Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1

A new strain of Trichoderma harzianum WL1 was isolated from the Western Ghats region of Tamilnadu, India and assayed for laccase activity by ABTS oxidation. Supplementation of CuSO4 (1 mM) in liquid medium yielded high amounts of laccase (4.36 U ml−1) at an incubation period of 4 days. Laccase enzyme produced by T. harzianum in cultures supplemented with copper sulphate has been purified by ultrafiltration, Sephadex G-100 column chromatography and Concanavalin-A affinity chromatography with a final purification fold of 151.7 and a yield of 0.39%. The purified enzyme was identified as a glycoprotein with a molecular mass of 79 kDa by SDS gel electrophoresis. The UV–vis spectrum of the purified laccase had a peak at 608 and 325 nm suggesting the presence of types I and III Cu centers. The redox potential of the enzyme was found to be 692 mV for the type 1 Cu (T1) site. The optimum pH and temperature for enzyme activity were 4.5 and 35 °C, respectively. Laccase was stable for 24 h at 35 °C and had half-life of 60 min at 65 °C. Purified laccase showed Km values of 180 and 60 μM, respectively, and Vmax values of 3.95 and 1.42 U mg−1 protein, respectively, for the substrates ABTS and guaiacol. The susceptibility of laccase towards several putative inhibitors and metal cations was also assessed. The enzyme activity was completely inhibited by sodium azide (NaN3) at the concentration of 20 μM. Laccase activity was also inhibited by several metal cations, especially Hg2+.