Spacer-mediated display of active lipase on the yeast cell surface

Citation
M. Washida et al., Spacer-mediated display of active lipase on the yeast cell surface, APPL MICR B, 56(5-6), 2001, pp. 681-686
Citations number
22
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
ISSN journal
0175-7598 → ACNP
Volume
56
Issue
5-6
Year of publication
2001
Pages
681 - 686
Database
ISI
SICI code
0175-7598(200109)56:5-6<681:SDOALO>2.0.ZU;2-6
Abstract
We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding R hizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha -factor leader sequence and the C-terminal half of alpha -agglutinin inclu ding the glycosylphosphatidylinositol-anchor attachment signal. The constru cted gene was overexpressed under the control of the glyceraldehyde-3-phosp hate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gl y/Ser repeat sequence were inserted at the C-terminal portion of ROL to enh ance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surf ace was confirmed by immunofluorescence microscopy. The ROL displayed on th e yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1- ol tributyl ester (BALB) and insoluble triolein. The insertion of linker pe ptides effected the activity towards BALB, thereby demonstrating that the o ptimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the ce ll wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lip ase displayed on the cell surface. Furthermore, insertion of a linker pepti de of the appropriate length as a spacer may be an improved method to effec tively display enzymes, especially those having the active region at the C- terminal portion, on the cell surface.