ABSTRACT Influence of fenugreek gum on bread and in vitro physiological effects Keisha T. Roberts Advisors: University of Guelph Dr. S.W. Cui Professor T.E. Graham This thesis examined the effect of fenugreek gum, from Canadian grown fenugreek on bread quality, when substituted for wheat flour at 5 % and 10 %, and the in vitro physiological effects of these breads, based on models of acute and long - term feeding. Study I determined bread could be produced with 10 % fenugreek gum, while maintaining quality parameters of volume and texture, comparable to a control. This was accomplished through the development of a novel bread production method, using the lamination procedure for puff pastry production. The behavior of fenugreek gum and starch (wheat flour) was determined by rapid visco analysis (RVA), farinograph and dynamic rheological measurement, while scanning electron microscopy of bread found fenugreek gum could be identified within the bread matrix. Study 2 in vitro starch digestion found fenugreek gum at 5 % and 10 % reduced glucose liberated from bread, with 10 % fenugreek gum causing a reduction of over 30 %. RVA of fenugreek breads highlighted differences in viscosity between breads and wheat flour substituted with the gum. This was substantial as viscosity measurements by RVA are conducted on raw ingredients and not the food as consumed, which reflects the possible reduction in viscosity with food processing. This study also determined extruding fenugreek gum may have caused morphological changes to the gum, which may possibly contribute to attenuation of glucose liberated in vitro. Study 3 evaluated the accumulation of short chain fatty acids (SCFA) from the fermentation of three substrates: Extruded fenugreek gum, bread with 10 % extruded gum and control bread, based on fecal microbiota from three donors. SCFA profiles varied with substrates and donors, with fenugreek gum having the highest accumulation of SCFA after 12 hours. Donors were a caucuasian Canadian, a black Jamaican and a black Trinidadian who was the only donor culturally exposed to fenugreek. This Trinidadian’s SCFA profiles were consistently higher for fenugreek gum than the other donors. These studies collectively showed fenugreek gum, though viscous could be successfully incorporated into bread and have potential as a functional food and nutraceutical.

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.

Petroleum powered vehicles emit volatile organic compounds (VOCs) through combustion that contributes to the pollution of the environment. A technique in the 1970s was developed to decrease these emissions, especially for nitrogen oxides (NOx) and sulphuric oxides (SOx) which is called exhaust gas recirculation (EGR). The technique works by recirculating a portion of the combusted gas back into the engine, this limits the NOx and SOx emissions because of lower temperatures and less available oxygen. The problems that follow these effects is the formation and condensation of acids that corrode the material of the EGR system, which are created by many different reactions. It is of importance to understand how the compounds in the EGR system behaves through analysis of authentic and simulated condensates, which is why a quantitative method for these compounds are of interest. The aim of the project was to develop a simple quantitative analysis method for formic acid, acetic acid, and lactic acid in aqueous solution, which was done at Gränges Sweden AB. The technique used for detection and quantification was gas chromatography (GC) coupled to a flame ionization detector (FID) and a water compatible polyethylene glycol (PEG) column. Fractional factorial design (FFD) was used for determination of adequate operating parameters of the GC method and the sample preparation. Sample preparation only required filtration and pH adjustment prior to direct aqueous injection (DAI) to the chromatographic instrument. Detection of the analytes was very difficult because of non-compatibility with the FID, and quantification of asymmetric peak shapes made this problem worse, omitting lactic acid from further analysis. Limit of detection (LOD) and limit of quantification (LOQ) was 490 and 1640 ppm for formic acid and 120 and 400 ppm for acetic acid, with an injection volume of 0.3 μL and split ratio 10:1. Limits were too high for every EGR sample leaving no peaks detected for the sample preparation used. Further development should be done with complementary techniques and sample reprocessing in order to quantify the compounds.

No description available.