Abstract
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 V
max and k
cat values of 1389 μmol min−1 mg protein−1 and 630 s−1, respectively. The apparent K
m and k
cat/K
m 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.