Defense Date


Document Type


Degree Name

Doctor of Philosophy


Chemical Engineering

First Advisor

Dr. Rachel R. Chen

Second Advisor

Dr. Gary S. Huvard


Whole-cell biocatalysts are preferred in many biocatalysis applications. However, cell envelope often represents a formidable permeability barrier. As a result, reactions catalyzed by whole-cells are reportedly orders of magnitude slower than those of by their free enzyme counterparts. The present research addresses this critical issue by using membrane engineering approaches. Two E. coli strains with genetically altered outer membrane structures were used in the study, a lipopolysaccarides (LPS) mutant SM101 and a Braun's lipoprotein mutant E609L. The effects of outer membrane mutation on the permeability of substrates differing substantially in size and hydrophobicity were investigated by combining the mutant cells with model enzymes. The reduction of the outer membrane permeability barrier by these mutations led to significant accelerations (2 to 14 fold) in reaction rates of all whole-cell catalyzed reactions investigated. In the case of tetrapeptide, LPS mutation of the outer membrane can render the outer membrane completely permeable to substrate, a barrier-less condition that maximizes the reaction rate. For reaction rates of toluene dioxygenase (TDO)-catalyzed reactions, a dramatic increase of up to six fold was observed with the lipoprotein mutant for each of the three small, hydrophobic substrates tested. Mutations in either the LPS or in the Braun's lipoprotein are effective for accelerating reactions with UDP-glucose, resulting in a striking acceleration (up to 14 fold) of reaction rate. The magnitude of reaction rate acceleration was found to be dependent upon the substrate concentrations, the enzyme expression level, and on the nature of the mutations and substrates. In addition, the mutations have been demonstrated to be far more superior to common permeabilization procedures like freeze-thaw (FT) or treatment with the chelating agent EDTA (ethylene diamine tetraacetic acid). Importantly, lipoprotein mutant E609L exhibited a normal growth rate and expressed the recombinant multi-component enzyme as well as the isogenic parent. The exact nature of lpp lipoprotein mutation in E609L was further studied and deletion of lpp was successfully introduced into E. coli strain with different genetic background for whole-cell biocatalysis applications. An example was provided by introducing an lpp deletion into an E. coli O44K74 strain to achieve a higher yield for L-carnitine production. This research and the results outlined in this dissertation demonstrate a valid strategy for addressing permeability issues in whole-cell biocatalysis. The work also highlights a need for accessing substrate permeabilities in biocatalysis research and development.


© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008