Gene cloning of Streptomyces phospholipase D P821 suitable for synthesis of phosphatidylserine

A strain, P821, with phospholipase D activitywas isolated from soil and identified as a Streptomycesspecies. The phospholipase D enzyme was purified from aprecipitation and DEAE-Sepharose, phenyl-Sepharose, andSuperose 12 HR column chromatographies. The purified enzymeexhibited an optimum temperature and pH of 55oC and 6.0,respectively, in the hydrolysis of phosphatidylcholine andremained stable up to 60oC within a pH range of 3.5-8.0. Theenzyme also catalyzed a transphosphatidylation reaction toproduce phosphatidylserine with phosphatidylcholine and serinewere 30oC and pH 5.0, indicating quite diferent optimumconditions for the hydrolysis and transphosphatidylationreactions. The gene encoding the enzyme was cloned bySouthern hybridization and colony hybridization using a DNAprobe designed from the conserved regions of other knownphospholipase D enzymes. The resulting amino acid sequencewas most similar to that of the PLD enzyme from Streptomyceshalstedii (89.5%). Therefore, the enzyme was confirmed to bephosphatidylserine. KCI Citation Count: 6