热敏型核酸酶在毕赤酵母中的分泌表达及催化特性

Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris

摘要:

核酸酶在生物工程领域有着重要的应用价值。本研究在优化北极虾核酸酶 (Shrimp nuclease，SNU) 基因序列的基础上，构建SNU的毕赤酵母分泌表达载体SNU-pPICZα A并转化酵母，以高拷贝整合转化子为基础，优化酶表达的条件，并对该酶的催化特性进行分析，结果显示SNU可在毕赤酵母SMD1168H中高效分泌表达，最佳诱导表达条件为：培养基BMMY pH 6.0，甲醇浓度为1%，诱导时间为72 h，诱导后粗酶液比活力为1.4×105 U/mL。经过DEAE Sephadex阴离子交换层析可纯化获得高纯度的目标蛋白，每升菌液可纯化15 mg目标蛋白，比活力达到6.291×106 U/mg，该酶表观分子量为50 kDa，PNGaseF酶切证实该酶存在糖基化现象。二价金属离子Ca2+、Mn2+、Co2+、Mg2+及还原剂DTT、β-ME能显著地提高其水解活性，但Zn2+、Cu2+、SDS、高浓度NaCl抑制该酶的活性，SNU为Ca2+/Mg2+依赖型核酸酶。70 ℃处理10 min可使该酶不可逆的失活。

Abstract:

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×105 U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×106 U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca2+, Mn2+, Co2+ and Mg2+, but inhibited by Zn2+, Cu2+ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.