Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.

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Scytalidium thermophilum is a termophilic fungus that effectively produces the extracellular enzyme catalase phenol oxidase (CATPO). The enzyme is distinct among catalases with its bifunctionality of oxidising phenolic compounds in the absence of H2O2. CATPO is capable of oxidizing catechol, chlorogenic acid, caffeic acid and catechin which are ortho &ndash / diphenolic compounds. Diphenolic compounds are known as strong antioxidants. Catalase is one of the important antioxidant enzymes. Therefore, in this thesis the effect of CATPO on the final antioxidant capacity of the oxidized products was analysed. Antioxidant capacity measurements of oxidized and unreacted phenolic compounds were done using the two widely used methods TEAC and FRAP. CATPO oxidized catechol showed 2.4 fold increase when compared to its nonoxidized form, which was highest among others. Catechol was followed by caffeic acid, chlorogenic acid, and catechin. This finding is new to the literature and may be of importance to the antioxidant mechanism of organisms. Results have also shown that the most well known phenol oxidases, laccase and tyrosinase, do not result in such high increases in antioxidant capacity upon oxidation of the substrates tested. Due to this finding, as a possible means of applying CATPO to increase the antioxidant capacity of products daily consumed, tea was selected. Tea is the second most consumed beverage after water and it is known to possess high amounts of flavanols. Green tea is rich in catechins whereas black tea is a rich source of theaflavins and thearubigins. Fermentation is a critical process for production of good quality tea and is the key step differing between green and black tea production. During this process phenol oxidases catalyze the oxidation of polyphenolic compounds present in tea leaves to their corresponding o-quinones. Utilization of CATPO in tea samples resulted in an increase in antioxidant capacity and its effect was enhanced by an increase in brewing time. Interestingly, the addition of sugar decreased antioxidant capacity. Laccase and tyrosinase were ineffective in increasing the antioxidant capacity of tea samples.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.

The main object of the presented study was investigation of the use of cross-linked enzyme (tyrosinase) aggregates (CLTA) obtained from crude mushroom extract for a rapid phenolic content analysis in wines. In addition, a comparison of phenolic characteristics of Turkish red wines was performed. Reproducible and reliable results in total phenolic measurement were obtained with CLTAs similar to pure tyrosinase and tyrosinase obtained from crude mushroom extract. Measurement of total phenolic content is possible both in standard solutions and in complex matrices, such as wine. In a very short time period, 10 seconds, phenolics content in red and white wines produced from grapes of Turkey were investigated by using CLTAs. Results were consistent when compared to a well known phenolic measurement method, Folin-Ciocalteau. CLTAs exhibited very high operational stability and retained more than 90% of its activity after 30th use. Moreover, it showed good shelf-life stability for about 2 months storage by maintaining 90% of its maximum activity. So, use of CLTAs prepared from crude mushroom extract is an effective, fast and cheap alternative in total phenolics measurements in wines. Moreover, a novel catalase phenoloxidase (CATPO) produced by a fungal microorganism, Scytalidium thermophilum, was studied to check its capabilities in phenolics measurements. This novel catalase phenol oxidase showed similarly good results, exhibiting widesubstrate selectivity.

Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

The work presented in this thesis describes the occurrence and properties of two multicopper oxidases derived from lichens. Despite numerous data on laccases and tyrosinases in fungi and flowering plants, this is the first report of the occurrence of these enzymes in lichenized ascomycetes. Extracellular laccase and tyrosinase activity was measured in 50 species of lichens from different taxonomic groupings and contrasting habitats. Out of 27 species tested from suborder Peltigerineae, all displayed laccase and tyrosinase activity that correlated to each other, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases and tyrosinases was confirmed by the ability of lichen thalli or leachates to readily metabolize substrates such as 2,2’-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in case of laccase and L-dihydroxyphenylalanine (L-DOPA), Ltyrosine and epinephrine in case of tyrosinase in the absence of hydrogen peroxide. The activities of both enzymes were highly sensitive to cyanide and azide, and tyrosinase activity was sensitive to hexylresorcinol. Laccase activity had typical pH and temperature optima and an absorption spectrum with a peak at 614 nm. Tyrosinases could be activated by sodium dodecyl sulphate (SDS) and had typical tyrosinase molecular masses of approx. 60 kDa. The diversity of laccase isoforms in 20 lichen species from suborder Peltigerineae was investigated. The molecular masses of the active forms of most laccases varied between 135 and 190 kDa, although some lichens within the family Peltigeraceae had laccases with higher masses, typically varying from 200 to over 350 kDa. Most species contained one oligomeric laccase isoform. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. The ability of laccases to decolorize dye is a classic attribute of laccases, and one with biotechnological potential. The ability of eight lichen species to decolourize different types of dyes was therefore tested. Interestingly, results showed that not only species belonging to suborder Peltigerineae but also species from other lichen group effectively decolourised dyes after 48 h suggesting that other oxidases appear to have ability to decolorize. Hopefully, our work could contribute to the better knowledge and application of lichen multicopper oxidases. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

ABSTRACT UTILIZATION OF SCYTALIDIUM THERMOPHILUM PHENOL OXIDASE IN BIOORGANIC SYNTHESES Kaptan, Yelda M.S., Department of Biotechnology Supervisor: Prof. Dr. Z&uuml / mr&uuml / t B. &Ouml / gel Co-supervisor: Prof Dr. Ufuk Bakir September 2004, 90 pages In this study, the ultimate aim was to utilize phenol oxidases of Scytalidium thermophilum in bioorganic syntheses. For this purpose, studies were conducted towards enhancing the production of phenol oxidases by Scytalidium thermophilum, developing a suitable method for laccase activity assays, analyzing the effects of organic solvents on phenol oxidase activity and analysis of the biotransformation of a number of organic substrates by phenol oxidases of Scytalidium thermophilum. In order to enhance the production of phenol oxidases, induction experiments were carried out with gallic acid, syringaldazine and chlorogenic acid. Gallic acid was found as the most effective inducer for phenol oxidase production. Inductive effect of edible mushroom Agaricus bisporus was also assayed, however, the phenolic compounds released by mushroom did not represent any induction for phenol oxidase activity of Scytalidium thermophilum. Different substrates were tested and catechol was determined as the most suitable substrate rather than syringaldazine and ABTS. Molar extinction coefficient (e) of catechol was calculated as 3450 M-1 cm-1 and 3700 M-1 cm-1 by using &ldquo / substrate blank&rdquo / and &ldquo / enzyme blank&rdquo / respectively at 420 nm. Kinetic parameters, Km and Vmax for the enzymatic reactions in which catechol was used as substrate were calculated as 52.03 mM and 0.253 U/ml respectively from Lineweaver-Burk plot and as 41.25 mM and 0.2055 U/ml from Hanes-Woolf plot. Effect of some organic solvents on phenol oxidases of Scytalidium thermophilum was assayed and DMSO was found as an appropriate solvent for the organic substrates. Phenol oxidase containing culture supernatant could oxidize benzoin, hydrobenzoin and benzoyl benzoin.

In an interdisciplinary study requiring the synergistic association of historical evidence and chemical and biochemical analyses, this thesis investigates the properties and characteristics of historically modified soils known as anthrosols. These soils, developed through the anthropogenic addition of high volumes of organic-rich municipal waste materials to land, including human and animal waste, as part of the waste management practices in medieval urban communities in Scotland at St Andrews, Roxburgh and Elgin, offer an insight to the state and dynamics of these organic material. Soil is one of the most sensitive environmental domains to transformation. These transformations are visible from the alterations to the physical and chemical properties of soil. Anthropogenic activities may leave behind signatures in the soil in the form of artefacts, ecofacts, elemental enrichment or depletion, enhancement in soil magnetic properties and organic matter content. In the historical dimension of this study, the observable features and measurable properties of soil profiles are exploited to reveal past organisation and functions of cultural landscapes by carefully studying the stratigraphic units of soil profile, and examining the association of each unit with settlement artefacts and soil properties. Through comparison with historical records of past events on the respective study sites, the relationship between the soils record of past human activities is observed through physical, chemical and biochemical properties. The historical record is used to assess if such evidence can be used reliably to develop the account of site use for the medieval burghs of Scotland. In the environmental aspect, investigation focuses on the physical and chemical conditions of these soils in terms of their carbon content, composition, residence time estimates and their role in global C cycle and terrestrial carbon budgeting. Past investigations of anthopogenically-deepened soils have been interpreted with respect to historical site use, however, the environmental implications of the resultant accumulated organic material or residue have not previously been considered in much detail. A particular novelty of this aspect of the project is that it is an in-depth examination of anthropogenic soils with known histories extending into the medieval period. This time-depth allows a new understanding of the processes and products of decomposition of known organic materials that were added to soil. The biophysicochemical data obtained from these soils such as their extant organic carbon content and variability with depth, the composition of the various carbon species that together constitute soil organic matter, and biological community and activity (microorganisms and enzymes) provides critical information on the relative recalcitrance, state of decomposition, and the mechanism of stabilisation of these materials in the soil.

Introduction: Dark spots appearing on the skin caused by hyperpigmentation results from the action of tyrosinase, an enzyme whose activity leads to the production of the skin pigment melanin. Extracts of the plant Glycyrrhiza glabra, also known as liquorice, are commonly used to treat a range of conditions including skin hyperpigmentation. This study aimed at isolating and identifying compounds in extracts from South African liquorice root and assaying these compounds as to their antioxidant activity, their ability to inhibit the tyrosinase enzyme and their level of cytotoxicity. Methods: The ability of plant extracts to scavenge free radicals was tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS) and the ferric ion reducing power (FRAP) tests. The polyphenolic content of extract fractions was determined and extract compounds were identified using UHPLC-QToF-20 MS. In vitro anti-tyrosinase activity was also investigated as well as cytotoxicity in HepG2 liver and SK-MEL-1 melanoma cells using the MTT cell viability assay. Results: Of the four fractions prepared from the 70% methanolic extract of liquorice root, fraction 3 (F3) showed increased polyphenolic content and antioxidant properties with IC50 of 56.1 ± 6.32, 39.14 ± 1.1 and 66.34 ± 1.4 μg/ml against DPPH, ABTS and FRAP, respectively. The anti-tyrosinase activity of this fraction showed an IC50 of 358.54 μg/ml compared to Kojic acid (0.75 mM) used as the control. In addition, this fraction showed reduced liver toxicity as a higher percentage cell viability was noted in the HepG2 cells compared to the SK-MEL-1 skin melanoma cells. However, both cell types showed higher percentage viability compared to acetaminophen that was used as cytotoxic control. The LC-MS analysis revealed the presence of a wide variety of compounds including 4-azido-3-benzyl-coumarin, ferulic acid, glycyrrhizin, quercitrin, cirsilineol, gentioflavine and 4'',6,7-trihydroxyisoflavone. The literature indicates the use of these compounds regarding antioxidant and anti-tyrosinase activity. Significantly, cularidine was identified in this study, a compound not previously reported in studies involving liquorice root. Conclusion: The results from this study concur with previous reports as to the anti-tyrosinase and antioxidant activities associated with liquorice roots, activities perhaps due to the relatively high polyphenolic content in extracts from South African liquorice root. / Life and Consumer Sciences / M. Sc. (Life Sciences)