Thesis (DTech(Biomedical Technology ))-- Cape Peninsula University of Technology, 2012 / Antioxidants are an important class of compounds that quench reactive free radical intermediates formed during oxidative reactions. They prevent oxidative reactions in food and protect biological tissues against oxidative damage. Plant phenols and phenolic acids are increasingly becoming a subject of intensive research due to their bioactive properties such as antioxidant, anti-mutagenic, anti-viral and anti-inflammatory activity. Modification of the structure of natural phenolic compounds can be achieved through the use of enzymes in biocatalysis reactions with the potential to enhance the antioxidant capacity of these natural phenolic compounds. The work reported here employed the oxidative enzyme, laccase from Trametes pubescens, in the modification of the antioxidant phenolic molecules, ferulic acid and 2,6-dimethoxyphenol (2,6-DMP) as a way of enhancing their antioxidant capacity. In addition, various phenolic compounds were focused upon for coupling reactions, with the aim to increase the antioxidant capacity of the compounds. The T. pubescens strain was cultured in a 4L airlift reactor and extracellular laccase production was monitored using the standard ABTS assay. The enzyme was isolated and purified from the culture filtrate once optimal enzyme production was detected. The enzyme was purified using standard ammonium sulphate precipitation and dialysis.

Yes / We report on the use of non-equillibrium biocatalytic self-assembly and gelation to guide the reductive synthesis of gold nanoparticles. We show that biocatalytic rates simultaneously dictate supramolecular order and presentation of reductive phenols which in turn results in size control of nanoparticles that are formed. / BBSRC funding (BB/K007513/1); European Research Council under the European Union’s Seventh Framework Programme, ERC (Starting Grant EMERgE) grant agreement no. 258775.

No description available.

A novel rapid method for the determination of biochemical oxygen demand (BOD) has been developed. By replacing oxygen, the terminal electron acceptor in the microbial oxidation of organic substrate, with the ferricyanide ion, a significant increase in the rate of the biochemical reaction could be achieved. This arises from the high solubility of the ferricyanide ion (compared to oxygen); therefore allowing for elevated microbial populations without rapid depletion of the electron acceptor. Therefore, the BOD of a sample can be determined within 1-3 hours compared to 5-days with the standard BOD5 assay. A range of microorganisms were shown to be able to use the ferricyanide ion as an alternative electron acceptor for the biodegradation of a range of organic compounds in the ferricyanide mediated BOD (FM-BOD) assay. The most suitable biocatalyst in the FM-BOD method, however, was shown to be a mixture of microorganisms that was capable of degrading large amounts and types of compounds. These mixed consortia of microorganisms included a synthetic mixture prepared in our laboratory and two commercially available consortia, BODseedTM and Bi-ChemTM. When these seed materials were employed in the FM-BOD assay, the method was shown to closely estimate the BOD5 values of real wastewater samples. The linear dynamic working range of the FM-BOD method was also greatly extended compared to the standard BOD5 assay (nearly 50 times greater) and other oxygen based BOD biosensors. The immobilisation of the microbial consortia by both gel entrapment and freeze-drying methods was shown to greatly reduce the preparation and handling time of the mixed consortia for use in the FM-BOD method. Immobilisation of the mixed microbial consortium in LentiKats®, a PVA hydrogel, resulted in a marked increase in the stability of the biocatalyst. Diffusion limitations resulting from the gel matrix, however, reduced the rate and extent of the bioreaction as well as the linear dynamic working range of the method. Freeze-drying techniques were shown to circumvent some of the limitations identified with gel entrapment for the immobilisation of the mixed consortia. The freeze-dried consortia could be used off-the-shelf and demonstrated reduced diffusional restrictions. A marked decrease in the viability of the microorganisms was observed directly following the freeze-drying process and in subsequent storage. Carrageenan, however, was shown to afford a significant degree a protection to the cells during the freeze-drying process.

The limited availability of fossil resourses mandates the development of new energy vectors, which is one of the Grand Challenges of the 21st Century [1]. Biocatalytic energy conversion is a promising solution to meet the increased energy demand of industrialized societies. Applications of biocatalysis in the gas-phase are so far limited to production of fine chemicals and pharmaceuticals. However, this technology has the potential for large scale biocatalytic applications [2], e.g. for the formation of novel energy carriers. The so-called solid/gas biocatalysis is defined as the application of a biocatalyst immobilized on solid-phase support acting on gaseous substrates [3]. This process combines the advantages of bio-catalysis (green chemistry, mild reaction conditions, high specicity & selectivity) and heterogeneous dynamic gas-phase processes (low diffusion limitation, high conversion, simple scale-up). This work presents the modifications of a PID Microactivity Reference reactor in order to make it suitable for solid/gas biocatalysis. The reactor design requirements are based on previously published laboratory scale solid/gas systems with a feed of saturated vapors [4]. These vapors are produced in saturation flasks, which were designed and optimized during this project. Other modifications included relocation of the gas mixing chamber, redesigning the location and heating mechanism for the reactor tube, and heating of the outlet gas line. The modified reactor system was verified based on the Candida antarctica lipase B catalyzed transesterication of ethyl acetate with 1-hexanol to hexyl acetate and ethanol and results were compared to liquid-phase model reactions. Products were analyzed on line by a gas chromatograph with a flame ionization detector. C. antarc- tica physisorbed on silica particles produced a 50% conversion of hexanol at 40 C in the gas-phase. A commercial immobilized lipase from Iris Biotech produced 99% and 97% conversions of hexanol in similar experiments. This project achieved its goal to design, establish and successfully verify a solid/- gas biocatalysis reactor. Future work will target optimization of the reactor's operating conditions and the development of whole cell catalysts for energy production reactions. Potential experiments include the study of hydrogenolytic carbon dioxide reduction to methanol by free enzymes or methanogenic organisms [5], and the investigation of hydrogen production by water splitting of algae or cyanobacteria.

Chiral amine motifs are found in many bioactive compounds and therefore strategies for their direct asymmetric synthesis are of great interest. Alongside traditional chemical methods, biocatalysis serves as an important tool for the formation of these compounds that can confer the benefits of sustainable catalyst supply and mild reaction conditions. This thesis describes the application of imine reductase (IRED) biocatalysts for the asymmetric reduction of pre-formed imines and the reductive amination of carbonyl compounds to produce chiral amines. These enzymes are relatively recent additions to the toolbox of biocatalysts for chiral amine synthesis and therefore their scope and application is still very much being explored. The research carried out as part of this PhD is presented as a series of manuscripts that have either been published or are planned for submission to peer-reviewed journals. The choice of presenting this thesis in journal format was made because a considerable body of the candidate's PhD research has been published, with the rest planned for publication in the near future. Furthermore, the compiled review articles and research papers lend themselves to a clear thesis narrative and, combined, have taken considerable time and effort to prepare, equal to that of a traditional thesis format. The contents are organised as follows: Chapter 1: an introduction to biocatalysis and its impact on sustainable chemical manufacturing; Chapter 2: a review assessing the current state of the art in imine reductase biocatalysts; Chapter 3: a perspective on the design and implementation of biocatalytic cascades; Chapter 4: a research article on the application of IREDs in a biocatalytic cascade for the synthesis of chiral piperidine and pyrrolidine frameworks; Chapter 5: aims of the PhD project; Chapter 6: a research article on the discovery and investigation of a reductive aminase (RedAm) found within the IRED family; Chapter 7: a research article on the screening of a diverse set of novel IREDs for their ability to facilitate reductive amination; Chapter 8: a research article on the synthesis of complex bulky dibenz[c,e]azepine compounds using IRED and transaminase biocatalysts; Chapter 9: a summary and outlook; Chapter 10: manuscript supporting information further detailing experimental work; Appendix: list of other publications resulting from this doctoral research.

This dissertation describes efforts to improve the oxidative capacity of n-alkane- induced Beauveria bassiana; a fungus and a versatile whole cell biocatalyst used in the biotransformation of steroids. n-Hexadecane was used as the carbon source during the growth of B. bassiana, presumably to induce the expression of oxidative enzymes, thus enhancing the oxidation of unactivated carbons. Dehydroepiandrosterone (DHEA) is an essential endogenous male-hormone and serves as a metabolic intermediate in the production of more potent androgens. Using DHEA as a substrate also provides the opportunity to study the hydroxylation of an unfunctionalized carbon, an attractive reaction that produces valuable intermediates for chemical synthesis. Results showed that exposing and inducing cells in n-hexadecane improves the synthesis of 11α-hydroxy derivatives. Reactions were carried out with cells grown on n-hexadecane, resulting in 65 ± 6.3 % conversion of DHEA to androstenediol (40.3% mM) and 3β,11∝,17β- trihydroxyandrost-5-ene (22.8% mM), as determined by HPLC, NMR and LCMS analyses. However, experiments with non-induced cells resulted in a poor substrate conversion (17%). To extend use of B. bassiana to pharmaceutical applications, it was necessary to optimize reaction conditions such as biocatalyst preparation, substrate concentration, agitation reaction temperature and pH. Higher substrate conversion, selectivity and yield of desired product were achieved with the reactor arrangement of “Resting Cells”. The apparent rate of reaction fits a Michaelis-Menten kinetic model with a maximum reaction rate of 4.45 mM/day, revealing that the transformation of intermediate androstenediol to desired 3β,11∝,17β-trihydroxyandrost-5-ene is the limiting step in the reaction. Interestingly, when a diluted amount of substrate was used, a higher yield of 11∞-hydroxy steroid was achieved. Also, reactions at 26°C with pH ranges between 6.0 and 7.0, resulted in the highest conversion (70%) and the higher product yield (45.8%). The maximum conversion of DHEA (71%) was achieved in experiments with high biomass loading, and the increment of desired product yield (11∝-hydroxy) was directly proportional to the amount of biomass used. Moreover, a high VMax/KM value was achieved with high biomass yields. Interestingly, the changes in biomass yield did not have a considerable effect on reaction selectivity. The main drawbacks of biocatalysis for production of steroids were addressed and approaches to minimize the drawbacks have been presented. The production of desired product (11∝-DHEA) was significantly improved using cells previously adapted to n-hexadecane.

An indigenous mixed culture of bacteria collected from a Wastewater Treatment Plant (Brits, North West Province, South Africa), biocatalytically reduced Cr(VI) in the presence of As(III). Both the reduced chromium (Cr(III)) and the oxidised arsenic (As(V)) readily form amorphous hydroxides that can be easily separated or precipitated from the aqueous phase as part of the treatment process. Treatment of Cr(VI) and As(III) before disposal of wastewater is critical since both compounds are known to be carcinogenic and mutagenic at very low concentrations, and acutely toxic at high concentrations. Batch experiments were conducted to evaluate the rate of Cr(VI) reduction under anaerobic condition in the presence of its co-contaminant As(III) typically found in the groundwater and mining effluent. Results showed near complete Cr(VI) reduction under initial Cr(VI) concentrations up to 70 mg/L in a batch amended with 20 mg/L As(III). However, increasing Cr(VI) concentrations up to 100 mg/L resulted in the inhibition of Cr(VI) reduction activity. Further investigation was conducted in a batch reactor amended with 70 mg/L Cr(VI) concentration at different As(III) concentrations ranging from 5-70 mg/L to evaluate the effect of varying As(III) concentration on Cr(VI) reduction efficiency. Results showed that Cr(VI) reduction efficiency increased as As(III) concentrations increased from 5-40 mg/L. However, further increase in As(III) concentration up to 50 mg/L resulted in incomplete Cr(VI) reduction and decrease in Cr(VI) reduction efficiency. These results suggest that the rate of Cr(VI) reduction depends on the redox reaction of As(III) and As(V) with Cr(VI). Moreover, the inhibitory effect observed at high Cr(VI) and As(III) concentration may also be attributed to the dual toxicity effect of Cr(VI) and As(III) on microbial cell. From the above batch kinetic studies lethal concentration of Cr(VI) and As(III) for these strains was evaluated and established. Initial evaluation of the bacteria using 16S rRNA partial sequence method showed that cells in the mixed culture comprised predominantly of the Gram-positive species: Staphylococcus sp., Enterobacter sp., and Bacillus sp. The biokinetic parameters of these strains were estimated using a non-competitive inhibition model with a computer programme for simulation of the Aquatic System “AQUASIM 2.0”. Microbial reduction of Cr(VI) in the presence of As(III) was further investigated in continuous-flow bioreactors (biofilm reactor) under varying Cr(VI) loading rates. The reactor achieved Cr(VI) removal efficiency of more than 96 % in the first three phases of continuous operation at lower Cr(VI) concentration ranging from 20-50 mg/L. However, 20 % decrease in Cr(VI) removal efficiency was observed as Cr(VI) concentration increase up to 100 mg/L. The reactor was able to recover from Cr(VI) and As(III) overloading phase after establishing the resilient nature of the microorganism. Similarly to the batch reactor studies the overall performance of the reactor also demonstrated that the presence of As(III) greatly enhance Cr(VI) reduction in a bioreactor. This was evident by near complete removal of Cr(VI) concentration up to 50 mg/L. The basic mass balance expressions on Cr(VI) along with the non-competitive inhibition model were used to estimate the biokinetic parameters in the continuous flow bioreactor system. Cr(VI) reduction efficiency along the longitudinal column was also evaluated in this study. Results showed that Cr(VI) efficiency increased as Cr(VI) concentration travels along the longitudinal column. Other important factors such as oxygen and pH during biological Cr(VI) reduction in the presence of As(III) oxidation were also evaluated. / Dissertation (MEng)--University of Pretoria, 2014. / tm2015 / Chemical Engineering / MEng / Unrestricted

Biocatalytic membranes (BMs) have promising applications in a diversity of fields including food, pharmaceutical and water treatment industries. Of particular relevance, Alcalase is a commercially important protease that has been applied for the production of peptides from the hydrolysis of proteins. In this study, two different approaches were applied for the modification of electrospun polyacrylonitrile nanofibrous membranes (EPNMs) for Alcalase immobilization. The first approach is alkali modification of EPNMs followed by EDC/NHS coupling for covalent bonding with Alcalase, whereas the other is based on polydopamine coating with or without glutaraldehyde grafting as a covalent linker. Immobilized Alcalase on these prepared BMs were studied and compared with free enzymes. It was found that the stabilities of Alcalase on BMs created using both approaches were improved, which enabled their reuse of 10 cycles with significant retention of enzymatic activity. A continuous reactor housing BMs were tested for hydrolysis of both model substrate, azo-casein and soybean meal protein (SMP). It was found that decreasing flux could improve the extent of hydrolysis and that a single-layer reactor can hydrolyze about 50% of the substrate to peptides with the molecular weight of 10 kDa or less. Hydrolysis of SMPs was demonstrated in a continuous five-layer BM reactor and both BMs showed excellent hydrolysis capacity. This study provides the groundwork for the development of high-efficiency BM for continuous and cost-effective protein hydrolysis for the production of value-added peptides.

Analytische Chemie heute meint nicht länger nur die große Messtechnik, die zeit- und kostenintensiv ist, die außerdem nur von qualifiziertem Personal zu bedienen ist und deren Resultate nur durch dieses Personal auswertbar sind. Meist erfordert diese sagen wir 'klassische analytische Messtechnik' auch noch spezielle Räumlichkeiten und oft eine relative große Menge an speziell vorbereiteten Proben. Neben dieser klassischen analytischen Messtechnik hat sich besonders in den letzten Jahren eine auf bestimmte Stoffgruppen und Anforderungen zugeschnittene Messtechnik durchgesetzt, die oft auch durch einen Laien bedient werden kann. Meist sind es sehr kleine Geräte. Auch die benötigten Probenvolumina sind klein und eine spezielle Probenvorbereitung ist nicht erforderlich. Ausserdem sind die Geräte einfach zu handhaben, billig sowohl in ihrer Herstellung als auch im Gebrauch und meist erlauben sie sogar eine kontinuierliche Messwerterfassung. <br /> <br /> Zahlreiche dieser in den letzten Jahren entwickelten Geräte greifen zurück auf 40 Jahre Forschung auf dem Gebiet der Biosensorik. Seit Clark und Lyons im Jahr 1962 in der Lage waren, mit einer einfachen Sauerstoffelektrode, ergänzt durch ein Enzym, Glucose zu messen, war die Entwicklung neuer Messtechnik nicht mehr aufzuhalten. Biosensoren, spezielle Messfühler, die aus einer Kombination aus biologischer Komponente (erlaubt eine spezifische Erkennung des Analyten auch ohne vorherige Reinigung der Probe) und einem physikalischen Messfühler (wandelt den primären physikochemischen Effekt in ein elektronisch messbares Signal um) bestehen, eroberten den Markt. <br /> <br /> Im Rahmen dieser Doktorarbeit wurden verschiedene Tyrosinasesensoren entwickelt, die je nach Herkunft und Eigenschaften der verwendeten Tyrosinase unterschiedliche Anforderungen erfüllen. Beispielsweise wurde einer dieser Tyrosinasesensoren für die Bestimmung phenolischer Verbindungen in Fluss- und Seewasserproben eingesetzt, und die mit diesem Sensor gemessenen Ergebnisse konnten sehr gut mit dem entsprechenden DIN-Test zur Bestimmung phenolischer Verbindungen korreliert werden. Ein anderer entwickelter Sensor zeigte eine sehr hohe Empfindlichkeit für Catecholamine, Substanzen die speziell in der medizinischen Diagnostik von Wichtigkeit sind. <br /> <br /> Ausserdem zeigten die ebenfalls im Rahmen dieser Doktorarbeit durchgeführten Untersuchungen zweier verschiedener Tyrosinasen, dass, will man in Zukunft noch empfindlichere Tyrosinasesensoren entwickeln, eine spezielle Tyrosinase (Tyrosinase aus Streptomyces antibioticus) die bessere Wahl sein wird, als die bisher im Bereich der Biosensorforschung verwendete Tyrosinase aus Agaricus bisporus. <br /> <br /> Desweiteren wurden erste Erfolge auf molekularbiologischem Gebiet erreicht, das heisst, dass Tyrosinasemutanten mit speziellen, vorher überlegten Eigenschaften, hergestellt werden sollen. Diese Erfolge können dazu genutzt werden, eine neue Generation an Tyrosinasesensoren zu entwickeln, Tyrosinasesensoren in denen Tyrosinase gerichtet gebunden werden kann, sowohl an den entsprechenden physikalischen Messfühler oder auch an ein anderes Enzym. Davon verspricht man sich deutlich minimierte Wege, die die zu bestimmende Substanz (oder deren Produkt) sonst zurücklegen müsste, was am Ende zu einer deutlich erhöhten Empfindlichkeit des resultierenden Biosensors führen sollte. / Today, analytical chemistry does not longer consist of only the big measuring devices and methods which are time consuming and expensive, which can furthermore only be handled by the qualified staff and in addition the results can also only be evaluated by this qualified staff. Usually, this technique, which shall be described in the following as 'classic analytic measuring technique', requires also rooms equipped especially and often a relative big quantity of the test compounds which should be prepared especially. Beside this classic analytic measuring technique, limited on definite substance groups and requests, a new measuring technique has gained acceptance particularly within the last years, which one can often be used by a layman, too. Often the new measuring technique has very little pieces of equipment. The needed sample volumes are also small and a special sample preparation isn't required. In addition, the new measuring instruments are simple to handle. They are cheap both in their production and in the use and they permit even a continuous measurement recording usually. <br /> <br /> Numerous of this new measuring instruments base on the research in the field of Biosensorik during the last 40 years. Since Clark and Lyon in the year 1962 were able to measure glucose with a simple oxygen electrode, completed by an enzyme the development of the new measuring technique did not have to be held back any longer. Biosensors, special pickups which consists of a combination from a biological component (permits a specific recognition of the analyte also without purification of the sample previously) and a physical pickup (convert the primary physicochemical effect into an electronically measurable signal), conquered the market. <br /> <br /> In the context of this thesis different tyrosinasesensors were developed which fulfilling the various requests, depending on origin and features of the used tyrosinase. One of the tyrosinasesensors for example was used for quantification of phenolic compounds in river and sea water and the results could correlated very well with the corresponding DIN-test for the determination of phenolic compounds. An other developed tyrosinasesensor showed a very high sensitiveness for catecholamines, substances which are of special importance in the medical diagnostics. <br /> <br /> In addition, the investigations of two different tyrosinases, which were carried out also in the context of this thesis, have shown, that a special tyrosinase (tyrosinase from Streptomyces antibioticus) will be the better choice as tyrosinase from Agaricus bisporus, which is used in the area of biosensor research till now, if one wants to develop in future even more sensitive tyrosinasesensors. <br /> <br /> Furthermore, first successes became reached on a molecular biological field, the production of tyrosinasemutants with special, before well-considered features. These successes can be used to develop a new generation of tyrosinasesensors, tyrosinasesensors in which tyrosinase can be bound directionally both to the corresponding physical pickup or also to another enzyme. From this one expects to achieve ways minimized which the substance to be determined (or whose product) otherwise must cover. Finally, this should result in an clearly visible increase of sensitivity of the Biosensor.