Truncation of the cellulose binding domain improved thermal stability of endo-beta-1,4-glucanase from Bacillus subtilis JA18.

The C-terminus region of endo-beta-glucanase Egl499 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 Egl330 was constructed and expressed. Compared with Egl499, Egl330 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(cat)/K(m)) of Egl330 increased for the substrate CMC compared to Egl499. A great improvement in thermal stability was observed in Egl330. The half life of Egl330 at 65 degrees C increased to three folds that of Egl499, from 10 to 29 min. After treated at 80 degrees C for 10 min, Egl330 could recover more than 60% of its original activity while Egl499 only recovered 12% activity. UV spectrometry analysis showed Egl330 and Egl499 differed in refolding efficiency after heat treatment.