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

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)22-28
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume338
Issue number1
DOIs
Publication statusPublished - Feb 1 1997
Externally publishedYes

Fingerprint

Arthrobacter
Acetylglucosaminidase
Cloning
amidase
Escherichia coli
Organism Cloning
Periplasm
Genes
Amino Acids
Chitinases
Peptides
Gene encoding
Asparagine
Enzyme activity
Protein Sorting Signals
Chryseobacterium
Nucleotides
Caenorhabditis elegans
Streptomyces
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Cloning, sequencing, and expression of Arthrobacter protophormiae Endo- β-N-acetylglucosaminidase in Escherichia coli. / Takegawa, Kaoru; Yamabe, Kayo; Fujita, Kiyotaka; Tabuchi, Mitsuaki; Mita, Masanori; Izu, Hiroyuki; Watanabe, Akira; Asada, Yasuhiko; Sano, Mutsumi; Kondo, Akihiro; Kato, Ikunoshin; Iwahara, Shojiro.

In: Archives of Biochemistry and Biophysics, Vol. 338, No. 1, 01.02.1997, p. 22-28.

Research output: Contribution to journalArticle

Takegawa, K, Yamabe, K, Fujita, K, Tabuchi, M, Mita, M, Izu, H, Watanabe, A, Asada, Y, Sano, M, Kondo, A, Kato, I & Iwahara, S 1997, 'Cloning, sequencing, and expression of Arthrobacter protophormiae Endo- β-N-acetylglucosaminidase in Escherichia coli', Archives of Biochemistry and Biophysics, vol. 338, no. 1, pp. 22-28. https://doi.org/10.1006/abbi.1996.9803
Takegawa, Kaoru ; Yamabe, Kayo ; Fujita, Kiyotaka ; Tabuchi, Mitsuaki ; Mita, Masanori ; Izu, Hiroyuki ; Watanabe, Akira ; Asada, Yasuhiko ; Sano, Mutsumi ; Kondo, Akihiro ; Kato, Ikunoshin ; Iwahara, Shojiro. / Cloning, sequencing, and expression of Arthrobacter protophormiae Endo- β-N-acetylglucosaminidase in Escherichia coli. In: Archives of Biochemistry and Biophysics. 1997 ; Vol. 338, No. 1. pp. 22-28.
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