谷氨酸棒杆菌过氧化氢酶的异源表达、纯化以及酶学性质
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国家自然科学基金 (Nos. 31801526, 31972061) 资助。


Expression, purification and characterization of catalase from Corynebacterium glutamicum
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National Natural Science Foundation of China (Nos. 31801526, 31972061).

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    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.

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杨弘宇,张雪,马振平,徐宁,刘君. 谷氨酸棒杆菌过氧化氢酶的异源表达、纯化以及酶学性质[J]. 生物工程学报, 2020, 36(8): 1568-1577

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  • 收稿日期:2019-12-08
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  • 在线发布日期: 2020-08-25
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