Steady-state enzyme kinetics in the Escherichia coli periplasm: a model ofa whole cell biocatalyst

This study provided analysis of in vivo enzyme kinetics in a model system w hich consisted of alkaline phosphatase in the periplasm of Escherichia coli . Modeling of complete substrate titration curves was achieved for a wide r ange of intraperiplasmic enzyme levels and outer membrane permeabilities. T he results helped to identify the features most important to optimize in vi vo reaction velocity. For many situations, a surprising finding was that ma ximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the lim iting step for the enzyme in the periplasm was substrate (para-nitrophenylp hosphate) diffusion through the outer membrane. In vivo reaction velocity w as directly proportional to substrate concentration, outer membrane permeab ility, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the a verage size of the pore. This model system therefore defined important feat ures of bacterial whole cell biocatalyst design, which may also apply to ot her reactors using intact cells as catalysts. (C) 1999 Elsevier Science B.V . All rights reserved.