Purification and Characterization of Glucosyltransferases from New Strains Leuconostoc Mesenteroides

ABSTRACT

The Leuconostoc species require sucrose in the culture medium as an inducer for the elaboration of glucosyltransferase (GTF) and glucans. Among all the reported putification methods, polyethylene glycol (PEG) is a effective and single step purification method for GTF from Leuconostoc mesenteroides.

The extracellular GTFs from Leuconostoc mesenteroides L.m.28 and mutant Leuconostoc mesenteroides L.m. M2860 were purified by phase partitioning using polyethylene glycol (PEG) of different molecular weights. The cell free extracts were subjected to fractionation by PEG-400, 1500, 4000 and 6000. In a single step precipitation, at a final concentration of 25% (w/v) PEG-1500, the enzyme from parent strain L.m.28 gave the maximum specific activity–12.17 U/mg protein. The purified enzyme by all using PEGs showed a single band corresponding to 180 kDa molecular size when run on SDS-PAGE for in situ activity detection by Periodic Acid Schiffs staining. The 20% (w/v) PEG-6000 gave GTF from mutant strain L.m. M2860 with maximum specific activity of 10.93 U/mg protein and a single band of protein corresponding to 180 kDa. In a single step precipitation, at a final concentration of 20% (w/v), 25% (w/v) and 30%(w/v) PEG-1500, the purified enzyme from L.m. M2860 showed a few molecular forms on SDS-PAGE corresponding to 180 kDa, 140 kDa, 120 kDa and 86 kDa. In addition, the purified GTF from L.m. M2860 showed higher thermal stability at 45 °C for 30 minutes.

Keywords:

• glucan
• glucosyltransferase
• Leuconostoc mesenteroides
• polyethylene glycol