Laccases are holoenzyme, monomeric, dimeric or tetrameric glycoprotein (Kunamneni et al 2008), with a molecular weight of around 60 – 85kDa of which 15 – 20% is carbohydrate (Thurston, 1994).
Extracellular laccase from Ganoderma sp was partially purified. Crude laccase was precipitated by acetone. Acetone precipitated enzyme was loaded on to a Sephadex G 100 column. Active fractions with laccase activity were collected and pooled together for further characterization study.
Purity of laccase was checked by SDS PAGE (Fig) and zymogram analysis (Fig). On SDS PAGE laccase showed a single band of molecular mass approximate (66 – 75kDa) compared with standard protein (BSA). Majority of the basidiomycete’s laccase have molecular weight in the range of 55 – 72 kDa (Thurston., 1994; Petroski et al., 1980). Presence of laccase enzyme in Ganoderma sp was confirmed by Zymogram. Laccase activity was detected by staining with guaicol.
The enzyme was able to oxidize the substrate (guaicol) and develop reddish brown color. Laccase had been partially purified 4.93 fold with a yield of 3.9%. A specific activity of 15,635.
3 U/mg protein was found using guaicol as substrate under standard assay conditions. In order to assess the enzyme suitability for industrial application, both purification and characterization are essential steps. Several methods of enzyme precipitation were used by workers, considered acetone precipitation as the best method of precipitation for laccase purification (Sun et al., 2013; Bryjak et al.
, 2010; Marques De Souza and Peralta, 2003 and Brazkova et al., 2016). Kumar and Srikumar, 2012 also reported laccase precipitation through acetone with 14.
8 fold purification and a yield of 21.4% using DEAE Sephadex A-50 column for laccase from Cereiis pterngonus. D’Suza – Ticlo et al., 2009 reported precipitation through membrane filtration techniques. Ammonium sulphate precipitation is also a widely used method for purification (Saito et al 2003; More et al 2011). Gahlout et al., 2013 reported 8.
92 fold purification and a specific activity of 8078 J/mg of protein from Ganoderma cupreum AG-1 with a yield of 21.41%. Patel et al 2016 reported 1.53 fold purification and a specific activity of 4.95 x 105 U/mg after performing ammonium sulphate precipitation and two step chromatography for laccase from T. giganteum AGHP.
To achieve more fold purification he performs gel chromatography using Sephadex G-75 which resulted into 3.33 fold purification with a specific activity of 1.07 x 105 U/mg and 10.49% yield). In the next chromatographic step (DEAE Sephadex A-50 ion exchange chromatography he reported10.
80 fold purification with a specific activity of 3.49 x 105 U/mg and a yield of 8.50%.
Our results are comparable with the results reported by Yan et al.2014, reported 1.37 fold purification with a yield of 5.78% and 4.
07 fold purification and a yield of 11.64% from the laccase of T. trogii S0301 after performing anion exchange and Sephadex G – 75 chromatographic technique respectively. More et al.
, 2011 reported 72.2 fold purification of laccase from Pleurotus sp with a yield of 22.4%, after performing a series of chromatographic techniques (DEAE Cellulose column chromatography followed by gel permeation using Sephadex G-100 column chromatography). Zhang et al.2010 reported 2.96 fold purification with a 14.97% of overall recycling rate of enzyme activity from Gladiolus Curvularia trifol.
Viswanath et al. 2008 reported 70 fold laccase purification after performing ammonium sulfate precipitation followed by Sephadex G-100 column chromatography from Stereum ostrea. Manavalan et al 2013 reported 5.
57 fold protein purification from G. lucidum with a yield of 32% and a specific activity of 145 U/mg by using ammonium sulfate precipitation and two chromatographic steps (Sephadex G-100 followed by DEAE Cellulose). Chaurasia et al 2014, reported purification fold of 10.42 from Trametes hirsuta MTCC-1171 laccase with a yield of 12.57% by using DEAE cellulose column chromatography. Diaz et al 2010 reported 32.
7 and 31.2 fold purification for lac1 and lac II isoenzyme from Coriolopsis rigida with a yield of 2.2 and 2.6% respectively. More than one isoenzyme has been detected in several white rot fungi with molecular weight ranging between 60 to 100 kDa (Bertrand et al., 2015).