Functional esterase surface display by the autotransporter pathway in Escherichia coli

Bacterial surface display is a promising tool for a wide variety of biotechnological applications. In this work, a carboxylesterase, EstA from Burkholderia gladioli was translocated to the surface of Escherichia coli using the autotransporter pathway. For this purpose, an artificial gene was constructed by PCR, that encodes a fusion protein of EstA and the essential autotransporter domains. Esterase activity of whole cells expressing the EstA-autotransporter fusion protein could be detected by a filter overlay assay using alpha-naphthylacetate as substrate as well as by an agar plate pH-assay using p-nitrophenylacetate as a substrate. The specific esterase activity of whole cells was determined to be 1.7 mU/mg protein with p-nitrophenylacetate, After differential cell fractionation, the specific esterase activity of the outermembrane fraction was determined to be 23 mU/mg protein, using the same substrate. Western blot analysis of the different cell fractions with an EstA specific antibody yielded positive signals only in the outer membrane fraction. Furthermore, the detected protein band was in the correct size, as it was predictable from the amino acid sequence of the fusion protein. In activity staining of SDS-gels using alpha-naphthylacetate as a substrate, it was the identical band that exhibited esterase activity. Surface exposure could be demonstrated by proteinase K digestion of the esterase domain, whereby the protease was externally added to intact cells. These results indicated that EstA is targeted to the surface of E. coli by the autotransporter pathway in its active form. (C) 2002 Elsevier Science B.V. All rights reserved.