Electrophilic Aromatic Substitution (EAS) is one of the most frequently used aryl substitution methods. Aside from the fact that most EAS reactions require an acid and an oxidizer to proceed, the reactions involving activated aryls typically produce a mixture of ortho- and para- products as well as an ortho-/para- disubstituted product. Regiospecificity in aromatic substitution is key in the production of many compounds in a variety of disciplines. Since EAS is one of the most often used substitution methods, it is extremely important to develop an efficient method for regiospecific substitutions. Previous research developed a method of ortho-substitution by using hydrocarbon media, a less hazardous, greener medium, which was modified to develop a method of p-iodination (bromination), but with extensive time periods. The research presented here not only reveals an expedient, rapid method for regiospecific p-bromination, but also does so without the need for an acid or an oxidizer. The conditions for p-bromination involve the use of acetone (sometimes with cyclohexane) and NBS resulting in GC yields of p-brominated product approaching 100% in a cost and time efficient manner without the concerns of hazardous materials or byproducts like Br2 or HBr. The reaction mechanism is briefly examined as well.

In a 2D cell culture, the cells are mainly grown on flat surfaces which are usually made of polystyrene plastic. Cells are able to attach to these surfaces, forming individual cell formations or colonies. In this study, we have been looked at many different platforms to improve cell growth, adhesion, attachment and proliferation on two different promising cell lines. These cell lines are the human neural stem cells (hNSCs) and human liver hepatocellular carcinoma cells (HepG2). Researchers have been very interested in studying these cell lines in the recent years as they have very useful potentials in the long run to aid and cure many of the disorders, diseases and possibly replace infected or injured organs as well. This can be done using actual clinical applications for cell therapies and tissue transplantation. Based on the studies conducted for this thesis, we have been able to show that cells can be maintained in a 2D culture setting with increasing growth and adhesion factors. The conditions used for these studies were a way to not use the traditional materials for cell attachment and growth. This was pursued due to the fact that most stem cells for their continuity require a microenvironment that will support their physical and chemical properties of an effective extra cellular matrix (ECM). To reiterate, presently most ECM molecules are human or animal derived for effective cell culture applications but not clinical. This is a major problem as each batch varies, they are difficult to isolate and most contain biological components that have been known to limit their use in clinical applications. Hence, this study concentrated on developing synthetic polymer based ECMs as they do not have the problems of the human or animal derived ECMs, but also as they are relatively low-cost, reliable and easily fabricated. Through many experimental trials we have successfully developed synthetic polymer based ECM molecules that sustain stem cell growth for HepG2 liver hepatocellular carcinoma and hNSC human neural stem cell lines. The different substrates developed were a peptide fabricated in our lab; different concentrations and solutions of Poly 4-vinylphenol (P4VP) that were used on a flat hollow fiber membrane made using Polyacrylonitrile (PAN) doped in a solution containing PAN/N, N-dimethylformamide (DMF) having a high biocompatibility. This hollow fiber membrane study was maintained with eight different conditions over a period of 6 weeks.

The biodesulfurization 4S-pathway is a promising complementary enzymatic approach to remove sulfur from recalcitrant thiophenic derivatives in petroleum products that remain from conventional hydrodesulfurization method without diminishing the calorific value of oil. The final step of this pathway involves the carbon-sulfur bond cleavage from HBPS, and the production of the final products 2-hydroxybiphenyl (HBP) and sulfite, has been recognized as the rate-limiting step, partially as a result of product inhibition. However, the mechanisms and factors responsible for product inhibition in the last step have not been fully understood. In this work, we proposed a computational investigation using molecular dynamic simulations and free energy calculations on 2’-hydroxybiphenyl-2-sulfinate (HBPS) desulfinase (DszB) with different bound ligands as well as different solvent conditions to develop a fundamental understanding of the molecular-level mechanism responsible for product inhibition. Based on available crystal structures of DszB and biochemical characterization, we proposed a “gate” area close to substrate binding site of DszB is responsible for ligand egress and plays a role in product inhibition. We have conducted biphasic molecular dynamic simulations to evaluate the proposed gate area functionality. Non-bonded interaction energy analysis shows that hydrophobic residues around the gate area produce van der Waals interactions inhibiting translocation through the gate channel, and therefore, the molecules are easily trapped inside the binding site. Umbrella sampling molecular dynamics was performed to obtain the energy penalty associated with gate conformational change from open to close, which was 2.4 kcal/mol independent of solvent conditions as well as bound ligands. Free energy perturbation calculations were conducted for a group of six selected molecules bound to DszB. The selections were based on functional group representation and to calculate binding free energies that were directly comparable to experimental inhibition constants, KI. Our work provides a fundamental molecular-level analysis on product inhibition for the biodesulfurization 4S-pathway.

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.

The objective of this thesis was to investigate BiOWiSHTM-Aqua, a commercial dry solid formulation containing a consortium of bacteria and yeast, as a biopesticide for treatment of Botrytis cinerea, a gray mold that affects strawberries. BiOWiSHTM-Aqua was compared with another commercial product specifically used as a fungicide and bacteriocide, Serenade® Garden Disease Control Spray (concentrated Bacillus subtilis strain QST 713). Both laboratory tests as well as in vivo lab tests were conducted. BiOWiSHTM-Aqua results varied widely from plate to plate, regardless of experimental conditions. In some of these plates, inhibition zones were observed around colonies from BiOWiSHTM-Aqua, indicating efficacy. The organism responsible for the inhibition zones of B. cinerea growth was isolated from BiOWiSHTM-Aqua, and 16s rRNA analysis identified this culture as a strain of B. subtilis. This strain was designated as B. subtilis ssp. KLB. The B. subtilis KLB concentration required to completely inhibit B. cinerea was 9.1x104 CFU/mL when B. subtilis KLB was inoculated 48 hours before B. cinerea, 1.3x105 CFU/mL at 24 hours, and 3.2x106 CFU/mL when both were inoculated at the same time. Various preliminary experiments using B. subtilis KLB were also conducted to investigate its economic feasibility, to characterize the organism, and to test its post-harvest in vivo viability. B. subtilis KLB cell concentration was 1.6x109 CFU/mL in a bioreactor with LB at the end of the log growth phase. B. subtilis KLB achieved cell concentrations as high as 5x109 CFU/mL in shake flasks with food-grade tapioca as a carbon source. Inoculation of B. subtilis KLB on post-harvest strawberries did not have an effect on Botrytis infection rates compared to the negative control. These various experiments were the first step in research to potentially produce B. subtilis KLB on a commercial scale.

The potential value of the chitin biomass (e.g. food waste) is recently considered being ignored by landfill. Chitin can be a potential cheap carbon source for converting into value-added chemicals by microorganisms. Serratia marcescens is a chitinolytic bacterium that harbors endogenous chitinase systems. With goals of characterzing S. marcescens chitinolytic capabilities and applying S. marcescens to chemical production from chitin, my dissertation main content includes five chapters: 1) Chapter 1 highlights background information of chitin source, S. marcescens and potential metabolic engineering targets using chitin as a substrate; 2) Chapter 2 demonstrates that ChiR is a key regulator in regulating 9 chitinase-related genes in S. marcescens Db11 and manipulation of chiR can be a useful and efficient genetic target to enhance chitin utilization; 3) Chapter 3 reports the production of N-acetylneuraminic acid (Neu5Ac) from chitin by a bottom-up approach of engineering the nonconventional chitinolytic bacterium, Serratia marcescens, including native constitutive promoter characterization and transcriptional and translational pathway balancing; 4) Chapter 4 describes improvement of S. marcescens chitinolytic capability by an adaptive evolution approach; 5) Chapter 5 elucidates S. marcescens intracellular metabolite profile using a constraint-based genome-scale metabolic model (iSR929) based on genomic annotation of S. marcescens Db11. Overall, the dissertation work is the first report of demonstrating the concept of chitin-based CBP using S. marcescens and the computational model and genetic molecular tools developed in this dissertation are valuable but not limited to design-build-test of S. marcescens for contributing to the field of biological science and metabolic engineering applications.

Photonic crystals serve as powerful building blocks for the development of lab-on-chip devices. Currently they are used for a wide range of miniaturized optical components such as extremely compact waveguides to refractive-index based optical sensors. Here we propose a new technique for analyzing and characterizing cells through the design of a micro-flow cytometer using photonic crystals. While lab scale flow cytometers have been critical to many developments in cellular biology they are not portable, difficult to use and relatively expensive. By making a miniature sensor capable of replicating the same functionality as the large scale units with photonic crystals, we hope to produce a device that can be easily integrated into a lab-on-chip and inexpensively mass produced for use outside of the lab. Using specialized FDTD software, the proposed technique has been studied, and multiple important flow cytometry functions have been established. As individual cells flow near the crystal surface, transmission of light through the photonic crystal is influenced accordingly. By analyzing the resulting changes in transmission, information such as cell counting and shape characterization have been demonstrated. Furthermore, correlations for simultaneously determining the size and refractive indices of cells has been shown by applying the statistical concepts of central moments.

Photosystem I (PSI) from plants, algae, and cyanobacteria can mediate H2 evolution in vivo and in vitro. A simple, self-platinization procedure that permits stable PSI-mediated H2 evolution in vitro has been developed. The H2 evolution capabilities of PSI from Thermosynechococcus elongatus have been characterized. This organism utilizes cytochrome c6 (cyt c6) as the e- donor to P700. Using a solution-based, self-organized platinization of the PSI nanoparticles, this study demonstrates a sodium ascorbate-cyt-PSI-Pt-H2 electron transport and proton reduction system that yields light-dependent H2. The system was thermostable with H2 evolution increasing up to 55°C. In addition, stability studies have shown the H2 evolution to be very stable, with no significant decrease over the 80 days investigated. Through simple optimization a H2 production rate of ~5.5 mol H2/h/mg Chl [micro-mole H2 per hour per milligram chlorophyll] was attained. To further optimize the H2 production Asc-cyt-PSI-Pt-H2 system, response surface methodology (RSM) was employed. The process parameter studied included temperature, light intensity and platinum salt concentration. The results showed that experimental data had a good fit to the proposed model (R2=0.99 and p < 0.001). Platinum salt concentration, temperature and the interaction between platinum salt concentration and temperature showed significant effects on the total H2 yield. Light intensity had minimal effect of the total H2 yield within the region studied. The optimum parameters for H2 photoproduction were light intensity of 240 μE/m2/s, [micro-eistien per square meter per second], platinum salt concentration of 636 μM [micro-mol/liter] and temperature of 310C. Finally, studies that will improve the H2 yield by increasing the kinetics of electron transfer were done. A hybrid protein was formed by engineering a gene to express a fusion of the membrane-bound [Ni-Fe] hydrogenase from Ralstonia eutropha H16 and the stromal-exposed subunits PsaE and PsaD of PSI from T. elongatus. A PsaE-free mutant of PSI was simultaneously formed by genetically disrupting the expression of the PsaE subunit of a native PSI; that will allow in vitro reconstitution of the desired PsaE-hydrogenase fusion protein with PsaE-free PSI.

As nontoxic biobased surfactants derived from plant oils and cellulose or starch, saccharide-fatty acid esters are widely used in cosmetics, food, and pharmaceutical industries due to their biocompatibility, biodegradability as well as antimicrobial activity. Generally, saccharide-fatty acid esters are synthesized chemically under high pressure, temperature and the presence of alkaline or acid catalysts leading to low-quality products (chemo-degradation of double bonds and oxygenated moieties) and large amounts of byproducts. In contrast, biocatalytic synthesis enhances sustainability: near-ambient pressure and temperature, the absence of toxic, acids and bases catalysts, and improved selectivity of products. For lipase-catalyzed synthesis under nearly anhydrous conditions, the major hurdle to be overcome is the poor miscibility of the acyl donor and acceptor substrates, resulting in slow reaction rates. Although several approaches such as, the employments of organic solvents, complexation agents, and ionic liquids, have been reported in the literature, a robust solution is desperately needed. This study focused on employing immobilized lipases under completely solvent-free conditions to synthesize saccharide-fatty acid esters using the ester products to enhance miscibility. Experimentally, metastable saccharide particles with a diameter of 10-100 micron-sized suspensions of saccharide were formed in oleic acid-rich ester mixtures initially for synthesis of saccharide-fatty acid esters in packed bed bioreactor containing immobilized lipases. Water, a by-product that limits ester yield by promoting hydrolysis, was removed via free evaporation. In this dissertation, a bioreactor system was developed for the eco-friendly solvent-free, immobilized lipase-catalyzed synthesis of biobasaed surfactants utilizing suspensions as reaction medium with 88 wt% in 6 days; the performance of the bioreactor systems developed for Objective 1 was optimized through water concentration control and interval time with 91 wt% in 4.8 days; and to improve design of bioreactor system developed in Objective 1 by in-line filter and derive a mathematical model to describe the production of esters by the bioreactor systems developed. Finally, 84 wt% ester content was achieved in 8.4 days.

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.