Aims:  Molecular characterization of commercially important group of xylanase producing thermophilic/thermotolerant fungi. Methods and Results:  DNA from 16 thermophilic/thermotolerant fungal isolates was amplified by PCR using three sets of primers: (i) internal transcribed spacer sequence (ITSI‐5·8S‐ITSII), (ii) D1/D2 hyper variable region of 26S rDNA and (iii) 18S rDNA region. The amplified products of internal transcribed spacers (ITS) and D1/D2 region were sequenced and analysed using ClustalX, whereas, amplified 18S rDNA region was subjected to RFLP analysis based on restriction digestion with RsaI, MboI and Hinf I. Conclusions:  The sequence based analyses of ITSI‐5·8S‐ITSII as compared with D1/D2 region of 26–28S rDNA was found to be a better tool for phylogenetic resolution of thermophilic/thermotolerant fungi. The ITSI‐5·8S‐ITSII sequence‐based dendrogram indicates an early divergence of the alkaline active xylanase producing thermophilic fungal strains. Significance and Impact of the Study:  This study was the first report on phylogenetic characterization of thermophilic/thermotolerant fungi.

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A thermotolerant Aspergillus fumigatus strain isolated from composting pile of mixed industrial waste was found to produce a spectrum of cellulase and hemicellulases when cultured on rice straw solidified substrate. The two-dimensional electrophoresis (2DE) resolved the secretome into 57 distinct protein spots. The zymograms developed against 2DE gels identified the presence of three β-glucosidases and five CBHI/EGI isoforms in the secretome. The peptide mass fingerprinting of 17 protein spots by liquid chromatography mass spectrometry characterized the secretome into different glycosyl hydrolase families. The enzyme cocktail produced by A. fumigatus was capable of efficient hydrolysis of alkali pretreated rice straw (at 7% and 10% w/v) resulting in 95% and 91% saccharification, respectively.

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Thirty heterokaryons, formed by protoplast fusion of Aspergillus nidulans and Aspergillus tubingensis, were selected on the basis of their ability to grow on 2-deoxyglucose (0.2 %, w/v) and intermediate spore color. These heterokaryons were studied for cellulase production using shake flask and solid substrate cultures at 40 °C. Fusants 51 and 28 exhibited appreciably higher levels of endoglucanase, cellobiohydrolase, β-glucosidase, and FPase activities when compared with parental strains. Employing proteomic-based approaches, the differential expression of proteins in secretome of fusants and parental strains were analyzed using two-dimensional electrophoresis. The expression of some of the proteins in the fusants was found to be up/downregulated. The upregulated proteins in the fusant 51 were identified by liquid chromatography-mass spectroscopy as endoxylanase, endochitinase, β-glucosidase, as well as hypothetical proteins. The cellulases produced by fusants 28 and 51 showed improved saccharification of alkali treated rice straw when compared with the parental strains.