Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography

A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg−1 and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol−1. The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (Kcat/Km) in the order of 7.25   > 0.67  > 0.27 mM−1 min−1 for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn2+, Cd2+, Ca2+, Na+, Fe2+, Co2+ and Cu2+ while weakly inhibited by Hg2+. The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.