Use of recombinant tagged proteins for isotopomer analysis and in enzyme immobilization

This work describes the use of recombinant proteins for isotopomer analysis and enzyme immobilization. The first part of this thesis focuses on the use of recombinant tagged proteins for isotopomer analysis to study metabolism of individual strains in mixed cultures and also to study stationary phase metabolism of a single microbe. The second portion of this report describes a method of immobilizing enzymes by recombinant expression of these proteins/enzymes tagged with silica-precipitating peptides and their subsequent purification and entrapment in silica matrices.;For isotopomer analysis, amino acids are obtained by hydrolyzing total protein obtained from total labeled biomass. For mixed culture analysis, it is difficult to determine from which organism an amino acid was derived. Using current methods, separation of microbes prior to analysis is a tedious and inefficient task. During this study, it was proved that an enriched protein from a microbe can provide the same data as total protein.;The gene expressing a his-tagged green fluorescent protein (Gfp) was overexpressed in the model organism Escherichia coli. It was demonstrated that production of Gfp did not affect growth kinetics of E. coli or the isotopomer distribution in key metabolites. The isotopomer labeling patterns of amino acids obtained from purified Gfp and total cell protein were indistinguishable. While the origin of an amino acid is ambiguous and cannot be assigned to a sub-population in a mixed culture, the origins of a protein on the other hand can be fully assigned. As a result, the use of a single protein for isotopomer-based flux analysis allows the study of a microbe in its community.;Expression of tagged Gfp was induced when E. coli reached stationary phase and isotopomer data of amino acids from purified Gfp was obtained. Since isotopomer data of a single protein serves as proxy for total protein, this analysis gave important information about flux in the stationary phase. The results indicated that the metabolic pathways leading to serine, alanine, glutamate/glutamine and aspartate/asparagine have the highest biosynthesis activity in E. coli during the stationary phase. The second part of this thesis covers the technique of tagging proteins/enzymes with silica-precipitating peptides for immobilizing these biomolecules in silica matrices. Silica is essential for the generation of stable structural materials in most living organisms and a large number of silica matrices in nature are found in diatoms. Diatoms, unicellular photosynthetic algae, create ornate species-specific cell walls made of amorphous silica at mild conditions using silicic acid from the environment. Several classes of diatoms exhibit fractal pore patterns wherein progressively smaller pores are nested to form self-similar patterns. Since industrial production of silica commonly involves elevated temperatures, pressures or alkaline conditions, the creation of biomimetic materials would be advantageous. Heavily post-translationally modified peptides called silaffins are involved in silica deposition in the diatom Cylindrotheca fusiformis. Phosphate groups are attached to serine residues of silaffins and the lysine residues are modified with polyamine side-chains. While the presence of these modifications is important for silica precipitation at acidic pH in the diatom, it was discovered that the unmodified R5 silaffin peptide (SSKKSGSYSGSKGSKRRIL) can deposit silica at neutral pH.;As proof of concept, green fluorescent protein (Gfp) and enzymes such as phosphodiesterase and organophosphate hydrolase were produced as translational fusions with R5 (autosilification domain) by linking the two proteins in frame with each other in an expression vector. E. coli was used as the host for expressing the recombinant fusion proteins. These proteins had the capacity of precipitating silica on the order of minutes in the appropriate buffer at ambient conditions. High immobilization efficiency as well as high loading of enzyme on silica were obtained. These enzymatically-active nanomaterials displayed improved stability. Kinetic analysis of the immobilized system was performed and found to be similar to free enzymes. It was also possible to alter the morphology of the resulting silica matrices by varying the autosilification domain. (Abstract shortened by UMI.)...