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

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,vmaxand kcatof 12.5mm,735 μmol mg-1 min-1 and 471.15s-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 (ah∗ 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-1,71.97 kj mol-1 and -64.7 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. © 2015 bentham science publishers.