The Permeability
of Microorganims as Whole-Cell Biocatalysts
One of the major disadvantages of using whole microorganisms as complex (multi-step) biocatalysts is their reduced reactivity due to the diffusion barrier of their cell membrane (to transport of substrates into the cells or efflux of reaction products) and low specific activity (enzyme content per cell). Unfortunately, most current metabolic engineering pathway modeling and simulation studies still use the kinetics of purified enzymes (determined in vitro ) to approximate the in vivo kinetics of these same enzymes. This often leads to erroneous model predictions because in vivo enzyme kinetics are very different. We have begun to develop methods to accurately measure and model the in vivo kinetics of enzymes in order to improve the ability to eventually engineer microorganisms as industrial catalysts with very high reactivity.
The impact of substrate diffusion to immobilized microorganisms has been modeled by several research groups to predict product formation kinetics by including cell density, specific enzyme activity within each cell, enzyme catalytic efficiency, substrate charge, and cell membrane permeability. However these previous models do not accurately predict the observed whole-cell kinetics. We have begun to investigate the in vivo factors in more detail that affect enzyme activity using alkaline phosphatase and other enzymes located in the E. coli periplasm. We have developed methods to accurately measure the in vivo kinetics of enzymes in the periplasmic space because the diffusion barrier of the E. coli outer membrane can be determined (and even genetically altered); the volume of the periplasm is known and therefore we can accurately measure in vivo enzyme concentration. This system is surprisingly amenable to precise modeling over a wide range of conditions. In this system in vivo reaction velocity correlates well with substrate diffusion-limitation due to the outer cell membrane, however enzyme number per cell, the effect of enzyme inhibitors, and the temperature dependence of the reaction have little effect on altering the observed rate. These findings suggest a theoretical basis for engineering whole-cell biocatalysts to significantly improve their specific reactivity, demonstrate that the in vivo kinetics of enzymes in microorganisms are indeed significantly different from the properties determined in vitro, and that the in vivo kinetics of almost any enzyme can be accurately determined if the protein can be expressed in active form in the E. coli periplasm.
Current collaborators on this project: G. L. Nelsestuen, M.B. Martinez
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