Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1927. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 103-110).

Magister Scientiae - MSc / The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with “universal” archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of “universal” archaeal primers A571F (5’-GCYTAA AGSRIC CGT AGC-3’) and UA1204R (5’-TTM GGG GCA TRCIKA CCT-3’) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermaystems. Upon sequencing of amplicons it was discovered that Chinese and. New Zealandsamples contained novel nanoarchaeal phylotypes. The preliminary nanoarchaeal phylotypes were used to design nanoarchaeal-specific primer N989R (5'-GGT TTC CGG TGT CAG TTC-3'), which was coupled with A571F and used in screening of nanoarchaeotes. The nanoarchaeal phylotypes identified with these primers were further screened by amplified ribosomal DNA restriction analysis (ARDRA), which was used to explore the diversity of these phylotypes. The novel nanoarchaeotes cluster into 9 cosely related clades which may represent separate species. Three of the New Zealand phylotypes form one separate clade which is closely related to the published nanoarchaeotes. The following nanoarchaeal sequences were submitted to the GenBank, TC9F (AY572420), TC11-5 (AY571283), TC11-B6 (AY727890), TC11-B7 (AY727887), TC11-C4 (AY727886), TC11-C6 (AY727889), TC11-C8 (AY727888), AND TC11-D4 (AY727891). Fluorescence in situhybridization was also used to simultaneously visualize, identify and localize nanoarchaeotes. / South Africa

Magister Scientiae - MSc / Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus. The G. pallidus RAPc8 NHase is a heterotetramer that has a 28 kDa α subunit and a 29 kDa β subunit, with a α2β2 configured functional unit. The G. pallidus RAPc8 NHase operon has been cloned, sequenced and expressed at high levels in E. coli, with subsequent crystallisation for X-ray diffraction analysis. Current work is based on the study of the reaction mechanism of NHase as proposed by Mitra and Holz (2006). This mechanism implicated βY72 and βW76 in the hydration of nitriles. The study involved the site directed mutation of the β subunit of G. pallidus RAPc8 NHase, resulting in the generation of a βY72FβW76V mutant. This was followed by sequencing, expression and purification of the recombinant protein. The pH-activity and temperature- activity profiles of the mutant showed the enzyme to be optimally active at pH 6.4 and 55oC, respectively. Further kinetic analysis of the mutant found optimal kcat/KM values of 94.7 + 7.5 at 50oC and pH 5.8. The determination of the kinetic parameters under differing environmental conditions such as changes in temperature and pH revealed the ionisation constants of the enzymesubstrate complex (pKES1 and pKES2) to be 5.9 and 7.7, respectively. These results suggest that the active site sulphinic acid as opposed to the active site tyrosine may be responsible for catalysis. The active site tyrosine probably plays a substrate-stabilising role. The fluctuation of the kcat, KM and kcat/KM values demonstrated the need to use an alternative measure of enzyme efficiency. A more appropriate term could be the enzyme efficiency function, Ef, which considers the dependence of the enzyme efficiency on substrate concentration. / South Africa

Magister Scientiae - MSc / Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50°C. AXE activity was stable for at least 1.5 hours between 30°C and 40°C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30°C-40°C suggests potential for industrial applications that utilise mesophilic fermentations. / South Africa

No description available.

The thermophilic bacterium designated NT-7 was shown to reduce acetylene to ethylene at 35 C. It was found that the organism does not reduce acetylene when it is grown in Burk's medium with 0.3 per cent (w/v) NH4 N0 3 . Reduction of acetylene at 55 C could not be demonstrated due to insolubility of acetylene in Burk's medium at this temperature. It was shown that the bacterium NT-7 can not grow in the absence of atmospheric nitrogen at 55 C when combined nitrogen is not supplied with the nutrient medium. All these characteristics were used to prove that NT-7 is a nitrogen fixing bacterium. Identification procedures confirmed a previous finding that the organisms are rod shaped cells possessing endoepores. Further tests showed that NT-7 is obligately aerobic and motile.

No description available.

No description available.

The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo / universal&rdquo / archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo / universal&rdquo / archaeal primers A571F (5&rsquo / -GCY TAA AGS RIC CGT AGC-3&rsquo / ) and UA1204R (5&rsquo / -TTM GGG GCA TRC IKA CCT-3&rsquo / ) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.