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Purification and characterization of an intracellular levansucrase derived from Bacillus methylotrophicus SK 21.002
Author(s) -
Li Runjing,
Zhang Tao,
Jiang Bo,
Mu Wanmeng,
Miao Ming
Publication year - 2015
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1334
Subject(s) - levansucrase , hydrolysis , chemistry , fructose , size exclusion chromatography , chromatography , enzyme , biochemistry , nuclear chemistry , bacteria , biology , bacillus subtilis , genetics
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 K m and V max 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.