Posoongnoen Saijai, Thummavongsa Theera
Division of Chemistry, Faculty of Science and Technology, Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima, 30000, Thailand.
Division of Biology, Faculty of Science and Technology, Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima, 30000, Thailand.
Plant Biotechnol (Tokyo). 2020 Mar 25;37(1):31-38. doi: 10.5511/plantbiotechnology.19.1209b.
The thermostable α-amylase from germinating sword bean ( (Jacq.) DC.) seeds (CgAmy) was successfully purified by a combination of ammonium sulphate fractionation and Epoxy-activated Sepharose 6B affinity chromatography. The purified α-amylase showed 507.8 fold with a specific activity of 750.0 U/mg. SDS-PAGE of the purified enzyme revealed a single protein band of 50.0 kDa. Purified enzyme was confirmed as α-amylase type by LC-MS/MS analysis and activity on specific substrate of ethylidene-pNP-G7. The CgAmy revealed extreme activity at a high temperature of 50.0-70.0°C with optimum activity at 70.0°C. The optimal pH of enzyme activity was observed at 6.0. The CgAmy exhibited stability in pH range of 5.0-8.0 and highly thermostable with a temperature of 40.0-60.0°C. The kinetic parameters for hydrolysis of starch were found to be 3.12 mg/ml. The α-amylase activity was enhanced in the presence of Co and β-mercaptoethanol. While, Na, K, Ca, Mg, Zn, Ba, Fe and Cd slightly inhibited α-amylase activity. Interestingly, the CgAmy displayed stability towards some organic solvents and detergents. Stability at high temperature and some metal ions, organic solvents and detergents indicated that this enzyme has potential for various applications.
通过硫酸铵分级分离和环氧活化琼脂糖6B亲和层析相结合的方法，成功纯化了发芽刀豆（(Jacq.) DC.）种子中的耐热α-淀粉酶（CgAmy）。纯化后的α-淀粉酶比活为750.0 U/mg，纯化倍数为507.8倍。纯化酶的SDS-PAGE显示有一条50.0 kDa的单一蛋白条带。通过LC-MS/MS分析以及对亚乙基-pNP-G7特异性底物的活性，确认纯化后的酶为α-淀粉酶类型。CgAmy在50.0 - 70.0°C的高温下具有极高活性，在70.0°C时活性最佳。酶活性的最适pH值为6.0。CgAmy在pH 5.0 - 8.0范围内表现出稳定性，在40.0 - 60.0°C的温度下具有高度耐热性。淀粉水解的动力学参数为3.12 mg/ml。在Co和β-巯基乙醇存在下，α-淀粉酶活性增强。而Na、K、Ca、Mg、Zn、Ba、Fe和Cd对α-淀粉酶活性有轻微抑制作用。有趣的是，CgAmy对一些有机溶剂和洗涤剂表现出稳定性。在高温以及一些金属离子、有机溶剂和洗涤剂存在下的稳定性表明该酶具有多种应用潜力。
