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

Michael B. Martinez, Michael C. Flickinger, Gary L. Nelsestuen

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the limiting step for the enzyme in the periplasm was substrate (para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, 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 average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)59-66
Number of pages8
JournalJournal of Biotechnology
Volume71
Issue number1-3
DOIs
StatePublished - May 28 1999

Bibliographical note

Funding Information:
Supported in part by Grant HL15728 from the National Institutes of Health. M.M. was supported by a supplement for students under-represented in science and by a grant from the Merck Co.

Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.

Keywords

  • Biocatalysis
  • Bioreactor
  • In vivo enzyme kinetics
  • Periplasm
  • Whole cell reactor

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