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Introduction

Purification of fossil fuels is a necessary and very expensive step in fuel processing. Hydrodesulfurization is the current method for the removal of sulfur containing compounds, but this is an expensive process which leaves many sulfur containing compounds in the fuel. This costly method’s failings necessitate a more effective treatment method that can clean cheaply and is capable of a high level of sulfur removal which has been termed ‘deep desulfurization’. Enzymes could be attractive catalytic agents for desulfurization of fossil fuels because of their high activity at ambient conditions. Enzymes have been shown to have activity in non-aqueous media, though considerably lower than the activity in native aqueous conditions. The reason for such decreased activity is enzyme unfolding in the presence of a non-aqueous solvent. It has also been shown that enzymes attached to nanoparticles are stabilized in conditions that would normally negatively affect the activity of an enzyme. Thus, there are good reasons to believe that immobilization on nanoparticles would considerably improve the existing approaches of enzyme immobilization for catalysis in non-aqueous solvents while maintaining the beneficial effects associated with immobilization on conventional surfaces. The ultimate goal of this project was to use enzymes stabilized on nanoparticles in organic solvents to remove sulfur compounds by oxidative desulfurization.

During the first year, we investigated a model system, consisting of enzyme chloroperoxidase (CPO) immobilized on polystyrene latex nanoparticles and demonstrated that enzyme immobilized on nanoparticles showed similar or slightly higher activity with a model colorimetric substrate, N,N,N',N'-tetramethyl-p-phenylenediamine in 99.5% ethanol. The goal of the second year was testing activity of enzyme-nanoparticle conjugates in conditions approaching those that would be used in actual fuel desulfurization processes. Specifically, Dimethyldibenzothiophene (DBT, a major sulfur-containing contaminant of diesel fuels) was used as the substrate, and hexane was used as the non-aqueous solvent.

Materials and Methods

Two enzymes were used in this study. Chloroperoxidase from Caldariomyces fumago (CPO) was chosen because of it ability to exhibit oxidative desulfurization in non-aqueous media. CPO catalyzes oxidation reactions with the heme molecule in the active site that coordinates the reaction. The second enzyme is myoglobin (MYO), which also has an active site heme, was chosen because of its availability as potential candidate for industrial processes. Latex nanoparticles with a diameter of ~40 nm surface modified with chloromethyl reactive groups were used in the experiments. Attachment to nanoparticles was performed in 20mM HEPES overnight at room temperature. Enzyme-nanoparticle conjugates were separated from unattached enzymes using centrifugation at 12,000 g for 2 h. Absorbance of CPO and MYO at 405 nm was used to quantitatively evaluate enzyme attachment to nanoparticles and for leaching studies. After attachment and purification the conjugates and free enzyme were lyophilized overnight with 100mM TRIS added as a lyoprotectant. A particle size analyzer (Brookhaven Instruments) was used to determine the diameter of the conjugates and their zeta potential. Enzyme activity assays were performed both in aqueous and non-aqueous media. All reactions were started by the addition of 2 mM hydrogen peroxide. 4,6-Dimethyldibenzothiophene (DBT) was used for aqueous activity assays with DBT added via 20% acetonitrile as it is poorly soluble in aqueous solutions. Non-aqueous activity assays were performed in hexane. DBT oxidation was monitored by decrease of absorbance at 240nm both in aqueous and non-aqueous solvents. Free lyophilized enzyme was used as control in all activity assays; concentrations of free and immobilized enzyme were kept identical in all kinetic experiments.

Results and Discussion

Adsorption isotherms were obtained for both CPO and MYO on chloromethylated latex nanoparticles. Saturation occurred at approximately monolayer enzyme coating in both cases. Activity assays were performed both in water and in hexane. Reaction rates with DBT for conjugated CPO in aqueous media were not significantly different from those for free enzyme at the same concentration. Similarly, reaction rates with DBT in hexane were not significantly different for immobilized and free CPO. Stability studies showed that activity of free enzyme deteriorates much faster than that of the conjugates. Reaction rate for CPO conjugated to nanoparticles was approximately 5-times higher than that of the free enzyme after 2 day storage in aqueous media. Free MYO outperformed MYO immobilized on nanoparticles in both aqueous and hexane solutions; however immobilized enzyme showed considerable level of activity with reaction rates in hexane approximately twice as low as those for free enzyme. Results with MYO are particularly encouraging because this protein, as well as similar heme-containing protein hemoglobin could be readily available in large quantities from animal processing industry.

Conclusions

We were able to demonstrate that both free enzymes and enzymes immobilized on nanoparticles can oxidize DBT, one of the major sulfur-containing compounds in diesel, in non-aqueous media. The result that both CPO and MYO are able to catalyze this desulfurization in hexane is the most exciting as it shows the potential our system possesses to purify diesel fuels. The ability to easily remove the nanoparticle combined with the increased stabilization of the enzyme over time makes this a good prospect for a recyclable desulfurization agent. Steps can now be taken to apply the developed approach to decontamination of diesel fuels.

Impact

Principal investigator: PRF grant was my first award from a nationally competitive funding program. Through PRF funding, I learned what it takes to prepare, manage and implement a successful grant application. Currently, I serve as a PI and co-PI in a number of federally funded research projects from such agencies as NIH (RR024449), NSF (EFRI-0937985), and DoD (LC090033). My total funding exceeds $1 M. My research group consists of seven graduate students and a postdoctoral fellow. Experience obtained through PRF funding helped me tremendously in building our successful research program.

Graduate student: John Barry, Clemson Bioengineering Ph.D. candidate was partially funded through this PRF grant. Through this experience, he mastered experimental methods for enzyme immobilization on nanoparticles and learned the methodology of enzyme catalysis in non-aqueous media. He gave an oral presentation at an international meeting (2010 Society for Biomaterials) and is currently preparing a manuscript for a peer-reviewed publicaion.