来自Staphylococcus aureus N315的2-脱氧-D-核糖5-磷酸醛缩酶的工程菌构建、表达纯化与性质鉴定

利用基因挖掘在数据库中发现一种来自Staphylococcus aureus N315的潜在DERA(SaDERA),将其基因密码子优化后实现了在大肠杆菌中的表达,重组酶经纯化后研究了其催化性质。结果表明:构建的工程菌具有较高的可溶表达量(占总蛋白的70%),通过简单的一步纯化即可得到电泳纯的酶;SaDERA是一种同源二聚体的酶(5.7×104),其最适反应条件是pH 7.7和45℃;SaDERA具有良好的碱耐受性,在pH 11.0、25℃的条件下温浴24 h后仍有93%的残余活力;SaDERA具有良好的乙醛耐受性,在0.3 mol/L乙醛浓度、25℃下,30 min内保持了70%以上的残余活力;乙醛连续自缩合产物被纯化并鉴定,所得产品为两次缩合产物。

Cloning,purification and characterization of promising 2-deoxy-D-ribose 5-phosphate aldolase from Staphylococcus aureus N315

The genome mining was used to find a promising DERAs from the genomic database. After the optimization of the codons ,a DERA from Staphylococcus aureus N315 （SaDERA）was overexpressed in E. coli BL21 （DE3） , purified, and characterized. The purified SaDERA was obtained after a simple one-step simple purification. The homodimeric enzyme （57 kDa） has the optimal activity at pH 7.7 and 45 ℃. It showed quite high stability at alkaline pH（ pill 1.0）and 89% of activity remained after incubation（25 ℃ for 24 h）. It showed a resistance to acetaldehyde and more than 70% of activity remained after exposure to 0. 3 mol/L acetaldehyde for 30 min at 25 ℃. To confirm its synthetic potential, the double aldol product was synthesized, purified and identified.