Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis

Abstract

The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50-60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0-8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat) were determined by using a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.

Keywords: Pleurotus nebrodensis; biochemical properties comparison; enzyme characterization; laccase isoenzymes.

Figure 1

Chromatographic profiles of laccase isoforms. Black dotted line: absorbance at 280 nm; grey dotted line: activity of laccase isoforms; dashed line: NaCl gradient. (A) Ion exchange charomatography on DEAE-cellulose column. Lac1 and Lac2 resided in fraction D₂ and Lac3 resided in D₃; (B) Ion exchange chromatography of fraction D₂ on CM-cellulose column. Two peaks with activity: Lac1 resided in peak D₂C₁ and Lac2 resided in peak D₂C₂; (C) Ion exchange chromatography of fraction D₃ on CM-cellulose column. Lac3 resided in the only peak with activity; (D) Ion exchange chromatography of fractions Lac1 (D₂C₁), Lac2 (D₂C₂) and Lac3 (D₃C) on Q-Sepharose column (showing in the same figure, results of three separate fractionation experiments for Lac1, Lac2 and Lac3, respectively).

Figure 2

SDS-PAGE of isolated laccases. Lane 1: Molecular markers (GE Healthcare). From top to bottom: phosphorylase b (94 KD), bovine serum albumin (67 KD), ovalbumin (43 KD), carbonic anhydrase (30 KD), soybean trypsin inhibitor (20 KD) and lactalbumin (14.4 KD; Lane 2: Lac1; Lane 3: Lac2; Lane 4: Lac3).

Figure 3

Effect of temperature on the activities of purified P. nebrodensis laccase isoforms. Measurements were carried out in triplicate (standard deviations for all data points < 5%).

Figure 4

Stability of purified P. nebrodensis laccases at different temperatures (A) Effect on Lac1; (B) Effect on Lac2; (C) Effect on Lac3. “-x-” curves: treatment at 30 °C; “-●-” curves: treatment at 40 °C; “-▲-” curves: treatment at 50 °C; “-♦-” curves: treatment at 60 °C; “-■-” curves: treatment at 70 °C. Measurements were carried out in triplicate (standard deviations for all data points < 5%).

Figure 4

Stability of purified P. nebrodensis laccases at different temperatures (A) Effect on Lac1; (B) Effect on Lac2; (C) Effect on Lac3. “-x-” curves: treatment at 30 °C; “-●-” curves: treatment at 40 °C; “-▲-” curves: treatment at 50 °C; “-♦-” curves: treatment at 60 °C; “-■-” curves: treatment at 70 °C. Measurements were carried out in triplicate (standard deviations for all data points < 5%).

Figure 5

Effect of pH on the activities of purified P. nebrodensis laccase isoforms. Measurements were carried out in triplicate (standard deviations for all data points < 5%).

Figure 6

Stability of purified P. nebrodensis laccases after exposure to different pH values for 24 h. Measurements were carried out in triplicate (standard deviations for all data points < 5%).