PURIFICATION, OPTIMIZATION OF ASSAY, AND STABILITY STUDIES OF DEXTRANSUCRASE ISOLATED FROM Weissella cibaria JAG8

Dextransucrase-producing (Gen Bank accession no. KC110687) Weissella cibaria JAG8 was isolated from apple. The cell-free extract containing dextransucrase with specific activity of 1.0U/mg was purified by polyethylene glycol (PEG). A concentration of 33% (v/v) PEG-400 fractionation gave a specific activity of 20.0U/mg, whereas 15% (w/v) PEG-1500 resulted in a specific activity of 10.6U/mg. The PEG-400-purified enzyme was further purified by chromatography using a Sephacryl S-300HR column, which resulted in 37-fold purification with 37U/mg. The non-denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of column-purified enzyme showed a single homogenous band of 177 kDa by silver staining. The production of dextran was confirmed by in situ detection of the activity band using periodic acidSchiff's base staining. The optimum assay conditions for dextransucrase were 35 degrees C, pH 5.4, and 5.0% (w/v) sucrose concentration. The enzyme followed MichaelisMenten kinetics with Km of 13mM and Vmax 27.5U/mg. The enzyme was stable in 10500mM sodium acetate buffer, pH 5.4. A 22% increase in enzyme activity was observed with 2mM magnesium chloride; 64% loss in enzyme activity was observed with 10mM ethylenediamine tetraacetic acid (EDTA), whereas a complete loss in activity was observed with 5 M urea. The dextransucrase was stable up to 35 degrees C and pH of 5.4 for 1hr.