Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host

Title:Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host

Volume: 22 Issue: 9

Author(s): Ikram ul Haq, Bushra Muneer, Zahid Hussain, Mahmood A. Khan, Sumra Afzal, Sana Majeed, Fatima Akram and Saira Akmal

Affiliation:

Keywords: Endo-1, 4-, saccharification, thermostable, thermodynamics, Thermotoga petrophila.

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 K_m, V_max and K_cat 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^-1K^-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.

Cite this article as:

Haq ul Ikram, Muneer Bushra, Hussain Zahid, Khan A. Mahmood, Afzal Sumra, Majeed Sana, Akram Fatima and Akmal Saira, Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host, Protein & Peptide Letters 2015; 22 (9) . https://dx.doi.org/10.2174/0929866522666150630105035