PRODUCTION AND CHARACTERIZATION OF AN ACID PGASE FROM Aspergillus kawachii CLONED INTO S.cerevisiae

ROJAS NL; ORTIZ GE ; GRAMISCI B; CAVALITTO SF ; GHIRINGHELLI PD

San Migel de Tucumán

Congreso; 45 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2009

Polygalacturonase (PG; EC 3.2.1.15) catalyses the hydrolysis ofpectin and/or pectic acid and it is useful for juice clarification andpectin extraction. Aspergillus kawachii produces an acidic PGcalled PG1. This enzyme was cloned into S. cerevisiae.Heterologous expression and growth of recombinant yeast in asynthetic medium was studied in batch and fed batch cultures. Itwas determined a maximum of PG1 production between 20 and 24hours of batch growth (2 U/ml and 15 ppm of secreted proteins).Specific growth rate was determined as 0,28 h-1 and carbon balancewas 1.05, which indicates that there is no other product but ethanolgenerated. When grown on fed batch system, PG1 activity obtainedwas 16 U/ml. A purification process was developed. At pH 3.0, theacid PGase was adsorbed to a cationic matrix; meanwhile majorcontaminating proteins were eliminated. After that, a size exclusionchromatography was performed, recovering the purified protein.From SDS PAGE and IEF analysis it was determined a molecularweight of 60 kDa and an Ip of 3.6. Optimum pH was 4.0. Aftertrypsin hydrolysis and MALDI ToF-ToF analysis, interrogationagainst the NCBI nr database identified peptide similarities with aPPase-AS from A. Awamori, a PG from A. kawachii and a PG fromA. niger. These results indicated successful expression of the geneencoding PG1 activity from A. kawachii by S. Cerevisiae.