Selection of a pH- and temperature-stable laccase from Ganoderma australe and its application for bioremediation of textile dyes

By virtue of screening, purification, and properties characterization, this study captures a new pH- and temperature-stable laccase, designated Galacc-F, from Ganoderma australe for dye bioremediating applications. The enzyme was purified to homogeneity by salt precipitation, ionic exchange, and size exclusion chromatography with a final specific activity of 22.214 U mg(-1), yielding a purification fold of 23.989 and recovery of 38.44%. Its molecular weight was estimated to be 48.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, Sephadex G-100 column, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which confirmed its monomeric nature. Galacc-F exhibited high levels of activity and stability over wide ranges of pH (5.0-8.0) and temperature (10-60 degrees C), which are highly valuable properties in industrial processes. Broad substrate specificity was observed, wherein a better affinity was found for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with a low value of K-m (164.137 mu M) and higher k(cat)/K-m ratio (1.663 s(-1) mu M-1). Activity was stimulated by Cu2+ and beta-mercaptoethanol but inhibited by ethylenediaminetetraacetic acid, diethylpyrocarbonate, iodoacetic acid, phenylmethylsulfonyl fluoride, and Hg2+, indicating that Galacc-F is a metalloprotease containing a typical histidine-cysteine-serine catalytic triad. It had high tolerance to surfactants, oxidants, and salts. Additionally, a fabricated protocol for native Galacc-F immobilization onto Fe3O4@Chitosan composite nanoparticles using glutaraldehyde as a crosslinker was developed. Most importantly, the enzyme was determined to be ideal for use in efficient treatment of dye effluents as compared with the laccases requiring redox mediators.

Automated summary

This study discovered a new pH- and temperature-stable laccase, Galacc-F, from Ganoderma australe, with high activity, broad substrate specificity, and stability in industrial processes. The enzyme was successfully immobilized onto Fe3O4@Chitosan composite nanoparticles using glutaraldehyde as a crosslinker, showing high tolerance to various conditions.