Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum

Abstract

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many laccase isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have been purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative gel electrophoresis, and electroelution. Their estimated molecular weights are 65-68 kDa, and they contain 7-10% N-linked carbohydrates. The three isozymes have identical N-terminal amino acid sequences: G-I-G-P-T. The optimum pH and temperature both for each isozyme singly and the isozyme mixture are pH 3.5 and 20 degrees C, respectively. One isozyme (GaLc 3) is quite stable at pH 4.0-10.0, and shows good stability when incubated at temperatures lower than 40 degrees C. The Km values of GaLc 3 for o-tolidine and 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) are 401.6 microM and 3.7 microM respectively, and the Vmax of GaLc 3 for these substrates is 0.0198 OD min(-1) unit(-1) and 0.0142 OD min(-1) unit(-1), respectively.

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

“…This is as expected as these enzymes originate from different organisms and their physicochemical characteristics have been shown to be different. The other physicochemical characteristics, for example, pH and temperature optima, of the laccase isozyme isolated from G. lucidum- CDBT1 were similar but not necessarily identical to those previously reported [25, 47]. The isolated enzyme was thermally stable (70°C for up to 1 h) and exhibited broad pH stability (pH 3.0–7.0).…”

“…The major isozyme has been successfully purified in this study. This enzyme has characteristics similar to the enzyme isolated by Ko and associates [25] although the molecular mass is different. Molecular weight of the laccase isozyme-3 (Figure 4), reported herein (43 kDa as judged by SDS-PAGE), is in agreement with the value reported by Murugesan and associates [49] based on SDS- and native-PAGE of crude extracts stained for enzyme activity.…”

“…This has been supported by the fact that G. lucidum is a mesophilic fungus and others have shown that G. lucidum secretes maximal laccase at 25°C [25]. …”

“…The fractions with high laccase activity were subjected to dialysis overnight against 0.1 M sodium phosphate buffer pH 7.0. Finally, the desalted enzyme factions were subjected to a DEAE Sepharose anion exchange column chromatography at pH 7.0 as described previously [25]. The protein fractions collected were analyzed for laccase activity, and the fractions with high laccase activity were pooled and subjected to SDS- and native-PAGE to assess its purity and determine its molecular mass.…”

Fungal Laccases and Their Applications in Bioremediation

“…Extracellular laccases of white-rot fungi exist in isozymes that may be inducible or constitutive. Ganoderma lucidum produced more than three laccase isozymes in liquid culture [72]. Pleurotus pulmonarius produced three laccase isozymes among which the lcc 1 and lcc 2 isoforms were produced in noninduced cultures, while lcc 3 was found only in induced-culture filtrates [73].…”