Characterization and cloning of phospholipase A1 produced by microorganism isolated from soil

Abstract

A simple and rapid screening method for microorganisms with phospholipase $A_1$, $A_2$ and C activities using agar plate and gas chromatography (GC) method was successfully carried out. In agar plate method, soy bean lecithin and taurocholic acid were used as carbon source and emulsifier, respectively. In this agar plate method, microorganisms with phospholipase $A_1$ and $A_2$ or C activity produce a halo around the colony and two kinds(A``s and C) of microorganisms are clearly distinguished by turbidity of the halo. Microorganisms with phospholipase $A_1$ and $A_2$ activity is simply distinguished by GC using a synthetic phospholipid containing different fatty acid at sn-1 and sn-2 position. Using this screen method, a bacterium which has phospholipase $A_1$ activity was isolated from soil. This microorganism was aerobic, motile, oxidase negative and had flagella. The G + C content of the DNA base was 58.1 mol%. The major isoprenoids of cell wall were Q-8 and MK-8. The main cellular fatty acids were saturated straight chain(n-16) and cyclic(17:△) fatty acid. Based on their morphological, physiological and chemotaxonomic characteristics, this strain was placed in the genus Serratia and named MK1. Nutritional factors affecting enzyme production were explored. Xylose and ammonium sulfate were found to be the best carbon and nitrogen sources, respectively. Ferrous ion exerted a positive effect on enzyme production considerably. The optimal pH and temperature for phospholipase $A_1$ production were determined to be about 7.0 and 30℃, respectively. A novel type of extracellular phospholipase $A_1$ was isolated from Serratia sp. MK1 and purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration. The purified enzyme was a monomer with a molecular mass of about 43,000 Da. This enzyme showed the highest lipolytic activity toward phosphatidylserine among the phosphoglycerides tested, and preferentially catalyzed the hydro...