利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

Display of Serratia marcesens lipase on surface of Escherichia coli using N-terminal domain of ice nucleation protein

摘要:

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合, 构建成脂肪酶表面展示载体, 并转化大肠杆菌BL21 (DE3)。【结果】重组菌以终浓度0.05 mmol/L 异丙基硫代-D-半乳糖苷(IPTG)、25 °C条件下诱导培养, 16 h后表面展示脂肪酶活力达到最大值1 852 U/g 细胞干重。表面展示酶的最适pH为9.0, 最适反应温度为40 °C, 表面展示酶热稳定性较游离酶有较大提高, 在40 °C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。

关键词:表面展示, 脂肪酶, 粘质沙雷氏菌, 大肠杆菌

Abstract:

[Objective] We constructed an Escherichia coli strain displaying an active lipase on the cell surface by cell surface engineering and characterized the displayed lipase. [Methods] Escherichia coli surface display vector of lipase was constructed by fusing sequence encoding the N-terminal domain of ice nucleation protein with Serratia marcesens lipase gene, and the recombinant vector was transformed into Escherichia coli BL21(DE3). [Results] The highest lipase activity was observed when the recombinant cells were induced with 0.05 mmol/L isopropy-β-D-thiogalactoside (IPTG) and cultured at 25 °C for 16 h. Optimal pH and optimal temperature for cell surface-displayed lipase was 9.0 and 40 °C, respectively. The thermal stability of surface-displayed lipase was improved compared with that of free lipase, and the residual activity was above 90 percent of initial activity after incubated at 40 °C for 1 h. [Conclusion] The above results suggest that the bacterial surface display technology offers a promising alternative approach for immobilization of lipases.

Key words:Surface display, Lipase, Serratia marcesens, Escherichia coli