Molecular cloning and characterization of a novel bacteriophage-associated sialidase

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

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 σ 70. 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.

Original languageEnglish (US)
Pages (from-to)62-68
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume90
Issue number1
DOIs
StatePublished - Jul 2000
Externally publishedYes

Fingerprint

Bacteriophages
Cloning
Neuraminidase
Molecular Cloning
Enzymes
Escherichia coli
Open Reading Frames
Amino acids
Gels
Proteins
Amino Acids
Genomic Library
Polypeptides
Molecular mass
Sewage
Nucleotides
Polyacrylates
Electrophoresis
Purification
Gel Chromatography

Keywords

  • Bacteriophage
  • Escherichia coli K1
  • Polysialic acid
  • Sialidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering

Cite this

Molecular cloning and characterization of a novel bacteriophage-associated sialidase. / Machida, Yuichi; Hattori, Kouji; Miyake, Katsuhide; Kawase, Yuji; Kawase, Mitsuo; Iijima, Shinji.

In: Journal of Bioscience and Bioengineering, Vol. 90, No. 1, 07.2000, p. 62-68.

Research output: Contribution to journalArticle

Machida, Yuichi ; Hattori, Kouji ; Miyake, Katsuhide ; Kawase, Yuji ; Kawase, Mitsuo ; Iijima, Shinji. / Molecular cloning and characterization of a novel bacteriophage-associated sialidase. In: Journal of Bioscience and Bioengineering. 2000 ; Vol. 90, No. 1. pp. 62-68.
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