CLONING, SEQUENCING, AND EXPRESSION OF ARTHROBACTER-PROTAPHORMIAE ENDO-BETA-N-ACETYLGLUCOSAMINIDASE IN ESCHERICHIA-COLI

The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was det ermined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptide of 24 amino acids a nd a mature protein of 621 amino acids was found. The primary structur e of Endo-A exhibited significant homology with F01.10 gene product fr om Caenorhabditis elegans and weak homology with tide-N-4-(N-acetyl-be ta-D-glucosaminyl)-asparagine amidase from Flavobacterium meningosepti cum and chitinase from Streptomyces olivaceoviridis. However, the enzy me had no significant homology with any previously reported, endo-beta -N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity w as 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-t erminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed ve ry similar optimum pH profiles and transglycosylation activity. (C) 19 97 Academic Press.