亚心形四爿藻氢酶的稳定性及初步纯化

Stability of the hydrogenase from Tetraselmis subcordiformis and its preliminary purification

亚心形四爿藻Tetraselmis subcordiformis是一种具有高产氢能力的海洋绿藻,在厌氧环境中经暗诱导调控,可进行一定时间的连续产氢。氢酶是亚心形四爿藻进行光合产氢的关键酶,本文研究了在厌氧环境中连二亚硫酸钠、β-巯基乙醇和丙三醇等试剂对氢酶的稳定性影响,考察了硫酸铵分级沉淀对氢酶的纯化效果及回收情况。结果表明,连二亚硫酸钠和丙三醇均能对氢酶起到较好的保护作用。60%～70%饱和度硫酸铵沉淀出的蛋白样品比酶活较高,且所含其他蛋白较少,可用于氢酶的进一步纯化。

Tetraselmis subcordiformis, a marine green alga, can produce hydrogen by photobiologically hydrolyzing seawater with hydrogenase. In this study, the preliminary purification of the enzyme was explored by ammonium sulfate precipitation, and the impact of sodium dithionite, β-mercaptoethanol and glycerol on the enzyme stability during the process was investigated. The experimental results illustrated that sodium dithionite provided significant protection on the hydrogenase by depleting oxygen, while glycerol, a protectant against the structure instability of the enzyme, also presented protection. Crude enzyme with specific activity of 0.557 U/mg protein was extracted using 60%-70% saturated ammonium sulfate solution supplemented with 200 mmol/L sodium dithionite and 5% glycerol, and the hydrogenase recovery yield was about 30%.