Lipase‐catalyzed acidolysis and phospholipase D‐catalyzed transphosphatidylation of phosphocholine

Abstract

Lipase-catalyzed acidolysis and phospholipase D-catalyzed transphosphatidylation of phosphocholineTwo approaches on enzymatic phospholipid modification were studied: (1) transphosphatidylation of the 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and ethanolamine in biphasic and anhydrous organic solvent systems by phospholipase D (PLD) and (2) incorporation of oleic acid into the sn1-position of DLPC in organic solvents with different immobilized lipases at controlled water activity. First, DLPC was chemically synthesized from glycerophosphocholine and lauric acid. Next, PLD-catalyzed head group exchange of DLPC with ethanolamine was studied using an enzyme from Streptomyces antibioticus expressed recombinantly in E. coli. A comparison of the free PLD with the biocatalyst activated by a salt-activation technique using KCl showed that the salt-activated enzyme (PLD-KCl) was 10-12 folds more active based on the amount of protein used. Thus, DLPC was quantitatively converted to 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine in an anhydrous solvent system within 12 h at 60 7C. For the acidolysis of DLPC with oleic acid, among the four lipases studied (CAL-B, Lipozyme TL IM, Lipozyme RM IM and lipase D immobilized on Accurel EP-100), Lipozyme TL IM showed the highest activity and incorporation of oleic acid. A quantitative incorporation was achieved at 40 7C using a 8-fold molar excess of oleic acid in n-hexane at a water activity of 0.11.

Phospholipase D (PLD) catalyzed synthesis of phosphatidyl-glucose in biphasic reaction system

“…Reaction temperature affects substrate solubility, as well as enzyme activity and stability (Wongsakul et al, 2004). Both substrate solubility and enzyme activity increased with reaction temperature.…”

“…In the PLD-catalyzed reaction, it has been widely reported that the presence of excess water caused unwanted hydrolysis, resulting in formation of phosphatidic acid (PA) (Wongsakul, Bornscheuer, & H-Kittikun, 2004;Yamamoto et al, 2008).…”

Phospholipase D and its application in biocatalysis

“…(Asahi) has been designed by using anhydrous solvents and a cation-exchanger resin as scavenger for choline released from PC (Rich & Khmelnitsky 2001). In addition, these authors apply a salt activation technique to increase the catalytic activity of PLD, which was successfully used also in the transphosphatidylation of PC by Streptomyces antibioticus (Wongsakul et al 2004).…”

Synthesis of a series of new racemic [2,3-bis(acyloxy)propyl]phosphonocholines

“…9 Phosphonolipids, as amphiphilic molecules, can be components of membranes and cell walls, and thus they can be applied in food, cosmetic and pharmaceutical products or in gene transfer therapy. [10][11][12] There is a distinct possibility to employ phosphonolipids as lipidic prodrugs.…”