Graduation Semester and Year

2013

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Roshan Perera

Abstract

This study focuses on the process development for a nanobioreactor for the oxidation of aliphatic C-H bond by the dependent activation of molecular oxygen and hydrogen peroxide dependent catalysis of cytochrome P450cam and P450 BM3 and their genetically encoded 3-NH2tyrosine mutant enzymes.Oxidation of aliphatic carbons is an enormous challenge in classical synthesis due to their inert nature. However, it is significant in the production of fine chemicals and commodity products, which are precursors for the production of other valuable compounds.Among these reactions is the conversion of cyclohexane to cyclohexanol which is an intermediate in the production of nylon 6,6, a compound in high demand worldwide. Current industrial methods to carry out these reactions are energy intensive and suffer from low selectivity. In this context, industrial cyclohexanol production uses heavy metals like cobalt and manganese, requires pressures around 12 bar, and temperatures over 140 °C resulting in quite low yields (4-10%). These processes are inefficient and produce high amounts of toxic waste. Thus, there is clearly a need for a more efficient and greener technology. The direct biooxidation of hydrocarbons would change drastically the current chemical catalysis due to a number of advantages, such as the direct use of hydrocarbons for the synthesis of valuable intermediates, the reduction of the steps required to reach the final product, hence reduction of waste generation, etc. However, several major challenges exist in biocatalytic processes. Among them, denaturation of catalyst, difficulty in attaching the catalyst on the surface, low solubility of the substrate in water, etc. Cytochrome P450s (CYP) are heme metalloenzymes capable of selectively activating aliphatic C-H bonds and oxidizing them by inserting an oxygen atom under very mild conditions. Among these P450s is P450cam, a water soluble protein that enables the selective and efficient conversion of cyclohexane to cyclohexanol, utilizing H2O2 as the oxidant at ambient temperatures producing water as the only by-product. Therefore, the use of CYP proteins to carry out hydrocarbon oxidation reactions is promising in this field. Therefore, chapters 2 and 3 discuss the cloning and expression of P450cam as well as P450 BM3 proteins. Furthermore, attempts to prepare solid phase bioreactors for catalysis have faced enormous challenges due to the non-availability of a good protein immobilization technique where denaturation of the protein during the immobilization process is common. In this investigation, we have developed a method for nanoparticle-bound cytochrome P450. It involves covalent attachment of the protein to the support via a benzoxazine ring formation through a Diels-Alder reaction in water and a genetically encoded 3-NH2Tyr amino acid through site directed mutagenesis as discussed in chapter 3, 4, and 5. The method produces a homogeneous monolayer with retention of the protein conformation, and the site of the mutation dictates the orientation. Consequently, the mutation was strategically introduced in a specific site to control protein orientation. The immobilized protein was found to retain its inherent activity. As a result, fully functional protein microarrays, with monolayer arrangements and complete control over their orientations, were generated using this strategy. Iron oxide magnetic nanoparticles and silica nanoparticles have been synthesized and functionalized for site-oriented protein attachment. Other supports such as glass, polypropylene and gold electrodes have been successfully used for that purpose. In this research we also investigated the random immobilization of WT P450cam via EDC bioconjugation for its use in cyclohexane oxidation.In chapter 6, we have successfully used P450cam bound nano-particles as a recyclable catalyst for the oxidation of cyclohexane to cyclohexanol with 30% conversion in the presence of hydrogen peroxide at room temperature. Our by-product was water, and hence it is a greener process. This also provides a strategy for cost effective production of valuable chemicals on a larger scale. Furthermore, the genetically encoded 3-NH2Tyr amino acid has not only served as an anchor point for the protein to control its orientation, but it has also incorporated redox properties to the protein that may play an important role during electron transfer processes. It is noteworthy that in the case of the mutated Q343(3-NH2Tyr) P450cam protein, it has shown a four fold increase in the activity over the WT for camphor oxidation. The 3-NH2>Tyr moiety may facilitate the electron transfer from the putidaredoxin to the P450cam protein as it is probably in the electron transfer pathway of these proteins. Similar immobilization techniques have been used to immobilize P450 BM3, to produce a promising protein microarray for the field of bioreactors for alcohol generation as well as P450 assay platform for drugs during drug discovery process.

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

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