Exploring the Potential for Using the BrkA Autotransporter to Surface Express Functional Subtilisin E in Escherichia coli BL21 DE3

Bacillus subtilis enzyme subtilisin E is a widely used industrial protease with applications in biotechnology and manufacturing. Specifically, subtilisin E is an important element in many detergents and food manufacturing processes due to its ability to hydrolyze proteins under harsh conditions such as high temperature and alkaline pH. Currently, purification of subtilisin E is expensive and time-consuming, and the generation of a whole-cell biocatalyst system based on a well-studied model organism, Escherichia coli, presents an attractive avenue for efficient subtilisin E production in Gram-negative bacteria for industrial purposes. In this study, we explored the surface expression of a functional subtilisin E in E. coli BL21 DE3 using the Bordetella resistance to killing (BrkA) autotransporter. We constructed a recombinant BrkA-subtilisin E plasmid (pMEGA) and quantified surface expression of subtilisin E with trypsin accessibility assay and western blot. E. coli BL21 DE3 transformed with pMEGA were unable to surface express subtilisin E. Premature release and transient surface expression of BrkA-subtilisin E and functionality was evaluated via a casein hydrolysis assay using skim milk agar plates, and no protease activity was detected. Our findings demonstrate that further optimization is needed to investigate the potential of the BrkA autotransporter as a system for surface expression of subtilisin E. Overall, this work contributes to the characterization of heterologous protein expression for industrial applications via the BrkA autotransporter system.