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 journalArticle

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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

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endoglycoceramidase
Cloning
Molecular Cloning
Sequence Analysis
Genes
Amino Acids
Amino Acid Sequence
Enzymes
Sphingomyelin Phosphodiesterase
Rhodococcus
Glycosphingolipids
Gene encoding
Genomic Library
Oligonucleotide Probes
Ceramides
Consensus Sequence
Hydrolases
Protein Sorting Signals
Substrate Specificity
Oligosaccharides

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene fron Rhodoccocus species strain M-777. / Izu, Hiroyuki; Izumi, Yoshiya; Kurome, Yoko; Sano, Mutsumi; Kondo, Akihiro; Kato, Ikunoshin; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 272, No. 32, 08.08.1997, p. 19846-19850.

Research output: Contribution to journalArticle

Izu, Hiroyuki ; Izumi, Yoshiya ; Kurome, Yoko ; Sano, Mutsumi ; Kondo, Akihiro ; Kato, Ikunoshin ; Ito, Makoto. / Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene fron Rhodoccocus species strain M-777. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 32. pp. 19846-19850.
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