title: CenC, a multidomain thermostable GH9 processive endoglucanase from Clostridium thermocellum: cloning, characterization and saccharification studies.
abstract: The growing demands of bioenergy has led to the emphasis on novel cellulases to improve efficiency of biodegradation process of plant biomass. Therefore, a thermostable cellulolytic gene (CenC) with 3675 bp was cloned from Clostridium thermocellum and over-expressed in Escherichia coli strain BL21 CodonPlus. It was attested that CenC belongs to glycoside hydrolase family 9 (GH9) with four binding domains, a processive endoglucanase. CenC was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 137.11 kDa, by purification steps of heat treatment combined with ion-exchange chromatography. Purified enzyme displayed optimal activity at pH 6.0 and 70 °C. CenC had a half-life of 24 min at 74 °C, was stable up to 2 h at 60 °C and over a pH range of 5.5-7.5. Enzyme showed high affinity towards various substrates and processively released cellobiose from cellulosic substrates. It efficiently hydrolyzed carboxymethyl cellulose (30 U/mg), β-Glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-β-D-cellobioside (18 U/mg), birchwood xylan (19 U/mg), beechwood xylan (17.5 U/mg), avicel (9 U/mg), whatman filter paper (11 U/mg) and laminarin (3.3 U/mg). CenC exhibited Km, Vmax, Kcat, Vmax Km(-1) and Kcat Km(-1) of 7.14 mM, 52.4 µmol mg(-1) min(-1), 632.85 s(-1), 7.34 min(-1) and 88.63, respectively used CMC as substrate. Recombinant CenC saccharified pretreated wheat straw and bagasse to 5.12 and 7.31%, respectively at pH 7.0 and 45 °C after 2 h incubation. Its thermostability, high catalytic efficiency and independence of inhibitors make CenC enzyme an appropriate candidate for industrial applications and cost-effective saccharification process.
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan. ravianiib@yahoo.com.
authors.forename: Ikram ul
authors.initials: Iu
authors.lastname: Haq
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan. fatima_iib@yahoo.com.
authors.forename: Fatima
authors.initials: F
authors.lastname: Akram
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan.
authors.forename: Mahmood Ali
authors.initials: MA
authors.lastname: Khan
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan.
authors.forename: Zahid
authors.initials: Z
authors.lastname: Hussain
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan.
authors.forename: Ali
authors.initials: A
authors.lastname: Nawaz
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan.
authors.forename: Kaleem
authors.initials: K
authors.lastname: Iqbal
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan.
authors.forename: Ali Javed
authors.initials: AJ
authors.lastname: Shah
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002480:Cellulase
keywords: Clostridium thermocellum; Module protein; Processive endoglucanase; Saccharification; Thermostable