Journal of Bioscience and Bioengineering, Vol.117, No.4, 422-430, 2014
Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri
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 degrees C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60 degrees C) and within a narrow pH range near pH 7. The enzyme was stable at 50 degrees 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 degrees C and pH 72, apparent V-max and turnover number (k(cat)) were determined to be 24.7 mu mol 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. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
Keywords:Glycerophosphocholine cholinephosphodiesterase;Purification;Characterization;Molecular cloning;Expression;New metabolic pathway;Streptomyces