title: Production and Characterization of Organic Solvent-Tolerant Cellulase from Bacillus amyloliquefaciens AK9 Isolated from Hot Spring.
abstract: A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ∼47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg-1. The purified cellulase retained its activity over a wide range of temperature (50-70 °C) and pH (3-7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Irfan
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
authors.forename: Ammara
authors.initials: A
authors.lastname: Tayyab
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
authors.forename: Fariha
authors.initials: F
authors.lastname: Hasan
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
authors.forename: Samiullah
authors.initials: S
authors.lastname: Khan
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
authors.forename: Malik
authors.initials: M
authors.lastname: Badshah
authors.affiliation: Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan. alishah@qau.edu.pk.
authors.forename: Aamer Ali
authors.initials: AA
authors.lastname: Shah
mesh_terms: D000069977:Bacillus amyloliquefaciens / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism*; D018533:Biomass; D002480:Cellulase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D046508:Culture Techniques; D004795:Enzyme Stability / Q000187:drug effects; D045482:Hot Springs / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007221:Industry; D008670:Metals / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology*; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
chemical_list: D002614:Chelating Agents; D008670:Metals; D009930:Organic Chemicals; D012997:Solvents; D013501:Surface-Active Agents; D002480:Cellulase
keywords: Bacillus amyloliquefaciens AK9; Cellulase; Characterization; Organic solvents; Purification; SDS-Page; Surfactants