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-p PICZαA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 p H 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1.4×10~5 U/m L。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6.291×10~6 U/mg,该酶表观分子量为50 k Da,PNGase F酶切证实该酶存在糖基化现象。二价金属离子Ca^(2+)、Mn^(2+)、Co^(2+)、Mg^(2+)及还原剂DTT、β-ME能显著地提高其水解活性,但Zn^(2+)、Cu^(2+)、SDS、高浓度Na Cl抑制该酶的活性,SNU为Ca^(2+)/Mg^(2+)依赖型核酸酶。70℃处理10 min可使该酶不可逆的失活。
Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene（SNU） from Northern Shrimps was inserted into p PICZα A vector, and expressed extracellularly in strain SMD1168 H. 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 1.4×10~5 U/m L. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 k Da. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10~6 U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca^（2＋）, Mn^（2＋）, Co^（2＋） and Mg^（2＋）, but inhibited by Zn^（2＋）, Cu^（2＋） and high salt. Meanwhile, it was irreversibly inactivated at 70 for℃ 10 min.
出处
《生物工程学报》
CAS
CSCD
北大核心
2016年第7期991-995,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金(Nos.30400282
31171606)
西北农林科技大学基本科研业务费(No.2452015214)资助~~
关键词
北极虾Ca2+/Mg2+依赖型核酸酶
毕赤酵母
分泌表达优化
催化特性
Northern Shrimp Mg2＋/Ca2＋-independent nuclease
Pichia pastoris
secretory expression optimization
catalytic characteristics
