Cloning, sequencing, and expression of Arthrobacter protophormiae 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 determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1997-02, Vol.338 (1), p.22-28
Main Authors: Takegawa, K, Yamabe, K, Fujita, K, Tabuchi, M, Mita, M, Izu, H, Watanabe, A, Asada, Y, Sano, M, Kondo, A, Kato, I, Iwahara, S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Arthrobacter - enzymology
Arthrobacter - genetics
Base Sequence
Cloning, Molecular
DNA Primers - genetics
DNA, Bacterial - genetics
Escherichia coli - genetics
Gene Expression
Genes, Bacterial
Glycosylation
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - genetics
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism
Molecular Sequence Data
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
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Summary:The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with FO1F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme 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 was 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-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.
ISSN:0003-9861
DOI:10.1006/abbi.1996.9803