Kim D, Robyt J F
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Enzyme Microb Technol. 1994 Dec;16(12):1010-5. doi: 10.1016/0141-0229(94)90134-1.
Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28 degrees-30 degrees C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4 degrees C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100 degrees C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.
肠系膜明串珠菌B-512FMC是一种葡聚糖蔗糖酶的组成型突变体，在葡萄糖、果糖或蔗糖上生长。在不同浓度（0.6%和3%）下，三种糖产生的细胞相关葡聚糖蔗糖酶量大致相同。在葡萄糖培养基中产生的酶可吸附于葡聚糖凝胶G-100和G-200，但在蔗糖培养基中产生的酶吸附量要少得多。当在葡萄糖中产生的酶与分子大小大于10 kDa的葡聚糖预孵育时，葡聚糖凝胶的吸附作用减弱。缓冲液（20 mM乙酸盐，pH 5.2）在28℃-30℃时从葡聚糖凝胶中释放葡聚糖蔗糖酶活性的能力最强。向酶中添加葡聚糖可刺激葡聚糖合成，但对温度或pH稳定性影响很小。通过硫酸铵沉淀、羟基磷灰石层析和葡聚糖凝胶G-200吸附纯化的葡聚糖蔗糖酶不含任何碳水化合物，且能合成葡聚糖，表明启动葡聚糖合成不需要引物。纯化后的酶在SDS-PAGE上的分子大小为184 kDa。在4℃下放置30天后，天然酶解离为65 kDa、62 kDa和57 kDa的三种无活性蛋白。然而，184 kDa的活性酶在100℃热变性后，在SDS-PAGE上得到了63 kDa和59 kDa的两条蛋白带。63 kDa蛋白的量约为59 kDa蛋白的两倍。天然酶被认为是由两个63 kDa和一个59 kDa单体组成的三聚体。
