A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. / A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible for-ration of significant amounts of 160 -kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the umolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was unchanged in the presence of the leading antischistosomal drug Praziquantel, which bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between the m-v,llue and the size of the protein indicates that the amount of protem. surface exposed to solvent upon unfolding is the major structural de.erminant for the dependence of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained by differential scanning calorimetry also fitted to a two-state irreversible unfolding transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779 cal mol-IK-I , ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02) and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically determined values, implied that the thermal denaturation of Sj26GST did not result in complete unfolding of the protein, / Andrew Chakane 2018

by Suen Tsang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 96-103). / Abstract also in Chinese. / Chapter Chapter 1: --- Literature review --- p.1 / Chapter 1.1 --- "Straw mushroom, Volvariella volvacea" --- p.1 / Chapter 1.2 --- Problems which restrict the market of straw mushroom --- p.3 / Chapter 1.3 --- Non-enzymatic browning --- p.5 / Chapter 1.4 --- Enzymatic browning --- p.7 / Chapter 1.5 --- Impact of browning --- p.12 / Chapter 1.6 --- Mechanism of inhibition of PPO --- p.13 / Chapter 1.7 --- Sulfites --- p.13 / Chapter 1.8 --- Classification of PPO inhibitors based on chemical property --- p.14 / Chapter 1.9 --- Classification of PPO inhibitors based on inhibitory mechanism --- p.17 / Chapter 1.10 --- Physical methods for prolonging shelf-life --- p.18 / Chapter 1.11 --- Significance of this research --- p.20 / Chapter Chapter2: --- Characterization of PPO in straw mushroom --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.22 / Chapter 2.2.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.24 / Chapter 2.2.3 --- PPO isoenzymes in straw mushroom --- p.25 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.29 / Chapter 2.3.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.29 / Chapter 2.3.3 --- PPO isoenzymes in straw mushroom --- p.32 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter3: --- Several attempts to solve browning problem of straw mushroom --- p.55 / Chapter 3.1 --- Inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1 --- Investigation of inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1.1 --- Materials and methods --- p.55 / Chapter 3.1.1.2 --- Results --- p.56 / Chapter 3.1.2 --- The potential of using a combination of different PPO inhibitors --- p.58 / Chapter 3.1.2.1 --- Materials and methods --- p.58 / Chapter 3.1.2.2 --- Results --- p.59 / Chapter 3.1.3 --- Direct application of PPO inhibitors --- p.61 / Chapter 3.1.3.1 --- Materials and methods --- p.61 / Chapter 3.1.3.2 --- Results --- p.62 / Chapter 3.1.4 --- PPO and lipase content in straw mushroom under post harvest storage --- p.62 / Chapter 3.1.4.1 --- Materials and Methods --- p.74 / Chapter 3.1.4.2 --- Results --- p.75 / Chapter 3.2 --- Vacuum packaging --- p.75 / Chapter 3.2.1 --- Materials and methods --- p.75 / Chapter 3.2.2 --- Results --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4: --- Future work --- p.87 / Chapter 4.1 --- Suggested improvements of experiments --- p.87 / Chapter 4.2 --- Suggested experiment in future: application of calcium chloride --- p.88 / Chapter Chapter 5: --- Conclusion --- p.94 / References --- p.96

No description available.

Human pyruvate kinase performs the last step in glucose glycolysis in all cells and organisms and can be a key regulator of glycolytic flux. Pyruvate produced by PYK is transported into the mitochondria to fuel the TCA cycle, which enables the production of ATP; the main energy source of the cell. Human PYK contains four isoforms: M1 (found in muscle, heart and brain), M2 (in foetal cells and tumours), L (liver), and R (red blood cells) PYK. M2PYK plays a crucial role in tumour cell proliferation; by down-regulating metabolic flux, upstream metabolites can be used for protein and DNA synthesis. Reprogramming the metabolism of fast proliferating cells is called the 'Warburg effect'. The biological relevance of the different isoform activities is also discussed. For example RPYK in red blood cells is exposed to slowly altering metabolite concentrations, especially after intestinal absorption in plasma and RBCs uptake some of the metabolites. This thesis describes biochemical and biophysical studies of human M1PYK, M2PYK, LPYK, and RPYK. PYK is allosterically regulated by a range of metabolites. A comparative enzyme kinetics study of the four isoforms was performed to examine the mechanisms of activation and inhibition of these small molecule regulators, including all 20 amino acids and the thyroid hormone T3. The redox state of the environment was also found to be an important regulator of PYK activity. All four PYK isoforms were successfully expressed and purified. Interestingly, only M2PYK and RPYK were strongly regulated by amino acids and metabolites. We also found that the redox state regulates the activity of all four PYK isoforms as well as the sensitivity of M2PYK in response to natural regulators. These studies also confirmed the dissociation of tetrameric PYK into inactive monomers as an important mechanism of regulation, particularly for M2PYK activity. Nuclear magnetic resonance (NMR) and Small-angle X-ray scattering (SAXS) studies were performed to investigate the conformational behaviour of PYK isoforms in solution and to compare the effects of ligand binding. NMR data of all four isoforms reveal a conserved binding mechanism between isoforms and specific amino acids. SAXS data of all four isoforms demonstrate that ligands affect tetramerisation of PYK isoforms.

Faculty of Science School of Chemistry 9800165e govendor@aurum.wits.ac.za / Pyranonaphthoquinones are biologically important molecules found in a wide variety of bacteria, microbial fungi and plant species. Their biological activity is proposed to be a consequence of their ability to function as bioreductive alkylating agents. This class of compounds, which include monomeric and dimeric examples, contain the basic naphtho[2,3-c]pyran-5,10-dione skeleton, usually with substituents at the C-1 and C-3 positions of the pyran ring. The aim of the first part of the project was to develop a novel method for the synthesis of enantiomerically pure 5,8-dimethoxy-isochroman-4-ol, which will provide a handle for stereoselectively adding substituents to the C-1 and C-3 positions of the pyran nucleus. In the second part of the project we wished to attempt to synthesize the naturally occurring compound, cardinalin 3, the dimer of ventiloquinone L previously synthesized in the Wits laboratories. The synthesis of the enantiomerically pure isochromanol began with 2,5-dihydroxybenzoic acid, which was subjected to a diallylation followed by a Claisen rearrangement. The phenols were protected by a methylation reaction and the ester moiety was reduced to give (2-allyl-3,6- dimethoxyphenyl)methanol. It was then allylated to produce a suitable precursor for a one pot/two step ruthenium mediated isomerisation/ring closing metathesis reaction to produce 5,8-dimethoxy-1H-isochromene in an overall yield of 47%. It was converted to racemic 5,8-dimethoxy-isochroman-4-ol through a hydroboration-oxidation reaction in a yield of 84%. The separation of the enantiomers was achieved by acetylating the alcohol to form 5,8-dimethoxy-3,4-dihydro-1H-isochromen-4-yl acetate and then a lipase enzyme was used to stereospecifically deacetylate one enantiomer, while leaving the other enantiomer untouched. The second part of the dissertation discusses the progress towards the synthesis of cardinalin 3. This project began with the formation of the C-C biaryl axis starting from 1,3-dimethoxybenzene. The synthesis then continued with the diformylation of the biphenyl to give 2,2’,6,6’-tetramethoxy[1,1′-biphenyl]-3,3′-dicarbaldehyde. This was subjected to a Stobbe condensation and a Friedel-Crafts acylative cyclisation to produce diethyl [4,4′-diacetoxy-6,6′,8,8′-tetramethoxy-7,7′-binaphthalene]-2,2′- dicarboxylate. The synthesis will be continued in the PhD, using methodology previously developed for the formation of the monomer, as well as methodology developed here.

This thesis investigates an application of immobilized lipase for pre-treating wastewater containing fats and oils, which is difficult to treat practically. The kinetics of soluble lipase was studied for establishing background of the lipase. The immobilization of lipase was adopted in order to repeatedly use the expensive lipase. The developed immobilization methods were based on the characteristics of carriers, but covalent bonding of lipase was preferred because of strong adsorption nature. Three types of materials, nylon membrane and polystyrene-divinylbenzene and silica gel beads, were used for studying the lipase immobilization characteristics. The lipase from Canada rugosa was chosen because of its relatively high catalytic activity and commercial availability. The oily wastewater sources used were a simulated mixture of olive oil and distilled water as well as actual restaurant oily wastewater. A packed bed reactor packed with immobilized lipase was suitable for the study. Moreover, a comparative study of anaerobic digestion of lipase treated and un-treated oily wastewater was undertaken to evaluate the efficiency of the lipase pre-treatment method due to lack of the relevant literature in the enzymatic wastewater treatment field. The kinetics of lipase catalyzed hydrolysis reactions was investigated in a stirred tank reactor. The experimental results confirmed that the lipase catalyzed reaction obeyed Michaelis-Menten model. The optimal pH and temperature of the lipase catalysed hydrolysis reaction were 7 and 37°C, respectively. The conversion of oil to fatty acid was dependent on the reaction time and mass of the enzyme used. The lipase activities depended on the concentrations of some selected additives. Calcium ion improved lipase activity significantly amongst the additives used. / The immobilization of lipase was carried out using different materials, nylon membranes, polystyrene-divinylbenzene beads, and silica gel. Covalent adsorption was simple and successful for immobilizing the lipase onto nylon membrane which was pre-treated with HC1 solution for releasing amino groups. The adsorption of lipase was completed after only a 2-hour reaction time. It was much more practical for this shorter adsorption time (2 hours) rather than the 24 hours required for physical capillary adsorption of lipase. The properties of the immobilized lipase and the performance of the reactors we compared amongst the soluble and immobilized lipase forms. The immobilization, particularly for covalent bonding, made lipase more resistant to thermal deactivation. It was evident that the optimum temperature was shifted from 37°C for the soluble lipase to 45 and 40°C for immobilized lipase adsorbed onto nylon and polystyrene-divinylbenzene beads, respectively. The immobilized lipase could be used repeatedly with only little activity loss. The repeatedly operational stability made the reuse of the immobilized lipase possible. Comparison was also made between two types of beads, polystyrene-divinylbenzene beads and silica gels. Though polystyrene-divinylbenzene beads showed higher lipase activity and shorter adsorption time when compared to silica gels, the forme beads were not suggested for large scale study because of high cost of the beads. On improvement achieved in this work was that the 24 hours required for silanization of silica gel was reduced to only a few hours using evaporating 3-APTES in acetone instead of refluxing 3-APTES in toluene. / It is worthwhile to point out that much higher enzyme activity was obtained using the packed bed reactor as against the membrane reactor when aqueous oil emulsion was fed into the reactors. The lipase activity was 64.2% of soluble lipase activity for the immobilized lipase in the packed reactor but its activity was hardly detectable in the membrane reactor. Moreover, the operation of the packed bed reactor solved the of separating problem that severely hampered the lipase catalytic activity in the membrane reactor in aqueous phase. This result suggests that the packed bed reacts with the immobilized lipase is applicable in treating oily wastewater. The intrinsic parameters, Vmax and Km, were evaluated to study the internal diffusional effects of the porous spherical silica gel on the immobilized lipase. The changes of Vmax and Km for the immobilized lipase from those of the soluble lipase indicated that some alteration in the lipase intrinsic properties was caused by the immobilization of lipase. However, the magnitude of Thiele modulus suggested the immobilized lipase was most likely reaction controlling. In addition, good agreement for Vmax and Km from experiments and numerical model estimations seemed to suggest that the numerical model could be used for estimating Vmax and Km for the immobilized lipase. / An application was tried for conducting the hydrolysis of oily restaurant wastewater by soluble and the immobilized lipase. Enzyme activity of both forms was severely inhibited by the oily wastewater. The enzymatic activity was only 20% and 15% for soluble and the immobilized lipase, respectively, when compared to the initial activity value for the hydrolysis of olive oil by soluble lipase. Evaluation of the efficiency for the proposed lipase pre-treatment method was carried out by monitoring the performance of two anaerobic digesters. These two digesters were fed with lipase treated and untreated restaurant wastewater that was neutralised with KOH solution prior to feeding. The oil-floating problem was minimised by this saponification of fatty acids with potassium hydroxide. However, there was no clear sign of an improvement for the treatment efficiency of the anaerobic digesters in terms of COD removal and methane production rate resulted in digesting lipase treated oily wastewater when compared to the one without lipase pre-treatment.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Microfluidic technology is a useful tool to help answer unsolved problems in multidisciplinary fields, including molecular biology, clinical pathology and the pharmaceutical industry.Current microfluidic based devices with diverse structures have been constructed via extensively used soft lithography orphotolithography fabrication methods. A layer-by-layer stacking of 2D planar microchannel arrays can achieve limited degrees of three dimensionality. However, assembly of large-scale multi-tiered structures is tedious, and the inherently planar nature of the individual layers restricts the network’s topological complexity. In order to overcome the limitations of existing microfabrication methodswe demonstrate several novel methods that enable microvasculature networks: electrostatic discharge,global channel deformation and enzymatic sculpting to fabricate complex surface topologies. These methods enable construction of networks of branched microchannels arranged in a tree-like architecture with diameters ranging from approximately 10 μm to 1 mm. Interconnected networks with multiple fluidic access points can be straightforwardly constructed, and quantification of their branching characteristics reveals remarkable similarity to naturally occurring vasculature. In addition, by harnessing enzymatic micromachining we are able to construct nanochannels, microchannels containing embedded features templated by the substrate’s crystalline morphology, and an irregular cross section of microchannel capable of performing isolation and enrichment of cells from whole blood with throughput 1 – 2 orders of magnitude faster than currently possible. These techniques can play a key role in developing an organ-sized engineered tissue scaffolds and high-throughput continuous flow separations.

Lignocellulosic biomass (e.g., poplar wood) provides a unique and sustainable resource for environmentally safe organic fuels and chemicals. The core of this study is the pretreatment step involved in bioconversion processes. Pretreatment is required to realize high yields vital to commercial success. The focus of the pretreatment step is to methodically change key features of the biomass to favor enzymatic hydrolysis. This work assesses the compositional changes due to oxidative and non-oxidative longterm lime pretreatment of poplar wood (up to 4 weeks of pretreatment) at mild temperatures (25ºC to 65ºC), and their effect on the enzymatic yield of glucan and xylan. The most important pretreatment yield of lignin was 54 g lignin remaining/100 g lignin in raw biomass, and was accomplished for 4-week lime pretreatment at 65ºC in oxidative conditions. The corresponding pretreatment yields of glucan and xylan were 85.9 g glucan recovered/100 g glucan in raw biomass and 80.2 g xylan recovered/100 g xylan in raw biomass respectively. For poplar wood oxidatively pretreated with lime for 4 weeks at 65ºC and enzymatically hydrolyzed with an enzyme loading of 15 FPU/g glucan in raw biomass during a 3-day period, the best overall yields of glucan and xylan, were 80.7 g glucan hydrolyzed/100 g glucan in raw biomass and 66.9 g xylan hydrolyzed/100 g xylan in raw biomass respectively. The corresponding hydrolysis yields were 94.0 g glucan hydrolyzed/100 g glucan in treated biomass and 83.5 g xylan hydrolyzed/100 g xylan in treated biomass respectively. Because there is a previous study of long-term lime pretreatment of corn stover (Kim, 2004), the data obtained in this work show the effect of using woody lignocellulose as substrate. From the comparison, resulted that in the case of poplar wood oxidatively pretreated at 65ºC for 4 weeks, less lignin was removed and more carbohydrates were solubilized, however the hydrolysis yield of glucan was almost equal and the hydrolysis yield of xylan was higher than the reported by Kim for corn stover oxidatively pretreated at 55ºC for 4 weeks. The overall yield of glucan resulted lower in the case of poplar wood because of the lower pretreatment yield of glucan. Thus, it is important to complete the mass balances including an analysis on the pretreatment liquor to determine if the solubilized glucan was degraded.