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

Abstract

A novel metal ion-independent phospholipase B (PLB 684 ) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 UÁmg protein À1 ). 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. PLB 684 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 mmolÁmin À1 Ámg protein À1 and 1.02 9 10 4 s À1 , 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 PLB 684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB 684 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 PLB 684 and that the active site may include residues Ser330 and His332.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

“…In particular, a secreted PLA 2 produced by Streptomyces violaceoruber A-2688 isolated from a soil sample, was described as the first PLA 2 identified in prokaryote (Sugiyama et al, 2002; Takemori et al, 2012). Similarly, a PLA 1 and a phospholipase B have also been reported in the Streptomyces genus (Sugimori, Kano & Matsumoto, 2012; Matsumoto et al, 2013). Some authors have also reported the presence of lipases in actinomycete strains (Sztajer, Maliszewska & Wieczorek, 1988) while others did not observe the production of lipolytic enzymes (Basha et al, 2009).…”

Cloning, expression and characterisation of phospholipase B fromSaccharomyces cerevisiaeand its application in the synthesis ofl-alpha-glycerylphosphorylcholine and peanut oil degumming

“…The optimal pH for the rPLB enzyme was 5.5 (Figure 2b), which is more acidic than the optimum pH for the PLB from Streptomyces sp. (pH 8.4) [23]. In addition, rPLB retained more than 35% of its maximum activity over a broad pH range of 4.5-8.5 (Figure 2), whereas other PLBs display no or low activity at pH values ranging from 5.0-9.0 [6,8,10,11].…”

A Thermolabile Phospholipase B from Talaromyces marneffei GD-0079: Biochemical Characterization and Structure Dynamics Study

“…Doi and Nojima reported that lysophospholipase (Phospholipase B) activity was inhibited by 10 mM Fe +3 , and Al +3 . He also reported that Ca +2 also affects the phospholipase B activity and stability [4,44]. Divalent metal ions affects phospholipids by forming complexes and affects the hydration of phosphate group [4,[45][46][47] (p > 0.05).…”

“…They showed high preference for substrate from lysophospholipids to diacylphospholipids and hydrolyzes diacylphospholipids without forming lysophospholipids. Some of the phospholipase B enzyme having lysophospholipase-transacylase activity which transfers free fatty acids to lysophospholipids and forms diacylphospholipid [4].…”