title: Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.
abstract: 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 (K(cat)/K(m)) 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 Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). 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.
authors.affiliation: Department of Biochemistry, University of Lucknow, Lucknow 226007, U.P., India.
authors.forename: Nivedita
authors.initials: N
authors.lastname: Jaiswal
authors.affiliation: Department of Biochemistry, University of Lucknow, Lucknow 226007, U.P., India.
authors.forename: Veda P
authors.initials: VP
authors.lastname: Pandey
authors.affiliation: Department of Biochemistry, University of Lucknow, Lucknow 226007, U.P., India. Electronic address: upendradwivedi@hotmail.com.
authors.forename: Upendra N
authors.initials: UN
authors.lastname: Dwivedi
mesh_terms: D029441:Carica / Q000201:enzymology*; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D042845:Laccase
keywords: Alkaline laccase; Papaya; Thermostability