重组磷脂酰丝氨酸合成酶的分离纯化及酶学性质研究

Purification and Biochemical Properties of Phosphatidylserine Synthase Produced by Bacillus subtilis DB104(pBES-pss)

利对重组枯草芽孢杆菌（pBES-pss）表达的磷脂酰丝氨酸合成酶进行分离纯化及酶学性质研究。pBES-pss发酵后的粗酶液经硫酸铵盐析、中空纤维膜除盐浓缩、SP—SepharoseHP离子交换层析和Sephadex G-75凝胶层析。基本获得电泳纯的重组磷脂酰丝氨酸合成酶，比活力可达13．62U／mg，分子量约为53kD。酶学性质研究表明，该酶催化卵磷脂水解反应的最适pH8．0，最适温度为35℃。稳定性研究表明：该酶在pH6．5～9．5区间和低于45℃温度下稳定。表面活性剂及金属离子对该酶水解活性的影响结果表明，SDS、Tween20、Tween80对该酶有抑制作用，Triton X-100对该酶有增强作用；Mg^2＋、Zn^2＋、K^＋对该酶有抑制作用，Ca^2＋、Mn^2＋和EDTA对该酶有增强作用。

Phosphatidylserine synthase excreted from Bacillus subtilis DB104 （pBES-pss）which was reconstructed. The phosphatidylserine synthase was purified by the procedures including amoninium sulfate precipitation,ultrafiltration,SP- Sepharose HP chromatography and Sephadex G-75 chromatography. By multi-step purification,the phosphatidylserine synthase was purified to 39.59 folds and the specific activity of the Phosphatidylserine synthase was up to 13.62 U/mg. Analysed by SDS-PAGE, the molecular weight of purified phosphatidylserine synthase was showed as 53 kD. The optimum pH and temperature for hydrolysis of phosphatidylcholine were 8.0 and 35℃ respectively. There were some factors that can inhibit enzyme activities,induding SDS,Tween20,Tween80,while some ions such as Ca^2＋,Mn^2＋,EDTA and TritonX- 100 can improve its activity.