Lipase inactivation

Harper and Gould (1959) indicate that besides the protective effect of fat on lipase inactivation, the solids-not-fat content is also a factor. A higher solids-not-fat concentration, within limits, affords some protection. 

To restore nutrient digestion in exocrine pancreatic insufficiency, sufficient enzymatic activity must be administered into the duodenal lumen simultaneously with meal substrates. Intraluminal lipid digestion in postprandial chyme requires lipase activity of at least 40-60 IU/mL throughout the digestive period, which translates into 25,000 to 40,000 IU intraduodenal lipase for digestion of a regular meal. Because plain enzyme preparations undergo rapid lipase inactivation due to acid and proteolytic destruction, it is necessary to administer up to 10-fold more lipase orally to achieve these quantities within the duodenum. 

When tuna oil underwent ethanolysis with more than 2/3 mol EtOFI for FAs in tuna oil using immobilized C. antarctica lipase, the lipase inactivated because a part of EtOFI existed as micelles in the oil. To avoid such inactivation, first-step ethanolysis was conducted in a mixture of tuna oil and 1/3 mol EtOH using 4wt% immobilized lipase. After complete consumption of EtOH, the second and third 1/3 mol of EtOH were added to the reaction mixture. The three-step ethanolysis achieved the conversion of more than 95% of tuna oil to its corresponding FAEEs, and maintained the high degree of conversion for 54 cycles (108 days) (Watanabe et al., 1999). 

The lipoxygenase and peroxidase enzymes also have a negative impact on the oxidative state of the bran (Table 9). Further degradation of the oil occurs as reflected in an increase in peroxide and thiobarbituric acid value and a decrease in iodine value. Both lipoxygenase and peroxidase enzymes are inactivated with lipase inactivation. 

Extrusion (dry heat) cookers have been ideal for stabihzation because excess moisture is not added, eliminating the need for drying. The heating of the bran occurs through conversion of mechanical energy of the screw drive to heat the bran. Temperatures used for stabilization vary from 100° to 140°C. The bran is kept hot for 3-5 minutes after extrusion to ensure lipase inactivation. The hot bran is then cooled using ambient air. 

Wet heating is more effective for bran stabilization for oil extraction than is dry heating. Lipase is inactivated in 3 minutes at 100°C (37). The equipment that can be used include steam cookers, blanchers, autoclaves, and screw extruders with injected steam and water (30). Extrusion with steam injection and up to 10% added water reduces the temperature required for lipase inactivation. Temperatures are reduced to 100-120°C. Product may be held at 100°C for 1.5-3.0 minutes before drying to a stable moisture content. Bran expands as it exits the extruder, and water flashes to steam (8). Porous pellets assist in solvent percolation during oil extraction. Fines are agglomerated as well. 

Addition of water/steam to bran during wet extrusion requires drying after stabilization. Hot air is simply passed through a bed of pellets. Although this increases the cost of stabilization, lipase inactivation is permanent with less nutritional damage to the bran. The recovered oil is lighter in color with lower rehning losses. 

Marguet, F., Cudrey, C., Verger, R. and Buono, G. (1994) Digestive lipases Inactivation by phosphonates. Biochim. Biophys. Acta 1210, 157-166... 

Fig. 2.37. Thermal inactivation of enzymes of milk, 1 Lipase (inactivation extent, 90%), 2 alkaline phosphatase (90%), 3 catalase (80%), 4 xanthine oxidase (90%), 5 peroxidase (90%), and 6 acid phosphatase...

Rice bran oil with a high FFA content is often used as an industrial oil, because FFA removal often leads to a reduction in the content of desirable antioxidant/ bioactive components. A short discussion on methods to control FFA formation is therefore warranted. Lipase inactivation becomes significant when rice bran oil cannot be extracted from rice bran immediately after the milling process. As mentioned above, the rate of FFA formation within bran lipids can reach 5-7% per day (Nasirullah et al., 1989), with consequent reductions in oil yield and quality. Pretreatment of the rice bran by physical methods has been the primary means to inactivate rice bran lipase prior to oil extraction. 

A stepwise addition of methanol was the most common strategy to avoid lipase inactivation (Chen et al., 2009). Using a different acyl acceptor as methyl acetate or ethyl acetate, the lipase inactivation is also avoided (Jeong and Park, 2010). Another strategy for solving the problem of Upase inactivation by methanol is the use of organic solvents (Iso et al., 2001), but difficulties in solvent recovery make these methods less competitive at an industrial scale. 

In addition to having the required spedfidty, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortunately many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. 

Increased lipid synthesis/inhibi-tion of lipolysis Activation of lipoprotein lipase (LPL)/induc-tion of fatty acid synthase (FAS)/inactivation of hormone sensitive lipase (HSL) Facilitated uptake of fatty acids by LPL-dependent hydrolysis of triacylglycerol from circulating lipoproteins. Increased lipid synthesis through Akt-mediated FAS-expression. Inhibition of lipolysis by preventing cAMP-dependent activation of HSL (insulin-dependent activation of phosphodiesterases )... 

In adipose tissue, the effect of the decrease in insulin and increase in glucagon results in inhibition of lipo-genesis, inactivation of lipoprotein lipase, and activation of hormone-sensitive lipase (Chapter 25). This leads to release of increased amounts of glycerol (a substrate for gluconeogenesis in the liver) and free fatty acids, which are used by skeletal muscle and liver as their preferred metabolic fuels, so sparing glucose. 

Neither lipase nor lipoxygenase (inactivated enzymes) have an effect on the LA transfer. The presence of TL, 1.3-dilinolein and monolinolein does not affect transfer of LA from organic to aqueous phase. 

Fixed-bed reactors employed for lipase-catalyzed hydrolysis and interesterification reactions are highly efficient and have been used on a large scale (Table 5). The two phases may flow through the reactor in the opposite or same directions. If no solvents are used, the effect of viscosity of some substrates (i.e., oil) may be minimized by employing high temperatures which lead to faster rates of inactivation of lipases. 

The regulation of fat metabolism is relatively simple. During fasting, the rising glucagon levels inactivate fatty acid synthesis at the level of acetyl-CoA carboxylase and induce the lipolysis of triglycerides in the adipose tissue by stimulation of a hormone-sensitive lipase. This hormone-sensitive lipase is activated by glucagon and epinephrine (via a cAMP mechanism). This releases fatty acids into the blood. These are transported to the various tissues, where they are used. 

Lipase Inhibitor Orlistat (Xenical, Roche) is prescribed for the treatment of obesity. It inhibits the gastrointestinal lipase enzymes by binding to the lipase through the serine site and inactivates the enzyme. Fat in the form of triglycerides cannot be hydrolyzed by the lipase and converted to free fatty acids and monoglycerides. Thus, there is no uptake of fat molecules into the cell tissue. 

Pancreatic enemies (B) from slaughtered animals are used to relieve excretory insufficiency of the pancreas ( disrupted digestion of fats steatorrhea, inter alia). Normally, secretion of pancreatic enzymes is activated by cholecystokinin ancreozymin, the en-terohormone that is released into blood from the duodenal mucosa upon contact with chyme. With oral administration of pancreatic enzymes, allowance must be made for their partial inactivation by gastric acid (the lipases, particularly). Therefore, they are administered in acid-resistant dosage forms. 

Selected entries from Methods in Enzymology [vol, page(s)] Inhibitory properties, 68, 212 inactivator, of ribonucleases, 65, 681 lipase modification, 64, 390 nuclease inactivation, 79, 63 ribonuclease inactivation, 79, 52, 112-113, 267. 

Mecfianism of Action A gastric and pancreatic lipase inhibitor that inhibits absorption of dietary fats by inactivating gastric and pancreatic enzymes. Therapeutic Effect Resulting caloric deficit may positively affect weight control. 

Large differences in sensitivity toward interfacial inactivation were observed between a-chymotrypsin and Candida rugosa lipase [56]. The lipase was most rapidly inactivated by 1-butanol and tolerated the hydrophobic hydrocarbons quite well, while the opposite was true for a-chymotrypsin. A detailed study of interfacial inactivation by 12 different solvents, all having log P values around 4, revealed... 

Baginsky ML, Brown WV (1979) A newmethodfor the measurement of lipoprotein lipase in postheparin plasma using sodium dodecyl sulfate for the inactivation of hepatic triglyceride lipase. J Lipid Res 20 548-556... 

Lipase was first isolated from skim milk and characterized by Fox and Tarassuk in 1967. The enzyme was optimally active at pH 9.2 and 37°C and found to be a serine enzyme (inactivated by organophosphates). A lipoprotein lipase (LPL activated by lipoprotein co-factors) was demonstrated in milk by Korn in 1962 and was isolated by Egelrud and Olivecrona in 1972. LPL is, in fact, the principal indigenous lipase in milk and most recent work has been focused accordingly. The molecule has been characterized at the molecular, genetic, enzymatic and physiological levels (see Olivecrona et al, 1992). 

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