Isolation and Physicochemical Characterization of Laccase fromGanoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal

Abstract

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, K m, V max, and K cat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process.

Evaluation of laccase production by Ganoderma lucidum in submerged and solid‐state fermentation using different inducers

“…Also, by combining tamarind shell with ethanol (3%, v/v), CuSO 4 (0.4 mM), and gallic acid (1 mM) the value of 74.84 U/ml was achieved after 15 days, which was 416 times higher in comparison with the control . On the other hand, Shresta et al achieved 93 U/ml of laccase activity with a much lower CuSO 4 concentration of 30 μM, after 10 days of cultivation.…”

“…Kraft lignin, also called alkali lignin, is largely produced by pulp and paper industry during the cooking process . Shrestha et al reported the potential of lignin isolated from rice for induction of laccase activity on G. lucidum. Sitarz et al evaluated the ability of G. lucidum , Polyporus brumalis , Polyporus ciliatus , and Trametes versicolor to grow on malt extract and minimal media containing alkali lignin and only G. lucidum showed the ability to grow on these conditions.…”

Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

“…B7 on guaiacol agar and α-napthol agar with dark brown and deep purplish colours around the colonies indicating laccase production. Several authors have reported the oxidation of ABTS, guaiacol and α-napthol by laccase-producing fungi to produce dark green, reddish brown and deep purplish halos respectively, around colonies on solid media incorporated with the indicators [11,38,40]. Isolates which did not show any colour change lacked laccase activity and were not considered for further work.…”

“…Studies have reported the use of ABTS, guaiacol and α-naphthol in plate screening for visual identification of laccases [8]. Oxidation of guaiacol presents reddish brown colour, α-naphthol a deep purple colour, and 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) a dark green halo around laccaseforming colonies on agar [11]. Most fungal laccases prefer ABTS as the ideal substrate [8].…”

An Efficient Method for Qualitative Screening of Ligninolytic Enzyme Potential of Ganoderma lucidum

“…These enzymes catalyze the oxidation of various phenolic and nonphenolic compounds and complementarily reducing the molecular oxygen to water (Casciello et al, 2017). The enzymes from wood-rotting basidiomycetes possess significant interest because of their ability to utilize a wide spectrum of carbon sources, including intermediates of lignin degradation, phenols, and heterocyclic compounds (Shrestha et al, 2016).…”