Membrane assisted coupled enzyme system for phospholipid modification

Phospholipase D obtained from a culture of Streptomyces sp. strain PMF, was immobilised in a hollow fiber membrane by ultrafiltration. The reactor was used to contact an organic solution containing phosphatidylcholine (PC) and a water phase; the rate of formation of the hydrolysis product (phosphatidic acid, PA) depending on different parameters (pH, temperature, PC concentration and enzyme units) was measured. Subsequently, the water phase was replaced with a 2-3 M solution of different alcohols (L-serine, glycerol and ethanolamine) and the rate of transphosphatidylation was studied and finally optimized using glycerol as model alcohol. The ratio of formation of phosphatidylglycerol (PG) (transphosphatidylation reaction) over PA (hydrolysis reaction) was minimized working at pH 4.5 where hydrolysis rate is lower. The operational stability of the system was excellent during a period of several months. Purification of the reaction mixture from PA was effected by selectively hydrolysing it with phosphatase (Pase). Co-immobilization of PLD and Pase resulted in effective PA removal. Although the space-time yield of the transphosphatidylation reaction is lower than the one observed in a biphasic system, product purification is unnecessary, product isolation easier and enzyme stability higher.