Purification, characterization, molecular cloning and extracellular production of a phospholipase A₁ from Streptomyces albidoflavus NA297

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 Vmax and kcat values of 1389 μmol min−1 mg protein−1 and 630 s−1, respectively. The apparent Km and kcat/Km 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.

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

“…This catalytic site resembles that of SsEst (1ESC) with a Ser-His dyad and the carbonyl group of Trp280. The active site of PLB 684 may correspond to that of SaPLA 1 , which was previously reported by Sugimori et al [20,33]. Molecular modelling indicated that His73 may stabilize the phosphoric group of the bound phospholipid, thereby imparting the substrate specificity that PLB 684 has towards phospholipids over 4-nitrophenyl esters.…”

“…PLB 684 has a much higher turnover rate and somewhat low affinity toward phospholipid, indicating that the formation of the ES may be the rate limiting step. The k cat /K m value (703 mM À1 Ás À1 ) of PLB 684 is higher than that of SaPLA 1 (265 mM À1 Ás À1 ) [20] and is comparable to that of SMC (756 mM À1 Ás À1 ) [43].…”

“…The k cat value of PLB 684 is higher than the k cat values of these enzymes. However, the K m (14.5 mM) value of PLB 684 is higher than those of fungal PLBs (0.05-2.9 mM) [6,9,15,33,44], SMC (0.458 mM) [43] and SaPLA 1 (2.38 mM) [20]. PLB 684 has a much higher turnover rate and somewhat low affinity toward phospholipid, indicating that the formation of the ES may be the rate limiting step.…”

“…NA684 probably produces PLB 684 to utilize glycerophospholipids as energy and phosphate sources from the biomass in the environment. [20], respectively. The k cat value of PLB 684 is higher than the k cat values of these enzymes.…”

“…For internal amino acid sequencing, the proteins from the excised SDS/PAGE gel were digested by trypsin (Sequencing Grade Modified Trypsin; Promega Corp.) and the resulting peptides were extracted in accordance with the method of Shevchenko et al [55]. The extracted peptides were separated using a nano ACQUITY UPLC Ò BEH130 C18 column (Waters Corp., Milford, MA, USA; 75 lm 9 150 mm 9 1.7 lm) and a Xevo QTOF MS as described previously [20]. The collision gas was argon and the collision energy was varied to monitor the product ions of interest.…”