Nakano H, Moriwaki M, Washino T, Kino T, Yoshizumi H, Kitahata S
Osaka Municipal Technical Research Institute, Japan.
Biosci Biotechnol Biochem. 1994 Aug;58(8):1430-4. doi: 10.1271/bbb.58.1430.
An unicellular green alga identified as Lobosphaera sp. by morphological observations was selected as a source of trehalase. The alga grew well heterotrophically and produced intracellular trehalase using Polypepton, yeast extract, and glycerol as nutrients. The enzyme was highly purified by ammonium sulfate fractionation, column chromatography on DEAE-Toyopearl, Sepharose CL-4B, and SP-Toyopearl. The molecular mass was estimated to be 400 kDa by gel filtration. SDS-PAGE indicated that the enzyme consisted of two subunits with a molecular mass range of 180-220 kDa and it contained carbohydrates. The enzyme was most active at pH 5.5 and at 65 degrees C and stable between pH 4-9 and below 65 degrees C. Fe3+ inactivated the enzyme. Sucrose was a competitive inhibitor with a Ki of 7.5 mM. The enzyme specifically hydrolyzed trehalase with a Km of 0.6 mM.
通过形态学观察鉴定为球孢藻属（Lobosphaera sp.）的一种单细胞绿藻被选为海藻糖酶的来源。该藻类在异养条件下生长良好，并以蛋白胨、酵母提取物和甘油作为营养物质产生细胞内海藻糖酶。通过硫酸铵分级沉淀、DEAE- Toyopearl柱色谱、Sepharose CL - 4B柱色谱和SP - Toyopearl柱色谱对该酶进行了高度纯化。通过凝胶过滤估计其分子量为400 kDa。SDS - PAGE表明该酶由两个亚基组成，分子量范围为180 - 220 kDa，并且含有碳水化合物。该酶在pH 5.5和65℃时活性最高，在pH 4 - 9和65℃以下稳定。Fe3+使该酶失活。蔗糖是一种竞争性抑制剂，Ki为7.5 mM。该酶特异性水解海藻糖，Km为0.6 mM。
