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Purification, Characterization, Molecular Cloning and Extracellular Production of a Phospholipase A1 From Streptomyces Albidoflavus NA297

Purification, Characterization, Molecular Cloning and Extracellular Production of a Phospholipase A1 From Streptomyces Albidoflavus NA297

Daisuke Sugimori, Kota Kano, Yusaku Matsumoto

FEBS Open Bio. 2012 Sep 28;2:318-27.

PMID: 23772365

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 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9008304-D	L-α-Lysophosphatidylcholine from egg yolk		9008-30-4	Price

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