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

【摘要】 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 about38kDa as determined by SDS-PAGE.The Km value and optimal temperature for optimal enzyme activities of S-I,S-II and S-III were1.2mg/mL and60℃,0.9mg/mL and55℃,1.1mg/mL and60℃,respectively.Their best performances were given at an optimal pH10.5,and sodium polygalacturonate was found to be the best substrate.The isoelectric points of S-I,S-II and S-III were5.4,7.4,and8.2,respectively.Surfactants of Tween-80and Tween-20and metal ions such as Mg2+and Ca2+stimulated the activity of S-I,S-II and S-III,whereas S-III was inhibited by Ca2+,and Mn2+and Zn2+ions inhibited the activity of the three enzymes.更多还原

【Abstract】 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 Mg2+ and Ca2+ stimulated the activity of S-I, S-II and S-III, whereas S-III was inhibited by Ca2+, and Mn2+ and Zn2+ ions inhibited the activity of the three enzymes.更多还原