Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp strain C9

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidi c Linkage between oligosaccharides and ceramides of various glycosphingolip ids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active Site of EGCase of Rhodococcus sp, strain M-777. This paper descr ibes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly di stinguishable from M-777 by 16S rDNA analysis, C9 EG:Case possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respe ctively. Interestingly, C9 EGCase showed the different specificity to the M -777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly t han the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.