Recovery of laccase from Agaricus bisporus waste by aqueous two-phase system

The laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) are members of the multi-copper protein family that are capable of oxidation phenolic compounds to phenoxyl radicals. In the presence 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonate) (ABTS) mediator, laccases has potential of oxidation of nonphenolic compounds. First time, laccases were isolated from plants but were founded in fungi and some bacteria. Fungal laccases facilitated several processes such as lignin degradation, sporulation, pigment production, fruiting body formation and plant pathogenesis. Laccases are known as the first and main ligninolytic enzymes secreted by Agaricus bisporus in spent mushroom stipe. In our work, to recovery of laccase from the residual mushroom stipe of Agaricus bisporus, a low cost aqueous two-phase systems (ATPS) were evaluated. ATPS was assembled from a mixture of polymers such as poly ethylene glycol (PEG) and salts. By evaluation of molecular mass, concentration of PEG, type of salt and system pH parameters, the best laccase concentrates in the aqueous two-phase systems (ATPS) were determined. In this study, PEG 1000 and Na2SO4 ATP System established to be appropriate for the primary recovery of laccase. An extraction of spent mushroom stipe from % 12.21 dry matter, amount of protein 3.9 mg/ml, enzyme activity 13.713 U/ml, ATPS stage comprising volume ratio <1.0, PEG 1000 10% (w/w), Sulphate 12.5% (w/w), system pH of 4.6 and loaded with 1% (w/w) of crude extract from residual mushroom stipe allowed the laccase recovery. The ATPS resulted in one-single primary recovery stage process produced an overall yield of 86%. Amount of protein 0.46 mg/ml, enzyme activity 16.144 U/ml, purification fold 1.20 and enzyme recovery %94.06 were determined. After ultra filtration, amount of protein and enzyme activity were resulted 1.1 mg/ml and 24.171 U/mg, respectively. According to these results, final enzyme extract were 1.76 fold purified more than crude enzyme extract.