Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase

Abstract

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.

Senescence of the Lentinula edodes Fruiting Body After Harvesting

“…Therefore, the Lcc2 purified by Nagai et al (2003) is designated Lcc4 in this paper. Expression of lcc1 is high in vegetative mycelia (Sakamoto et al, 2008), but lcc4 is expressed only in the fruiting body and increases after harvest . Furthermore, Lcc1 cannot oxidize DOPA (Nagai et al, 2002), but Lcc4 can (Nagai et al, 2003;Yano et al, 2009).…”

“…Two Lccs (Lcc1 and Lcc2) have been purified from L. edodes (Nagai et al, 2002 and, an Lcc-encoding gene has been cloned and characterized, and six Lcc encoding genes (lcc1-lcc6; lcc1: AB035409; lcc2: AB035410; lcc3: AB046713; Lcc4: AB446445; Lcc5: AB543788; Lcc6: AB543787) have been deposited in the DNA data bank (DDBJ) (Zhao and Kwan, 1999;Sakamoto et al, 2008Sakamoto et al, , 2009Yano et al, 2010). Lcc1 is secreted from vegetative mycelia in culture (Nagai et al, 2002) and is encoded by lcc1 (Sakamoto et al, 2008).…”

“…Lcc1 is secreted from vegetative mycelia in culture (Nagai et al, 2002) and is encoded by lcc1 (Sakamoto et al, 2008). Lcc2 is expressed in the brown gills of fruiting bodies after harvesting (Nagai et al, 2003).…”

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes Involved in Lignin- Degradation by Lentinula edodes on Native-PAGE

“…These two isozymes were identified as being laccases (Lcc1; Fig. 4d), and are known to be an extracellular laccase produced by L. edodes (Nagai et al, 2002;Sakamoto et al, 2008). These results, combined with enzyme assay data and results of isozyme detection using PAGE, indicate that MnP and Lcc isozyme detection using the improved LccS+EDTA, PerS+EDTA and MnPS enzymatic staining methods can be used to successfully distinguished between each of the phenol-oxidizing enzymes (Saeki et al, 2011).…”

“…These observations supported the hypothesis that these enzymes, particularly MnP, play an important role in degrading sawdust during cultivation, and corroborating reports that the expression and properties of these enzymes is likely to influence mycelial growth and fruit body development (Smith et al, 1988;Wood et al, 1988). Several reports have been published on the purification and characterization of the lignin-degrading enzymes secreted by L. edodes using sophisticated biochemical procedures (Forrester et al, 1990;Nagai et al, 2002Nagai et al, , 2003Nagai et al, , 2007Sakamoto et al, 2008Sakamoto et al, , 2009). However, these methods are impracticable for routine isozyme analysis during breeding trials.…”