Optimizing Laccase Activity and Stability

Optimum temperature and pH were determined by performing enzymatic assays at different temperatures (20–80°C) and pH levels (3–8), respectively. The pH level was adjusted using the following buffers: 0.1 M citrate buffer (pH 3–5), 0.1 M phosphate buffer (pH 6–8), and 0.1 M carbonate buffer (pH 9). The stability of the purified laccase at various temperatures was investigated by preincubating the purified laccase at different temperatures between 4 and 70°C for 1 h, followed by determination of the residual activity. The effect of pH on the laccase stability was determined by incubating the purified enzyme at 4°C in different pH levels for 24 h and determining the residual activity. Substrate concentration ranges of 50–500 l M, 500–1500 l M, and 1000–2500 l M were used for kinetic studies against ABTS, DMP, respectively. The effects of metal ions including Cu²⁺_, Ba²⁺_, Mg ²⁺_, Mn²⁺_, Fe²⁺_, and Hg²⁺ and inhibitors (EDTA, 1 and 10 mM; NaN₃, DTT, Urea, and SDS on laccase activity were investigated by incorporating them in to assay mixture prior to determination of residual activity [21 (link)].