by Kwong-bun Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 260-295.

Enzymes play an important role in the environment, they breakdown natural-occurring and anthropogenic molecules so that they can be transported into cells and utilized. Enzyme assays are routinely used in soil science and oceanography to measure the activities of specific processes and to serve as general indicators of microbial activity. Conventional enzyme assays are conducted as batch incubation of sediment and water samples. During these assays the concentration of product is measured and enzyme activity is then determined as the rate of product formation. Few studies have measured enzyme activities of groundwater. This work investigates the use of β-glucosidase and phosphatase assays for quantifying in situ enzyme activities in groundwater. Improvements to conventional enzyme assays using p-nitrophenyl substituted compounds were made by developing a high performance liquid chromatography method to improve quantitation limits of the product and to quantify concentrations of both the substrate and the product. An in situ single-well push pull test was then conducted to measure β-glucosidase activity in situ and to estimate the Michaelis constant (K[subscript m]) and the maximum reaction velocity (V[subscript max]) in petroleum-contaminated groundwater at a field site near Newberg, Oregon. An important feature of the single-well push pull test is the nonlinear drop in pore water velocity that the test solution experiences as it moves out from the injection point. The nonlinear drop in pore water velocity is of particular interest because enzyme-mediated reactions are very fast and changes in the hydraulic properties during the test may give rise to mass-transport limitations. Fast reactions lead to the simultaneous depletion of substrate and accumulation of product at the site of the reaction so substrate and product concentrations near the enzyme can be different then the concentrations in bulk solution. And the rates obtained from a single-well push pull tests may be a combination of the rates at which substrate diffuses to the microorganism and at which the reaction occurs. Laboratory experiments with sediment-packed columns were conducted with a range of pore water velocities typically achieved in the subsurface during as push-pull test as a means for examining the potential effects of inhibition and diffusion on phosphatase enzyme kinetics. In this set of column experiments rates of phosphatase-mediated reactions were investigated instead of β-glucosidase, which is an inducible enzyme. Numerical investigations were then conducted to examine the importance of diffusion limitations for describing the influence of transport processes on the observed rates of reaction. The theoretical investigation was conducted by formally upscaling the proposed sub-pore-scale processes to develop a macroscale (or Darcy scale) description of the transport of the substrate. These results indicate that mass-transfer limitations due to the diffusion of the substrate to the enzyme cause an increase in the apparent K[subscript m] but have no effect on V[subscript max]. In this study an analytical method was developed to measure rates of enzyme-mediated reaction in situ so that the measured rates reflected actual rates of microorganism in their natural environment. More carefully controlled laboratory experiments demonstrated that rates of enzyme-mediated reactions measured at low substrate concentrations depended on the flow properties of the test solution. / Graduation date: 2006

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 102-112). Also available in electronic version. Access restricted to campus users.

Gaucher disease (GD) is caused by an inherited deficiency of the human lysosomal enzyme acid β-glucosidase (GBA, EC 3.2.1.45). Absence of functional enzyme results in lysosomal glycolipid accumulation. This disorder primarily affects organs of the reticuloendothelial system and disease severity ranges from mild hepatosplenomegaly to extreme neurological degeneration. Disease symptoms have been shown to be greatly ameliorated by enzyme replacement therapy (ERT). Limitations to therapy include the high cost of current ERT and its inability to treat neurological symptoms. In the present study I sought to produce a GBA-fusion enzyme in an economical manner that can be used to treat neurological GD. I explored the use of Pichia pastoris as an economical recombinant protein expression system for production of human GBA. In addition, I synthesized a protein transduction domain (PTD)-GBA fusion protein for its potential to be used as a neurotherapeutic. The results show that GBA-PTD4 can be expressed and purified from P. pastoris. Hydrophobic interaction chromatography and gel filtration chromatography were successful in purifying GBA-PTD4. Further optimization of expression and purification techniques is required for effective large scale production of recombinant enzyme. / Graduate

Thesis (M.S.)--University of Michigan, 1971.

Thesis (M.S.)--University of Michigan, 1971.

Myrosinase is the beta-thioglucosidase enzyme which catalyses the hydrolysis of glucosinolates, a group of naturally occurring plant metabolites. Glucosinolates are S-glucosides which occur predominantly in the family Cruciferae, and are abundant in the Brassica vegetables. The hydrolysis reaction, specifically activated by L-ascorbic acid, gives beta-D-glucose and an aglucone fragment, which then rearranges to give sulfate and an isothiocyanate. The aim of this study was the investigation of the chemical mechanism of myrosinase, in order for it to be compared with those of the much more widely studied (3-glycosidases. Myrosinase catalysed transglycosylation reactions were examined as a potential synthetic method. However, no transglycosylation was detected, even though a wide range of glycosyl acceptors was examined, including simple alcohols and thiols and examples with charged side chains to mimic the glucosinolate. Such reactions are commonly observed with beta-glycosidases. However, the stability and activity of myrosinase was not substantially affected by the presence of the acceptors. A small amount of transglycosylation was observed using azide, a charged glycosyl acceptor. This is consistent with the other transglycosylation reactions failing due to the lack of a suitable basic residue at the active site to deprotonate the acceptor. A range of potential substrates was synthesised, although only the nitrophenyl-p-D-glucosides showed any substrate activity. All S-glucosides were inactive. Myrosinase therefore appears to be very specific. Inhibition studies revealed that D-glucono-y-lactone, a potent competitive inhibitor and transition state mimic of p-glucosidases, was a poor noncompetitive inhibitor of myrosinase. It was proposed that this inhibitor binds at the L-ascorbic acid activator site. The inhibitory properties of a number of other compounds were examined, including reaction products and potential substrates (S-glucosides). [1-2H]-Sinigrin, p-nitrophenyl- and 2,4-dinitrophenyl-beta-D-[1-2H]-glucoside were prepared to measure the secondary deuterium isotope effects. The isotope effect obtained for 2,4-dinitrophenyl-p-D-[1-2H]-glucoside (DV= 1.26 +/- 0.11) indicated an sp2 hybridised intermediate via an SN1-Iike mechanism. The solvent kinetic isotope effect for sinigrin hydrolysis was determined (DV = 1.54 +/- 0.07, DV/K = 1.24 +/- 0.15) implying that a proton transfer was partially rate limiting.

Le bélier a été choisi comme modèle animal en tant que reproducteur saisonnier pour l'étude de l'α-glucosidase dans le plasma séminal. Son activité spécifique est basse dans les cas d'immaturité du spermatozoïde suggérant ainsi qu'elle serait un marqueur de la fonction épididymaire. L'étude de l'enzyme dans les fluides du rete testis et de 10 zones de l'épididyme démontre que cette glande contribue majoritairement au contenu en α-glucosidase du plasma séminal. En vue d'élucider le rôle possible de cet enzyme dans le processus de maturation du spermatozoïde, l'α-glucosidase a été purifiée 10,000 fois avec un rendement de 5%. C'est une glycoprotéine très mineure de 105 KDa et de structure monomérique. / Montréal Trigonix inc. 2018

by Lee Chi-Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 74-78). / Abstracts in English and Chinese. / Acknowledgment --- p.ii / Table of Contents --- p.iii / Abstract --- p.v / Abstract (Chinese Version) --- p.vi / Abbreviation --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General Background --- p.1 / Chapter 1.1.1 --- Valienamine --- p.2 / Chapter 1.1.2 --- Valienamine Derivatives --- p.4 / Chapter 1.2 --- Mechanistic Aspects of Glycosidase Inhibition --- p.6 / Chapter 1.2.1 --- General Background --- p.6 / Chapter 1.2.2 --- Mechanism of Enzyme Catalyzed Hydrolysis of Glycosides --- p.6 / Chapter 1.2.3 --- Types of Glucosidase Inhibitors --- p.7 / Chapter 1.2.4 --- Inhibition of Glycosidases by Valienamine Derivatives --- p.8 / Chapter 1.3 --- Previous Synthesis of Valienamine --- p.9 / Chapter 1.3.1 --- Enantiospecific Synthesis of Valienamine by Vasella and co-workers --- p.9 / Chapter 1.3.2 --- Enantiospecific Synthesis of Valienamine by Tatsuta and co-workers --- p.10 / Chapter 1.3.3 --- Synthesis of N-Alkyl Derivatives of Valienamine --- p.12 / Chapter 1.4 --- Previous Syntheses of Valienamine-containing Pseudodisaccharides and its Diastereomers --- p.12 / Chapter 1.4.1 --- Epoxide aminolysis --- p.12 / Chapter 1.4.2 --- Condensation of amine with ketone --- p.15 / Chapter 1.4.3 --- Synthesis of pseudoaminodisaccharide by Kapp and co-workers --- p.16 / Chapter 2. --- Results and Discussion --- p.18 / Chapter 2.1 --- General Strategy --- p.18 / Chapter 2.2 --- Syntheses of coupling precursors --- p.20 / Chapter 2.2.1 --- Syntheses of protected valienamine 65 and its 2-epimer80 --- p.20 / Chapter 2.2.1.1 --- Synthesis of Diol68 --- p.20 / Chapter 2.2.1.2 --- Synthesis of Diol67 --- p.21 / Chapter 2.2.1.3 --- Synthesis of protected valienamine 65 and its 2-epimer80 --- p.22 / Chapter 2.2.2 --- Syntheses of 6-deoxyaminosugars 63 and118 --- p.24 / Chapter 2.2.2.1 --- Synthesis of benzyl ether91 --- p.24 / Chapter 2.2.2.2 --- Synthesis of β-diol103 --- p.28 / Chapter 2.2.2.3 --- Synthesis of α-alcohol107 --- p.30 / Chapter 2.2.2.4 --- Synthesis of β-diol113 --- p.31 / Chapter 2.2.2.5 --- Syntheses of amines 63 and118 --- p.33 / Chapter 2.2.3 --- Syntheses of allylic chlorides --- p.34 / Chapter 2.3 --- Syntheses of pseudoaminodissaccharides --- p.36 / Chapter 3. --- Conclusion --- p.42 / Chapter 4. --- Experimental --- p.44 / Chapter 5. --- References --- p.74 / Chapter 6. --- Appendix --- p.79 / List of spectra --- p.79

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2007. / Includes bibliographical references.