Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14, UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic pathway and acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid (C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis describes the results of biochemical, structural and mechanistic studies of B. subtilis PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high purity and yield. Enzyme activity and kinetic constants were measured using reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at the University of St. Andrews various commercial and in-house screens were used to obtain diffraction-quality crystals suitable for X-ray crystallography. This also included the generation of seleno-methionine (SeMet) labelled PCAS, as well as heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate pimelic acid and the pimeloyl-adenylate intermediate and product PPi were determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily of adenylate forming enzymes. The enzyme is a homodimer composed of two domains, a short N-terminus and a large C-terminal domain and the ligand-bound structures revealed the residues potentially involved in substrate specificity and enzyme catalysis. The enzyme uses an internal ruler composed of a number of conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct dicarboxylic acid substrate. The X-ray structures guided the production of a number of site directed mutants to identify residues involved in the catalytic mechanism and stabilising the acyl-adenylate intermediate. This also allowed rational engineering of the PCAS active site to generate mutants with altered substrate specificity. Mutant PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the synthetic potential of PCAS. The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin pathway, a pyridoxal 5'phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase (AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give AON which is converted to biotin by the action of three other enzymes. We used genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion protein was expressed and purified from E. coli and initial studies suggest that it forms a functional, fused, dimeric enzyme.

Abstract not available

Includes bibliographical refences

Oil sand deposits in the Athabasca Basin in Alberta represent one of the largest global oil reserves. The bitumen contents of oil sand shallow deposits are recovered by surface mining using modified version of the Clark hot water process. Extraction of bitumen results in extremely large volumes of process water, which are contaminated with naphthenic acids. Various ex-situ treatment techniques including ozonation, advanced oxidation, adsorption, and bioremediation have been evaluated for the treatment of these waters. Previous studies conducted by Paslawski et al. (2009) investigated aerobic biodegradation of naphthenic acids in properly designed and carefully operated bioreactors. In the current work, anaerobic biodegradation of naphthenic acids under denitrifying condition was examined as a potential approach to eliminate the aeration cost in ex-situ treatment and as an alternative for application of in-situ treatment of oil sand process water in stabilization ponds was examined. Using trans-4-methyl-1-cyclohexane carboxylic acid (trans-4MCHCA), a microbial mixed culture developed in earlier works (Paslawski et al., 2009), and nitrate as an electron acceptor, anaerobic biodegradation of trans-4MCHCA were studied in batch and continuous bioreactors: continuous stirred tank reactor (CSTR) and biofilm system. Effects of naphthenic acid concentration, temperature, and loading rate on biodegradation process were investigated. The batch studies showed that initial concentration of trans-4MCHCA influenced the biodegradation rate where the increase in initial concentration of trans-4MCHCA from 100 to 250 mg L-1 led to a higher rate but further increase in concentration did not have a marked effect. Moreover, batch experiments at temperatures ranging from 10° to 35°C demonstrated that the optimum temperature was in the range of 20 - 24°C. Continuous anaerobic biodegradation in the CSTR showed that increase in loading rate of trans-4MCHCA caused an increase in removal rate of both trans-4MCHCA and nitrate. Rates were decreased as the system approached the cell washout. The maximum biodegradation rate and nitrate removal rate, achieved at trans-4MCHCA loading rate of 157.8 mg L-1 h-1, were 105.4 mg L-1 h-1 and 144.5 mg L-1 h-1, respectively. A similar dependency between the loading and removal rates was also observed in the biofilm reactor. The maximum removal rate of trans-4MCHCA and nitrate in the biofilm reactor, operated at room temperature (24 ± 2ºC) were 2,028.1 mg L-1 h-1 and 3,164.7 mg L-1 h-1, respectively and obtained at trans-4MCHCA loading rate of 2,607.9 mg L-1 h-1. Comparison of the results from aerobic batch systems obtained by Paslawski et al. (2009) and the current results showed similar profile where increase in initial concentration of naphthenic acid increased the biodegradation rate of trans-4MCHCA. As far as the effect of temperature is concerned, room temperature (20 - 24ºC) was identified as optimum temperature regardless of mode of biodegradation. Under continuous mode of operation (CSTR and biofilm reactors), anaerobic biodegradation was much faster than its aerobic counterpart. For instance the maximum anaerobic removal rate of trans-4MCHCA in the CSTR was 105.4 mg L-1 h-1, while the highest removal rate achieved in the aerobic CSTR was 9.6 mg L-1 h-1. Similarly, anaerobic biofilm reactor achieved a higher maximum removal rate of 2,028.1 mg L-1 h-1 compared to a 924.4 mg L-1 h-1 removal rate in the aerobic biofilm reactor. The overall finding indicated that biodegradation of trans-4MCHCA can be achieved effectively under anaerobic condition with the rates markedly higher than those for aerobic system.

In our earlier work, silica-based diatom frustules were successfully converted to 3-dimesional F-doped titania-based replicas via shape-preserving gas/solid displacement reactions, and experiments showed that the hydrolysis of organophosphorous ester pesticides, methyl paraoxon (MOX) and methyl parathion (MTH) was significantly faster in the presence of these 3-D titania nanostructures than in the presence of other commercial titania nanoparticles. The enhancement effect of titania frustules appeared to be strongly related to the amount of F-doping on these materials. In this work, a wider range of titania frustule replicas with various F-doping were prepared and characterized, and compared in the hydrolysis of MOX and MTH as well as three carboxylic acids (methyl salicylate, methyl benzoate and methyl 4-(aminomethyl)benzoate). A strong relationship between the amount of F-doping and the enhancement effect on the hydrolysis of organophosphorous esters was still observed. However, such enhancement effect did not occur in the hydrolysis of the carboxylic acids. It was discovered that fluorine-leaching from the titania frustules was significant and yielded high concentration of fluoride ions in the reaction solutions. Dissolved fluoride ions alone could significantly catalyze the hydrolysis of organophosphorous esters but not that of carboxylic acids in the oxide-free systems. It is believed that fluoride ions act as nucleophilic catalysts to accelerate the hydrolysis of organophosphorous esters. Comparison in the hydrolysis product formation from the two potential hydrolysis pathways (i.e., the P-O bond and the C-O bond cleavages) in the studied systems also supports the direct involvement of dissolved fluoride ions in the observed catalytic effect.

Chiral polyfunctionalized 1,5-dicarbonyl compounds are important synthetic intermediates and starting materials for many biologically active compounds so their synthesis has a great importance in the literature. In the first step, 1,3-cyclohexandione and other b-diketone derivatives are protected under acid catalyzation and their corresponding b-keto enol ether derivatives are obtained. These b-keto enol ethers are then converted to a-acetoxy enones in racemic form by Mn(OAc)3 mediated oxidation. Enzymatic kinetic resolution is applied to the racemic acetoxy enones by using different lipases and enantiomerically pure a-acetoxy and hydroxy enones are obtained. Then, dicarbonyl derivatives are obtained by hydrolizing racemic a-acetoxy enones. Oxidative cleavage of racemic a-acetoxy diketones in the presence of oxone gives corresponding racemic 1,5-dimethyl ester derivatives. By using this reaction as a reference, same reactions are applied to the chiral a-acetoxy and hydroxy diketones in order to synthesize chiral a-acetoxy and hydroxy 1,5-diester derivatives.

<p>The interaction of carboxylic acids with copper is a phenomenon found both outdoors and, more commonly, indoors. The influence on copper of some carboxylic acids (formic, acetic, propionic, and butyric) have so far been studied at concentrations levels at least three or four orders of magnitude higher than actual indoor conditions (< 20 ppb, volume parts per billion), and with only limited emphasis on any mechanistic approach. In this licentiate study a unique analytical setup has been successfully applied for <i>in situ </i>characterization and quantification of corrosion products formed during initial atmospheric corrosion of copper in the presence of acetic, formic or propionic acid. The setup is based on monitoring mass changes by the quartz crystal microbalance (QCM) and simultaneously identifying the chemical species by infrared reflection-absorption spectroscopy (IRAS). Post-analysis of corrosion products was performed by coulometric reduction (mass of copper (I) oxide formed), grazing incidence x-ray diffraction (phase identification) and atomic force microscopy (surface topography).</p><p>The absolute amounts of mass of individual constituents in the corrosion products, mainly copper (I) oxide or cuprite, copper (II) carboxylate and water or hydroxyl groups, have been deduced<i> in situ</i> during exposure in 120 ppb of carboxylic acid concentration, 95% relative humidity and 20ºC. An overall result is the consistency of analytical information obtained. For copper (I) oxide the quantified data estimated from IRAS, QCM or coulomeric reduction agrees with a relative accuracy of 12 % or better.</p><p>The interaction of copper with the carboxylic acids seems to follow two spatially separated main pathways. A proton-induced dissolution of cuprous ions followed by the formation of copper (I) oxide, and a carboxylate-induced dissolution followed by the formation of copper (II) carboxylate. The first pathway is initially very fast but levels off with a more uniform growth over the surface. This pathway dominates in acetic acid. The second pathway exhibits a more constant growth rate and localized growth, and dominates in formic acid. Propionic acid exhibits low rates for both pathways. The difference between the carboxylic acids with respect to both total corrosion rate and carboxylate-induced dissolution can be attributed to differences in acid dissociation constant and deposition velocity.</p>

Title from title page of PDF (University of Missouri--St. Louis, viewed Mar. 3, 2010). Includes bibliographical references.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1964. / Includes bibliographical references (p. 64-66).

Determining the presence of an analyte of interest, and finding the enantiomeric purity of chiral molecules are challenging tasks. This work in molecular recognition is carried out routinely by many different researchers, including both academic as well as industrial research groups. The following dissertation presents original research directed toward two different areas of interest to the molecular recognition community: enantioselective sensing in solution, and sensing at a defined interfacial environment. This work begins with a review of the non-chromatographic ways that the enantiomeric purity of chiral carboxylic acids is determined, presented in Chapter 1. Carboxylic acids are important functional groups, both for organic synthesis as well as pharmaceutical drug development. Chapter 2 presents efforts that have been made to rapidly assess both the enantiomeric purity and identity of chiral carboxylic acids, utilizing the technique of exciton-coupled circular dichroism (ECCD). A twist is imparted on a complex, and can be correlated with the absolute configuration of the stereocenter. The enantiomeric composition can be rapidly determined. After creating the assay, the focus of the work shifted toward applying this system to new classes of analytes. Chapter 3 covers chemo- and enantioselective differentiation of [mathematical symbol]-amino acids, and continues to discuss the expansion to [mathematical symbol]-homoamino acids. Then a synthetic substrates was tested, and a series of reactions screened to determine if any enantioselectivity had been imparted by a Baeyer-Villiger oxidation. Finally, the enantiomeric composition of a biaryl atropisomer, a compound lacking a stereocenter, was determined. The signal produced from this assay is at a relatively short wavelength, and efforts were undertaken to push this signal to longer wavelength. Chapter 4 is a compendium of the lessons that were learned upon attempting to create a self-assembled sensing system. The final chapter details work that was done in collaboration with Professor Katsuhiko Ariga at the National Institute of Materials Science in Tsukuba, Japan. In this chapter, an indicator displacement assay was carried out for the first time at the air-water interface. This contribution opens the door for sensing to be carried out at defined regions, rather than free in bulk solution. / text