Characterization of a Novel Fe2+ Activated Non-Blue Laccase from Methylobacterium extorquens

Abstract

Herein, a novel laccase gene, Melac13220, was amplified from Methylobacterium extorquens and successfully expressed in Escherichia coli with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu²⁺ and Co²⁺ can slightly increase enzyme activity, whereas Fe²⁺ could increase Melac13220's activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe²⁺ could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from Methylobacterium extorquens shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH.

Keywords: Fe2+ ion; Methylobacterium extorquens; characterization; laccase.

Figure 1

Expression and analysis of the molecular weight of Melac13220 via SDS-PAGE using SP cationic exchange column. Lane M: molecular weight marker; Lane 1: purified Melac13220. The band of Melac13220 is marked with black arrow.

Figure 2

Characterization of purified recombinant Melac13220 from Methylobacterium extorquens; (a) optimal temperature in reaction with ABTS as a substrate; (b) optimal pH in reaction with ABTS and 2,6-DMP. All experiments were performed in triplicate, and the results represent mean ± standard deviation.

Figure 3

The pH stability of Melac13220 was assayed at 50 °C after the incubation of laccase in different pH levels buffers for 1 h using ABTS as substrate. All experiments were conducted 3 times, and the results represent mean ± standard deviation.

Figure 4

The thermostability of Melac13220 after incubation of the purified laccase at 40, 50 °C, 60 °C (a) and 65 °C (b) with different durations. All the results represented as the means ± standard deviation from at least three independent experiments.

Figure 5

Activity of Melac13220 after 1 h of pre-incubation in the presence of various metallic ions in the reaction mixture (a) and at 0.1 mM, 0.5 mM, 1 mM, 5 mM and 10 mM concentration of Fe²⁺, and Cu²⁺ (b). The enzyme activity in the absence of metal ions was regarded as 100%. The data represented as the mean ± standard deviation from at least independent experiments.

Figure 6

Activity of Melac13220 after 1 h of pre-incubation in the presence of organic solvents at 10% (v/v) concentration. The enzyme activity in the absence of organic solvents was regarded as 100%. Values represent the mean ± standard deviation of three measurements.

Figure 7

UV/visible absorption spectra of pure Melac13220 (0.6 U/mg) from in 20 mM Tris-HCl buffer (pH 7.5). The dotted lines show increasing Fe²⁺ concentrations from bottom to top (A–L, from 0.012 mM to 0.112 mM).

Figure 8

Isothermal titration calorimetry for the interaction of Fe²⁺ and Melac13220. Experiment conditions: C_Fe²⁺ = 1 × 10⁻⁴ M, C Melac13220 = 6.3 × 10⁻⁶ M, T = 298 K.

Figure 9

The photoluminescence spectra of Melac13220 laccase from Methylobacterium extorquens in 20 mM Tris-HCl buffer (pH 7.5). The dotted lines show the increasing Fe²⁺ concentration from top to bottom (A–W, from 0.01 mM to 0.47 mM).