Purification, characterization and molecular cloning of a novel endo-beta-1,4-glucanase AC-EG65 from the mollusc Ampullariacrossean.

A novel endo-beta-1,4-glucanase, AC-EG65, with a molecular mass of 65 kDa, was purified from the gastric juice of the mollusc, Ampullaria crossean, by ammonium sulfate fractionation, anion exchange, gel filtration, hydrophobic interaction and a second round of anion exchange chromatography. AC-EG65 showed specific carboxymethyl cellulose hydrolytic activity of 13.3 U/mg protein and the optimal pH and temperature of the activity were pH 5.5-6.5 and 50-55 degrees C, respectively. From the cDNA library of A. crossean stomach tissue, eight endo-beta-1,4-glucanase genes with high similarity were successfully cloned based on the partial amino acid sequences of AC-EG65 and were classified into 3 groups: eg65-a, eg65-b, and eg65-c. The open reading frames of the groups eg65-a, eg65-b, and eg65-c were 2142 bp, 2171 bp, and 2169 bp in length, encoding 713, 723 and 722 amino acids, respectively. The eight deduced proteins consisted of a family II carbohydrate-binding module (CBM2) and a glycosyl hydrolase family 9 (GHF9) catalytic domain. More than 98% amino acid identities were shared within the same group and more than 87% sequence identities among the groups. The endogenous origins of these EGase genes were supported by PCR amplification using ovary genomic DNA as template.