Production, Biochemical Characterization, and Application of Laccase from Halophilic Curvularia lunata MLK46 Recovered from Mangrove Rhizosphere

Abstract

Laccase production was evaluated in 108 fungal isolates recovered from the eastern coast of Saudi Arabia, a critical element in environmental biodegradation and biotransformation. The most active isolate was identified as Curvularia lunata MLK46 (GenBank accession no. PQ100161). It exhibited maximal productivity at pH 6.5, 30 °C, and incubation for 5 d, with 1% sodium nitrate and 1% galactose as the preferred nitrogen and carbon sources, respectively. Productivity was enhanced by NaCl, CuSO₄, and FeCl₃ supplementation, with a maximum at 0.3 mM, 0.2 mM, and 61.7 mM concentrations, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme through diethylaminoethyl (DEAE)-Sepharose chromatography revealed a prominent band at 71.1 kDa with maximum activity at pH 6 and stability at pH 6-9. Furthermore, it was optimally active at 50 °C and thermally stable at 50-80 °C with a half-life time (T_1/2) of 333.7 min to 80.6 min, respectively. Its activity was also enhanced by many metallic ions, especially Fe³⁺ ions; however, it was inhibited by Hg²⁺ and Ag⁺ ions. The enzyme demonstrated significant degradation of specific substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine, and 2,6-dichlorophenol, with a kinetic efficiency constant which ranged from 40.95 mM^-1 s^-1 to 238.20 mM^-1 s^-1. UV spectrophotometry confirmed efficient oxidation peaks by electron transition against guaiacol (at 300 nm), o-dianisidine (at 480 nm), ABTS (at 420 nm), and 2,6-dichlorophenol (at 600 nm). The results collectively demonstrate the potential of laccase from C. lunata MLK46 as a promising agent for the effective biodegradation of several industrial pollutants under extreme conditions.

Keywords: biodegradation; environmental sustainability; fungi; laccase; pollutant.

Figure 1

Comparable laccase productivity detected for some fungal isolates using bromophenol blue plate assay (a) and ABTS plate assay (b). The white arrowhead shows a yellow halo area due to the degradation of bromophenol dye, while the black arrowhead shows a green halo zone due to the degradation of ABTS by laccases produced from these fungi.

Figure 2

Macroscopic and microscopic structures of the most potent laccase producers stained with lactophenol cotton blue, highlighting distinctive features such as conidia, sporangia, and hyphal arrangements. The isolates include A. terreus EQ75 (a), A. alternata EK12 (b), A. alternata EK81 (c), A. alternata EK59 (d), A. nidulans EK107 (e), C. lunata MLK46 (f), and Acrophialophora levis EK56 (g). The phylogenetic tree of the most promising laccase-producing strain C. lunata MLK46 (accession number PQ100161.1) is shown in panel (h). The neighbor-joining phylogenetic tree shows the most promising fungal species isolated in this study, which are highlighted in yellow.

Figure 3

Chromatogram of the purified laccase from C. lunata MLK46 after elution through a DEAE-Sepharose column (1 × 15 cm) previously equilibrated with 50 mM sodium phosphate buffer (pH 6.0) (a). SDS-PAGE analysis of purified enzyme shows a distinct band at 71 kDa (b), indicating a successful purification procedure. Lane M represents the molecular weight markers, while lane E represents the purified protein.

Figure 4

Effect of pH (a) and high temperatures on the activity (b) and stability (c) of tested laccase.

Figure 5

Effect of laccase reagents on enzymatic activity. The * symbol denotes the control preparation where no reagent was added to enzyme.

Figure 6

Effect of metal ions on the activity of laccase from C. lunata strain MLK46. The effect of cobalt (a), manganese (b), zinc (c), calcium (d), copper (e), ferric iron (f), mercury (g), and silver (h) were tested at the concentration range of 2–64 mM.

Figure 7

Spectral changes in the oxidation of various substrates by laccase. These substrates include 2,6-dichlorophenol (a), o-dianisidine (b), ABTS (c), guaiacol (d), and α-naphthol (e). The black lines represent the tested substrate without enzymatic treatment, while the green lines indicate substrates after laccase treatment. In the middle of the panels, the kinetic analysis of laccase activity against each substrate is represented in the form of Lineweaver–Burk plots.