Purification and characterization of acidic endo-polygalacturonase encoded by the PGL1-1 gene from Saccharomyces cerevisiae

Abstract

The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encode polygalacturonase. Cloning of the PGL1 open reading frame behind the ADH1 promoter allowed overexpression of polygalacturonase activity in S. cerevisiae. This enzyme was purified to apparent homogeneity from cultures of recombinant S. cerevisiae on synthetic medium using one-step purification by anionic exchange chromatography. The enzyme, named Pgl1P, had an apparent M r of 42 kDa as shown by SDS-PAGE. Pgl1P was active from pH 3 to 5.5, with an optimum temperature at 25³C. This enzyme hydrolyzed polygalacturonic acid as an endo-polygalacturonase as demonstrated by independent methods. The purified protein was N-glycosylated. However, the activity remained in the N-deglycosylated form.

Comparative biochemical and structural characterizations of fungal polygalacturonases

“…• C (Gainvors et al 2000) and several unusual temperature stable PGs have also been reported (Mill & Tuttobello 1961;Kumar & Palanivelu 1999). Thermophilic fungal strains such as Thermoascus aurantiacus, Aspergillus aculeatus and Fusarium oxysporum produce different PGs with optimal activity at temperatures between 60-65…”

“…The physicochemical properties show that the majority of fungal PGs have pH optimum between pH 3 and pH 6 De Lourdes et al 1991;Waksman et al 1991;Devi & AppuRao, 1996;Rao et al 1996;Gainvors et al 2000;Niture & Pant 2004). Interestingly, some PGs, for example, the PG of Corticium rolfsi shows maximum activity at pH 2.5 (Kaji & Okada 1969) and the one produced by Aspergillus kawachii shows maximum activity between pH 2-3 (Contreras Esquivel et al 1999).…”

Influence of pectin, glucose, and pH on the production of endo- and exo-polygalacturonase by Aspergillus oryzae in liquid medium

“…This reaction was carried out at 30 °C for 30 minutes and the viscosity of the reaction mixture was determined using a DV-II+ viscosimeter (BROOKFIELD ENGINEERING, USA). One endo-PG unit was defined as the quantity of enzyme that causes a 50% reduction in solution viscosity under the standardized conditions (Gainvors et al, 2000). The exo-PG activity was estimated in a reaction mixture containing 50 µL of appropriately diluted sample and 2 mL of polygalacturonic acid (SIGMA, USA) in 0.1 M acetate buffer (pH 4).…”

Influence of medium composition and pH on the production of polygalacturonases by Aspergillus oryzae

“…For this estimation 3.2 mL of diluted sample were mixed with 14.8 mL of a 1% (w/v) solution of citric pectin (DELAWARE, Porto Alegre, Brazil) in 0.05 M acetic acid-acetate buffer (pH 4) and the reaction mixture incubated at 30°C for 30 min, after which the viscosity was measured in a DV-II + viscometer (BROOKFIELD ENGINEERING, Middleboro, USA). One unit of endo-PG activity was defined as the quantity of enzyme which caused a 50% reduction in viscosity of the reaction mixture after 30 min incubation at 30°C (Gainvors et al, 2000). For the evaluation of exo-PG, 50 µL of diluted sample of culture media were added to 2 mL of a 0.25% (w/v) of polygalacturonic acid (SIGMA) in 0.05 M acetic acid-acetate buffer (pH 4) and the reaction mixture incubated at 35°C for 30 min, after which the quantity of reducing sugars liberated was measured by the Somogyi (1952) method, using D-galacturonic acid (SIGMA) as a standard.…”