Bioprocess Engineering Group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Dept. of Cell & Molecular Biology, Shahid Beheshti University, Tehran, Iran.
Int J Biol Macromol. 2024 Nov;279(Pt 1):135135. doi: 10.1016/j.ijbiomac.2024.135135. Epub 2024 Aug 27.
Amylopullulanase (EC. 3.2.1.41/1) is an enzyme that hydrolyzes starch and pullulan, capable of breaking (4 → 1)-α and (6 → 1)-α bonds in starch. Here, the Amy1136 gene (2166 base pairs) from the thermophilic bacterium Cohnella sp. A01 was cloned into the expression vector pET-26b(+) and expressed in Escherichia coli BL21. The enzyme was purified using heat shock at 90 °C for 15 min. The expression optimization of Amy1136 was performed using Plackett-Burman and Box-Behnken design as follows: temperature of 26.7 °C, rotational speed of 180 rpm, and bacterial population of 1.25. The Amy1136 displayed the highest activity at a temperature of 50 °C (on pullulan) and a pH of 8.0 (on starch) and, also exhibited stability at high temperatures (90 °C) and over a range of pH values. Ag significantly increased enzyme activity, while Co completely inhibited amylase activity. The enzyme was found to be calcium-independent. The kinetic parameters K, V, k, and k/K for amylase activity were 2.4 mg/mL, 38.650 μmol min mg, 38.1129 S, and 0.09269 Smg mL, respectively, and for pullulanase activity were 173.1 mg/mL, 59.337 μmol min mg, 1.586 S, and 1.78338 Smg mL, respectively. The thermodynamic parameters K, t, Ea, ΔH, ΔG and ΔS were calculated equal to 0.20 × 10 (m), 462.09 (min), 16.87 (kJ/mol), 14.18 (kJ/mol), 47.34 (kJ/mol) and 102.60 (Jmol K), respectively. The stability of Amy1136 under high temperature, acidic and alkaline pH, surfactants, organic solvents, and calcium independence, suggests its suitability for industrial applications.
支链淀粉酶（EC.3.2.1.41/1）是一种能够水解淀粉和普鲁兰的酶，能够断裂淀粉中的（4→1）-α 和（6→1）-α 键。本文从嗜热菌 Cohnella sp. A01 中克隆了支链淀粉酶基因 Amy1136（2166 个碱基对），并将其表达在大肠杆菌 BL21 中。通过 90°C 热休克 15min 对酶进行纯化。通过 Plackett-Burman 和 Box-Behnken 设计对 Amy1136 的表达进行优化，条件分别为：温度 26.7°C、转速 180rpm、细菌浓度 1.25。Amy1136 在 50°C（普鲁兰）和 pH8.0（淀粉）时表现出最高活性，并且在高温（90°C）和较宽的 pH 值范围内具有稳定性。Ag 显著提高了酶的活性，而 Co 则完全抑制了淀粉酶的活性。该酶是钙非依赖性的。酶的动力学参数 K、V、k 和 k/K 对于淀粉酶活性分别为 2.4mg/mL、38.650μmolminmg、38.1129S 和 0.09269SmgmL，对于普鲁兰酶活性分别为 173.1mg/mL、59.337μmolminmg、1.586S 和 1.78338SmgmL。热力学参数 K、t、Ea、ΔH、ΔG 和 ΔS 分别计算为 0.20×10（m）、462.09（min）、16.87（kJ/mol）、14.18（kJ/mol）、47.34（kJ/mol）和 102.60（JmolK）。Amy1136 在高温、酸性和碱性 pH、表面活性剂、有机溶剂和钙非依赖性下的稳定性表明，它适合工业应用。
