title: [Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris and its characterization].
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. dai.min_1987@yahoo.com.cn
authors.forename: Min
authors.initials: M
authors.lastname: Dai
authors.forename: Changtao
authors.initials: C
authors.lastname: Ji
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhi
authors.forename: Hua
authors.initials: H
authors.lastname: Shao
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D004795:Enzyme Stability; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008670:Metals / Q000494:pharmacology; D009930:Organic Chemicals / Q000737:chemistry; D010843:Pichia / Q000166:cytology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008562:Membrane Glycoproteins; D008670:Metals; D009930:Organic Chemicals; C077394:SED1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008049:Lipase