Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• 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℃. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60℃) and within a narrow pH range near pH 7. The enzyme was stable at 50℃ 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 Mg^<2+> however, Cu^<2+>, Zn^<2+>, and Co^<2+> remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca^<2+>, Na^+, EDTA, DIT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37℃ and pH 7.2, apparent V_<max> and turnover number (k_<cat>) were determined to be 24.7 μmol min^<-1> mg-protein^<-1> and 29.0 s^<-1> respectively. The apparent K_m and k_<cat>/K_m 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.

著者

• Sugimori Daisuke

  Department Of Chemistry And Biology Engineering Fukui National College Of Technology

• Ogasawara Junki

  Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University

• Okuda Koki

  Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University

• Murayama Kazutaka

  Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University

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