Purification and Characterization of Three Alkaline Endopolygalacturonases from a Newly Isolated Bacillus gibsonii

A newly isolated Bacillus gibsonii, designated as S-2 （CGMCC1215）, was cultivated for production of alkaline pectinases utilizing sugar beet pulp as growth substrate. Purification of three alkaline endopolygalacturonases （endoPGs） from the crude pectinases extract was carried out by ultra-filtration, ammonium sulphate fractionation and ion-exchange chromatography, and their enzyme activities characterized. The three purified alkaline endoPGs, designated as S-Ⅰ, S-Ⅱ, and S-Ⅲ, had a molecular weight about 38 kDa as determined by SDS-PAGE. The Km value and optimal temperature for optimal enzyme activities of S-Ⅰ, S-Ⅱ and S-Ⅲ were 1.2 mg/mL and 60 ℃, 0.9 mg/mL and 55 ℃, 1.1 mg/mL and 60 ℃, respectively. Their best performances were given at an optimal pH 10.5, and sodium polygalacturonate was found to be the best substrate. The isoelectric points of S-Ⅰ, S-Ⅱ and S-Ⅲ were 5.4, 7.4, and 8.2, respectively. Surfactants of Tween-80 and Tween-20 and metal ions such as Mg^2＋ and Ca^2＋ stimulated the activity of S-Ⅰ, S-Ⅱ and S-Ⅲ, whereas S-Ⅲ was inhibited by Ca^2＋, and Mn^2＋ and Zn^2＋ ions inhibited the activity of the three enzymes.