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Item Laccases as biocatalysts for the biosynthesis of hybrid antioxidants(2021) Ngubane, Sandile; Kudanga, Tukayi; Permaul, KugenThe past century has seen an overwhelming upsurge in research interest concerning natural antioxidants, primarily due to rising awareness and knowledge regarding the carcinogenicity of the previously used synthetic antioxidants. Owing to their functional role in numerous redox systems, natural phenolic antioxidants can be applied in diverse areas such as pharmaceuticals, food products, dietary supplements, cosmetics and many other products. However, some natural antioxidants have been shown to exhibit undesirable properties such as low solubility (leads to instability in certain solvents), low bioavailability, low heat stability, low antioxidant capacity and pro-oxidant activity when present at high concentrations alongside transition metal ions such as Cu2+ and Fe3+. Structural modification of these natural compounds is accomplished by chemical or enzymatic means. Biocatalysis has attracted notable attention as a viable way to modify and synthesise bioactive compounds. Laccases are better suited for this function since they can be applied in a plethora of environmentally benign organic synthesis mechanisms through bond formation reactions such as oxidative decomposition, nuclear amination, thio bond formation, oxidative coupling, and C-C bond forming reactions. In this study, the biotransformation of natural phenolic compounds using laccases from Trametes pubescens CBS 696.94 was investigated. Before its application, laccase was biochemically characterised and had its thermodynamic parameters determined. Catechol, gallic acid, quercetin and nordihydroguaiaretic acid were identified as promising substrates and were used in subsequent hetero-coupling studies. Hetero-coupling reactions were carried out in a mixture of a water-miscible and a buffer. Products were monitored using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), purified using preparative TLC and column chromatography, and their molecular weight determined using liquid chromatography-mass spectrometry (LCMS). The antioxidant activities of the products were determined by using the ferric reducing antioxidant power (FRAP), 2,2- diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging assays. The antibacterial activities of the products were assessed against selected American Type Culture Collection (ATCC) bacteria, and their minimum inhibitory concentrations (MICs) were determined. T. pubescens CBS 696.94 produced high titres of extracellular laccase (2330 ± 50 U/l). The enzyme (~58 kDa) had an optimum activity at 60°C while optimum pH varied with the substrate used. The activity was shown to drop drastically at temperatures above 60°C, while the enzyme was most stable between pH 4.5 and 5.0. Enzyme activity was enhanced when the enzyme was pre-incubated in 20 mM CuSO4. The kinetic constants (Km) values for ABTS, syringaldazine (SGZ), 2,6-dimethoxy phenol (2,6-DMP) and guaiacol were 198 µM, 211 µM, 168 µM, and 102 µM, respectively. The kcat values were 103 s-1, 32 s-1, 12 s-1, and 13 s-1 with corresponding catalytic efficiency values (kcat/ Km) of 5.2×105 s-1 M-1, 5.8×104 s1 M-1, 1.9×105 s-1 M-1 and 1.2×105 s-1 M-1, respectively. The t1/2 values of the T. pubescens CBS 696.94 laccase at 50°C, 60°C and 70°C were 7.8 h, 3.8 h, and 0.72 h, respectively. The enzyme deactivation energy (Ed) was 109.362 kJ/mol while ΔG, ΔH, and ΔS for thermal inactivation of the T. pubescens CBS 696.94 laccase were all positive. The enzyme was susceptible to non-competitive (in the presence of sodium azide and sodium dodecyl sulphate) or uncompetitive modes of inhibition (in the presence of L-cysteine, hydrogen peroxide and dithiothreitol). Three heterodimers (catechol + quercetin, quercetin + nordihydroguaiaretic acid (NDGA) and gallic acid + nordihydroguaiaretic acid) and a single heterotrimer (2× quercetin and 1× catechol) were successfully produced, purified and partially characterised. The large scale catechol/quercetin coupling reaction yielded 15.6 ± 1.26% and 9.8 ± 1.12% of the heterodimer and heterotrimer, respectively. The best yields of the catechol/quercetin product were achieved in a monophasic system consisting of 50% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The products heterodimer showed inferior antioxidant activity, while the heterotrimer displayed enhanced antimicrobial activity against Listeria monocytogenes and Staphylococcus aureus at minimum inhibitory concentrations of 200 and 150 µg/ml, respectively. Large scale reaction of the quercetin/NDGA coupling reaction yielded 14.71 ± 0.59% of the heterodimer. The optimum yield was achieved in a monophasic system consisting of 50% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The heterodimer showed superior antioxidant activity, exhibiting 1.3 and 1.9-fold increases in the ABTS radical scavenging capacity, 1.3- and 2.0-fold increases in DPPH radical scavenging activity, and 1.14- and 1.6-fold increases in FRAP units when compared to quercetin and nordihydroguaiaretic acid, respectively. It also showed enhanced antimicrobial activity against L. monocytogenes, S. aureus, Escherichia coli and Enterobacter cloacae at minimum inhibitory concentrations of 200, 100 and 50 µg/ml. The large scale reaction of the gallic acid/NDGA coupling reaction yielded 14.12 ± 0.53% of the heterodimer. The optimum yield was achieved in a monophasic system consisting of 60% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The heterodimer displayed superior antioxidant activity, exhibiting 1.7- and 2.2-fold increases in the ABTS radical scavenging capacity, 2.1- and 3.0-fold increases in DPPH radical scavenging activity, and 1.4- and 1.8-fold increases in FRAP units when compared to nordihydroguaiaretic acid and gallic acid, respectively. In conclusion, two antioxidant and antibacterial compounds were successfully produced, purified and characterised. Overall, this study has demonstrated that laccases from T. pubescens CBS 696.94 can facilitate the cross-coupling of phenolic compounds to form hybrid compounds with enhanced antioxidant and antibacterial activity.