Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae.

Abstract

The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.