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

Objective: 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.
Results: 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 ⁺ , Ca ²⁺ , Mn ²⁺ , Mg ²⁺ , and Cr ⁺ had an apparent stimulating effect on enzyme activity, especially in divalent ions Ca ²⁺ and Mn ²⁺ , 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.
Conclusion: All these data collectively indicate that B-2 glucansucrase is a novel one, which have good properties and may applied to new food areas.