Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities

Abstract

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.