Production of laccase enzyme from Curvularia lunata MY3: purification and characterization

Abstract

Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn²⁺ and Mg²⁺ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg²⁺. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

Keywords: Characterization; Curvularia lunata; Laccase; Purification.

Fig. 1

Qualitative screening of laccase producing fungi. Isolate MY3 grown on potato dextrose agar supplemented with tannic acid (a), guaiacol (b), and Remazol Brilliant Blue R (c), while d, e, and f represent the negative results

Fig. 2

Phylogenetic trees showing relationship of strain Curvularia lunata isolate MY3 with other related fungal species retrieved from GenBank based on their sequence homologies of 18S rRNA

Fig. 3

Physical parameters of amylase production: effect of different media (a), incubation period (b), different pH (c), and incubation temperature (d) on laccase production by fungal isolate MY3

Fig. 4

Elution profile for the chromatography of C. lunata MY3 laccase diammonium sulfate fraction on a Sephacryl S-200 column. b Sephacryl S-200 active fractions for C. lunata MY3 laccase on DEAE-Sepharose column. Absorbance at 280 nm (o^___o) and laccase activity (●^____●)

Fig. 5

Electrophoretic analysis of C. lunata MY3 laccase. a Molar mass on 10% SDS-PAGE in (A) molar mass marker proteins and (B) purified laccase. b Native-PAGE in (A) purified laccase and (B) crude extract

Fig. 6

Temperature effects on the activity (x——x) and stability (•——•) of C. lunata MY3 laccase. Activation energy was estimated from Arrhenius plot shown in the inset

Fig. 7

Effect of different pH on purified C. lunata MY3 laccase a activity and b stability: citrate buffer (o–o), acetate buffer (•-•), and phosphate buffer (x-x)

Fig. 8

Effect of a 10 mmol/L of each metal cations and b different inhibitors on the activity of C. lunata MY3 laccase. The enzyme without any addition was taken as 100%