Gilvocarcin V (GV) and ravidomycin (RMV) exhibit excellent antitumor activities in the presence of near-UV light at low concentration maintaining a low in vivo cytotoxicity. Although, the exact molecular mechanism for in vivo actions of these antibiotics has yet to be determined, a [2+2] cycloaddition reaction of the vinyl side chain with DNA thymidine residues in addition to the inhibition of topoisomerase II and DNAhistone H3 cross-linking are reported for the GV’s mechanism of action. Such activities have made these molecules interesting candidates for the biosynthetic investigation to generate analogues with improved activity/solubility. Previous biosynthetic studies have suggested that the GV biosynthetic pathway involves a number of synchronously occurring transformations leading to the oxidative C-C bond cleavage and other intriguing biosynthetic reactions, such as the vinyl side chain formation, methylations, Cglycosylation and dehydrogenation. Although gene inactivation results identified many candidate genes whose corresponding enzymes are involved in these biochemical transformations, their exact functional roles and the identity of their natural substrates remained elusive. To provide more insights into these complex biochemical tranfrormations, three specific aims were set up. Specific aim 1 was to clone and characterize the RMV biosynthetic gene cluster. Through the comparison of GV cluster with the RMV cluster, the genes encoding the biosynthesis of sugar and tetracyclic aromatic moieties were identified. RavGT, the sole glycosyltransferase of the RMV cluster has demonstrated to have unprecedented sugar donor substrate flexibility, transferring an amino-pyranose sugar as well as a neutral furanose sugar. Specific aim 2 was to characterize all of the TDP-D-ravidosamine biosynthetic enzymes. The aim also included to a one-pot enzymatic synthetic protocol for the routine production of TDP-D-ravidosamine. Specific aim 3 focussed on a total enzymatic synthesis of defucogilvocarcin M (defucoGM), the polyketide-derived core of GV and RMV. This aim clearly identified the minimal enzymes required to biosynthesize the complex architecture of defucoGM from the simple building blocks acetate and malonate. In addition, the GV-pathway enzyme GilR was fully characterized. Through in vitro studies, GilR was shown to catalyze the dehydrogenation of hemiacetal moiety of the penultimate intermediate pregilvocarcin V to the lactone moiety of GV at the last step.

In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.

While considerable progress has been made towards understanding the effect that membrane-based layer-by-layer (LbL) immobilizations have on the activity and stability of enzymatic catalysis, detailed work is required in order to fundamentally quantify and optimize the functionalization and operating conditions that define these properties. This work aims to probe deeper into the nature of transport mechanisms by use of pressure-induced, flow-driven enzymatic catalysis of LbL-functionalized hydrophilized poly(vinyldiene) (PVDF)-poly(acrylic acid) (PAA)-poly(allylamine hydrochloride) (PAH)-glucose oxidase (GOx) membranes. These membranes were coupled in a sealed series following cellulose acetate (CA) membranes for the elimination of product accumulation within the feed-side solution during operation. At pH = 6 and T = 21oC, the enzymatic catalysis of LbL-immobilized GOx from Aspergillus niger performed remarkably well in comparison to the homogeneous-phase catalysis within an analogous aqueous solution. On average, the enzymatic turnover was 0.0123 and 0.0076 mmol/(mg-GOx)(min) for the homogeneous-phase catalysis and the LbL-immobilized catalysis, respectively. Multiple consecutive permeations resulted in replicable observed kinetic results with R2 > 0.95. Permeations taking place over the course of a three week trial period resulted in a retention of >90% normalized activity when membranes were removed when not in use and stored at -20oC, whereas the homogenous-phase kinetics dropped below 90% normalized activity in under one day.

Increased amounts of pesticide production and application of pesticides for agriculture, plant protection and animal health has resulted in soil, water and air pollution, consequently relating a serious risk to the environment and also to human health. Pesticides include several groups of compounds, herbicides, insecticides, rodenticides and fumigants consisting of several hundred individual chemicals. Herbicides are an integral pan of modem agriculture and for industries requiring total vegetation control. Most herbicides are soil applied and more and more concern is raised that herbicides not only affect target organisms but also the microbial community present in soil. The ESKOM sub-station Zeus, in Mpumalanga (South Africa) used to apply an industrial weed control program for the eradication of vegetation, which led to the contamination of soil by several herbicides. These herbicides consisted of Bromacil, Tebuthiuron and Ethidimuron which are all photosynthesis inhibitors, more specifically, they disrupt the plastoquinone protein during electron transport at photosystem I1 (PSII). In this study the effect of biostimulation and bio-augmentation of a specific bioremediation agent (B350) as prescribed by ESKOM, on residual herbicides, Bromacil, Tebuthiuron and Ethidimuron was evaluated by monitoring the soil physical and chemical properties, microbial attributes, including potential microbial activity and community structure, as well as the physiological effect experienced by plants (Cynodoh dactylon and Zea mays). Results from soil physical and chemical analyses were correlated with results obtained for the functional and structural diversity of microbial communities. All results were investigated through statistical and multivariate analysis and the most prominent soil physical and chemical parameters that influence the biological and biochemical properties of the soil were identified. Results obtained from this study indicated that there were no significant difference (p < 0.05) between the treatments, with bioremediation agent, irradiated agent and without the agent based on results obtained from soil microbial properties and plant physiology. Before the trial started the uncontaminated soil showed an active microbial function, characterised by dehydrogenase, urease and arylsulphatase activity, but community structure was not very diverse. The contaminated soil, irradiated contaminated soil and silica sand showed less enzymatic function and was characterised by phospholipid fatty acid groups, mid-branched saturated fatty acids, terminally branched saturated fatty acids, normal saturated fatty acids and monosaturated fatty acids which are indicative of microorganisms that survive better in harsh environments. Three weeks after the addition of the specific bioremediation took place, the uncontaminated soil showed an increase in P-glucosidase activity and percentage organic carbon (%C), which could be a result of the presence of available plant material. Furthermore, an increase in major PLFA groups were seen, suggesting that an increase in diversity within the soil community occurred. The contaminated soil, irradiated contaminated soil and silica sand once again was characterised by a low microbial function and diversity, showing no improvement. Fluorescence data clearly show a decline in PS 11 function that result in the decline of the rate of photosynthesis, which was seen from COz gas exchange rates. Furthermore, the decrease in photosynthetic activity after three weeks was too severe to supply additional information about the mechanism within photosynthesis or the photoprotective mechanisms. A detailed study was conducted in which a 3: 1 dilution of contaminated soil with silica sand, was also monitored for changes within plant physiology. Results revealed that inhibition of PS I1 function already takes place within a few days time and the decline in photosynthesis is as a result of electron transport that does not supply adenosine triphosphate (ATP) and P-nicotinamide adenine dinucleotide (NADPH) to the Calvin cycle (or Reductive Pentose Phosphate pathway). It does not appear that rubulose-1,sbisphosphate carboxylase-oxygenase (Rubisco) is affected within the Calvin cycle. As a result of PS I1 function failure, reaction centres are damaged by the production of harmful singlet oxygen and photoprotective mechanisms (xanthophyll cycle) can not be activated. Thus, except for dealing with ineffective electron transport, additional damage is caused to physiological functions. After six weeks a decrease in the estimated viable biomass for all growth mediums was found. Results of the of trans- to cis- monoenoic fatty acids and cyclopropyl fatty acids to their monoenoic precursors ratios indicated that the soil microbial community for the contaminated growth mediums, all experienced nutritional stress throughout this trail. The specific bioremediation agent (B350) used, seemed to have no effect on the microbial function and community structure within soil and as agent had no effect on the residual herbicides or the plant physiology which experienced an extreme decline in major metabolic functions. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2

Mining activities cause severe disturbance to the soil environment in terms of soil quality and productivity and are of serious concern worldwide. Under South African legislation, developers are required to ecologically rehabilitate damaged environments. The application of agronomic approaches for the rehabilitation of coal discard sites has failed dismally in the arid areas of southern Africa. It is obvious that compliance with mitigation and rehabilitation requirements cannot be enforced without a thorough understanding of the ecological principles that ensure ecological stability and subsequent sustainability of soil ecosystems. Soil micro organisms are crucial role-players in the processes that make energy and nutrients available for recycling in the soil ecosystem. Poor management practices and other negative impacts on soil ecosystems affect both the physical and chemical properties of soil, as well as the functional and structural properties of soil microbial communities. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, plant reestablishment and long-term stability. In this regard, an extensive overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, enzymatic activity and the analysis of microbial community structure by the quantification of specific signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between the physical and chemical characteristics and different biological indicators of soil quality in the topsoil covers of seven coal discard sites under rehabilitation in South Africa, as well as three reference sites was investigated. Through the assimilation of basic quantitative data and the assessment of certain physical, chemical and biological properties of the topsoil covers obtained from the various coal discard sites as well as the reference sites, the relative success or progress of rehabilitation and the possible correlation between the biological indicators of soil quality and the establishment of self sustaining vegetation covers was determined. Results from soil physical and chemical analyses and percentage vegetation cover were correlated with the results obtained for the functional and structural diversity of microbial communities at the various sites. All results were investigated through statistical and multivariate analysis and the most prominent physical and chemical parameters that influence the biological and biochemical properties of the soil and possibly the establishment of self-sustainable vegetation cover on these mine-tailing sites were identified. Results obtained from this study indicated no significant difference (p>0.05) between the various discard sites based on conventional microbiological enumeration techniques. However, significant differences (p<0.05) could be observed between the three reference sites. All enzymatic activities assayed for the rehabilitation sites, with the exception of urease and alkaline phosphatase displayed a strong, positive association with the organic carbon content (%C). Ammonium concentration had a weak association with all the enzymes studied and pH only showed a negative association with acid phosphatase activity. A positive association was observed between the viable microbial biomass, vegetation cover and the organic carbon content, ammonium, nitrate and phosphorus concentrations of the soil. The various rehabilitation and reference sites could be differentiated based on the microbial community structure as determined by phospholipid fatty acid (PLFA) analysis. It is hypothesised that the microbial community structure of the Hendrina site is not sustainable when classified along an r-K gradient and that the high percentage of vegetation cover and high levels of estimated viable microbial biomass are an artificial reflection of the current management practices being employed at this site. Results obtained during this study, suggest that an absence or low percentage of vegetation cover and associated lower organic matter content of the soil have a significant negative impact on soil biochemical properties (enzymatic activity) as well as microbial population size. Furthermore, prevailing environmental physico-chemical and management characteristics significantly influences the vegetation cover and subsequently the microbial community structure. The results indicate that the microbial ecosystems in the coal discard sites could become more stable and ecologically self-regulating, provided effective management to enhance the organic carbon content of the soil. This could enhance nutrient cycling, resulting in changes of soil structure and eventually an improved soil quality which could facilitate the establishment of self sustaining vegetation cover. Results obtained during this study suggest that a polyphasic assessment of physical and chemical properties; microbial activities by enzymatic analysis; the characterisation of microbial community structure by analysis of phospholipid fatty acids; and the multifactorial analysis of the data obtained can be used as complementary assessment criteria for the evaluation of the trend of rehabilitation of mine tailings and discard sites. Strategic management criteria are recommended based on the soil quality environmental sustainability indices to facilitate the establishment of self sustainable vegetation covers. The contribution of this research to soil ecology is significant with regards to the intensive investigation and explanation of characteristics and processes that drive ecological rehabilitation and determine the quality of the soil environment. The multidisciplinary approach that is proposed could, furthermore, assist in the successful rehabilitation and establishment of self-sustaining vegetation covers at industrially disturbed areas, as well as assist in improving degraded soil quality associated with both intensive and informal agriculture. Additionally, this approach could negate the negative social and environmental impacts frequently associated with these activities. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2004.

The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

The scalable syntheses of functional, porous nanostructures with tunable three-dimensional morphologies is a significant challenge with potential applications in chemical, electrical, electrochemical, optical, photochemical, and biochemical devices. As a result, several bio-enabled and synthetic approaches are explored in this work (with an emphasis on peptide-enabled deposition) for the generation of aligned nanotubes of nanostructured titania for application as electrodes in dye-sensitized solar cells and biofuel cells. As part of this work, peptide-enabled deposition was used to deposit conformal titania coatings onto porous anodic alumina templates under ambient conditions and near-neutral pH to generate aligned, porous-wall titania nanotube arrays that can be integrated into dye-sensitized solar cells where the arrays displayed improved functional dye loading compared to sol-gel-derived nanotubes. A detailed comparison between synthetic and bioorganic polyamines with respect to titania film properties deposition rate provided valuable information for future titania coating experimental design given specific applications. The development of template-based approaches to single-wall titania nanotube arrays led to the development of a new synthetic method to create aligned, multi-walled titania nanotube arrays. Lastly, peptide-enabled deposition methods were extended beyond inorganic mineral and used for enzyme immobilization by cross-linking the peptide with the multicopper oxidase laccase. Peptide-laccase hybrid enzyme coatings improved both the amount of enzyme adsorbed onto carbon nanotube “buckypaper” and allowed the enzyme to retain more activity upon immobilization onto the surface.

Bioethanol is an appealing alternative to petroleum-based liquid fuel due to drivers such as environmental regulations and government mandates. Second generation lignocellulosic feedstocks are abundant, but their resistance to hydrolysis continues to be problematic. Different pretreatments have been proposed to increase cellulose reactivity. Softwood pine autohydrolyzed at different severities was subjected to further treatment to increase fibre reactivity. Liquid hot water is most effective at removing barriers, with the highest increase in sugar yield after enzymatic hydrolysis. Alkaline (NaOH) is found to be the worst option compared to dilute acid and organosolv. In addition, higher chemical concentrations and longer treatment times do not guarantee higher enzymatic hydrolysis yield. Process modifications such as fiber washing and multistage enzymatic hydrolysis are observed to be effective at increasing yield. However, more research is required to bring the enzymatic hydrolysis yield to a level where commercialization is feasible.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.