Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene fron Rhodoccocus species strain M-777

Hiroyuki Izu, Yoshiya Izumi, Yoko Kurome, Mutsumi Sano, Akihiro Kondo, Ikunoshin Kato, Makoto Ito

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

Endoglycoceramidase (EGCase (EC 3.2.1.123)) is a hydrolase that hydrolyzes the linkage between the oligosaccharide and ceramide of various glycosphingolipids. This paper describes the molecular cloning and expression of EGCase II, one of the isoforms of EGCases. The gene encoding EGCase II was obtained by screening of a genomic DNA library from Rhodococcus sp. strain M- 777 constructed in pUC19 with oligonucleotide probes deduced from a partial amino acid sequence of the enzyme protein. Recombinant Escherichia coli cells in which the EGCase II gene was expressed produced 14 units of the enzyme per liter of culture medium but did not produce sphingomyelinase. Recombinant EGCase II was a functioning enzyme with substrate specificity identical to that of the wild-type enzyme. Sequence analysis showed the presence of an open reading frame of 1470 base pairs encoding 490 amino acids. The N- terminal region of the deduced amino acid sequence had the general pattern of signal peptides of secreted prokaryotic proteins. Interestingly, the consensus sequence in the active site region of the endo-1,4-β-glucanase family A was found in the amino acid sequence of EGCase II.

Original languageEnglish
Pages (from-to)19846-19850
Number of pages5
JournalJournal of Biological Chemistry
Volume272
Issue number32
DOIs
Publication statusPublished - Aug 8 1997

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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