Purification and Characterization of an Extracellular Dextransucrase from Pediococcus pentosaceus Isolated from the Soil of North East India

Seema Patel, Damini Kothari and Arun Goyal*

Department of Biotechnology, Indian Institute of Technology Guwahati, 781039 Guwahati, Assam, India

Article history:
Received September 9, 2010
Accepted January 25, 2011

Key words:
dextransucrase, polyethylene glycol, SDS-PAGE, activity staining, Pediococcus pentosaceus

Summary:
The extracellular dextransucrase produced from Pediococcus pentosaceus, a new isolate from the soil in Assam, India, was purified and characterized. The enzyme activity of cell-free supernatant was 3.4 U/mL and specific activity was 0.6 U/mg. The crude enzyme was purified by a single-step fractionation using polyethylene glycols of different molecular mass. The specific activity achieved was 18 U/mg with 31-fold purification by PEG 400 and 26 U/mg with 45-fold purification by PEG 1500. The molecular mass of dextransucrase determined by non-denaturing SDS-PAGE was approx. 180 kDa. The dextran formation activity of the enzyme was confirmed by activity staining. Optimum conditions for dextransucrase activity were: pH=5.4, reaction temperature 30 °C, 5 % sucrose and 20 mM sodium acetate buffer. A concentration of 1 mM MgCl₂ and 6 mM CaCl₂ enhanced dextransucrase activity by 5 and 150 %, respectively. The chaotropic agent urea (7 M) and chelating agent EDTA (1 mM) resulted in the residual enzyme activity of 98 and 80 %, respectively. The organic solvents such as ethanol (50 %), DMSO (90 %), acetone (50 %) and acetonitrile (20 %) decreased the dextransucrase activity by 80, 91, 94 and 80 %, respectively.

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