Molecular cloning and characterization of a novel bacteriophage-associated sialidase

Yuichi Machida, Kouji Hattori, Katsuhide Miyake, Yuji Kawase, Mitsuo Kawase, Shinji Iijima

研究成果: Article査読

10 被引用数 (Scopus)

抄録

Bacteriophage 63D, previously isolated from sewage, is associated with α-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for σ70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.

本文言語English
ページ(範囲)62-68
ページ数7
ジャーナルJournal of Bioscience and Bioengineering
90
1
DOI
出版ステータスPublished - 2000
外部発表はい

ASJC Scopus subject areas

  • バイオテクノロジー
  • バイオエンジニアリング
  • 応用微生物学とバイオテクノロジー

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