谷氨酸棒杆菌过氧化氢酶的异源表达、纯化以及酶学性质

Expression, purification and characterization of catalase from Corynebacterium glutamicum

摘要:

过氧化氢酶能催化过氧化氢分解为水和氧气，在工业上有着较为广泛的应用。然而，纺织和造纸工业的特殊的高碱性环境，使得开发碱性过氧化氢酶有着重要的应用价值。利用大肠杆菌表达来自于谷氨酸棒杆菌的过氧化氢酶，对其表达条件进行了优化，并通过镍柱亲和层析的方法分离纯化重组蛋白，然后表征纯酶的酶学性质。最适表达条件为：诱导剂IPTG浓度0.2 mmol/L，诱导温度25 ℃，诱导时间11 h。过氧化氢酶比酶活达到55 266 U/mg，具有较高的催化活性。该酶具有相当宽泛的pH值适应范围，在pH 4.0–11.5范围内均具有较高的酶活性，并在pH 11.0条件下表现出最高的酶活性。将纯酶在pH 11.0的溶液中处理3 h时剩余酶活为93%，说明该酶在高碱条件下有良好的稳定性。该酶最适温度为30 ℃，在25–50 ℃热稳定性较好。其动力学参数Km为25.89 mmol/L，Vmax为185.18 mmol/(min×mg)。抑制剂十二烷基硫酸钠 (SDS)、尿素、NaN3、β-巯基乙醇、EDTA对酶活有不同程度的抑制作用。来源于谷氨酸棒杆菌的过氧化氢酶具有较高的催化效率、良好的碱耐受性，在工业生产中有较好的应用前景。

Abstract:

Catalase catalyzes the decomposition of H2O2 to H2O and O2, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(min×mg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN3, β-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.