PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR EXOPOLYGALACTURONASE FROM GEOTRICHUM-LACTIS

Geotrichum lactis ATCC 48590 produced extracellular polygalacturonase (EC 3.2.1.67) in media containing pectate, pectin, or galacturonic acid as inducers. The synthesis of the enzyme was strongly repressed by glucose. The polygalacturonase was purified 80-fold by ammonium sulphate precipitation, Sephadex G-100 filtration, and DEAE Sephadex ion-exchange chromatography. Polyacrylamide gel electrophoresis with copolymerized substrate indicated that the isolated material was a single enzyme with polygalacturonase activity. The main product of enzyme action was galacturonic acid. The enzyme shows a molecular weight close to 53 000 by gel filtration and degrades preferentially de-esterified substrates. K(m) values for pectate and pectin (64% esterified) were 0.09 and 0.49 mg mL-1, respectively. The optimum pH for enzyme activity was 5.0, while the optimum temperature was 40-degrees-C. The polygalacturonase was precipitated by concanavalin A - Sepharose, and treatment with endoglycosidase H reduced its precipitation by the lectin, suggesting that the enzyme is a glycoprotein. In addition to being found extracellularly, the polygalacturonase is also present in the periplasm of the cells. A different form of the polygalacturonase showing a lower molecular weight was located inside the cells.