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Kinetic isotope effect (KIE) study of enzymatic mechanisms has the potential for aiding the design of tight binding inhibitors, but is hampered by the need for isotopically labeled substrates. Recently, however, methods for measuring ¹³C and ²H KIEs at natural abundance by NMR spectroscopy have been developed, allowing KIEs to be measured at every NMR resolvable nucleus without isotopic substitution. Until this study, this technique had yet to be applied to an enzymatic system. KIEs provide information about transition states (TS) and since enzymes tightly bind structures resembling the TS, TS analogs can be used as powerful inhibitors and potential drugs. Glycosidases are enzymes that hydrolyze the acetals of carbohydrates. Inhibition of glycosidases has a large potential for therapeutic value. Methyl glucoside hydrolysis was used as a model substrate in the measurement of natural abundance KIEs. ¹³C KIEs were successfully measured on the acid and glucosidase catalyzed hydrolysis of methyl glucosides. The values of the primary ¹³C KIEs show that hydrolysis of β-methyl glucoside by β-glucosidase a more concerted ANON reaction. KIEs on the corresponding α-anomer suggest the opposite result, a ON*AN reaction. The experimental KIEs also matched well with calculated equilibrium isotope effects, lending support for the accuracy of the measurements. / Thesis / Master of Science (MSc)

Polymer flooding is a commercial enhanced oil recovery (EOR) method used to increase the sweep efficiency of water floods. Hydrolyzed polyacrylamide (HPAM), a synthetic commercial polymer, is widely used in commercial polymer floods and it is also used for mobility control of chemical floods using surfactants such as surfactant-polymer flooding and alkaline-surfactant-polymer flooding. The increase in the degree of hydrolysis of HPAM at elevated temperature or pH with time affects the polymer solution viscosity and its adsorption on rock surfaces. A polymer hydrolysis model based on published laboratory data was implemented in UTCHEM, a chemical EOR simulator, in order to assess the effect of hydrolysis on reservoir performance. Both 1D and 3D simulations were performed to validate the implementation of the model. The simulation results are consistent with the laboratory observations that show an increase in polymer solution viscosity as hydrolysis progresses. The numerical results indicate that hydrolysis occurs very rapidly and impacts the near wellbore region polymer injectivity. / text

Viscoelastic surfactants (VES) have been widely used in acidizing and acid fracturing. They are used as diversion agents during matrix acid treatments and leakoff control agents during acid fracturing. At high temperatures, viscoelastic surfactants hydrolyze, resulting in phase separation after a certain time. Their viscosities significantly decrease and it is much easier for them to flow back causing much less damage to the formation. In this study, 4 to 8 wt% of a new VES-acid system was tested at temperatures of up to 250°F over hydrolysis times of 0 to 6 hours. Then, the solutions were neutralized by calcium carbonate until the pH reached 4.5. An HP/HT rheometer was used to measure the viscosity of the spent acids. Mass spectrometry (MS) was conducted to analyze the hydrolysis products of the VES. Coreflood tests were also conducted on Indiana limestone to determine the effects of the hydrolysis products on the permeability of these cores. The temperature was set at 250°F and the flow rate at 2.5 cm^(3)/s. The viscosities of all VES-acid systems remained high at the beginning of hydrolysis, which was good for acid diversion. After that, the VES acid systems experienced a significant viscosity reduction due to phase separation; it became much easier for the spent acid to flow back. Coreflood experiments caused little damage to the Indiana limestone. MS results indicated hydrolysis of peptide bonds. Fatty acids formed the top oil layer, and amine-based molecules formed the aqueous phase. This study will summarize and discuss the details of viscosity changes of the acid systems of this kind of viscoelastic surfactant, the damage caused by hydrolysis products, and how this kind of viscoelastic surfactant can be used to improve treatments.

Products of hydrolytic polymerization of Cr(III) hydroxide were investigated in Milli-Q water and in natural water matrices. Products were first fractionated on Sephadex column using eluents of increasing strength. Ion chromatography (IC) with UV detection at 436 nm was then used to separate the ionic species.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The goals of this project were to (i) determine maximum extractable neutral invertase (NI) activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI), (iii) determine SNI copy number in the genome, (iv) describe SNI transcript and protein expression patterns throughout the plant, and (v) attempt to determine the contribution of hydrolysis to sucrose accumulation. SNI and sugars were extracted from the developing culm tissues of sugarcane, commercial variety N19. Tissues were divided according to developmental stage (internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or storage tissues). The lowest sucrose content was found in the core of the bottom of each of the internodes. The ratio between hexoses and sucrose was highest in the young internodes. In these internodes hexose content was higher in the bottom than the top. There was a significant correlation between sucrose content and NI. Fluxes involved in sucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructose were supplied as a carbon source for tissue discs of young and maturing internodal tissues of sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher in maturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates via invertase were higher in maturing internodes of US6656-15 than N19. Taking metabolic compartmentation into account, hydrolysis of sucrose via invertase made a significant contribution to the net turnover of sucrose. Along with this, it would appear that the ability to partition sucrose between the vacuole and cytosol causes a significant difference in sucrose content between varieties. A full-length cDNA for SNI was sequenced. This expressed gene showed significant homology to known NI sequences on both nucleic and amino acid levels. The SNI sequence did not contain the putative invertase catalytic amino acid sequence, suggesting it developed separately from the other classes of invertases. Approximately 1.8 kb of the SNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcane tissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcript expression was observed in all tissues of the sugarcane plant: roots, internodes, leaf roll and leaves. In culm tissues where sucrose content was low and hexose contents were high, SNI transcript and protein levels were high. This suggests that SNI is involved in growth metabolism. / AFRIKAANSE OPSOMMING: Die doel van die projek was om (i) maksimum ekstaheerbare neutrale invertase (NI) aktiwiteit in die suikerriet stingel te bepaal, (ii) die volgorde van 'n eDNA wat vir suikerriet NI (SNI) kodeer te bepaal, (iii) die SNI kopie-getal in die genoom te bepaal, (iv) SNI m- RNA en proteïenuitdrukkingspatrone deur die plant te beskryf, en (v) te poog om die bydrae van hidrolise op sukrose akkumulering te bepaal. SNI en suikers is geëkstraheer uit 'n kommersiële varieteit, N19. Weefsels was volgens ontwikkelingstadiums (internodes 3, 6 en 9) en anatomiese verskille (verryking vir groeiende, vaat- en bergings-weefsels) verdeel. Die laagste sukrose inhoud is in die middel van die onderste helfte van elke internode gevind. Die verhouding van heksoses tot sukrose was die hoogste in die jong internodes. Die inhoud heksoses was hoër in die onderste deel van die internode as die boonste deel. 'n Betekenisvolle korrelasie tussen sukrose inhoud en SNI is gevind. Flukse betrokke by sukrose sintese en hidrolise is ondersoek. Glukose en fruktose is as koolstofbron aan stingelweefsel van twee variëteite (US6656-15 and N19) toegedien. Sukrose-inhoud het tienvoudig tussen volwasse weefsels van die twee variëteite verskil. Hidrolise via invertase was hoër in ouer weefsels van US6656-15 as N19, en het In noemenswaardige bydrae tot sukroseomset gemaak. Die verdeling van sukrose tussen die vakuool en die sitosol kan moontlik 'n groot rol speel in die vermoë van die sel om sukrose te akkumuleer. Die volgorde van 'n volledige SNI eDNA is bepaal. The uitgedrukte geen het, op beide In nukleïen- en aminosuur vlak, betekenisvolle ooreenkoms getoon met ander bekende plant NI volgordes. Die SNI volgorde bevat nie die kenmerkende invertase katalitiese setel nie, wat daarop kan dui dat dit onafhanklik van ander klasse invertases ontwikkel het. Min of meer 1.8 kb van die SNI eDNA is in 'n vektor geskik vir bioliestiese transformering van suikerrietweefsel, geïnkorporeer. Southern klad analise het gewys dat die ensiem 'n lae kopiegetal op geen vlak het. SNI mRNA uitdrukking is waargeneem in elke weefseltipe van die suikerriet plant: wortels, internodes, blaarrol en blare. In stingelweefsels met lae sukrose- en hoë heksose-inhoud, was die vlakke van beide SNI-mRNA en -proteïen hoog. Dit dui daarop dat SNI moontlik betrokke is by groei-metabolisme.

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