Molecular cloning, purification, and characterization of a novel, acidic, pH-stable endoglucanase from Martelella mediterranea
A highly alkaline endo-1,4-β-glucanase (Egl) was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-N252. The optimal pH for activity was as high as 10, and the optimal temperature was 55°C. The molecular mass and isoelectric point were around 50 kDa and pH 4.2, respectively. The enzyme hydrolyzed carboxymethyl cellulose in a random fashion. Unlike previously reported Egls, the enzyme was highly active on p-nitrophenyl cello-oligosaccharides and acid-swollen cellulose, and its activity was stimulated by cellobiose at high concentrations. The entire gene for the enzyme contained a 1,476-bp single open reading frame encoding 492 amino acids, including a 29-amino-acid signal peptide. The mature enzyme (463 amino acids: 51,174 Da) exhibited moderate homology to other family 5 alkaline Egls. In the C-terminal region, a carbohydrate-binding module that belongs to family XII was repeated. Furthermore, four and six repeats of Pro-Pro-Ser/Thr-Glu/Asp-Pro-(Glu) were found immediately before the first and second carbohydrate-binding modules, respectively.