INSTITUTUL NAÅ¢IONAL DE CERCETARE-DEZVOLTARE - ICECHIM

4 - Protection and Environmental Engineering - PSTUDIES ABOUT THE SINERGISTIC EFFECT OF CELLOBIOHYDROLASE ANDβ-GLUCOSIDASE IN THE HYDROLYSIS OF CELLULOSEGURGU Leontina 1,2 , Julia MARIN-NAVARRO 2 , Julio POLAINA 21Faculty of Food Science and Engineering, ”Dunarea de Jos” University of Galati,Domneasca Street, No. 111, 800 008, Galati, Romania2 Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, SpainCellulose degradation requires the combined action of at least three types of enzymes: endoβ-1,4-glucanases(EG, EC 3.2.1.4), cellobiohydrolases (CBHs, EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). From a structural point of view, there are CBHs with differentmolecular architecture which are classified as glycoside hydrolases belonging to differentfamilies (1). A first objective of this study was to express a cellobiohydrolase encoding gene(cbhB) from A. niger into S. cerevisiae as a preliminary step to obtain yeast strains withimproved properties for cellulose fermentation. Another objective was to obtain a yeast strainwith cellobiohydrolase and cellobiase activities, by simultaneous expression of cbhB and theβ-glucosidase gene bgl1 from Saccharomycopsis fibuligera (2, 3). The ORF sequence ofcbhB, which encodes an enzyme belonging to family GH7 of the glycoside hydrolaseclassification, was amplified from genomic DNA of A. niger, fused to the sequence of thesignal peptide of the glucoamilase Sta1 from S. cerevisiae var diastaticus and expressed in S.cerevisiae under the control of a galactose inducible promoter. We have obtained physical evidenceof the simultaneous expression of cbhB and bgl1 in S. cerevisiae by analysis of the proteins secreted by thedouble transformant. While the cellobiase activity could be proved by using p-nitrophenyl glucoside as thesubstrate, cellobiohydrolase activity could not be measured effectively due to the absence of anappropriate substrate.Bibliography1.Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-ActiveEnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233-238.2.Marín-Navarro J, L Gurgu, S Alamar and J Polaina (2011) Structural and functional analysis of hybridenzymes generated by domain shuffling between Saccharomyces cerevisiae (var. diastaticus) Sta1 glucoamylaseand Saccharomycopsis fibuligera Bgl1 β-glucosidase. Applied Microbiology and Biotechnology 89:121-130.3.Gurgu L, A Lafraya, J Polaina and J Marín-Navarro (2011) Fermentation of cellobiose to ethanol by industrialSaccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera. BioresourceTechnology doi: 10.1016/j.biortech.2011.01.062.Financial support and acknowledgmentsLeontina Gurgu has benefited from financial support through the 2010 POSDRU/89/1.5/S/52432 project, ORGANIZINGTHE NATIONAL INTEREST POSTDOCTORAL SCHOOL OF APPLIED BIOTECHNOLOGIES WITH IMPACT ONROMANIAN BIOECONOMY, project cofinanced by the European Social Fund through the Sectoral OperationalProgramme Human Resources Development 2007-2013.