Cloning, Sequencing, and Expression ofArthrobacter protophormiaeEndo-β-N-acetylglucosaminidase inEscherichia coli

The gene encoding endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae(Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology withF01F.10gene product fromCaenorhabditis elegansand weak homology with peptide-N4-(N-acetyl-β-d-glucosaminyl)asparagine amidase fromFlavobacterium meningosepticumand chitinase fromStreptomyces olivaceoviridis.However, the enzyme had no significant homology with any previously reported endo-β-N-acetylglucosaminidases. TransformedEscherichia colicells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.