Purification, characterization, cloning and sequencing of phospholipase D from Streptomyces septatus TH-2

Streptomyces septatus TH-2 produced 34 mg/l extracellular phospholipase D (PLD) when cultured in a medium containing glucose and citrate at 34 °C for 4 days. The enzyme was purified to homogeneity by a one-column ion exchange procedure. PLD from S. septatus TH-2 gave Km values of 0.99 and 0.67 mM for an artificial substrate, phosphatidyl-p-nitrophenol (PpNP), and kcat values of 86.7 and 258.8 s−1 in hydrolytic and transphosphatidylation reactions, respectively. The PLD gene from S. septatus TH-2 was cloned and sequenced. Although the primary sequences of PLDs from S. septatus TH-2 and a commercially available PLD from Streptomyces sp. showed a high homology, these two PLDs showed similar kcat values and different Km values for PpNP and ethanol in transphosphatidylation.