title: Purification, optimization of assay, and stability studies of dextransucrase isolated from Weissella cibaria JAG8.
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.
authors: Department of Biotechnology, Indian Institute of Technology Guwahati , Guwahati, Assam, India.
authors: T Jagan
authors: TJ
authors: Mohan Rao
authors: Arun
authors: A
authors: Goyal
mesh_terms: D002498:Centrifugation; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003911:Dextrans / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D027845:Malus / Q000382:microbiology*; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D058836:Weissella / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
chemical_list: D003911:Dextrans; D015636:Magnesium Chloride; D011092:Polyethylene Glycols; D005964:Glucosyltransferases; C020025:dextransucrase