It has been observed that thermophilic enzymes are often more sluggish at lower temperatures but comparable active as their mesophilic homologues at their corresponding living temperatures. Although these thermophilic enzymes exhibit high structural stability, the increased stability leads to a decreased flexibility of the thermophilic enzymes in return. To yield further advances in analysis of the interrelationships between flexibility and activity of enzymes, also the molecular basis of enzyme adaptation, we used a pair of thermo-meso acylphosphatase homologues with high level of similarity isolated from hyperthermophilic archeaon Pyrococcus horikoshii (PhAcP) and human (HuAcP) as model to study this issue. Despite the fact that their active-site residues are highly conserved, activity (kcat) of PhAcP is remarkably reduced compared with HuAcP at low temperatures. Based on crystal structure comparison, an extra salt bridge was formed between active site residue and C-terminus of PhAcP. To examine the role of salt bridge plays in catalytic reaction of AcPs, we designed a mutant PhG91A to disrupt the salt bridge in thermophilic PhAcP. In parallel, a salt bridge was re-engineered into mesophilic HuAcP to create HuA99K. Interestingly, the thermophilic variant PhG91A exhibited a more mesophilic-like manner in terms of activity and thermodynamic parameters. On the contrary, mesophilic HuA99K displayed a more thermophilic-like character. This is supplemented by detailed molecular dynamics (MD) simulations, revealing good qualitative agreement with experimental findings. Both theory and experiment results had provided evidences that the presence of a specific salt bridge is directly associated with the temperature adaptation of AcPs by reducing the catalytic site flexibility. / Lam, Yan. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3364. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (Ph.D.) - University of St Andrews, November 2006.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Bruce Banks; submitted to the Dept. of Chemistry. Title from PDF t.p. (viewed Jul. 31, 2009). Includes bibliographical references (p. 95-102).

Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Bommarius, Andreas S.; Committee Member: Doyle, Donald F.; Committee Member: Koros, William J.; Committee Member: Moore, Jeffre C.; Committee Member: Prausnitz, Mark R. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: John W. Peters. Includes bibliographical references (leaves 155-169).

Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.

The tunneling fold (T-Fold) superfamily is a small superfamily of enzymes found in organisms encompassing all kingdoms of life. Seven members have been identified thus far. Despite sharing a common three-dimensional structure these enzymes perform very diverse chemistries. QueF is a bacterial NADPH-dependent oxidoreductase that catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to a primary amine (preQ1) in the queuosine biosynthetic pathway. Previous work on this enzyme has revealed the mechanism of reaction but the cofactor binding residues remain unknown. The experiments discussed herein aim to elucidate the role of residues lysine 80, lysine 83, and arginine 125 (B. subtilis numbering) in NADPH binding. The biological role of the disulfide bond between the conserved residues cysteine 55 and cysteine 99 observed in several crystal structures is also examined. Characterization of QueF mutants K80A, K83, R125A and R125K revealed lysine 80, lysine 83 and arginine 125 are required for turnover. Further analysis of turnover rates for R125K are consistent with this residue and both lysines being involved in cofactor binding presumably by interacting with the negatively charged phosphate tail of NADPH and are therefore involved in cofactor binding. Based on bond angles and energies, the disulfide bond between Cys55 and Cys99 was characterized as non-structural. Enzyme oxidation assays were consistent with the bond serving to protect QueF against irreversible oxidation of Cys55, which would render the enzyme inactive. This is the only known example of a stress protective mechanism in the Tunneling-fold superfamily. QueF-like is an amidinotransferase found in some species of Crenarchaeota and involved in the biosynthesis of archaeosine-tRNA. The work presented here is focused on the preliminary characterization of this enzyme, including the elucidation of the natural substrate as well as the source of ammonia. The structure of the enzyme was solved and is also discussed. Substrate analysis for QueF-like indicated this enzyme is capable of binding both preQ0 and preQ0-tRNA and reacting to form a thioimide intermediate analogous to QueF but only the latter serves as a substrate for the reaction. This makes QueF-like the first example of a nucleic acid binding enzyme in the Tunneling-fold superfamily. Ammonia, glutamine and asparagine were tested as nitrogen sources and unlike most known amidotransferases, QueF-like can only use free ammonia to produce the archaeosine-tRNA product. The crystal structure of P. calidifontis QueF-like indicates the functional enzyme is a dimer of pentamers pinned together by a large number of salt bridges. The structure presents a high degree of similarity to that of QueF albeit the higher twist of the QueF-like pentamers with respect to QueF results in a more compact structure.

Cellulomonas find was grown on minimal media with casamino acids and yeast extract added. Avicel was found to be the best cellulosic carbon source for the production of cellulase enzymes. The Millipore Ultrafiltration System was found to be the most efficient method of concentrating the enzyme preparations. Unpretreated sawdust samples of four different softwood species were degraded between 12 and 16 percent over a 15-day treatment. Increasing the concentration of substrate lead to a lower percent degradation but a higher overall degradation. Chemical pretreatment did not appreciably increase degradability of the samples. Physical pretreatments decrease the degrada-bility of the sawdust samples. The lotech pretreatment, which is a combination of chemical and physical pretreatment, gives a substrate that is degraded by the Cellulomonas fimi enzyme preparation. The pretreatment makes 50% of the sample water soluble. In 3 days, a further 35% is degraded from large insoluble chunks into insoluble small particles which remain in suspension. There is 6% degraded into soluble state. This leaves only 9% of the initial sample left in the pellet. With longer incubation, one would expect the degradation to continue. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate