疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的表面展示及酶学性质

Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris and its characterization

摘要:

摘要:【目的】构建疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase，TLL)在毕赤酵母GS115中的细胞表面展示体系，筛选展示成功且酶活力及展示率较高的重组子作为全细胞催化剂，并研究其酶学性质。【方法】克隆TLL 基因tll，以酿酒酵母细胞壁蛋白Sed1p 为锚定蛋白，构建表面展示载体pPICZαA-TLS。重组载体经SacⅠ线性化后转入毕赤酵母GS115 中，经三丁酸甘油酯平板检测及摇甁发酵筛选获得高酶活力的毕赤酵母重组子，采用抗FLAG 标签一抗和R-PE 荧光素标记的二抗处理细胞后，进行荧光显微镜检测和流式细胞仪分析，并考察全细胞催化剂的最适反应温度和pH、金属离子耐受性等酶学性质。【结果】成功构建TLL 毕赤酵母细胞表面展示体系，筛选到1 株具有三丁酸甘油酯和橄榄油水解活力的克隆子，经1% 的甲醇诱导发酵120 h后，水解橄榄油酶活力达257.8U/g干细胞。经抗体处理后的重组菌发酵细胞在荧光显微镜下呈现强烈的红色荧光，流式细胞仪分析结果也证实脂肪酶被成功展示在酵母细胞表面，展示率达98. 36%。展示的TLL 作为全细胞催化剂水解对硝基苯酚丁酸酯(pNPB)的最适温度为30℃，最适pH为8.0，且具备良好的热稳定性和有机溶剂耐受性;K+、Ca2+、Mg2+对其有微弱的激活作用，Mn2+、Ni2+则有微弱的抑制作用，Cu2+的抑制作用较强，而EDTA、SDS、Tween 20 对酶活力影响不明显。【结论】首次将TLL脂肪酶成功展示在毕赤酵母细胞表面，获得具有较高水解活力和良好酶学特性的全细胞催化剂，为表面展示TLL 脂肪酶的规模化应用奠定了技术基础。

关键词:关键词:疏棉状嗜热丝孢菌脂肪酶，毕赤酵母，表面展示，全细胞催化剂，酶学性质

Abstract:

Abstract:［Objective］To construct a novel cell-surface display system of Thermomyces lanuginosus lipase (TLL) based on an efficient anchor protein Sed1p in Pichia pastoris，to screen recombinant strains with high enzyme activity and displaying rate，and further to characterize the enzyme． ［Methods］The lipase gene from T．lanuginosus was sub-cloned and fused with the anchor protein gene sed1 from Saccharomyces cerevisiae to construct a display vector pPICZαA- TLS．The vector pPICZαA-TLS was linearized by SacⅠand then transformed into P． pastoris GS115 by electroporation．After screening by tributyrin medium，a clone exhibiting the maximum lipase activity in shaking flask was chosen to treat with rabbit anti-FLAG-tag and R-PE-conjugated goat anti-rabbit IgG，and then its positive location on the cell wall was detected by fluorescence microscope and flow cytometer． The recombinant strain displaying TLL was further characterized as a whole-cell catalyst．［Results］A novel cell-surface display system of T． lanuginosus lipase was successfully established，and a clone with lipase activity of 257. 8U/g dry cells in shaking flask was obtained． The displayed TLL on the cell surface was confirmed by immunofluorescence，and the treated cells under the fluorescence microscope emitted brightly red fluorescence，and the displaying rate was 98. 36% detected by Flow Cytometer． The displayed TLL exhibited excellent thermostability and high tolerance to some organic solvents，and its maximal activity was observed at 30℃ and pH 8.0．The lipase activity was a little enhanced by K+，Ca2 + and Mg2 + and strongly inhibited by Cu2 + ，Mn2+ and Ni2+．However，ethylenediaminetetraacetic acid (EDTA)，Sodium lauryl sulfate (SDS) and Tween 20 showed little effect on the displayed TLL． ［Conclusion］The lipase TLL was successfully displayed on the cell surface of P． pastoris by the anchor protein Sed1p for the first time to obtain a whole-cell catalyst，which had high hydrolytic activity and excellent enzymatic characterization． Thus，we here established a solid foundation for industrial applications of the displayed lipase TLL．

Key words:Keywords: Thermomyces lanuginosus lipase， Pichia pastoris， surface display， whole-cell catalyst， enzymatic characterization