Periplasmic Expression as a Basis for Whole Cell Kinetic Screening of Unnatural Enzyme Reactivities

Abstract

Publisher Summary This chapter discusses the importance of periplasmic expression as a basis for whole cell kinetic screening of unnatural enzyme reactivities. The work on the modification of esterolytic activities of the serine protease subtilisin E by directed molecular evolution using a periplasmic expression strategy is presented. It is found that the periplasmic fraction of E. coli TOP10 and TOP10 F´ could not catalyze ester hydrolysis, and therefore, was free of interfering enzyme activities that would prevent the effective screening of mutant enzymes expressed in the periplasm. Growth pseudosynchronization was achieved by culturing the cells for a prolonged time in minimal medium at low cell density to ensure that the cells were in steady state, balanced growth. Preparation of bacterial cells and measurement of esterolytic activity of subtilisin E on Phe-NPE is performed. Sucrose esters have found application as monomers in the synthesis of biodegradable and biocompatible sugar-based hydrogels and polymers. The evolved mutants may also be useful in regioselective ester synthesis in nonaqueous media and thus find applications ranging from the selective synthesis of sugar-based polymers to synthetic nucleoside derivatives.