Production and characterization of an acid PGase from Aspergillus kawachii cloned into S.cerevisiae

ROJAS N.L.; ORTIZ, G. E; GRAMISCI, B.R.; CAVALITTO, S; GHIRINGHELLI, P. D

San Miguel de Tucumán

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

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. (SAIB).

Polygalacturonase (PG; EC 3.2.1.15) catalyses the hydrolysis of pectin and/or pectic acid and is useful for juice clarification and pectin extraction. Aspergillus kawachii produces an acidic PG called PG1. This enzyme was cloned into S. cerevisiae. Heterologous expression and growth of recombinant yeast in a synthetic medium was studied in batch and fed batch cultures. It was determined a maximum of PG1 production between 20 and 24 hours of batch growth (2 U/ml and 15 ppm of secreted proteins). Specific growth rate was determined as 0,28 h-1 and carbon balance was 1.05, which indicates there is no other product but ethanol generated. When grown on fed batch system, PG1 activity obtained was 16 U/ml. A purification process was developed. At pH 3.0, the acid PGase was adsorbed to a cationic matrix; meanwhile major contaminating proteins were eliminated. After that, a size exclusion chromatography was performed, recovering the purified protein. From SDS PAGE and IEF analysis it was determined a Molecular weight of 60 kDa and an Ip of 3.6. Optimum pH was 4.0. After trypsin hydrolysis and MALDI ToF-ToF analysis, interrogation against the NCBI nr database identified peptide similarities with a PPase-AS from A. Awamori, a PG from A. kawachii and a PG from A. niger. These results indicated successful expression of the gene encoding PG1 activity from A. kawachii by S. cerevisiae.