All fossil fuels contain varying levels of sulfur compounds which are undesirable because they cause environmental pollution, corrosion, acid rain and lead to health problems. There is strict international legislation for the permissible levels of sulfur compounds in fossil fuels. The aim of this research therefore was the biocatalyst development for biodesulfurisation using two approaches. In the first approach, Rhodococcus erythropolis IGTS8-5 and IGTS8-5G were immobilised in porous coke particles and tested in repeated cycles successfully. Both bacterial strains grew well in the chemically defined medium with glucose as the main carbon and energy source and the model sulfur compound dibenzothiophene (DBT) as the sole sulfur source. 0.8 g of cells was immobilized on 250 g of coke particles without refreshing the medium over 72 h while 1.8 g of cells were immobilised on 250 g of coke when the media was refreshed every 24 hours for 120 h after the initial immobilisation batch of 72h. The latter, were used repeatedly in twelve consequtive batch desulfurisation cycles during which the biodesulfurisation activity progressively decreased from over 95% removal of 100 ppm DBT to around 45% removal. DBT removal is often expressed in terms of 2-hydroxybiphenyl which is the end product of biodesulfurisation. The biodesulfurisation activityof immobilised bacteria was equivalent to 310 umol 2-HBP h-1g-1 dry cell weight during the first hour. Freely suspended cells on the other hand exhibited biodesulfurisation activity equivalent to 91 umol 2-HBP h-1g-1 dry cell weight. Unfortunately, after the first 24 h, the activity of the immobilised cells decreased to 12 umol 2-HBP h-1g-1 dry cell weight. Use of plant cell cultures for biodesulfurisation is the other novel aspect of this work. Armoracia rusticana (horse radish) cell culture was chosen as the novel biocatalyst since this plant is a well known source of peroxidase enzyme which is involved in the biodesulfurisation metabolism according to the literature on bacterial biodesulfurisation. Arabidospsis thaliana (thale cress) was also used since its genome is completely sequenced and it is a model organism in genomics studies. Our results indicate that cell suspensions of both plants did show biodesulfurisation activity by reducing the level of sulfur compounds, mainly DBT and other three derivatives from both aqueous and oil phase. When compared to the bacteria, in terms of DBT consumption, the activity of A. rusticana was calculated as 55 umol DBT h-1 g-1 DCW and 65 umol DBT h-1 g-1 DCW for A. thaliana while in bacteria it was 91 umol DBT h-1 g-1 DCW for IGTS8-5 and 73 umol DBT h-1 g-1 DCW for IGTS8-5G. Transcriptomics analysis of the plant cell cultures after exposure to the DBT when compared to control cultures showed alterations in gene expression levels several of which were related to sulfur metabolism and transmembrane transporters of sulfate.

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2’-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced in Escherichia coli BL21 and purified to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was competitively inhibited by the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminates 2’2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root mean square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that a combination of a molecule’s hydrophobicity and nonspecific interactions with nearby functional groups contribute to a competitively inhibitive mechanism that locks DszB in a closed conformation and precludes substrate access to the active site. Limitations in DszB’s potential applications in industrial sulfur fixation are not limited to turnover rate. To better characterize DszB stability and to gain insight into ways by which to extend lifetime, as well as to pave the way for future studies in inhibition regulation, we evaluated the basic thermal and kinetic stability of DszB in a variety of solvation environments. Thermal stability of DszB was measured in a wide range of different commercially available buffer additives using differential scanning fluorimetry (DSF) to quickly identify favorable changes in protein melting point. Additionally, a fluorescent kinetic assay was employed to investigate DszB reaction rate over a 48 hr time period in a more focused group of buffer to link thermal stability to DszB life-time. Results indicate a concerningly poor short-term stability of DszB, with an extreme preference for select osmolyte buffer additives that only moderately curbed this effect. This necessitates a means of stability improvement beyond alteration of solvation environment. To this end, a more general investigation of glycosylation and its impact on protein stability was performed. Post-translational modification of proteins occurs in organisms from all kingdoms life, with glycosylation being among the most prevalent of amendments. The types of glycans attached differ greatly by organism but can be generally described as protein-attached carbohydrate chains of variable lengths and degrees of branching. With great diversity in structure, glycosylation serves numerous biological functions, including signaling, recognition, folding, and stability. While it is understood that glycans fulfill a variety of important roles, structural and biochemical characterization of even common motifs and preferred rotamers is incomplete. To better understand glycan structure, particularly their relevance to protein stability, we modeled and computed the solvation free energy of 13 common N- and O-linked glycans in a variety of conformations using thermodynamic integration. N-linked glycans were modeled in the β-1,4-linked conformation, attached to an asparagine analog, while O-linked glycans were each modeled in both the α-1,4 and β-1,4-linked conformations attached to both serine and threonine analogs. Results indicate a strong preference for the β conformation and show a synergistic effect of branching on glycan solubility. Our results serve as a library of solvation free energies for fundamental glycan building blocks to enhance understanding of more complex protein-carbohydrate structures moving forward.

Biodesulfurization (BDS) is an alternative to hydrodesulfurization (HDS) as a method to remove sulfur from crude oil. Dibenzothiophene (DBT) was chosen as a model compound for the forms of thiophenic sulfur found in fossil fuels; up to 70% of the sulfur in petroleum is found as DBT and substituted DBTs; these compounds are however particularly recalcitrant to hydrodesulfurization, the current standard industrial method. My thesis deals with enhancing BDS through novel strains and through nanotechnology. Chapter highlights are: Chapter 2. My first aim was to isolate novel aerobic, mesophilic bacteria that can grow in mineral media at neutral pH value with DBT as the sole sulfur source. Different natural sites in Iran were sampled and I enriched, isolated and purified such bacteria. Twenty four isolates were obtained that could utilize sulfur compounds. Five of them were shown to convert DBT into HBP. After preliminary characterization, the five isolates were sent to the Durmishidze Institute of Biotechnology in Tbilisi for help with strain identification. Two isolates (F2 and F4) were identified as Pseudomonas strains, F1 was a Flavobacterium and F3 belonged to the strain of Rhodococcus. The definite identification of isolate F5 was not successful but with high probability it was a known strain. Since no new strains were apparently discovered, I did not work further in this direction. Chapter 3. In a second approach I studied the desulfurization ability of Shewanella putrefaciens strain NCIMB 8768, because in a previous investigation carried out at Cranfield University, it had been found that it reduced sulfur odour in clay. I compared its biodesulfurization activity profile with that of the widely studied Rhodococcus erythropolis strain IGTS8. However, S. putrefaciens was not as good as R. erythropolis. Chapter 4 and 5. I then turned to nanotechnology, which as a revolutionary new technological platform offers hope to solve many problems. There is currently a trend toward the increasing use of nanotechnology in industry because of its potentially revolutionary paths to innovation. I then asked how nanotechnology can contribute to enhancing the presently poor efficiency of biodesulfurization. Perhaps the most problematic difficulty is how to separate the microorganisms at the end of the desulfurization process. To make BDS more amenable, I explored the use of nanotechnology to magnetize biodesulfurizing bacteria. In other words, to render desulfurizing bacteria magnetic, I made them magnetic by decorating their outer surfaces with magnetic nanoparticles, allowing them to be separated using an external magnet. I used the best known desulfurizing bacterial strain, Rhodococcus erythropolis IGTS8. The decoration and magnetic separation worked very well. Unexpectedly, I found that the decorated cells had a 56% higher desulfurization activity compared to the nondecorated cells. I proposed that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product respectively. Supporting evidence for enhanced permeabilization was obtained by Dr Pavel Grigoriev, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino. In Chapter 6, to optimize attachment of the nanoparticles to the surface of the bacteria I created thin magnetic nanofilms from the nanoparticles and measured the attachment of the bacteria using a uniquely powerful noninvasive optical technique (Optical Waveguide Lightmode Spectroscopy, OWLS) to quantify the attachment and determine how the liquid medium and other factors influence the process.