Cloning, sequencing, and expression of Arthrobacter protophormiae Endo- β-N-acetylglucosaminidase in Escherichia coli

Kaoru Takegawa, Kayo Yamabe, Kiyotaka Fujita, Mitsuaki Tabuchi, Masanori Mita, Hiroyuki Izu, Akira Watanabe, Yasuhiko Asada, Mutsumi Sano, Akihiro Kondo, Ikunoshin Kato, Shojiro Iwahara

研究成果: ジャーナルへの寄稿記事

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The gene encoding endo-β-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 F01F. 10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-β-D- glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-β-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.

元の言語英語
ページ(範囲)22-28
ページ数7
ジャーナルArchives of Biochemistry and Biophysics
338
発行部数1
DOI
出版物ステータス出版済み - 2 1 1997
外部発表Yes

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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