Molecular cloning, expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1.
An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.
Takenak
Akio
A
Department of Animal Nutrition, National Institute of Animal Industry, Tsukuba Norindanchi, Ibaraki 305-0901, Japan.
D'Silva
Colin G.
CG
Kudo
Hiroshi
H
Itabashi
Hisao
H
Cheng
Kuo-Joan
KJ