No abstract

Two novel endo-beta-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 degrees and 60 degrees; for EG45 it was 50 degrees. The analysis on the stability of these two endo-beta-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 degrees, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 degrees for 24 h. However, less than 10% residual activity of EG45 was detected at 50 degrees. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-beta-D-cellobioside, salicin and starch.

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.