Purification, identification and functional characterization of glucansucrase from Leuconostoc dextranicum NRRL B-1146

Order of Publishing in Issue: 
4
Volume :2
Issue :4
October, 2008
Page No: 
493 -505
Authors: 
Avishek Majumder, Anshuma Mangtani and Arun Goyal*
Address: 
Department of Biotechnology Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India

In the present study the polyethylene glycols of different molecular weights were screened topurify exo-cellular glucansucrase from Leuconostoc dextranicum NRRL B-1146. Theglucan produced by this enzyme is unique in that it contains ?-(1-6) and ?-(1-4) linkages whichhave clinical applications. The cell free extract was subjected to fractionation by PEG-200, 400,4000 and 6000. The 30% (w/v) PEG-400 gave glucansucrase with maximum specific activityof 4.5 U/mg with 7.5 fold purification in a single step. Further purification of PEG-400 purifiedglucansucrase by gel-filtration gave glucansucrase with specific activity of 9 U/mgwith 15 fold purification. The purified glucansucrase confirmed the presence of glucan,after in-situ activity detection by Periodic Acid Schiff staining when run under non-denaturingon SDS-PAGE gels. The activity bands corresponded to the native and active form ofthe purified glucansucrase of approximately, 205 kDa molecular size, that appeared on thedenaturing gels stained with Coomassie Brilliant Blue. The purified glucansucrase gave a Km valueof 18.7 mM and a Vm of 9.4 U/mg. The addition of 0.8 mM EDTA led to 50% loss of enzymeactivity. Urea displayed deactivating effect on glucansucrase at all concentrations. Tween 80provided stability to the enzyme against activity losses.

Keywords: 
Glucansucrase, Leuconostoc dextranicum, Glucan
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