Triolein hydrolysis

As shown in Fig. (14), chondroitin sulfate inhibited the pancreatic lipase activity dose-dependently at the concentrations of 1-20 mg/ml in the assay system using triolein emulsified with phosphatidylcholine at 10 mg/ml it inhibited triolein hydrolysis by about 60%. On the other... 

Figure 2. Variation of the rate of lipase hydrolysis of triacetin and methyl butyrate with the concentration of the substrate. The concentration is expressed as a fraction of the saturation concentration (S) and the hydrolysis rate as a percentage of the rate of triolein hydrolysis under optimal conditions. Key O, impure lipase plus esterolytic activity lipase purified by electrophoresis.

Fig. 6. The effect of tauiocholate on the activation energy of triolein hydrolysis by pancreatic lipase. V = initial rate of reaction, T = absolute temperature — no taurocholate added, O—taurocholate added. The activation energy in the presence of taurocholate is 5240 cal/mole (214).

Willstatter ef al. 196, 301) found that activation could be observed in an alkaline medium but not in an acid medium. Inhibition of tributyrin hydrolysis by bile salts at pH values below 7.0 was also observed by Click and King (297), and Wills (302) found that, although triolein hydrolysis by lipase was stimulated by sodium taurocholate in an alkaline medium, it was inhibited at pH values below 7.0. The effect of the bile salt may also depend on the length of fatty acid chain of the triglyceride under investigation. Thus Click and King (297) showed that, at pH 7.0, sodium taurocholate inhibited tributyrin hydrolysis but stimulated triolein hydrolysis. Similar results were obtained by Wills (302), who found that triacetin hydrolysis was inhibited and triolein hydrolysis stimulated by equivalent concentrations of sodium taurocholate. 

A Chromobacterium viscosum lipase is microencapsulated in AOT reversed micelles in isooctane with a Wo=24 and used in the controlled hydrolysis of 50 mM triolein at pH 7.0 and 35°C, in a continous stirred membrane reactor, with a flow rate of 1 l.min 1 Design the reactor in order to achieve 95% of conversion. 

Fig. 3 Hydrolysis and esterification by modified lipase (1mg) at 37°C. (a) was obtained at triolein (3mL) and (7mL). (b) was obtained at oleic acid (0.2mL) and glycerol (5mL). Conversion was defined as the decrement of oleic acid concentration.

The LPL catalytic assay measures the hydrolysis of a [14C[- or [3H]-triolein emulsion producing the 14C- or 3H -labeled free oleic acid [6]. The 14C- or 3H-labeled oleic acid is isolated by a selective extraction procedure and its radioactivity is determined by liquid scintillation counting [40]. Lipase activity is calculated as nanomoles of oleic acid released per minute per milliliter of postheparin plasma [41]. 

Ten microliters of each sample will be added to 500 pi of the triolein emulsion in triplicate and the HL-catalyzed triglyceride hydrolysis will be allowed to proceed for 1 h at 28°C. The reaction is stopped by adding 5.33 ml of methanolichloroform heptane (56 50 4 by volume) and vigorous shaking. 

Trinitrobenzenesulfonic (TNBS) acid hydrate, for degree of protein hydrolysis, 144-146 Triolein, lipid oxidation, 536 Tristearin, lipid oxidation, 536 Tryptophan... 

Glowacz et al. (1996) Batch Hydrolysis of triolein and its partial glycerides Porcine pancreatic lipase... 

There are many pharmaceutical applications for the modification of one enantiomer over another, and to this end, many have studied these selective reactions in carbon dioxide. Glowacz et al. (1996) studied the enzymatic hydrolysis of triolein and its partial glycerides and found that stereoselectivity depends on reaction time and enzyme water content. They suggest that the water content varies the local environment of the enzyme in carbon dioxide and changes the local pH value. Rantakyla et al. (1996) also found that the hydrolysis of one stereoisomer over another was water-dependent. They studied the hydrolysis of 3-(4-methoxyphenyl)glycidic acid methylester and found that the 2S,3R enantiomer hydrolyzed more than fivefold faster than the 2R3S form. 

Glowacz, G. Bariszlovich, M. Linke, M. Richter, P. Fuchs, C. Morsel, J. T. Stereoselectivity of Lipases in Supercritical Carbon Dioxide. I. Dependence of the Regio- and Enantioselectivity of Porcine Pancrease Lipase on the Water Content During the Hydrolysis of Triolein and its Partial Glycerides. Chem. Phys. Lipids 1996, 79, 101-106. 

The positional specificity of Lipase B is questionable. The low activity of the immobilized enzyme, and consequently the low incorporation of lauric acid, makes the specificity determination uncertain. It is also difficult to determine the positional specificity of Lipase B before irrmobilization by the traditional analysis of 1,2- and 1,3-diolein obtained by hydrolysis of triolein as the diolein is very quickly degraded by the enzyme. 

Table V. Relative Maximal Rates V of Hydrolysis of Oleic Acid Esters by Pancreatic Lipase, Compared with Triolein, V = 1.0 (7)...

Hydrolysis has traditionally been used for the production of fatty acids and glycerols, which find widespread apphcation in soaps and detergents, cosmetics, pharmaceuticals, and food products (174). Hydrolysis of soybean (181), canola (147, 208, 209), sunflower (149, 181, 210), tuna (150), and blackcurrant oils (145), tri-palmitin (146), triolein (211), and ethyl stearate (202) in SCCO2 has been reported. These investigations employed a variety of lipases, including immobilized lipase from porcine pancreas (211), Novozyme 435 (146, 181), Lipozyme (147, 150, 208, 209), non-immobilized Candida rugosa (150, 181), Lipase OL (150), and Lipolase lOOT (149, 181, 210). The effects of water content, enzyme load, operating conditions (temperature and pressure), pH, enzyme/substrate ratio, oil/buffer ratio, and CO2 flow rate (for continuous reactions) on the hydrolysis reaction were reported. 

None of the venoms, however, showed any interaction with films of cholesterol or protein, nor did hydrolysis occur in films of tripalmitin, triolein, cerebron, or sphingomyelin. The reaction with lecithin is highly specific. Not only is it sensitive to the chemical structure of the film, but the reaction rate may also be greatly altered by slight changes in the orientation of the molecules in the film or by changes in the pH of the solution or of the concentration of venom. 

Initially, the enzyme preferentially hydrolyzed the triolein (or 1,3-diolein) at the sn- i position, but the selectivity decreased over time, possibly due to back-reaction and acyl migration. The enantioselectivity and activity were not functions of the water content of the SCF but were functions of the water content during the preparation of the enzyme. The hydrolysis of rac-1,2-diolein again showed a preference for hydrolysis at the sn- i position, resulting in an excess of unreacted OT-l,2-diolein in solution. 

Glowacz G, Bariszlovich M, Linke M, Richter P, Fuchs C, Morsel JT. Stereoselectivity of lipases in supercritical carbon dioxide. 1. dependence of the regio- and enantioselectivity of porcine pancreas lipase on the water content during the hydrolysis of triolein and its partial glycerides. Chem Phys Lipids 1996 79 101-106. 

Topical pancreatic lipase substrates like tributyrin and triolein emulsions are hydrolyzed by carboxylester lipase in the presence of bile salt but slowly—at a rate lower than 3% and 0.5%, respectively, of that observed with the lipase-colipase complex. On the other hand, the positional specificity is not restricted all three sn positions of triglycerides can be split by the carboxylester lipase. While long-chain phospholipids are resistant, short-chain phospholipids are readily attacked by carboxylester lipase [40]. The low substrate specificity of carboxylester lipase makes possible an essential role for this enzyme in the hydrolysis of triglycerides containing certain esterified polyunsaturated fatty acids, such as eicosapentaenoic, arachidonic, or linoleic acids [41], and which may be resistant to attack by pancreas lipase (see p. 190). 

Figure 11 Hydrolysis of an artificial triolein/phospholipid/cholesteiol oleate emulsion with either pancreatic phospholipase A2 (PLA2) and/or pancreatic carboxylester lipase (CEL) in the presence of 10 mM bile salt. Closed squares PLA2 + CEL open squares CEL alone. Hydrolysis is monitored using tritiated tracers. (From Ref. 61.)...

A lipase has been used to convert solid triolein (glycerol trioleate) to the monooleate by treatment with glycerol at 8°C.75 Other lipases have been used in the hydrolysis and transesterification of oils, as well as in the esterification of fatty acids without solvents.76 Peptides can be produced from eutectic mixtures of amino acid derivatives with the addition of a small amount of solvent.77 Immobilized sub-tilisin and thermolysin were used with 19-24% water or an alcohol to produce polypeptides. Subtilisin on celite (a di-atomaceous earth) was used to convert a mixture of L-phenylalanine ethyl ester and L-leucinamide containing 10% triethyleneglycol dimethyl ether to L-phenylala-nineleucinamide in 83% yield. Addition of 30% 2 1 ethanol/water reduced the time needed from 40 to 4 h. The enzyme could be used three more times. These reaction mixtures contained 0.13-0.75 g peptide per g reaction mixture compared with 0.015-0.035 when the reaction was... 

It is well known that dietary fat is not absorbed from the intestine unless it has been subjected to the action of pancreatic lipase [1], Previously, we found that basic proteins such as protamines, histones and purothionine inhibited the hydrolysis of triolein emulsified with phosphatidylcholine [2], The inhibition of hydrolysis of dietary fat may cause a decrease or delay in the intestinal absorption of fat and reduce blood chylomicron levels, an excess of which is known to induce obesity [3], Therefore, there was a possibility that inhibitory substances toward pancreatic lipase activity may prevent the onset of obesity induced by feeding a high fat diet to mice. Recently, we found that natural products such as tea saponin, platycodi radix saponin, chitin-chitosan and chondroitin sulfate inhibited the pancreatic lipase activity. In the following section, the anti-obesity effects of these natural products will be described in detail. 

Plou, F.J., Barandiaran, M., Calvo, M.V., Ballesteros, A., and Pastor, E. 1996. High-yield production of mono- and di-oleylglycerol by lipase-catalyzed hydrolysis of triolein. Enz Microb. Technol. 18 66-71. 

Sarda and Desnuelle (6) showed more recently that methyl butyrate was hydrolyzed by pure pancreatic lipase, provided the ester was present in an emulsion, but that the hydrolysis was very much slower than that of tributyrin. Similarly, methyl oleate is hydrolyzed by lipase at only one thirtieth the rate of triolein (6). 

Fig. 1. Variation of the rate of hydrolysis of simple glycerides with the chain length of the fatty acid. Each experiment was carried out on an emulsion with an optimum inteifacial area. Hydrolysis rates are expressed as a percentage of the rate of hydrolysis of triolein (241).

The fact that pancreatic lipase is active only in a heterogeneous system was shown by studying the rate of hydrolysis of triacetin emulsified in gum arabic (6). When the concentration of added triaeetin is very low a true solution is produced and the rate of hydrolysis is very slow, but this increases sharply as the coneentration of triacetin is increased to form a heterogeneous system (Fig. 7). Furthermore, if the rate of lipase hydrolysis of triolein or tributyrin emulsified in gum arabic is plotted against the interfacial area of the substrate, the resulting curves are very similar to those obtained by plotting velocity against... 

Fic. 8. Variation of the rate of lipase hydrolysis of triolein and tributyrin with the interfacial area of the substrate emulsions. 0=triolein, B=tributyrin (6),... 

Fig. 10. The effect of hexadecylpyridinium bromide on the hydrolysis of triolein by lipase. The rate of hydrolysis is expressed in arbitrary units, taking that of the control in the absence of detergent as 100. The rate of acid liberation in the control experiment was 0.32meq/hour (308).

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