Lipase membrane

Another emerging technique which deserves mention is the use of a supported ionic liquid membrane. This involves two liquid phases that both contain an enzyme and are separated by the membrane. Lipase-catalyzed esterification takes place in the feed phase to afford a mixture of the (R)-acid and the (S)-ester (Figure 10.22). The latter diffuses through the membrane and is hydrolyzed in the receiving phase to afford the (S)-acid [151, 152]. The methodology has been applied, for example, to the resolution of ibuprofen [151]. 

Early research on lipolytic enzymes in cows milk suggested that at least two major lipases were present a plasma lipase in the skim portion and a membrane lipase associated with the milk fat globule membrane (Tarassuk and Frankel, 1957) while later research indicated that there might be up to six different molecular species with lipase activity (Downey and Andrews, 1969). However, work by Korn (1962) showed that milk contained a lipoprotein lipase (EC 3.1.1.34) (LPL) with properties very similar to those of post-heparin plasma, adipose tissue and heart LPLs, particularly the enhancement of its activity on emulsified triglycerides by blood serum lipoproteins. It is now accepted that LPL is the major, if not the only, lipase in cows milk. Its properties have been reviewed by Olivecrona et al. (2003). 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

The values of the Michaelis-Menten kinetic parameters, Vj3 and C,PP characterise the kinetic expression for the micro-environment within the porous structure. Kinetic analyses of the immobilised lipase in the membrane reactor were performed because the kinetic parameters cannot be assumed to be the same values as for die native enzymes. 

The inhibition analyses were examined differently for free lipase in a batch and immobilised lipase in membrane reactor system. Figure 5.14 shows the kinetics plot for substrate inhibition of the free lipase in the batch system, where [5] is the concentration of (S)-ibuprofen ester in isooctane, and v0 is the initial reaction rate for (S)-ester conversion. The data for immobilised lipase are shown in Figure 5.15 that is, the kinetics plot for substrate inhibition for immobilised lipase in the EMR system. The Hanes-Woolf plots in both systems show similar trends for substrate inhibition. The graphical presentation of rate curves for immobilised lipase shows higher values compared with free enzymes. The value for the... 

The steps in the subsequent utilization of muscle LCFAs may be summarized as follows. The free fatty acids, liberated from triglycerides by a neutral triglyceride lipase, are activated to form acyl CoAs by the mediation of LCFAcyl-CoA synthetase which is situated on the outer mitochondrial membrane. The next step involves carnitine palmitoyl transferase I (CPT I, see Figure 9) which is also located on the outer mitochondrial membrane and catalyzes the transfer of LCFAcyl residues from CoA to carnitine (y-trimethyl-amino-P-hydroxybutyrate). LCFAcyl... 

Different enzymes - particularly lipases - immobilized in membrane reactors have been studied in the presence of two-liquid phases (Table 5). Organic and aqueous phases containing respectively hydrophobic and hydrophilic reactants are separated by a solid mem-... 

Membrane reactor Ethyl laurate hydrolysis Candida rugosa lipase 116... 

Hollow-fiber membrane reactor Hydrolysis of sunflower oil Lipase from Rhizopus sp. 122... 

Membrane reactor Synthesis of monoglycerides Lipase from Candida rugosa 75... 

Two possible pathways for the biosynthesis of 2-AG have been proposed (1) a phospholipase C (PLC) hydrolysis of membrane phospholipids followed by a second hydrolysis of the resulting 1,2-diacylglycerol by diacylglycerol lipase or (2) a phospholipase Ai (PLA,) activity that generates a lysophospholipid, which in turn is hydrolyzed to 2-AG by lysophospholipase C (Fig. 5) (Piomelli, 1998). Alternative pathways may also exist from either triacylglycerols by a neutral lipase activity or lysophosphatidic acid by a dephosphorylase. The fact that PLC and diacylglycerol lipase inhibitors inhibit 2-AG formation in cortical neurons supports the contention that 2-AG is, at least predominantly, biosynthesized by the PLC pathway (Stella, 1997). However, a mixed pathway may also be plausible. 

Anandamide amidase recognizes and hydrolyzes 2-AG (Goparaju, 1999 Di Marzo, 1999 Lang, 1999) however, there is evidence for the existence of another specific hydrolase [monoacylglycerol (MAG) lipase] that hydrolyzes 2-AG (D. Piomelli and A. Makriyannis, 2000, personal communication). In addition to this pathway, 2-AG diffuses rapidly into the cell membrane where it could be either hydrolyzed to arachidonic acid and glycerol or esterified back to phosphoglycerides (Di Marzo, 1999b). 

Release of active pancreatic enzymes directly causes local or distant tissue damage. Trypsin digests cell membranes and leads to the activation of other pancreatic enzymes. Lipase damages fat cells, producing noxious substances that cause further pancreatic and peripancreatic injury. 

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