Purification, characterization, molecular cloning and extracellular production of a phospholipase A1 from Streptomyces albidoflavus NA297

A novel metal ion-independent phospholipase A1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 °C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 °C and pH 7.2 gave apparent Vmax and kcat values of 1389 μmol min−1 mg protein−1 and 630 s−1, respectively. The apparent Km and kcat/Km values were 2.38 mM and 265 mM−1 s−1, respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.

▸ A novel metal ion-independent phospholipase A1 has been purified and characterized. ▸ We report the positional specific hydrolysis of glycerophospholipids. ▸ The enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine. ▸ The apparent Km, kcat, and Kcat/Km values were 2.38 mM, 630 s−1 and 265 mM−1 s−1. ▸ We demonstrate that the active site should be composed of Ser11, S216, and H218.