Purification and characterization of a novel endo-β-1,4-glucanase, AfEG22, from the giant snail, Achatina fulica frussac

In this study, we confirmed that at least three endo-beta-1,4-glucanases existed in the digestive juice of the giant snail, Achatina fulica ferussac, by Congo red staining assay. One of these enzymes, a novel endo-beta-1,4-glucanase (AfEG22), was purified 29.5-fold by gel filtration, anion exchange, and hydrophobic interaction chromatography. The carboxymethyl cellulose (CMC) hydrolytic activity of the purified enzyme was 12.3 U/mg protein. The molecular mass of AfEG22 was 22081 Da determined by MALDI-TOF. N-terminal amino acid sequencing revealed a sequence of EQRCTNQGGILKYYNT, which did not have significant homology with any proteins in BLAST database. The optimal pH and temperature for hydrolytic activity toward CMC were pH 4.0 and 50 degrees C, respectively. AfEG22 was stable between pH 3.0 and pH 12.0 when incubated at 4 degrees C for 3 h or at 37 degrees C for 1 h. The enzyme remained more than 80% activity between pH 4.5 and pH 7.0 after incubation at 50 degrees C for 1 h. AfEG22 possessed excellent thermostability as more than 70% activity was remained after incubation at 60 degrees C for 3 h. Substrate specific analysis revealed that AfEG22 was a typical endo-beta-1,4-glucanase. This is the first time to report a novel endo-beta-1,4-glucanase with high stability from the digestive juice of A. fulica.