Small laccases as catalysts for the synthesis of antioxidants
Authors:   , Nemadziva Blessing 
Published: August 2021

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.