A novel metal ion-independent phospholipase B (PLB684) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 U·mg protein-1). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50 °C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K m, Vmax and kcat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mm, 15.8 mmol·min-1·mg protein-1 and 1.02 × 104 s-1, respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1: sn-2) was 45: 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB684 and that the active site may include residues Ser330 and His332. Phospholipase B (PLB684) from Streptomyces sp. NA684 shows a new catalytic property producing lysophospholipid during hydrolysis of diacylglycerophospholipid. The catalytic functions of PLB684 are apparently different from those of fungal PLBs that accumulate no lysophospholipids. PLB684 is a new type of PLB that hydrolyzes diacylphospholipid with the order different twice reaction.
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