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Theses and dissertations (Applied Sciences)

Permanent URI for this collectionhttp://ir-dev.dut.ac.za/handle/10321/6

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Subject: search.filters.subject.Laccase

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    Laccase-mediated biotransformation of phenolic compounds for the synthesis of new antioxidants
    (2020) Mazibuko, Bodine; Kudanga, Tukayi
    The increased incidences, mortality rate and economic impact of noncommunicable diseases (e.g. high blood pressure and diabetes) associated with oxidative stress, have led to the higher demand for antioxidant supplements for their prevention. The use of naturally occurring antioxidants is becoming a more attractive option due to the health risks associated with synthetic antioxidants. Phenolic compounds from plants have been shown to have antioxidant properties with the potential to be used as substitutes to synthetic antioxidants. However, monomeric phenolic compounds have several short comings such as low bioavailability, poor solubility, and low antioxidant capacity while some have pro-oxidant properties at high concentrations. Hence there has been increasing research focused on the biotransformation of these phenolic antioxidants through enzymatic oligomerisation to higher molecular weight compounds with improved antioxidant capacity and stability. Of the investigated enzymes, laccases have shown the most promise owing to their green catalytic properties. Their reaction mechanism involves the use of molecular oxygen as a co- substrate in oxidising phenolic compounds to corresponding radicals, with water as the only by- product. This study focused on the synthesis of antioxidants with enhanced antioxidant capacity using a laccase from Trametes pubescens as biocatalyst. To establish the potential of the phenolic compounds for use as substrates for the coupling reactions, a preliminary screening process was done. Guaiacol, caffeic acid, vanillic acid, eugenol, catechol, gallic acid, ferulic acid and quercetin hydrate were identified as suitable substrates for the laccase enzyme. However, only products from eugenol, coumaric acid and quercetin could be isolated, hence coupling reactions were carried out using these substrates in monophasic systems. Reaction products were monitored using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purification was carried out using preparative TLC and characterisation using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The antioxidant capacities of reaction products were determined using ABTS (2,2’-Azinobis 3- ethylbenzthiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric- reducing antioxidant power) assays. Quercetin hydrate oxidation produced one product which was purified and characterised. The product had an Rf of 0.68, tR 13.567 and m/z 601 in negative mode, indicating that it was a dimeric form of quercetin. Oxidation of ρ-coumaric acid resulted in the production of two products designated P1 (Rf 0.47) and P2 (Rf 0.42). Further characterisation was done on product P2 since product P1 could not be successfully purified. P2 had a retention time of 11.295 and m/z 325, indicating that it was a dimer of ρ-coumaric. The ρ-coumaric dimer had an enhanced antioxidant capacity, approximately 2-fold, 3-fold and 6-fold higher compared to the substrate, as demonstrated by the ABTS, DPPH and FRAP assays, respectively. A symmetrical 5-5 eugenol dimer (m/z 325, [M] =326), bis-eugenol, was produced from eugenol oxidation. Maximum product formation (50% yield) was obtained in a monophasic system with 40% v/v dioxane as co-solvent after incubation for 18 h. The bis- eugenol dimer had an improved antioxidant capacity of up to three and four times that of eugenol as demonstrated by the ABTS and DPPH assays, respectively. In conclusion, two dimers with high antioxidant capacity were successfully produced, purified and characterised. The study has demonstrated the potential of the T. pubescens laccase as a catalyst for the synthesis of phenolic compounds with enhanced antioxidant capacity.
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    Small laccases as catalysts for the synthesis of antioxidants
    (2018) Nemadziva, Blessing; Kudanga, Tukayi; Le Roes-Hill, Marilize
    The rise in antioxidant demand for industrial applications has necessitated the need to investigate new methods for antioxidant production. Conventionally, antioxidants have been used in the food industry. However, newer applications in industries such as pharmaceuticals, cosmetics, medicine, nano-bioscience, as well as in chemical industries, have contributed to the increase in antioxidant demand. The market for antioxidants has been forecasted to increase by 6.42% compound annual growth rate (CAGR) between 2015 and 2022. Therefore, there is now a need to develop new processes for antioxidant synthesis to meet this rising demand. Biocatalysis has gained notable attention as a viable approach for antioxidant synthesis. Laccases are the preferred enzymes since their reaction mechanism involves the use of molecular oxygen to oxidise phenolic compounds to corresponding radicals, with water as the only by-product. Most laccase antioxidant synthesis research has employed fungal and plant laccases. However, bacterial laccases may be promising biocatalysts, considering the advances in molecular technology which make expression in bacterial hosts easier. This study focused on the biotransformation of natural phenolic compounds using small laccase (SLAC), a two-domain bacterial laccase native to Streptomyces coelicolor. Because of the low redox potential of the enzyme, a preliminary substrate screening process was conducted to identify phenolics oxidisable by the SLAC. Caffeic acid, 2,6-dimethoxyphenol, catechol, gallic acid, guaiacol, ferulic acid, and pyrogallol were identified as SLAC substrates and further coupling reaction studies were conducted using caffeic acid and gallic acid. Coupling reactions were carried out either in biphasic systems consisting of water-immiscible organic solvents and a buffer system or monophasic systems consisting of miscible organic solvents that form a homogenous phase with the buffer system. Coupling products were monitored using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), purified using preparative TLC and column chromatography, and characterised by liquid chromatography-mass spectrometry (LCMS) and nuclear magnetic resonance spectroscopy (NMR). Antioxidant capacity of the oxidation products were investigated by using the 2,2’-diphenyl-1- picrylhydrazyl (DPPH) and Trolox equivalence antioxidant capacity (TEAC) assays. Two oxidation products (one from caffeic acid and another from gallic acid) were successfully produced, purified and characterised. The oxidation product obtained from the SLAC-catalysed oxidation of caffeic acid was identified as a β-β dimer using LC-MS and NMR. When the reaction was carried out at a large-scale, a 32.8% yield of the dimer was achieved. Results showed that optimum yield of the dimer was achieved when the reaction was carried out for 6 h in a biphasic system consisting of 80% ethyl acetate and sodium acetate buffer pH 7.5. The dimer demonstrated superior antioxidant capacity, showing a 1.5- fold increase in DPPH radical scavenging capacity and a 1.8-fold improvement in TEAC. The dimer exhibited several positive physicochemical attributes, including improved solubility properties in aqueous media and remarkable stability in acidic pH (pH 2.2 and pH 5.5). One oxidation product from the SLAC-catalysed oxidation of gallic acid was successfully produced, purified and partially characterised. Optimum yield of gallic acid oxidation product was achieved when the reaction was conducted in a biphasic system consisting of 80% ethyl acetate and Tris-HCl buffer pH 8.0, using 0.5 U SLAC and a reaction time of 4 h. However, the oxidation product showed a lower antioxidant capacity than the substrate, as demonstrated by standard antioxidant assays (DPPH and TEAC). In conclusion, two antioxidant products were successfully produced, purified and characterised. Furthermore, selected physicochemical and antioxidant activities were determined. Overall, this study has highlighted the potential of the small laccase as a catalyst for the synthesis of antioxidants.
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    The effects of laccase and xanthan gum on the quality of glutten-free amadumbe bread
    (2018) Seke, Faith; Kudanga, Tukayi; Amonsou, Eric Oscar
    Celiac disease (CD) is an auto-immune disorder that is triggered by the consumption of gluten in predisposed individuals. The only remedy that has been proposed thus far is total exclusion of gluten from the diet. This may be the most difficult task to most celiac disease patients for most of the convenient and widely consumed baked products such as bread are prepared using ingredients that contain gluten. The replacement of gluten in the baking industry comes with some implications on the overall quality of the baked products, especially bread. It has been observed that gluten-free baked products currently on the market are of poor texture, less volume, not visually appealing and have a bad taste. Hence, the need for polymeric substances that can mimic gluten properties, yielding baked products with similar characteristics as the gluten-containing counterparts. Various crops such as rice, sorghum, sweet potato and cassava have been used and additives such as hydrocolloids, protein-based ingredients, emulsifiers and enzymes included to improve gluten-free bread quality. The use of carbohydrate-rich tubers and protein-rich legumes as gluten-free ingredients shows great potential in the food industry. Amadumbe (Colocasia esculenta) is a carbohydrate rich tuber which is highly underutilized in South Africa and contains vast amounts of mucilage, a hydrocolloid which can be of great help to improve dough rheology. Hydrocolloids have been reported in literature to have the ability of improving dough water holding capacity and improving dough viscosity hence facilitating gas retention and impacting on the overall quality of the baked product. However, despite the presence of mucilage, amadumbe is very low in protein and it is difficult to produce bread with properties that resemble gluten-containing bread. Hence the need for protein supplementation which may also potentially facilitate protein cross-linking during bread making. Legume proteins from crops such as soy bean and bambara groundnuts contain abundant quantities of lysine, tyrosine and cysteine which could potentially be manipulated through the use of enzymes such as laccase in order to initiate the formation of a network similar to gluten. The project investigated the effect of laccase and xanthan gum (a hydrocolloid) on the quality of gluten-free bread supplemented with bambara groundnut flour and soy protein isolate as protein sources. Flour blends were prepared using a ratio of 70:30 (amadumbe flour: bambara groundnut flour) and 88:12 (amadumbe flour: soy protein isolate) based on a targeted protein content of 16 g/100 g and the quality properties were determined. Colour analysis showed that amadumbe flour had a higher L* value compared to the other flours and the blends, showing that amadumbe can be used in applications where food colour contributes to food perceptions. However, when bambara groundnut flour and soy protein isolate were added the L* value decreased. The nutritional profile of the individual flours and the blends showed that amadumbe flour protein content was improved with the addition of bambara groundnut flour and soy protein isolate in the above-mentioned ratios. The protein content of amadumbe increased from 2.36 g/100 g to 15.87 g when bambara groundnut flour was added and to 16.10 g/100 g when soy protein isolate was added, values that were close to the targeted protein content. Incorporating bambara groundnut flour and soy protein isolate in amadumbe flour resulted in improved water absorption capacity, foam capacity and stability as well as emulsion capacity and stability of the amadumbe flour. However, there was no significant difference in oil absorption capacity between amadumbe flour and the blends. The blends were then used to formulate different bread samples incorporating the enzyme laccase (25 nkat/g flour) and a hydrocolloid, xanthan gum (1%). Laccase-mediated treatment of gluten-free amadumbe dough resulted in a 30% decrease in the free sulfhydryl groups and a 40% decrease in phenolic content indicating that crosslinking had occurred. Laccase action resulted in a 64% increase in bread specific volume and a 32% decrease in bread crumb hardness. Sensory analysis showed that laccase-treated bread samples were more acceptable compared to the non-treated bread samples in terms of appearance, texture, aroma and taste. The acceptability index varied between 46% and 86.2%. This study showed that there is great potential of laccase in gluten-free bread making. The addition of 1% xanthan gum to amadumbe dough supplemented with bambara groundnut flour and soy protein isolate resulted in gluten-free amadumbe bread with improved crumb texture and specific volume, and decreased the rate of moisture loss. Sensory analysis also revealed that gluten-free amadumbe bread with added xanthan gum was more acceptable compared to the bread samples without xanthan gum. The acceptability index of the bread samples ranged between 40% and 85%. The resulting bread with xanthan gum showed that hydrocolloids such as xanthan gum can be successfully used in the development of gluten-free baked products. Overall, this study has shown that the incorporation of laccase and xanthan gum to gluten-free amadumbe bread results in bread with improved and acceptable bread properties.