Production of lysophospholipid using phospholipase $A_1$ from serratia sp. MK1

Abstract

For the phospholipase $A_1$ preparation as a reaction catalyst, factors affecting phospholipase $A_1$ production by Serratia sp. MK1 has been investigated. Serratia sp. MK1 showed maximum phospholipase $A_1$ production at $30^\circ C$ and at pH 7.0 in 14 hours. The M9 minimal medium supplemented with fructose and $(NH_4)_2HPO_3$ as a carbon and nitrogen sources, respectively, showed improved enzyme production. $FeSO_4$ stimulated enzyme production and its optimal concentration was 1$\mu M$ in a growth medium. As a result of above optimization procedure, improved minimal medium could be formulated, and this medium resulted in about seven fold increase enzyme production as compared to the initial complex medium. General properties of phospholipase $A_1$ were also examined. The phospholipase $A_1$ showed its hydrolysis optimum pH and temperature at 8.0 and $40^\circ C$, respectively. The bile salts showed either an activating or inhibitoring effects, depending on the concentration. However, below 3mM concentration, addition of cholate and deoxycholate showed stimulatory effect on the enzyme action. Divalent metal ion was essential for enzyme action. Finally, the production of lysophospholipid by hydrolysis of phospholipid using phospholipase $A_1$ was investigated in two-phase and emulsion systems. Considering the yield of reaction products and efficiency of reaction, aqueous-solvent two-phase system was more appropriate than emulsion system for the lysophospholipid production. Among the 13 organic solvents tested as a solvent-phase, butyl acetate was selected as the most suitable solvent in terms of catalytic activity and enzyme stability. The optimal substrate concentration in butyl acetate was 5-10\%. Under the selected conditions, the reaction was carried out and the result of TLC/FID showed that phospholipid was hydrolyzed completely to the lysophospholipid in this system.