Preparative Biochemistry and Biotechnology 2013-01-01

Purification, optimization of assay, and stability studies of dextransucrase isolated from Weissella cibaria JAG8.

T Jagan Mohan Rao, Arun Goyal

Index: Prep Biochem Biotechnol. 43(4) , 329-41, (2013)

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Abstract

Dextransucrase-producing (Gen Bank accession no. KC110687) Weissella cibaria JAG8 was isolated from apple. The cell-free extract containing dextransucrase with specific activity of 1.0 U/mg was purified by polyethylene glycol (PEG). A concentration of 33% (v/v) PEG-400 fractionation gave a specific activity of 20.0 U/mg, whereas 15% (w/v) PEG-1500 resulted in a specific activity of 10.6 U/mg. The PEG-400-purified enzyme was further purified by chromatography using a Sephacryl S-300HR column, which resulted in 37-fold purification with 37 U/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 acid-Schiff's base staining. The optimum assay conditions for dextransucrase were 35°C, pH 5.4, and 5.0% (w/v) sucrose concentration. The enzyme followed Michaelis-Menten kinetics with Km of 13 mM and Vmax 27.5 U/mg. The enzyme was stable in 10-500 mM sodium acetate buffer, pH 5.4. A 22% increase in enzyme activity was observed with 2 mM magnesium chloride; 64% loss in enzyme activity was observed with 10 mM ethylenediamine tetraacetic acid (EDTA), whereas a complete loss in activity was observed with 5 M urea. The dextransucrase was stable up to 35°C and pH of 5.4 for 1 hr.