Biomedical subjects

Rachel R Chen

Publications and source records attributed to Rachel R Chen.

5 recordsLinked to original sources

PubMed2006

Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate.

In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun's lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze-thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.

Amino Sugars↗

PubMed2004

Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier.

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, due to permeability barriers imposed by cell envelopes, whole-cell catalyzed reactions are reportedly 10-100-fold slower than reactions catalyzed by free enzymes. In this study, we accelerated whole-cell biocatalysis by reducing the membrane permeability barrier using molecular engineering approaches. Escherichia coli cells with genetically altered outer membrane structures were used. Specifically, a lipopolysaccarides mutant SM101 and a Braun's lipoprotein mutant E609L were used along with two model substrates that differ substantially in size and hydrophobicity, nitrocefin, and a tetrapeptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The reduction of the outer membrane permeability by genetic methods led to significant increases (up to 380%) in reaction rates of whole-cell catalyzed reactions. The magnitude of increase in biocatalysis rates was dependent on the substrates and on the nature of mutations introduced in the outer membrane structure. Notably, mutations in outer membrane can render the outer membrane completely permeable to one substrate, a barrierless condition that maximizes the reaction rate. The impact of the mutations introduced on the permeability barrier of the membranes was compared to the impact of polymixin B nonapeptide, a known potent permeabilizer acting on lipopolysaccharides. Our results suggest that genetic modifications to enhance the permeability of hydrophilic molecules should target the Lipid A region. However, strategies other than reduction of Lipid A synthesis should be considered. As we have demonstrated with tetrapeptide, membrane engineering can be much more effective in reducing a permeability barrier than are exogenous permeabilizers. This work, to our knowledge, is the first use of a molecular membrane engineering approach to address substrate permeability limitations encountered in biocatalysis applications.

Catalysis↗

PubMed2004

A pH-sensitive assay for galactosyltransferase.

We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N-acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn(2+) concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes.

Clinical Enzyme Tests↗

PubMedDate not supplied

Lipoprotein mutation accelerates substrate permeability-limited toluene dioxygenase-catalyzed reaction.

One of the major problems in whole-cell biocatalysis is its low reaction rate. The underlying cause is the substrate permeation barrier presented by cell envelopes. The present research investigates mutation effects of the Braun's lipoprotein, the most abundant outer membrane structural protein in Escherichia coli, on toluene dioxyengase (TDO)-catalyzed reaction. Dramatic enhancement of the reaction rate, an increase of up to 6-fold, was observed with the mutant for all three small, hydrophobic substrates tested (toluene, ethylbenzene, and 2-indanone). The increase was observed over a wide range of substrate concentrations (0.1-5 mM). The mutant exhibited a normal growth rate and expressed the recombinant multicomponent enzyme as well as the isogenic parent strain. Taken together, the lipoprotein mutant expressing TDO is a much better whole-cell catalyst for the oxidation reaction. The beneficial effect of the lipoprotein mutation may be general for a broad range of substrates and enzyme systems as the mutation affects the global integrity of the cell membrane. A comparison of the mutation effect with a common permeabilizing procedure, the EDTA treatment, further illustrates the clear advantages of using genetic modification in cellular membrane engineering for improved whole-cell catalysts.

Benzene Derivatives↗

PubMedDate not supplied

An improved phage display procedure for identification of lipopolysaccharide-binding peptides.

We successfully implemented several modifications to the regular phage display procedure and significantly improved the lipopolysaccharides-binding properties of the peptides selected. Specifically, the number of biopannings was increased and peptides with consensus sequences were obtained. A dual selection procedure (referred to as subtractive panning) was used to simultaneously select for the desired target and deselect for an undesired target, thereby increasing the binding specificity. In addition, binding and washing conditions in the subtractive panning were also modified to favor the selection of peptides with higher binding strength. As a result, two peptides, ASFPPAF and SSHTISF, were identified with much improved binding properties compared to those selected with regular panning. The binding specificities of these two peptides, as measured by the ratio of phages bound to the desired and undesired targets, were severalfold higher than previously reported. These modifications could easily be implemented with many other target molecules, indicating the general applicability of the procedure.

Amino Acid Sequence↗