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.
Li
Yanhong
Y
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China.
Yin
Qiuyu
Q
Ding
Ming
M
Zhao
Fukun
F
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D005750:Gastric Juice / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008974:Mollusca / Q000201:enzymology*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D002480:Cellulase