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Abstract

The development of a biotechnological method for the production of new biologically active phosphatidylcholine containing monoterpene citronellic acid (CA) was the aim of this work. Incorporation of citronellic acid (CA) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed acidolysis process was studied. Isoprenoid acid CA was used as an acyl donor and five commercially available immobilized lipases were examined as biocatalysts. The effects of organic solvent, enzyme load, reaction time and molar ratio of substrates on the incorporation of citronellic acid (CA) into the phospholipids were evaluated. Modified phospholipid fraction enriched with CA in the sn-1 position (39% of incorporation) was obtained in high 33% yield using Novozym 435 as biocatalyst. In this study a biotechnological method for production of new phospholipid biopreparation enriched with citronellic acid, which can play an important role as a nutraceutical, was applied.

2.1. Screening of Lipases

The progress of the reactions was monitored by collecting samples of the product mixtures after allowing the reactions to proceed for chosen length of time (12, 24, 48 and 72 h). The products mixtures were subsequently subjected to the solid phase extraction (SPE) to aid separation of PLs from the acids. In the next step, the fatty acid composition in the PL fraction was analyzed by gas chromatography (GC), whereas the information about the proportion between the modified and native PC was provided by high-performance liquid chromatography (HPLC) analysis. In these initial studies the PL were not fractionated into individual PC and lysophosphatidylcholine (LPC), which is formed in this process due to partial hydrolysis of phosphatidylcholine during the acidolysis reaction. The time course of the incorporation of CA into PC by lipases is presented in . It can be observed that only lipase B from C. antarctica immobilized on a macroporus acrylic resin (Novozym 435) was able to produce the desired PC with high incorporation level among all tested biocatalysts. This enzyme exhibits the highest activity in PC modification giving 19% incorporation of CA into phospholipid fraction after 48 h of reaction. Preparations of Lipozyme®, Lipozyme TL IM and CALB, showed significantly lower activity and the CA incorporation into phospholipid fraction did not exceed 9%, whereas lipase A from C. antarctica was inactive in acidolysis process. Novozym 435 was therefore used as the biocatalyst on the synthesis of phospholipid biopreparation enriched with citronellic acid for the following experiments.

2.6. Identification of Acidolysis Products

The best method for identification of products of enzymatic modifications and the composition of modified phospholipid fractions in our experiments was HPLC equipped with a Corona charged aerosol detector (CAD). This method was chosen because we had the standards of products that can be formed during the acidolysis reaction: 1-citroneloyl-2-palmitoyl-sn-glicero-3′-phosphatidylcholine (1-CA-2-PA-PC), as an analogue of modified PC and 1-citroneloyl-2-hydroxy-sn-glicero-3′-phosphatidylcholine (1-CA-2-OH-LPC). Those products we have obtained previously by the chemical methods []. We used also as the standards native PC (PC-egg yolk) and product of its enzymatic hydrolysis (LPC-egg yolk). Below we present the HPLC chromatogram of products mixture (phospholipid fraction) () and fatty acids (wt %) profile of modified phospholipid fraction () for the best variant of the reaction of acidolysis of PC with CA with optimized parameters: organic solvent (toluene), lipase (Novozym 435, enzyme load 30%), substrate molar ratio (PC:CA, 1:60) and reaction time (48 h).