School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
School of Biological Science and Engineering, Hebei University of Science and Technology, Shijiazhuang, 050018, People's Republic of China.
Biotechnol Lett. 2020 Aug;42(8):1535-1545. doi: 10.1007/s10529-020-02881-6. Epub 2020 Apr 3.
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, Ca, Mn, Mg, and Cr had an apparent stimulating effect on enzyme activity, especially in divalent ions Ca and Mn, 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 and V 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.
虽然前期已经对胞外多糖进行了分析，但对其生化、酶学性质以及对葡聚糖蔗糖酶的刺激和抑制作用还不完全了解。
经过三步纯化，盐析、DEAE-Sepharose 和 Sephadex G-75，最终比活为 264.84 U/mg 蛋白，比活力提高了 4.31 倍。部分 SDS-PAGE 分析在染色凝胶中给出了 170.35 kDa 的单带。用 LC-MS/MS 对活性带进行分析，以鉴定葡聚糖蔗糖酶。从 Leu. citreum（ACY92456.2）获得的葡聚糖蔗糖酶的最高覆盖率为 55.60%，推测 Leu. citreum B-2 分泌的葡聚糖蔗糖酶可能是一种新型的葡聚糖蔗糖酶。纯化后的酶在 20-30°C 和 pH6.0-8.0 下具有最佳活性。金属离子 K、Na、Ca、Mn、Mg 和 Cr 对酶活性有明显的刺激作用，特别是在二价离子 Ca 和 Mn 中，残余活性高于 200%。相反，Hg、乙腈、SDS、盐和胍对酶残余活性有抑制作用。检测到 K 和 V 分别为 4.82 mM 和 0.97 U/mg。
所有这些数据表明，B-2 葡聚糖蔗糖酶是一种新型酶，具有良好的性质，可能应用于新的食品领域。
