Lipase-Catalysed Lipid Modifications in Supercritical Carbon Dioxide

Abstract: The production of ethyl esters (EE) from ethanol and cod-liver oil by an immobilised lipase in supercritical carbon dioxide (SCCO2) is described. This reaction results in a complex mixture of lipid components consisting of triglycerides, diglycerides, monoglycerides and ethyl esters. The primary goal has been to carry out enzymatic reaction and product separation simultaneously, by selectively extracting the ethyl esters formed during the reaction from the multicomponent mixture with supercritical carbon dioxide. The main focus of this work has been the investigation of the effects of process parameters such as pressure and flow rate of the carbon dioxide on the yield and the purity of the ethyl ester product. Two reactor configurations were used for this integrated enzyme reaction/product separation process: an extractive reactor and a simulation of a continuous reactor. For both reactors the ethyl esters could be selectively extracted at low process pressures (i.e. between 9 and 10 MPa at 40°C). As the pressure increased the amount of co-extracted lipid components increased. In addition, the conversion of the reaction was found to decrease with increasing pressure. In the extractive reactor, a fast carbon dioxide flow rate increased the extraction rate and the recovery of ethyl esters. This also resulted in a more selective extraction of ethyl esters from the reactor. In addition, by increasing the flow rate of the extraction fluid, the reaction was shifted towards synthesis. The production rate in this process was at best 0.44 mg EE/mg immobilised enzyme/h after 86% conversion. In the continuous process, the flow rate of the carbon dioxide did not strongly affect the total recovery of the product. In this process, a production rate of 0.76 mg EE/immobilised enzyme/h after 70% conversion was obtained. A study of the phase behaviour of the reaction system used with the extractive reactor showed that this system comprises three phases, i.e. a vapour, a liquid and a solid (enzyme) phase. This three-phase reaction system favours the selective extraction of ethyl esters from the reactor. The effects of the chemical properties of the immobilisation support surface on the lipolytic activity of immobilised lipases in SCCO2 were also studied. Lipases from Humicola lanuginosa lipase (HLL) and Candida antarctica lipase B (CALB) were adsorbed onto methylated glass beads with varying degrees of hydrophobicity. The immobilised lipases were examined for their activity in an alcoholysis reaction of cod-liver oil with ethanol in SCCO2. Moreover, the activity of the immobilised HLL in glyceride synthesis was studied. For HLL, the lipolytic activity was found to depend largely on the substrates and the hydrophobicity of the immobilisation support. In the alcoholysis reaction, a highly hydrophobic carrier enhanced lipase activity, whereas in glyceride synthesis the reverse effect was observed. For CALB the hydrophobicity of the support did not affect the activity of the lipase. The lipid class composition of the reaction mixture reflected the lipolytic activity of the lipases immobilised on the different supports tested. Thus, by choosing the hydrophobicity of the support the product selectivity could be influenced to a certain degree.