title: Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host.
abstract: The thermotolerant endo-1,4-β-glucanase gene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus. Enzyme was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 38 kDa, by purification steps of heat treatment combined with ion-exchange column chromatography. The purified enzyme was optimally active, with specific activity of 530 Umg(-1) against carboxymethyl cellulose (CMC), at pH 6.0 and 95°C and was also stable upto 8 h at 80°C. The enzyme also showed activity against β-glucan barley: 303 %, laminarin: 13.7 %, Whatman filter paper: 0.017 % with no activity against starch and Avicel. The recombinant enzyme exhibited Km, Vmax and Kcat of 12.5 mM, 735 µmol mg-1min-1 and 2351.23 s-1, respectively against CMC as a substrate. The stable recombinant enzyme manifested half life (t1/2) of 6.6 min even at temperature as high as 97°C, with free energy of denaturation (ΔG*D), enthalpy of denaturation (ΔH*D), and entropy of denaturation (ΔS*D) of 98.2 kJ mol(-1), 528.9 kJ mol(-1), and 1.17 kJ mol(-1)K(-1), respectively at 97°C. In addition, the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for hydrolysis of CMC substrate by endo-1,4-β-glucanase were calculated at 95°C as 48.2 kJ mol(-1), 54.6 kJ mol(-1) and -17.4 J mol(-1) K(-1), respectively. The recombinant enzyme saccharified pre-treated wheat straw and bagasse to 3.32 % and 3.2 %, respectively after 6 h incubation at 85°C. Its thermostability, resistance to heavy metal ions and specific activity make this enzyme an interesting candidate for industrial applications.
authors.forename: Ikram
authors.initials: I
authors.lastname: Ul Haq
authors.forename: Bushra
authors.initials: B
authors.lastname: Muneer
authors.forename: Zahid
authors.initials: Z
authors.lastname: Hussain
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore- 54000, Pakistan. chemistrym@hotmail.com.
authors.forename: Mahmood A
authors.initials: MA
authors.lastname: Khan
authors.forename: Sumra
authors.initials: S
authors.lastname: Afzal
authors.forename: Sana
authors.initials: S
authors.lastname: Majeed
authors.forename: Fatima
authors.initials: F
authors.lastname: Akram
authors.forename: Saira
authors.initials: S
authors.lastname: Akmal
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002480:Cellulase