Molecular Cloning, Expression, and Sequence Analysis of the Endoglycoceramidase II Gene from Rhodococcus Species Strain M-777
| Autor: | Yoshiya Onojyo-shi Izumi, Hiroyuki Izu, Akihiro Kondo, Yoko Kusatsu-shi Kurome, Mutsumi Sano, Ikunoshin Kato, Makoto Ito |
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| Rok vydání: | 1997 |
| Předmět: |
DNA
Bacterial Signal peptide Glycoside Hydrolases Sequence analysis Molecular Sequence Data Gene Expression Molecular cloning Biology Biochemistry Open Reading Frames Cellulase Hydrolase Consensus sequence Rhodococcus Amino Acid Sequence Cloning Molecular Molecular Biology Peptide sequence Gene chemistry.chemical_classification Base Sequence Sequence Analysis DNA Cell Biology Molecular biology Amino acid Isoenzymes chemistry Sequence Alignment |
| Zdroj: | Journal of Biological Chemistry. 272:19846-19850 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.272.32.19846 |
| Popis: | 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. RecombinantEscherichia 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. |
| Databáze: | OpenAIRE |
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