Abstract
Laccase and tyrosinases are naturally occurring enzymes that can be found in bacteria, fungi, plants, and other organisms. Laccase and tyrosinase are copper-containing phenoloxidases that catalyse the transformation of a variety of phenolic substrates using readily available O2 without the use of an additional cofactor. In this study, two commercially available enzymes; Trametes versicolor laccase and fungal tyrosinase were insolubilised combined crosslinked enzyme aggregates (Combi-CLEA). The two enzymes were precipitated with ammonium sulfate and crosslinked. The pH and temperature activity of the free Combi-CLEA enzyme was measured by ultra-violet (UV-Vis) Spectroscopy, where 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as a substrate for laccase and 3,4-dihydroxy-L-phenylalanine (L-DOPA) as a substrate for tyrosinase. The free laccase and combi-CLEA-laccase exhibited high enzymatic activity at pH 5 and pH 4, respectively. For the free tyrosinase and combi-CLEA-tyrosinase an optimum activity was found at pH 8 and pH 7, respectively. High enzymatic activity of temperature for the free laccase and Combi-CLEA-laccase was found 50°C and 40°C, respectively. While the optimum temperature for the free tyrosinase and Combi-CLEA-tyrosinase was found at 40°C and 20°C. The FTIR confirmed that the Combi-CLEA was successfully attached to the membrane; the presence of the C=N and C=O functional groups confirmed this. The increase in the concentration of the Combi-CLEA-Lac-Tyr modified membrane (0.5 to 2.0 mg/ml) caused a decrease in the pore size (0.034 to 0.012 μm). The pure polyethersulfone (PES) membrane had highest contact angle (81.20±9.82°) and upon functionalisation with Combi-CLEA-Lac-Tyr, the contact angle decreased to 58.67±7.01° at 2.0 mg/ml Combi-CLEA-Lac-Tyr. The optimum conditions for biodegradation of nevirapine and etravirine were 5 mg/L at pH 7 which led to a high degradation efficiency of 98.81% and 81.81%, respectively. The Ultra-Performance Liquid Chromatography (UP-LC) confirmed the ability of the biocatalytic membranes in the degradation of nevirapine to compounds such as 5,6-dihydroxy-2-indo-1-carboxylate, 1-ethylquinolium, 5,6-dihydroxy-2-indo-1-carboxylate, dinitroacetic acid, 3-hydroxy benzodiazepine and N-(4-Phenoxyphenyl)-1,3,5-triazine-2,4-diamine. The UP-LC analysis of etravirine did not show any by-products meaning that this pharmaceutical drug might have been
completely degraded or could not be detected due to preconcentration or chromatographic conditions.