Expression of an active polygalacturonase from Aspergillus kawachii in a heterologous system.

ORTIZ, G. E; ROJAS N.L.; FERNANDEZ, M.E; ELGUEA, L; CAVALITTO, S; GHIRINGHELLI, P. D

Villa Carlos Paz, Córdoba,

Congreso; 44ra Reunión de la Sociedad Argentina de Investigacion en Bioquímica y Biología Molecular (XLIII SAIB); 2008

Sociedad Argentina de Investigacion en Bioquímica y Biología Molecular

Pectinases catalyze the hydrolysis of pectin and/or pectic acid. Among pectinases,endopolygalacturonases (PGase; E.C. 3.2.1.15.) are the most importantbiocatalysts. A. kawachii expresses a PGase, namely PG1, active at acidic pHvalues. Low expression levels of wild type PG1 requires cloning and overexpression for its industrial applications. PG1 ORF was cloned in a pYES2(INVITROGEN ®) vector, generating the pYES2:PG1 construction. Since PG1primary transcript has an intron and S. cerevisiae is not able to remove it duringmaturation, the enzyme couldn´t be expressed when cloned with this intron. So, itwas deleted using partial PCR and digestion with restriction enzyme frompYES2:PG1; generating the pYES2:PG1DI construction. E. coli Top10 cells werethen transformed, and plasmid-containing cells were tested by PCR using specificprimers for Pg1. Nine positive clones showed no mutations and correct ORF. ThepYES2:PG1DI was used to transform S cerevisiae INVSc1. Transformed yeastclones were analyzed by colony PCR. Two positive clones were obtained andtested in terms of PG1 expression. Both clones expressed and exported an activePGase after 21 hours of induction with galactose. Expressed recombinant proteinwas recovered as an unique extracellular protein, has a MW of ~60 kDa and wasidentified as PG1 by hydrolysis of polygalacturonic acid at pH 2.5 but not at pH 5