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

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