Aqueous–Solid System for Highly Efficient and Environmentally Friendly Transphosphatidylation Catalyzed by Phospholipase D To Produce Phosphatidylserine

Abstract

The purely aqueous system of phospholipase D (PLD)-mediated transphosphatidylation using pre-existing carriers for the adsorption of phosphatidylcholine (PC) to act as an "artificial interface" was introduced to replace the liquid-liquid system. Toxic organic solvents are avoided during the reaction, and the free enzyme can be simply reused by centrifugation. Special attention has been paid to the effect of the pore diameter and surface area of silica gel 60H covered with PC molecules on the yield of phosphatidylserine (PS). Results indicated that the highest PS yield of 99.5% was achieved. Moreover, 73.6% of the yield of PS was obtained after being used for six batches. This is the first description of the remarkably high reusability of free enzymes for enzymatic synthesis of PS as well. The excellent results make the aqueous-solid system more promising candidates for the industrial production of PS.

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“…Samples (30 μ L) were taken from the reaction mixture at specified time intervals, intermittently mixed with 10 μ L of 1 N HCl to inactivate the enzyme. The phospholipid composition of the product was measured as described previously …”

“…The surface of NSD which acted as an “artificial interphase” between PC and PLD, allowed creation of a hydrophobic microenvironment and, thus, minimized hydrolysis. A similar phenomenon has also been observed in our previous work …”

“…Even if PC did not deposit on surfaces of carriers, the precipitant (acetone) would make them self‐aggregate and precipitate in solution, leading to no obvious difference of the PC loading in all experiments. Compared with reported PC loading (90.5%) during silica gel G as the carrier, the PC loading was improved in this work (98.4%) to avoided the waste of PC. It may be explained that NSD with a very small size can facilitate collisions between PC and carriers to decrease the mass transfer resistance and enhance the amount of PC adsorbed …”

“…Ten milligrams of NSD were added into the solution, and then 1 mL of acetone was added. The mixture was incubated at room temperature and 400 rpm for 3 h. The amount of PC adsorbed was determined as described previously . The NSD‐adsorbed PC was collected by centrifugation (3500 g, 20 min, 15°C).…”

“…With regard to PB as medicine for human being and increasing public health concerns, however, such toxic organic solvents should be avoided so as to decrease the residual solvent level and raise the safety of the product. In this field, a highly efficient and environmentally friendly aqueous–solid system for transphosphatidylation has been introduced in authors’ recent work . The surface of silica was employed as an “artificial interphase” between PC and PLD.…”