able to hydrolyze arabinan that was not transformed by the wild-type. Even if the mutant rPoAbf F435Y/Y446F was selected for its higher activity, it showed also a higher resistance to temperature and pH than the wild-type and it was shown to be a versatile enzyme. The biocatalysts rPoAbf wild-type and its evolved variant and rCelStrep from Streptomyces sp. G12 recombinantly expressed in Escherichia coli, were tested in the hydrolysis of lignocellulosic biomasses by using these enzymes in combination with other enzymatic activities involved in the hydrolysis of lignocellulose. The data suggest that the investigated biocatalysts act synergistically with bacterial and fungal enzymes. Moreover, rPoAbf and its evolved variant improved xylan and arabinan conversion of tested lignocellulosic biomasses highlighting the importance of accessory enzymes to obtain high xylan hydrolysis.