Botany and Microbiology Department, Faculty of Science, Damietta University, Egypt.
Botany and Microbiology Department, Faculty of Science, Damietta University, Egypt.
Int J Biol Macromol. 2019 Jul 1;132:1274-1281. doi: 10.1016/j.ijbiomac.2019.04.010. Epub 2019 Apr 3.
AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0-7.0 and 4.0-7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.
来自 Laceyella sp. DS3 的 AmyLa α-淀粉酶基因在大肠杆菌 BL21 中异源表达。在 37°C 下加入 0.02mM IPTG 4 小时后，大肠杆菌 BL21 最大程度地表达了 AmyLa。通过凝胶过滤和离子交换层析，将重组的 AmyLa α-淀粉酶纯化了 2.19 倍。我们将纯化的重组 AmyLa α-淀粉酶固定在四种载体上；壳聚糖的效率最高。重组游离和固定化 AmyLa α-淀粉酶的最适活性分别在 pH 值为 6.0-7.0 和 4.0-7.0 的范围内，最适温度分别为 55°C。游离酶的活化能、Km 和 Vmax 分别为 291.5 kJ、1.5 mg/ml 和 6.06 mg/min。固定化酶的活化能、Km 和 Vmax 分别为 309.74 kJ、6.67 mg/ml 和 50 mg/min。固定化酶是钙离子非依赖性的，对金属不敏感（相对于游离酶）。它还可以重复使用七次。
