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.
Keywords: CV, column volume; Characterization; DLS, dynamic light scattering; DMPA, 1,2-Dimyristoyl-sn-glycero-3-phosphate; DOPE, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; EGGL, lecithin from egg yolk; EcPLA1, phospholipase A1 from Escherichia coli; Expression; FFA, free fatty acid; LPC, l-α-Lysophosphatidylcholine; PC, l-α-phosphatidylcholine; PG, l-α-phosphatidylglycerol; PI, l-α-phosphatidylinositol; PLA1, phospholipase A1; PLA2, phospholipase A2; PLD, phospholipase D; POPA, 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate; POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol); PS, l-α-phosphatidyl-l-serine; Phospholipase A1; Purification; SBL, lecithin from soybean; SMPLA1, phospholipase A1 from Serratia sp. MK1; SaEst, esterase of Streptomyces albus J1074; SaPLA1, phospholipase A1 from Streptomyces albidoflavus; SsEst, esterase from S. scabies; Streptomyces albidoflavus; SxPLA1, phospholipase A1 from Serratia sp. xjF1; TSB, tryptic soy broth; pNPB, p-nitrophenyl butyrate; pNPD, p-nitrophenyl decanoate; pNPL, p-nitrophenyl laurate; pNPO, p-nitrophenyl octanoate; pNPP, p-nitrophenyl palmitate; pNPS, p-nitrophenyl stearate.