title: Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica.
abstract: The cell surface display of enzymes is of great interest because of its simplified purification stage and the possibility for recycling in industrial processes. In this study, we have focused on the cell wall immobilization of Yarrowia lipolytica Lip2 protein--an enzyme that has a wide technological application. By genome analysis of Y. lipolytica in addition to already characterized Ylcwp1, we identified five putative open reading frames encoding glycosylphosphatidylinositol-anchored proteins. Lip2 translation fusion with the carboxyl termini of these proteins revealed that all proteins were capable of immobilizing lipase in active form on the cell surface. The highest level of cell-bound lipase activity was achieved using C-domains encoded by YlCWP1, YlCWP3 (YALI0D27214g) and YlCWP6 (YALI0F18282g) comprising 16,173 ± 1,800, 18,785 ± 1,130 and 17,700 ± 2,101 U/g dry cells, respectively. To the best of our knowledge, these results significantly exceed the highest cell-bound lipase activity previously reported for engineered Saccharomyces cerevisiae and Pichia pastoris strains. Furthermore, the lyophilized biomass retained the activity and was robust to collecting/resuspending procedures. Nevertheless, in most cases, a substantial amount of lipase activity was also found in the growth medium. Further work will be necessary to better understand the nature of this phenomenon.
authors.affiliation: Russian State Collection of Industrial Microorganisms (VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow 117545, Russia. enja_t@mail.ru
authors.forename: Evgeniya Y
authors.initials: EY
authors.lastname: Yuzbasheva
authors.forename: Tigran V
authors.initials: TV
authors.lastname: Yuzbashev
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Laptev
authors.forename: Tatiana K
authors.initials: TK
authors.lastname: Konstantinova
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Sineoky
mesh_terms: D000595:Amino Acid Sequence; D002473:Cell Wall / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D007103:Immobilization; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D047071:beta-Glucans; C064197:beta-1,6-glucan; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase