Lipases PUFAs

Purified PUFAs can be converted to their ethyl esters by adopting lipase-catalyzed esterification. For example, the ethyl esterification of DHA (Shimada et al., 2001b, 2003d) with an equal mol of EtOH using immobilized C. antarc-... 

The two-step chemoenzymatic approach starting from glycerol to synthesize the symmetric MLM-type structured TAGs is illustrated in the scheme in Figure 24.2. In the first step lipase regioselectivity was exploited to synthesize symmetric 1,3-diacylglycerols (DAGs) of the MCFA. This was followed by chemical introduction of the PUFA into the mid-position. 

Despite its simplicity this route is not easily accessible. Lipases can act enantioselectively on glycerol derivatives, but their enantioselectivity is usually rather low. And, there is no such report for an efficient enough direct acylation of non-substituted glycerol. Lipases of the two type, on the other hand, are known, one of the most efficient being CAL as has been demonstrated in our previously described synthesis of structured MLM-type TAGs and ALM type ether lipids (Halldorsson et al., 2003 Haraldsson, 2005,2007). The third type of lipase is certainly not available when PUFAs such as EPA or DHA are involved. 

It is generally considered that PUFA in the acylglycerol form is nutritionally more favorable than the corresponding methyl or ethyl esters as impaired intestinal absorption of aUcyl esters of co3 fatty acids has been observed in laboratory animals (78-81). Although most methods produce PUFA concentrates in the form of free fatty acids or their corresponding alkyl esters, enzyme hydrolysis is a technique proposed to produce co3 fatty acids concentrates in the form of acyglycerols by hydrolyzing the TAG with lipase. Saturated and monounsaturated fatty acids can be easily hydrolyzed because they do not present any barriers to lipases such as the commercial microbial lipases (71). 

Stmctured lipids possessing n-3 PUFAs, such as EPA and DHA, located at mid position, with MCFAs at the end positions, have gained considerable attention as nutritional and health supplements. These TAGs provide rapid delivery of energy via oxidation of the MCFAs and, at the same time, supply metabolically functional fatty acids in the same molecule. Senanayake and Shahidi (51) used an immobilized in-1,3-specific lipase from Mucor miehei to incorporate capric acid into seal blubber oil containing EPA and DHA. On enzyme-catalyzed acidolysis, a stmctured lipid containing 27.1% capric acid, 2.3% EPA, and 7.6% DHA was achieved. Lipase from Mucor miehei incorporated capric acid predominantly at the in-1,3 positions of the stmctured lipid. 

Microbial lipases from Aspergillus niger (AN), Candida cylindracea (CC), Pseudomonas spp. (PS), Chromobacterium viscosum (CV), Rhizopus delemer (RD), and Rhizopus javanicus (RJ) have been widely used in modifying PUFA-rich oils (117). The fatty acid specificity of lipases (discrimination of PUFA over short-chain fatty acids) is a crucial factor when considering the application of enzymes to modify marine oils rich in PUFA (118-121). Lipases from Rhizopus spp are known to be 1,3-position specific (122). Wanasundara (79) and Wanasundara and Shahidi (123, 124) (seal blubber oil SBO and menhaden oil MHO) and Tanaka et al. (117)... 

TABLE 4. Enrichment of Total co3 PUFA in the Nonhydrolyzed Fraction of Seal Blubber (SBO) and Menhaden (MHO) Oils on Hydrolysis by Different Microbial Lipases. 

When inclusion of PUFA are concerned, direct esterification of glycerol with individual free fatty acids, including EPA and DHA, had been carried out by employing CV- and CC-lipases (128). Several researchers have reported that this method affords a high degree of incorporation of targeted fatty acids into the TAG molecule, which has been observed with microbial lipases for marine oils (128-130) and plant oils (131-133). All these studies have pointed out that the water content in the reaction medium is a crucial factor determining the extent of the esterification... 

The main response of plants to wounding either to mechanically injury but also by pathogen attack is activation of lipases, followed by those of lipoxygenases as outlined above. Lipoxygenases remove from PUFAs a hydrogen atom localized at a double allylically activated methylene group, forming a mesomeric radical (Scheme 2). 

Oxidation products in form of reactive oxygen species are suspected to induce cell death [70,148]. Since generation of an oxidative burst requires the activation of kinases [83] and these in turn cause activation of membrane bound lipases we assume that the triggering event is the production of free PUFAs, followed by their peroxidation after activation of lipoxygenases as outlined above. 

Urea inclusion compounds Fractionation of polyunsaturates from saturates Downstream processing of lipase-catalyzed PUFA-enriched FFA... 

ISOLATION OF PUFA VIA LIPASE-CATALYZED SELECTIVE ESTERIFICATION... 

Lipase-selective esterification has recently helped fractionate PUFA species present in the same mixture. Fish oil-derived FFA was esterified with glycerol using Lipozyme IM in solvent-free media to fractionate DHA and EPA. The unesterified FFA contained 78%o DHA and only 3% EPA, with a 79%o recovery of DHA the esters contained the majority of the EPA and the other acyl groups, with a 91%o recovery of EPA.f Lipases have also successfully fractionated the two most abundant species of CLAs 18 2-9c,llt and 18 2-10t,12c from an equimolar mixture derived from acid-catalyzed isomerization of ALA. ° ... 

TAG containing PUFA at the 2-position and medium-chain acyl groups at the 1- and 3-positions may be a useful nutraceutical, providing the benefits described above for Salatrim, Caprenin, and Caprucin , with the added benefit of an essential FFA being adsorbed by the body. Such a structured TAG has been suggested as a nutrition source for patients with pancreatic deficiencies. However, a problem recently discovered for structured TAG prepared by 1,3-selective lipases is the product s relatively low oxidative stability.l ... 

Synthesis of a structured TAG with PUFA confined to either the l-(3-) or 2-position requires 1,3-selective lipases to interesterify a TAG using FFA or fatty acid esters (reaction 1), or to transesterify two different populations of TAG molecules (Fig. 3). Alternatively, a two-step approach can be employed, where a TAG is first subjected to 1,3-selective lipolysis, resulting in 2-MAG that is carefully isolated, then crystallized, to prevent acyl migration. The second step is 1,3-selective esterification of the 2-MAG.f " Such an approach results in a 72-85% yield and >95%... 

Lipases have also been employed to isolate tocopherols and sterols, important antioxidants from deodorizer distillate, a by-product formed during the deodoriza-tion step of seed oil purification, by hydrolyzing MAG and DAG in the distillate, and by esterifying sterols into sterol esters. The latter serves as antioxidant in non-polar food products such as margarine (e.g., Benacol from the Raisio Group, Finland and Take Control from Unilever, Englewood Cliffs, New Jersey, U.S.A.). PUFA-sterol esters would provide the benefits of essential fatty acid intake and antioxidant protection. 

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