Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri

Daisuke Sugimori, Junki Ogasawara, Koki Okuda, Kazutaka Murayama

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583 U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66 kDa. The gene encoding the enzyme consists of a 1941-bp ORF that produces a signal peptide of 38 amino acids for secretion and a 646 amino acid mature protein with a calculated molecular mass of 70,447 Da. The maximum activity was found at pH 7.2 and 40°C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60°C) and within a narrow pH range near pH 7. The enzyme was stable at 50°C for 30 min and between pH 5-10.5. The enzyme exhibited specificity toward GPC and glycerol-3-phosphoethanolamine and hydrolyzed glycerol-3-phosphate and lysophosphatidylcholine. However, the enzyme showed no activity toward any diacylglycerophospholipids and little activity toward other glycerol-3-phosphodiesters and lysophospholipids. The enzyme was not inhibited in the presence of 2 mM SDS and Mg2+; however, Cu2+, Zn2+, and Co2+ remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca2+, Na+, EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37°C and pH 7.2, apparent Vmax and turnover number (kcat) were determined to be 24.7 μmol min-1 mg-protein-1 and 29.0 s-1, respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mM-1 s-1, respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes.

Original languageEnglish
Pages (from-to)422-430
Number of pages9
JournalJournal of Bioscience and Bioengineering
Volume117
Issue number4
DOIs
Publication statusPublished - 2014 Apr

Keywords

  • Characterization
  • Expression
  • Glycerophosphocholine cholinephosphodiesterase
  • Molecular cloning
  • New metabolic pathway
  • Purification
  • Streptomyces

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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