Cloning and Expression in Saccharomyces cerevisiae of Polygalacturonase 1 from Aspergillus. kawachii

ORTIZ, G. E.;; ROJAS, N. L; CAVALITTO, S.F.; GHIRINGHELLI, P. D

Mar Del Plata, Buenos Aires

Congreso; 43ra Reunión de la Sociedad Argentina de Investigacion en Bioquímica y Biología Molecular (XLIII SAIB).; 2007

SAIB

Polygalacturonase enzymes (Pg, E.C. 3.2.1.15.) catalyze the hydrolysis of pectin and/or pectic acid. Among the pectin hydrolyzing enzymes (endo) polygalacturonases are probably the most important biocatalysts. Low expression levels of wild type fungal enzymes require cloning and over expression of these enzymes for their industrial applications. The gene which encodes a polygalacturonase A (Pg1) from the filamentous fungus Aspergillus kawachii was amplified by PCR using specific primers for pg1 gene sequences and cloned (BamH1-EcoR1) into the pYES2 expression vector (INVITROGEN, Catalogue nº V825-20). E. coli TOP10F´ cells were then transformed. About 56 bacterial clones were tested by colony PCR using specific primers for pg1 gene sequences, obtaining 14 positive clones (named G1-G14). Two positive clones (G11 and G12) showed correct open reading frame by partially 5´ end sequencing. The pYES2 vector containing the pg1 gene was obtained from these clones and were transformed into Saccharomyces cerevisiae INVSc1. Transformed yeast clones were analyzed in terms of Pg1 expression of the protein, showing an active PG1 expressed by using INVSc1 as the host after 24 hours galactose induction. The recombinant protein expressed in these S. cerevisiae clones was identified as a Pg1 by hydrolysis of polygalacturonic acid measured as an increment in reducing ends.