The C-terminus region of endo-beta-glucanase Eg1499 from Bacillus subtilis JA18 was suggested to be a putative family 3 cellulose-binding domain (CBD) by computer analysis. To prove this proposal, C-terminus truncation mutant Eg1330 was constructed and expressed. Compared with Eg1499, Eg1330 lost the cellulose binding capability at 4 degrees C, confirming the C-terminus region was a CBD. Binding of the CBD to Avicel was inhibited by carboxymethylcellulose (CMC), but not by barley beta-glucan and glucose at concentration of 0.1% and 0.5%. Kinetic analysis showed both the turnover rate (k(cat)) and the catalytic efficiency (k(car)/K-m) of Eg1330 increased for the substrate CMC compared to Eg1499. A great improvement in thermal stability was observed in Eg1330. The half life of Eg1330 at 65 degrees C increased to three folds that of Eg1499, from 10 to 29 min. After treated at 80 degrees C for 10 min, Eg1330 could recover more than 60% of its original activity while Eg1499 only recovered 12% activity. UV spectrometry analysis showed Eg1330 and Eg1499 differed in refolding efficiency after heat treatment. (C) 2008 Elsevier Ltd. All rights reserved.