Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / (tm) after EM in title is for Trademark symbol. Typescript. Vita. Includes bibliographical references (leaves 120-142). Also available on the Internet.

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / (tm) after EM in title is for Trademark symbol. Typescript. Vita. Includes bibliographical references (leaves 120-142). Also available on the Internet.

Doctor of Philosophy / Department of Chemistry / Ping Li / Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which have received much attention due to their potential applications in degradation of lignin and anthraquinone dyes. In this research, studies of two types of DyPs are carried out and reported in the following three sections. The first section includes the identification and characterization of class-A TcDyP from Thermomonospora curvata, a thermophilic actinomycete found in composted manure. The TcDyP was found to be highly active toward a wide range of substrates including phenolic lignin model compounds. Transient- and steady state- kinetics involving wild-type (wt) and mutant TcDyPs revealed that Asp220 and Arg327 are essential for compound I formation and reduction of compound II to resting state is the rate-limiting step. Additionally, replacement of His312 and Arg327 shifted the redox potential (E°′) to a more negative value. In the second section, the residues involved in the radical generation and substrate oxidation were explored. TcDyP contains 7 tryptophans and 3 tyrosines, which are the likely candidates of protein radicals and substrate oxidation sites. Crystal structure of TcDyP solved at 1.75Å revealed Trp263, Trp376 and Tyr332 as surface-exposed protein radical sites. Further studies using site-directed mutagenesis, steady-state and stopped-flow kinetics determined that the Trp263 is also one of the surface-exposed substrate oxidation sites. The Trp376 was characterized as the residue essential for covalent crosslinking of the enzyme units and an off-pathway electron sink. The highly conserved Tyr332 was found to be unimportant for substrate oxidation due to its extremely narrow surface exposure. The final section involves mechanistic study of a class-B DyP from Enterobacter lignolyticus (ElDyP), a bacterium capable of growing on lignin anaerobically. The crystal structure of ElDyP revealed the presence of two heme access channels measuring at ~3.0 and 8.0 Å in diameter and a water molecule as the sixth ligand to the heme center. Bisubstrate Ping-Pong mechanism was found operational in the catalytic cycle of ElDyP, in which conformational change of the enzyme resting state was proposed as the final step and the rate limiting step in the presence of ABTS. Microscopic events leading to Compound I formation was analyzed using D₂O₂. A kinetic isotope effect (KIE) of 2.4 at pD 3.5 suggested that Compound 0 is formed initially with protonation/deprotonation as the rate-limiting step. Compound I was directly reduced to the enzyme resting state via a 2-electron process, for which the rate increases as the pH decreases. Based on viscosity effect and solvent KIE (sKIE) with the reducing substrate, aquo release was found to be mechanistically important. Distal aspartate was proposed as the key residue that modulates the acidic pH optimum in Compound I reduction. These findings will pave the way for engineering DyPs for their applications in the degradation of lignin and synthetic dyes.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities. / Science, Faculty of / Chemistry, Department of / Graduate

Nonspecific esterase, and in some cases a "Tween" lipase, were investigated histochemically in arteries and several other tissues of rats and rabbits under various conditions known to affect the development of atherosclerosis: age and sex differences, endocrine and metabolic factors (sex steroids, thyroid function, alloxan diabetes, and fasting), arterial injury, and acute and chronic lipemia. A study of the above enzymes was also made in atherosclerotic rabbit and human aortae. Of the several nonvascular tissues studied, only the enzymes of the male gonads and a male accessory sex organ (the prostate) were influenced by some of the experimental conditions tested: maturation greatly increased the activity of both enzymes in the Leydig cells of the testis, and stilbestrol markedly diminished the esterase activity in the prostate epithelium. Thus, in certain reproductive organs, the enzymes reflected the functional state of the cells under investigation. The following findings were made concerning the enzymes of the arterial wall: 1. Neither of the enzymes was influenced by ageing or sex difference in the rat, rabbit or human. 2. Neither of the enzymes was influenced by sex hormones, acute or chronic lipemia, or any of the metabolic conditions studied in the rat. 3. Esterase disappeared from the foci of acute arterial injury induced by Calciferol treatment in the rat and did not reappear six weeks after infliction of the injury. 4. Strong esterase activity appeared in the cytoplasm of lipo-phages within both human and experimental rabbit atherosclerotic lesions, in contrast to the normal arterial wall which exhibited no esterase activity at all in either species with the methods used. 5. Esterase was absent from the superficial fibrous cap of the human atherosclerotic lesion. It is concluded that: In the rat arteries, which normally exhibit appreciable esterolytic activity, this enzyme appears to be quite stable since it is not visibly modified by ageing or a series of endocrine and metabolic influences; it is, however, drastically diminished by acute vascular injury and this may account, at least in part, for the well known preferential accumulation of lipids in foci of acute arterial damage. In the rabbit arteries, which normally exhibit no histochemically demonstrable esterase, appreciable esterolytic activity appears only within the cytoplasm of cells that have taken up lipid after exposure to chromic lipemia (foam cells of atheromata). Similarly, in human atheromata esterase appeared only in foam cells and was absent from the fibrous cap of the atherosclerotic lesions; thus the absence of lipid from the cap cannot be attributed to increased enzyme activity in this part of the lesion. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Periodontal disease is the leading cause of tooth loss in adults. Bacterial biofilm on tooth surfaces is the primary initiator of periodontal disease. Various factors contribute to the severity of periodontal disease including the different virulence factors of the bacteria within the biofilm. In the progression of periodontal disease, the microflora evolves from a predominantly Gram positive microbial population to a mainly Gram negative population. Specific gram negative bacteria with pronounced virulence factors have been implicated in the etiology and pathogenesis of periodontal disease, namely Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola which form the red complex of bacteria. The orange complex bacteria become more dominant in the maturation process of dental plaque and act to bridge the early colonizers of plaque with the later more dominant red complex bacterial and consists of such bacteria as Campylobacter showae, Campylobacter rectus, Fusobacterium nucleatum and Prevotella intermedia. Perhaps the most investigated contributing factor is the relationship between smoking and periodontal disease. When examining the association between cigarette smoking and interproximal bone loss, greater bone loss is associated with higher cigarette consumption, longer duration (i.e., pack year history) and higher lifetime exposure. The presence of various virulence factors such as the production of a capsular material, as well as the proteolytic activity of the various periopathodontic bacteria has been associated with the pathogenesis of periodontitis. Even though many different enzymes are produced in large quantities by these periodontal bacteria, trypsin-like enzymes, chymotrypsin-like enzymes and elastase-like enzymes, as well as dipeptidyl peptidase-like enzymes, have been thought to increase the destructive potential of the bacterium and mediate destruction of the periodontal apparatus. More specifically, it is hypothesized that the proteolytic activity of other clinically important periodontal pathogens, such as Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas assacharolyticus, is increased in the presence of nicotine. The purpose of this study was to determine the effects of nicotine on F. nucleatum, P. intermedia and P. assacharolyticus proteolytic activity. Cultures were maintained on anaerobic blood agar plates containing 3% sheep blood. Bacterial cells were harvested from the plates and washed. Washed F. nucleatum, P. intermedia and P. assacharolyticus cells were incubated with 1 mg/ml of nicotine. Bacterial cells not incubated with nicotine were used as positive controls. Secreted enzymatic activity was measured using the synthetic chromogenic substrates glycyl-L-proline-p-nitroanilide (GPPNA), N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (SAAAPNA), N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPPPNA) and N-α-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) (Sigma-Aldrich Products, St. Louis, MO, USA). Appropriate means and standard deviations were determined for each of the enzymatic activities measured and analysis of variance (ANOVA) was used to compare the groups utilizing a 5% significance level for all comparisons. Results demonstrated that after 60 minutes of incubation of F. nucleatum, P. intermedia and P. assacharolyticus cells with 1 mg/ml of nicotine and the various synthetic substrates, had the following proteolytic activity for GPPNA: 0.83 ± 0.14, 0.72 ± 0.03 and 0.67 ± 0.10, respectively; SAAAPNA: 0.82 ± 0.06, 0.76 ± 0.05 and 0.68 ± 0.08, respectively; SAAPPPNA: 0.90 ± 0.13, 0.85 ± 0.17 and 0.72 ± 0.03, respectively; and BAPNA: 0.81 ± 0.15, 0.74 ± 0.13 and 0.74 ± 0.16, respectively. In conclusion, the results indicate that in the presence of 1 mg/ml of nicotine, the proteolytic activity of F. nucleatum and P. assacharolyticus was increased with all of the synthetic substrates (with statistical significance seen only in the increases with F. nucleatum and GPPNA, SAAAPNA and BAPNA). The proteolytic activity exhibited an increasing trend in activity for P. intermedia with SAAPPPNA and BAPNA but a decreasing trend in activity with GPPNA and SAAAPNA when incubated with 1 mg/ml of nicotine, once again demonstrating no statistical significance for any of the substrates. Therefore, it could be concluded that based on these results nicotine at a concentration of 1 mg/ml may increase the proteolytic activity of periodontal pathogens and thus may increase periodontal disease activity and subsequent periodontal breakdown. Further studies are needed to validate these results utilizing different concentrations of nicotine.

No description available.

This thesis describes the investigations of leaving group effects on enzymatic catalysis by Methylyoxal synthase (MGS). MGS is a glycolytic enzyme involved m bacterial metabolism which catalyzes the irreversible elimination of dihydroxyacetone phosphate (DHAP). In order to probe the effect of various leaving groups to MGS catalysts, a range of' ’mutant’ substrates have been synthesised where the phosphate group of natural substrate DHAP has been replaced by sulfate, thiosulfate and bromo groups yielding the substtates dthydroxyacetone sulfate (DHAS), dihydroxyacetone thiosulfate (DHATS) and bromohydroxyacetone (BHA). To quantify an overall enzymatic rate acceleration detailed kinetic analysis of the solution, non-enzymatic reactions of these substrates have been made. Rates for elimination and enolization via C-1 and C-3 deprotonation respectively have been quantified for each substrate using high resolution (^1)H NMR spectroscopy for a range of pD values. From the second order rate constants for buffer catalyzed deprotonation, k(_B) (M(^-1)s(^-1)) comparisons can be made with k(_car)/K(_M) (M(^-1)s(^-1)) for the enzymatic reactions at the same pD values. The non-enzymatic reactions of DHAS were monitored in the pD range of 7.3-9.2 using phosphate and quinuclidinone buffers. Second order rate constants, k(_B) (M(^-1)s(^-1)) of 5.60 x 10(^-4), 5.28 X 10(^-3), 5.82 x l0(_-3) and 7.40 x l0(^-3) M(^-1)s(^-1) for C-1 deprotonation and 1.41 x l0(^-3), 2.24 x l0(^-3) and 3.30 x 10(^-3) for C-3 deprotonation show that increasing the pD results m an increase in the rate of buffer catalyzed deprotonation (phosphate catalyzed C-3 deprotonation was not observed at this pD). This is indicative of a general base catalyzed deprotonation mechanism. First order rate constants, k(_M) (s(^-1)) for the buffer independent catalyzed deptotonation reactions of 1.39 x 10' (^5), 1.22 X 10(^-4), 1.26 X 10(^4) and 2.04 x l0(^-4) for C-1 deprotonation and 9.66 x l0(^-5), 1.10 x 10(^-4) and 9.30 X 10(^-5) for C-3 deprotonation show that an increase 0.7 pD units leads to only a 67 % and 12 respective tacrease in the mtes of deprotonation at the C-l md C-3 positions. This is reflected in the relatively level pD rate profile of the reaction. The background, non-enzymatic reactions of DHATS were carried out in acetic acid buffers and DCl solutions, In DCl solution an average K(_int) (s(^-1)) value of 1.33 x l0(^-5) s(^-1) was obtained. For the acetate catalyzed C-1 deprotonation reactions it was found that moving from pD 3.75 to 4.38 leads to an overall 4.5 fold increase in the value of k(_int) (s(^-1)) for the buffer independent deprotonation reaction of the C-1 protons of the molecule. Values of 9.03 x l0(^-5), 1.03 x 10(^-4) and 4.07 x 10(^-4) s(^-1) were obtained for the reactions in acetate 5, 10 and 20 % f(_B) respectively. Accordingly, in the same pD range the values for the average of first order rate constants, k(_av) (s(^-1)) increase by a factor of 4.9 fold. This is believed to represent a hydroxide dependent reaction. An average k(_B) (M(^-1)s(^-1)) of 5 x 10(^-2) M(^-1)s(^-1) was obtained for the acetate catalyzed reaction at the C-1 position of the molecule. Reactions of the C-3 protons of this molecule were not quantifiable by (^-1)H NMR and thus were followed by mass spectrometry which suggests that a potential dimerisation reaction occurs following deprotonation at this position. Reactions of the C-3 protons of BHA were monitored in quinuclidinone buffet, with values of k(_B)(M(^-1)s(^-1)) = 3.35 x 10(^-3)M(^-1)s(^-1) and k(_int) (s(^-1)) = 9.35 x l0(^-5) s(^-1) obtained for the buffer catalyzed reaction and buffer-independent reaction respectively. The C-1 deprotonation reactions of BHA were monitored in acetate buffers (70-90 % f(_B)). An average k(_B) (M(^-1)s(^-1)) of 9.17 X 10(^-5) M(^-1)s(^-1) was obtained for the buffer catalyzed reaction in this pD range (5.18-6.09). In the same pD range values of k(_int) (s') - 3.7 x 10(^-6). 4.7 x l0(^-6) and 1.19 x l0(^-5) s (^1)were obtained. The reactions of DHA and DHAS were monitored m the presence of MGS (19.6 and 78.4 µg/mL). While no reaction was observable for DHA in the presence of enzyme. DHAS was found to undergo C-1 and C-3 deprotonation leading to average k(_cat)/K(_M) values of 56.5 M(^-1)s(^-1) and 43.5 M(^-1)s(^-1) for exchange and elimination respectively. The rate for elimination is approximately 90,000 fold slower than that observed for natural substrate DEHAP, k(_cat)/k(_M) = 5.2 x 10(^-6) M(^-1)s(^-1) indicating that the phosphodianior group, of DHAP is for optimal cataysis.

Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de obter a melhor produção de peptidases e lipases. Essas enzimas foram microencapsuladas por spray drying, visando garantir sua estabilidade e investigar outros prováveis benefícios obtidos pela técnica. A utilização de planejamento experimental permitiu analisar os efeitos e interações entre as variáveis operacionais do processo (temperatura de secagem, proporção de adjuvantes e relação entre adjuvantes). A caracterização bioquímica e físico-química do extrato enzimático e das micropartículas também foram estudadas. O emprego de farelo de trigo como substrato demonstrou maior produção enzimática que o uso de farelo de algodão. A fermentação produziu uma serinopeptidase e uma lipase, ambas com característica alcalina, com alta estabilidade em ampla faixa de pH e certa estabilidade em diferentes temperaturas. Para ambas as enzimas, observou-se modulação positiva da atividade frente à maioria dos íons estudados e forte inibição pelo surfactante SDS, enquanto a lipase demonstrou superatividade frente a CTAB. A caracterização enzimática permite sugerir a aplicação dessas enzimas na formulação de detergentes enzimáticos, indústria de couro, indústria de papel, agroquímicos, síntese de biopolímeros e biodísel. No processo de microencapsulação, a temperatura foi a variável operacional mais importante para a estabilidade, enquanto a quantidade de adjuvantes em relação à quantidade de extrato enzimático influenciou nas condições de manipulação. O estudo demonstrou que a técnica de microencapsulação por spray drying resultou em grande benefício no armazenamento das enzimas, por aumentar consideravelmente sua estabilidade e melhorar as propriedades físicas do extrato. / Two variations of agroindustrial residue were analyzed as culture medium for the bioprocess of solid-state fermentation by the fungus Fusarium oxysporum, in order to achieve the best production of peptidases and lipases. These enzymes were microencapsulated by spray drying in order to ensure its stability and investigate other potential benefits obtained by the technique. The use of experimental design allowed us to analyze the effects and interactions between the operating variables of the process (drying temperature, proportion of adjuvants and relation among adjuvants). Biochemical and physico-chemical characterization of the enzymatic extract and of the microparticles were also studied. The use of wheat bran as substrate demonstrated a greater enzyme production then the use of cottonseed meal. The fermentation produced serinepeptidases and lipases, both alkaline, with high stability over a wide pH range and some stability at different temperatures. For both enzymes, there was up regulation of activity with most of the ions analyzed and strong inhibition against the surfactant SDS, whereas lipase demonstrated superactivity against CTAB. The enzymatic characterization suggests the application of these enzymes in the formulation of enzymatic detergents, leather industry, paper industry, agrochemicals, synthesis of biopolymers and biodiesel. In the microencapsulation process, the temperature was the most important operating variable for stability, while the amount of adjuvants in relation to the amount of enzymatic extract influenced in terms of handling. The study demonstrated that the technique of microencapsulation by spray drying resulted in benefits for the storage of enzymes, by increasing considerably its stability and improve the physical properties of the extract.

Yiu Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 106-117). / Abstract also in Chinese. / Abstract --- p.1 / Chinese abstract --- p.4 / Acknowledgment --- p.5 / Abbreviations --- p.7 / Content --- p.9 / Chapter I. --- Introduction --- p.11 / Chapter II. --- Literature Review --- p.14 / Chapter II. 1. --- Basic concepts --- p.14 / Chapter II.2. --- Clinical aspects of enzymology --- p.17 / Chapter II.3. --- Changes fetal enzymes in neonate --- p.34 / Chapter III. --- Materials and Methods --- p.48 / Chapter III. 1. --- Study population --- p.48 / Chapter III.2. --- Sample collection and storage --- p.49 / Chapter III.3. --- Determination of cardiac and hepatic enzymes in plasma --- p.51 / Chapter III.4. --- Data handling and statistical analysis --- p.67 / Chapter IV. --- Results --- p.68 / Chapter IV.1. --- Study population --- p.68 / Chapter IV.2. --- Enzyme reference values in cord blood --- p.72 / Chapter IV.3. --- Changes in fetal enzymes in relation to acid-base status at birth --- p.78 / Chapter IV. 4. --- Changes fetal enzymes in relation with other obstetric events --- p.80 / Chapter IV.5. --- Variables related to fetal enzymes levels --- p.85 / Chapter V. --- Discussion --- p.93 / Chapter V. 1. --- Reference value for cardiac and liver enzymes in cord blood for current study population --- p.93 / Chapter V.2. --- Changes fetal enzymes in relation to fetal acid-base status --- p.95 / Chapter V.3. --- Changes in fetal enzymes in relation with other obstetric variables --- p.99 / Chapter V.4. --- Analysis of inter-relationships between obstetric variables and fetal enzymes --- p.102 / Reference --- p.106