title: Molecular cloning and expression of thermostable glucose-tolerant β-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization.
abstract: 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.
authors.affiliation: Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625021, India.
authors.forename: Gurusamy
authors.initials: G
authors.lastname: Ramani
authors.forename: Balasubramanian
authors.initials: B
authors.lastname: Meera
authors.forename: Chinnathambi
authors.initials: C
authors.lastname: Vanitha
authors.forename: Jeyaprakash
authors.initials: J
authors.lastname: Rajendhran
authors.forename: Paramasamy
authors.initials: P
authors.lastname: Gunasekaran
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism / Q000600:supply & distribution; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D002475:Cellobiose; D000431:Ethanol; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D005947:Glucose