Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum

Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40 kDa) and CD structural studies showed a conserved alpha/beta structure at different temperatures (25 and 70 degrees C) and high thermoresistance (Tm of 88 degrees C). Interestingly, the enzyme showed high endo-beta-1,4-mannanase activity vs various mannans, but low endo-beta-1,4 glucanase activity towards carboxymethylcellulose. The KM and V-max of DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 mu mol/min mg and 1.83 mg/ml and 1.349 mu mol/min mg, respectively. Its optimal activity towards temperature and pH resulted to be 70 degrees C and pH 5.4, respectively. Further characterization highlighted good thermal stability (similar to 50% of enzymatic activity after 2 h at 70 degrees C) and pH stability over a broad range (> 90% of activity after 1 h in buffer, ranging pH 5-9); resistance to chemicals was also observed.