title: Purification and characterization of an intracellular levansucrase derived from Bacillus methylotrophicus SK 21.002.
abstract: An intracellular levansucrase from Bacillus methylotrophicus SK 21.002 was isolated, purified, and characterized. The final specific levansucrase activity was 135.40 U/mg protein with an 11.78-fold enrichment and a 9.28% recovery rate. The molecular weight of the enzyme was approximately 60,000 Da as evaluated by gel filtration and SDS-PAGE. Both the maximum transfructosylation and hydrolytic activities were observed at pH 6.5. The enzyme exhibited optimum transfructosylation activity at 40 °C, whereas the optimum temperature of hydrolytic activity was 45 °C. Cu(2+), Fe(2+), Zn(2+), and Ni(2+) inhibited both the transfructosylation and hydrolytic activities up to 100%, whereas Mn(2+) inhibited only hydrolytic activity. Ca(2+) and Mg(2+) stimulated both transfructosylation and hydrolytic activities. The chemical modifiers (n-bromosuccinimide and phenylmethanesulfonyl fluoride) strongly inhibited hydrolytic and transfructosylation activity of the levansucrase. The Km and Vmax values of the purified levansucrase were 117.2 mM and 33.23 μmol/mg·Min, respectively. When the fructose concentration was below 0.2 M, higher fructose concentrations promoted the transfructosylation and inhibited the hydrolytic activity.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Runjing
authors.initials: R
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Ming
authors.initials: M
authors.lastname: Miao
mesh_terms: D000818:Animals; D001407:Bacillus / Q000166:cytology* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D042541:Intracellular Space / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D005632:Fructose; D006602:Hexosyltransferases; C021513:levansucrase
keywords: Bacillus methylotrophicus SK 21.002; characterization; chromatography; levan; levansucrase; transfructosylation