An insight into transcriptome of Cyathus bulleri for lignocellulase expression on wheat bran.

To identify enzymes that can be effectively used for hydrolysis of lignocellulosic biomass, an attractive carbon source in biorefineries, transcriptome analysis was carried out of wheat bran grown fungus, Cyathus bulleri. A comprehensive set of transcripts, encoding carbohydrate active enzymes, were identified. These belonged to 55, 32, 12, 11 and 7 different families of the enzyme classes of Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Auxiliary Activities (AAs), Carbohydrate Esterases (CEs) and Polysaccharide Lyases (PLs) respectively. Higher levels of transcripts were obtained for proteins encoding cellulose and hemicellulose degrading activities (of the GH class) with the highest diversity found in the transcripts encoding the hemicellulases. Several transcripts encoding pectin degrading activity were also identified indicating close association of the pectin with the cellulose/hemicellulose in the cell wall of this fungus. Transcripts encoding ligninases were categorized into Cu radical oxidase, Glucose-Methanol-Choline oxidoreductase (with 37 different transcripts in the AA3 sub-family), Laccase and Manganese peroxidases. Temporal gene expression profile for laccase isoforms was studied to understand their role in lignin degradation. To our knowledge, this is the first analysis of the transcriptome of a member belonging to the family Nidulariaceae.

Identification and evaluation of bioremediation potential of laccase isoforms produced by Cyathus bulleri on wheat bran.

Multiplicity in laccases among lignin degrading fungal species is of interest as it confers the ability to degrade several types of lignocellulosics. The combination of laccases produced on such substrates could be beneficial for treatment of complex aromatics, including dyes. In this study, we report on production of high units (679.6Ug-1 substrate) of laccase on solid wheat bran (WB) by Cyathus bulleri. Laccase, purified from the culture filtrates of WB grown fungus, was effective for oxidation of veratryl alcohol, Reactive blue 21 and textile effluent without assistance of externally added mediators. De novo sequencing of the 'purified' laccase lead to identification of several peptides that originated from different laccase genes. Transcriptome analysis of the fungus, cultivated on WB, confirmed presence of 8 isozymes, that were re-amplified and sequenced from the cDNA prepared from WB grown fungus. The 8 isozymes were grouped into 3 classes, based on their sequence relationship with other basidiomycete laccases. The isoforms produced on WB decolorized (by ∼57%) and degraded textile effluent far more effectively, compared to laccase obtained from Basal salt cultivated fungus. The decolorization and degradation was also accompanied by more than 95% reduction in phytotoxicity.

Decolorization of complex dyes and textile effluent by extracellular enzymes of Cyathus bulleri cultivated on agro-residues/domestic wastes and proposed pathway of degradation of Kiton blue A and reactive orange 16.

In this study, the white-rot fungus Cyathus bulleri was cultivated on low-cost agro-residues, namely wheat bran (WB), wheat straw (WS), and domestic waste orange peel (OP) for production of ligninolytic enzymes. Of the three substrates, WB and OP served as good materials for the production of laccase with no requirement of additional carbon or nitrogen source. Specific laccase activity of 94.4 U mg-1 extracellular protein and 21.01 U mg-1 protein was obtained on WB and OP, respectively. Maximum decolorization rate of 13.6 µmol h-1 U-1 laccase for reactive black 5 and 22.68 µmol h-1 U-1 laccase for reactive orange 16 (RO) was obtained with the WB culture filtrate, and 11.7 µmol h-1 U-1 laccase for reactive violet 5 was observed with OP culture filtrate. Importantly, Kiton blue A (KB), reported not to be amenable to enzymatic degradation, was degraded by culture filtrate borne activities. Products of degradation of KB and RO were identified by mass spectrometry, and a pathway of degradation proposed. WB-grown culture filtrate decolorized and detoxified real and simulated textile effluents by about 40%. The study highlights the use of inexpensive materials for the production of enzymes effective on dyes and effluents.