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A partial peptide sequence of β-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with β-glucosidic, β-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 μmol/min/mg with the substrates p-nitrophenyl β-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant β-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.

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A cellobiohydrolase gene cbh1 was cloned from a cellulolytic fungus, Penicillum funiculosum NCL1. Nucleotide sequencing of cbh1 gene revealed that this gene was 1590 bp length encoding a putative protein consisting of 529 amino acids. The deduced amino acid sequence showed that the predicted molecular mass of the CBHI was 54.9 kDa, and showed significant homology to glycoside hydrolase family 7 cellobiohydrolases. The cbh1 gene was cloned using pET30b and expressed in E. coli BL21 (DE3). The expression analysis of the recombinant E. coli BL21 (pETC7) revealed the production of cbh1 transcript; however, functional cellobiohydrolase could not be detected. Therefore, the cbh1 gene was sub-cloned into GST tagged expression vector pGEX4t-3. The GST tagged cellobiohydrolase was purified to homogeneity using affinity chromatography. The recombinant enzyme exhibited optimum catalytic activity at pH 5.0 and 50 °C respectively. It was thermostable at 50 °C and retained 70% of its original activity after 30 min at 60 °C.

The purpose of this work was to study the production of cellobiohydrolase production by a Penicillium funiculosum NCL both in SSF and SMF using different agricultural residues (wheat bran, sugarcane bagasse, rice bran and rice straw) as substrates. Maximum cellobiohydrolase activity (13 U/gds) was produced by the culture grown on wheat bran substrate when compared to the cultures grown on sugarcane baggase, ricebran and rice straw. Time course of cellobiohydrolase production in SSF showed that maximum cellobiohydrolase activity of 13.2 U/gds was obtained in SSF at 144 h. Penicillium funiculosum NCL1 was grown under SSF and SmF conditions with different carbon sources. A maximum level of cellobiohydrolase was produced at 144 h in SSF whereas this strain produced maximum cellobiohydrolase at 96 h in SmF. Among the carbon sources tested wheat bran enhanced the cellobiohydrolase production by P. funiculosum NCL1. This strain produced higher level of cellobiohydrolase at acidic pH of the medium. The effect of various carbon sources on cellobiohydrolase was examined. Avicel was found to be a potent inducer for cellobiohydrolase production by P. funiculosum NCL1.