Cloning, functional expression and purification of endo-beta-galactosidase from Flavobacterium keratolyticus.

Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15000-fold to apparent homogeneity. The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli.