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.
Cucumber (Cucumis sativus) is one of the most widely used vegetable in the world,and different pesticides have been extensively used for controlling the insects and disease pathogens of this plant.However,little is known about how the pesticides affect the microbial community in cucumber phyllosphere.This study was the first attempt to assess the impact of pyrethroid insecticide cyperemethrin on the microbial communities of cucumber phyllosphere using biochemical and genetic approaches.Phospholipid fatty acid (PLFA) assay indicated that cyperemethrin insecticide treatment led to a significant increase in both total and bacterial biomass and a decrease in fungal biomass and the ratio of Gram-positive (GP) bacteria to Gram-negative (GN) bacteria within the cucumber phyllosphere.Principal-component analyses (PCA) suggested that the number of unsaturated and cyclopropane PLFAs (16:1ω9t,18:1ω7,cy17:0,cy19:0) increased with the insecticide treatment,whereas the saturated PLFA i16:0,i17:0 decreased.The increase of GN bacteria implied that the cypermethrin insecticide might be a nutrient for the growth of these phyllosphere microbes.Terminal restriction fragment length polymorphism (T-RFLP) reinforced the PLFA results.A significant change of bacterial community structure was observed in the separate dendrogram cluster between control and treated samples with the cucumber phyllosphere following cypermethrin insecticide treatment.Moreover,the increased terminal restriction fragments (T-RFs) (58,62,89,99,119,195,239,311,340,and 473 bp) indicated that some bacteria might play a significant role in the insecticide degradation within the cucumber phylosphere,whereas the disappeared T-RFs (44,51,96,223,306,and 338 bp) implied that some other bacteria might potentially serve as microbial indicator of cyperemethrin insecticide exposure.
Three endo-polygalacturonases(endoPGs) from a newly isolated Penicillum oxalicum(CGMCC 0907) capable of utilizing waste biomass as growth substrate were separated and purified to homogeneity by ultra-filtration,affinity adsorption chromatography,CM-cellulose column chromatography,and Sephadex G-100 gel filtration chromatography with the overall yield of 64.5% from the crude enzyme.The specific activities and recovery rates of endoPG-1,endoPG-2 and endoPG-3 were 1120 U/mg and 21.6%,1350 U/mg and 25.9%,and 1560 U/mg and 17.0%,respectively.The three purified endoPGs had a close molecular weight to 41 kDa as estimated by SDS-PAGE.The optimum temperature and pH for the function of them were 65℃ and 5.0,55℃ and 5.0,50℃ and 5.5,respectively.Their pI and Km values were 5.9 and 0.78 mg/mL,6.0 and 1.2 mg/mL,and 6.1 and 2.0 mg/mL,respectively.
