Purification and biochemical characterization of a novel glucansucrase from Leuconostoc citreum B-2

Although the extracellular polysaccharides have been analyzed in the previous period, the biochemical, enzymological characters and stimulation and inhibition effect on glucansucrase are not fully understood.

After three steps purification, salting out, DEAE-Sepharose and Sephadex G-75, the final specific activity was 264.84 U/mg protein with 4.31-fold. The SDS-PAGE analysis of fraction gave a single band 170.35 kDa in the stained gel. The active band was analyzed with LC-MS/MS to identify glucansucrase. The highest coverage rate of dextransucrase from Leu. citreum (ACY92456.2) was 55.60%, the results were speculated that the glucansucrase secreted from Leu. citreum B-2 may be a novel glucansucrase. The purified enzyme was optimally active at 20-30 °C and pH 6.0-8.0. Metal ions K⁺, Na⁺, Ca²⁺, Mn²⁺, Mg²⁺, and Cr⁺ had an apparent stimulating effect on enzyme activity, especially in divalent ions Ca²⁺ and Mn²⁺, the residual activities were higher than 200%. In a reverse, Hg⁺, acetonitrile, SDS, salt, and guanidine expressed inhibition effect on enzyme residual activity. The K_M and V_max were detected to be 4.82 mM and 0.97 U/mg, respectively.

All these data collectively indicate that B-2 glucansucrase is a novel one, which have good properties and may applied to new food areas.