A novel phospholipase B from Streptomyces sp. NA684 - Purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

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, V_max and k_cat 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.