Objective: To construct a novel cell-surface display system of Thermomyces lanuginosus lipase (TLL) based on an efficient anchor protein Sedlp 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 pPICZalphaA-TLS. The vector pPICZalphaA-TLS was linearized by Sac I and 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 257.8 U/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 degrees C 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.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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