Optimization of Tremetes Versicolor Laccase Reverse Micelles System for the Removal of Phenolic Environmental Pollutant Bisphenol A

Introduction Laccase (benzenediol: oxygen: oxidoreductases, (EC. 1.10.3.2), phylogenitically old enzyme of oxidoreductase family and major secretome of many of the white rot fungi. They are capable of oxidizing diphenols, polyphenols, different substituted phenols, diamines, aromatic amines, benzenethiols, polyphenols and even some inorganic compounds (Bakkiyaraj et al., 2013). It has been widely used in various applications including paper manufacturing (Flory et al., 2013), wood processing (Fackler et al., 2008), environmental bioremediation (Ashrafi et al., 2013), food industry (Dhillon et al., 2012), as well as in textile engineering (Basto et al., 2007). Bisphenol A [2,2 bis (4 hydroxyphenyl) propane] is widely used in the variety of industrial and residential applications such as the synthesis of polymers including polycarbonates, epoxy resins, phenol resins, polyesters and polyacrylates. BPA has been ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 39-49 http://www.ijcmas.com In the present study we have tried to optimize preparation of reverse micelle from Tremetes versicolor laccase using isooctane: AOT system for removal of Bisphenol A {2,2bis-(4 hydroxyphenyl) propane} (BPA) by statistical optimization using central composite design method. On the basis of the various diagnostic plots and point prediction, actual concentration of different variables were found to be 150mM for AOT, 1 mM for Dimethoxy phenol and 100 μg/ml for Enzyme at pH 4.5 with 3.33 fold increase in the enzyme activity in reverse micelles as compare to aqueous system. The disappearance of Bisphenol A was monitored by High Performance Liquid Chromatography and after the incubation at 50 C for 240 minutes 84.3% elimination of 200 ppm Bisphenol A was observed which 36.81% higher than elimination by free laccase. Owing to insolubility of compounds like Bisphenol A in aqueous media, system like laccase-RM provides better alternative over aqueous enzyme system to degrade such pollutants. K e y w o r d s Laccase-RM system, Bisphenol A, High Performance Liquid Chromatography, Central Composite Design Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 39-49 40 recognized as an Endocrine Disrupting Chemicals (EDC), thus it is necessary to assess its biodegradability or fate in the natural environment. Due to its mass production and widespread use, the environmental release and contamination of BPA have been found through permitted discharges of industrial wastewater treatment systems, sewage sludge, and leachate from waste plastic in landfills. BPA has become one of the major toxic environmental pollutants of concern due to its acute toxicity towards algae, invertebrates, fish within the range of 0.04 0.4μM, as well as its mutagenic and estrogenic effects on humans within the range of 0.1 10μM (Saiyood et al., 2010). In general, environmental pollutant such as BPA does not dissolve in aqueous media, owing to their high hydrophobicity, and hence non-aqueous catalysis can be employed to enhance biodegradability of phenolic environmental pollutant. This implies that the use of organic solvents inevitably allows the degradation reaction to proceed at a high concentration of environmental pollutants in a homogenous system. However, native enzymes do not exhibit significant catalytic activities inorganic media. The reverse micellar system was then introduced to enhance the activity of laccase in organic media. This technique enables us to perform biodegradation reaction in an organic solvent facilitating degradation of hydrophobic pollutant (Michizoe et al., 2001). In the present study, a reversed micellar (RM) system was optimized to entrap laccase enzyme by Central composite design followed by the oxidative removal of Bisphenol A by laccase-RM system. Materials and Methods Chemicals Laccase used in the current study was purchased from SIGMA Life Science which is extracted and purified from Trametes versicolor (12.9 u mg); 2,2-bis-(4 hydroxyphenyl) propane (Bisphenol A) was procured from LobaChemie (Mumbai), M.W.: 228.29; Bis (2-ethylhexyl) sulfosuccinate sodium salt (AOT) was purchased from Fluka Chemical Ltd.(Finland); 2, 6 dimethoxyphenol (2,6 DMP) was purchased from SIGMAM.W.154.16; Organic solvents used in this work were of analytical grade and were purchased from Merck Chemicals (Mumbai, India); All other chemicals used were of analytical grade and of highest purity available. Laccase activity determination Laccase activity was determined by measuring the oxidation of 2, 6 DMP with laccase enzyme dissolved in sodium acetate buffer (pH-5.0).Increase in absorbance for 3 min was measured spectrophotometrically (Shimadzu Corp. 01846) at 469 nm ( =27,500 cm M ). One unit of enzyme was defined as amount of enzyme that oxidized 1μM of substrate per minute. Laccase Difference in OD × Volume activity (u ml) = in cuvette × 106 Aliquot of enzyme× Extinction co-efficient×Time Preparation and reaction of reverse micelle system with laccase A reversed micellar solution containing laccase was prepared by direct injection of 40μl of purified laccase in aqueous solution pre-pared in 100 mM of sodium acetate buffer pH-5.0 in to 150 mM of AOT in isooctane. The mixture was vortexed till it became optically transparent. 40 μl of 2,6 dimethoxy phenol, 1.0 mM used as a model substrate was injected in to a micellar solution and vortexed for 3 min. Laccase activity in reverse micelles was determined by measuring the oxidation of 2,6 DMP. Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 39-49 41 Increase in absorbance for 3 min was measured spectrophotometrically (Shimadzu Corp. 01846) at 469 nm ( =27,500 cm M ). One unit of enzyme was defined as amount of enzyme that oxidized 1μM of substrate per minute. Response Surface Methodology (RSM) for optimization of the screened components Response Surface Methodology was used to optimize the screened components for DMP oxidation using central composite design (CCD).The behavior of the system was demonstrated by the following quadratic equation.