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

ORTIZ, GASTON EZEQUIEL; ROJAS, NATALIA LORENA; FERNANDEZ, MARIA ELENA; ELGUEA, LUCIA; CAVALITTO, SEBASTIÁN FERNANDO; GHIRINGHELLI, PABLO DANIEL

Hotel Portal del Lago, Villa Carlos Paz, Córdoba, Argentina

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 important biocatalysts. A. kawachii expresses a PGase, namely PG1, active at acidic pH values. Low expression levels of wild type PG1 requires cloning and over expression for its industrial applications. PG1 ORF was cloned in a pYES2 (INVITROGEN ®) vector, generating the pYES2:PG1 construction. Since PG1 primary transcript has an intron and S. cerevisiae is not able to remove it during maturation, the enzyme couldn´t be expressed when cloned with this intron. So, it was deleted using partial PCR and digestion with restriction enzyme from pYES2:PG1; generating the pYES2:PG1DI construction. E. coli Top10 cells were then transformed, and plasmid-containing cells were tested by PCR using specific primers for Pg1. Nine positive clones showed no mutations and correct ORF. The pYES2:PG1DI was used to transform S cerevisiae INVSc1. Transformed yeast clones were analyzed by colony PCR. Two positive clones were obtained and tested in terms of PG1 expression. Both clones expressed and exported an active PGase after 21 hours of induction with galactose. Expressed recombinant protein was recovered as an unique extracellular protein, has a MW of ~60 kDa and was identified as PG1 by hydrolysis of polygalacturonic acid at pH 2.5 but not at pH 5.