Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli.
The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C. Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode.
Wanker
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E
Institut für Biotechnologie, Technische Universität, Graz, Austria.
Huber
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A
Schwab
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H
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007444:Inulin / Q000378:metabolism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002412:Cations; D011993:Recombinant Fusion Proteins; D007444:Inulin; D006026:Glycoside Hydrolases; C021512:levanase