Lipase incubation

The enzymes for the SIF incubations were, in general, split into two groups proteases and amylase plus lipase. The incubations were performed only on compounds (I)-(VIII), (XI),and (XII) notall the anilines were investigated because of their instability in the stomach. Compounds were incubated in an SIF buffer (pH 7.S-7.9) containing either trypsin, chymotrypsin, carboxypeptidase A B, elastase or a-amylase and lipase. Compounds (I), (V)-(VII), and (XII) showed some degradation in the incubation with proteases which appeared to be enzyme mediated. Compounds (II), (III), (IV), (VII I), and XI were stable in the incubations. All the compounds investigated in the amylase/lipase incubations were stable. 

Table 9.9 The triglyceride content of palm oil pre- and post-incubation with lipase and stearic acid as described in the text.

Numerous workers have found that measurements of serum lipase activity are useful in the diagnosis of pancreatitis (83, 84, 85). Despite this, serum lipase determinations are not usually performed in clinical laboratories, probably due to inherent problems associated with the conventional methods, based on an emulsified lipid substrate. The methods are also not very suitable for manual batch analysis nor for automation due to laborious post incubation procedures. 

Cherry and Crandall in 1932 (86) used olive oil as substrate with gum acacia as the emufsTfier. This method has served as the basis for a number of modifications that increased the stability of the emulsion, decreased incubation time and gave better precision. When a serum sample is incubated with a stabilized olive oil emulsion, lipase acts at the interface of substrate and water to hydrolyze olive oil into fatty acid plus diglycerides, and to a small extent to monoglycerides and glycerol. The bile salt sodium deoxycholate activates the reaction. These methods measure the liberated fatty acids by titration with a standardized NaOH solution. An indicator such as phenolphatalein, thymolphthalein or methyl red or a pH meter are used to detect the end point. 

In the reaction, it was essential to use an IL as a co-solvent. Lozano, Iborra and co-workers recently reported an interesting stabilizing effect of two types of water-immiscible ILs ([emim][TFSI] and [BuMe3N][TFSI]) for CAL-B-catalyzed transesterification of vinyl butyrate. The synthetic activity and the stability of the enzyme in these IL solvent systems were markedly enhanced as compared to those in hexane. CAL-B maintained its activity higher than 75% after 4 days of incubation in [emim][TFSI] solvent, while it showed an activity of only 25% when incubated in both water and hexane media at 50°C. Comparison of the ratio of a-helix and (3-sheet by CD spectra showed the activity was closely related with a-helix content which reduced to 31% immediately after lipase was added to hexane and had reached only 2% after 4 days in hexane. On the contrary, no significant reduction of a-helix content was... 

Our final example is that of cyclic anhydrides, namely prochiral 3-sub-stituted glutaric anhydrides (7.101, R = Me, Et, or Pr). When incubated with lipase in an inert solvent in the presence of an alcohol (methanol, butan-l-ol, etc.), these compounds underwent nucleophilic ring opening with formation of a hemiester (7.102) of (/ -configuration (60-90% ee) [180]. This product enantioselectivity and, of course, the lack of reactivity in the absence of lipase show the enzymatic nature of the reaction. 

A further group of AT-[(acyloxy)methyl] pro-moieties contains acidic and/or lipid-like substituents. Here again, most published results concern phenytoin. Thus, some phenytoin-lipid conjugates such as 8.183 and 8.186 (with R = various fatty acyl moieties) were reported [233]. Such prodrugs are, of course, insoluble in water but formed dispersions when briefly sonicated in EtOH/water mixtures containing sodium taurodeoxycholate. No significant hydrolysis was seen in buffer or plasma. In contrast, incubation with pancreatic lipase yielded the bis-deacyl derivatives (i.e., 8.182 and 8.185, respectively), with subsequent liberation of phenytoin the time for 50% liberation of phenytoin varied from 20 to 200 min under the conditions of the studies [233][234], The intermediates 8.182, 8.184, and 8.185 were also substrates for human and rat plasma hydrolases. 

The immobilized lipase (0.1 g) in pH 7 phosphate buffer (25 mL) was added to 25 mL (20 mM) of ester stock solution in a 250 mL Erlenmeyer flask (reaction flask). The reaction flask was incubated in an incubator shaker at 40 °C with the agitation speed set to 200 rpm. Samples from the organic phase and aqueous phase were withdrawn at 24 h intervals over a 5-day reaction period. The samples collected were filtered using 0.45 pm nylon filter and injected into the HPLC system to determine the rate of resolution by monitoring both substrate ((/ ,5)-2-ethoxyethyl ibuprofen ester) and product (5-ibuprofen acid concentration). 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Figure 3. Effect of the particle size of PEA powders on the hydrolysis by R. delemar lipase. Reaction mixtures were incubated at 30 °C. Particle size -0-, 0-0.25 mm 0-1.00 mm,...

Figure 9 Effect of molar ratio of PCL and aromatic polyester on the biodegradability of CPE by R. dememar lipase, (a), (b), and (c) indicate PCL-PETG, PCL-PBT, and PCL-PEIP systems, respectively. Each reaction mixture for biodegradability assay contained CPE powder or its films ( 20 mg as polyester moiety) in a total volume of 1.0 ml. Reaction mixtures were incubated at 37 °C for l6 hours. Formation of the water-soluble TOC was in proportion to substrate amounts (up to 50 mg as PCL moiety) in this reaction system. (Reproduced from Reference l6. Copyright 1981 John Wiley. )...

Spectrophotometric assays can be used for the estimation of the enantiosel-ectivity of enzymatic reactions. Reetz and coworkers tested 48 mutants of a lipase produced by epPCR on a standard 96-well microtiter plate by incubating them in parallel with the pure R- and S-configured enantiomers of the substrate (R/S-4-nitrophenol esters) [10]. The proceeding of the enzyme catalyzed cleavage of the ester substrate was followed by UV absorption at 410 nm. Both reaction rates are then compared to estimate the enantiomeric excess (ee-value). They tested 1000 mutants in a first run, selecting 12 of them for development of a second generation. In this way they were able to increase the enantiomeric excess from 2% for the first mutants to 88% after four rounds of evolutive optimization. 

If the bonded water is extracted by dry CO2 the enzyme is denaturated and loses its activity. Therefore a certain amount of water is necessary in the supercritical fluid because acting with water-saturated CO2 again causes an inhibition of the enzyme and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Table 9.2-1 shows the residual activity of lipase from Candida cylindracea, esterase from Mucor mihei, and esterase from Porcine liver after a incubation time of 22 hours in supercritical CO2 at 40°C. It is obvious that higher water concentrations cause a strong reduction in the residual activity compared to the activity of the untreated enzyme, which was set as 100 %. Further, the system-pressure has an influence because at higher pressures the activity-loss is lower with a larger amount of water in the system [7,8],... 

Residual activity at different CO2 humidity at 40°C, 22 hours incubation time and 170 mL total volume of the system (I, lipase from Candida cylindracea II, esterase from Mucor mihev, III, esterase from Porcine liver) ... 

Most, if not all, milks contain sufficient amounts of lipase to cause rancidity. However, in practice, lipolysis does not occur in milk because the substrate (triglycerides) and enzymes are well partitioned and a multiplicity of factors affect enzyme activity. Unlike most enzymatic reactions, lipolysis takes place at an oil-water interface. This rather unique situation gives rise to variables not ordinarily encountered in enzyme reactions. Factors such as the amount of surface area available, the permeability of the emulsion, the type of glyceride employed, the physical state of the substrate (complete solid, complete liquid, or liquid-solid), and the degree of agitation of the reaction medium must be taken into account for the results to be meaningful. Other variables common to all enzymatic reactions—such as pH, temperature, the presence of inhibitors and activators, the concentration of the enzyme and substrate, light, and the duration of the incubation period—will affect the activity and the subsequent interpretation of the results. 

Thermal Inhibition, Heat treatment of milk is the most important practical means of inactivating its lipases. The temperature-time relationship necessary for partial or complete inactivation has been extensively studied, but a number of discrepancies have been apparent. These are probably due to several factors, including the sensitivity of the assay procedure, the length of the incubation period following heating, the presence and concentration of fat and solids-not-fat in the milk at the time of heating, and the type and condition of the substrate. In view of these variables, references to a number of early studies on heat inactivation have been omitted. 

The data of Nilsson and Willart (1960) indicate that heating at 80°C for 20 sec is sufficient to destroy all lipases in normal milk. Their studies included assays after 48 hr of incubation following heat treatment. At lower temperatures for 20 sec, some lipolysis was detected after the 48-hr incubation period after heating. Thus, 10% residual activity remained at 73 °C. Below the temperature of 68°C the amount of residual activity was enough to render the milk rancid in 3 hr temperatures below 60 °C had no appreciable effect on lipolysis. With holding times of 30 min, 40°C produced only slight inactivation, and at 55°C 80% inactivation was reported. 

An extensive study of the effects of formaldehyde in milk lipase inhibition showed that formaldehyde acts as a competitive inhibitor and, under the proper conditions, selectively inhibits the lipases of raw skim milk (Schwartz et al. 1956A). This study showed that the inhibitory effect of formaldehyde was dependent on such factors as pH, time of addition of the inhibitor, length of the incubation period, concentration... 

Lipases are sensitive to extremes of pH, and even in the vicinity of the pH optimum, where enzymes are supposedly more stable, marked inhibition may occur (Frankel and Tarassuk 1956B). Thus, it must also be borne in mind that the length of the incubation period and the prior history of the preparation can influence the range and perhaps the shape of the pH activity curve. 

The incubation of raw skim milk at pH 6.0 and at pH 8.9 for 1 hr at 37 °C in the absence of substrate was subsequently shown to cause a 47% and 40% decrease, respectively, in lipase activity when the milk was later incubated with milk fat. When tributyrin was the substrate the inhibition was even more marked. Although some of the inactivation was due to temperature, the majority of it was attributable to pH exposure. Stadhouders and Mulder (1964) have also demonstrated that milk lipase subjected to incubation at pH 5.0 is almost completely destroyed. 

The point on the acid side of the pH curve where milk lipase activity ceases is of considerable practical importance, but there is still controversy regarding it. Willart and Sjostrom (1962) found that milk lipase is active in the range pH 4.1 to 5.7, whereas Schwartz et al. (1956B) could detect no activity at pH 5.2 on butterfat. Although Peterson et al. (1948) found no milk lipase activity on tributyrin at pH 7.0, activity was reported on this substrate at pH 5.0 and even at pH 4.7 when 24-hr incubation periods were used (Stadhouders and Mulder 1964). 

Dissolve powders in warm water, add 0.8 M phosphate buffer (pH 8.0) and lipase, shake for 5 min. Incubate tubes at 37°C for 2 h in an ultrasonic bath, cool. Add EtOH/MeOH (95 5) + solid K,C03 + cholesteryl phenylacetate (internal standard) and extract twice with hexane. 

For some foods, incomplete extraction of color is obtained, probably due to the high binding affinity of dyes to the bulk of the food matrix, especially to proteins, lipids, and carbohydrates (156,161,162). This problem can be overcome by the use of selected solvents or enzymes to digest the food prior to extraction. Petroleum ether can be used to extract lipids (163). Acetone can be used to remove lipids and coagulate protein (164). Enzymes, such as amyloglucosidase (165,166), papain (167), lipase, pectinase, cellulase, and phospholipase, added to the sample and incubated under optimum pH and temperature conditions release synthetic colors bound to or associated with the food matrix. Furthermore, enzyme digestion can solubilize some foods, enabling analysis to be continued (156). 

Apply a standard error-prone PCR (epPCR see Chapter 2) to the wild-type lipase gene from Bacillus subtilis and express conventionally in E. coli [37] initiate by inoculation of the cultures in deep-well microtiter plates (96-well format). Use LB/M9 medium with 100 pL carbenicillin (lOOmgmL-1) per 100 mL of medium and incubate for 5-6 h at 37 °C while shaking. 

The effect of incubation temperature, initial cake moisture, and olive oil supplementation on lipase production was evaluated following a two-level experimental plan. The range of study of the variables was chosen based on previous results of the our group (2,7,10). 

Two 0.5-g samples were taken from each beaker for moisture and pH determination. Moisture content was measured by gravimetry, and pH was measured after adding 5 mL of deionized water to the sample, using a pH meter. The enzyme was extracted from the remaining fermented cake by adding 45 mL of sodium phosphate buffer (100 mmol/L, pH 7.0) following incubation at 35°C and 200 rpm for 30 min. After filtration the liquid phase was used for determination of lipase activity. 

Lipase activity was determined by adding 2 mL of the extract to an emulsion of olive oil (10% olive oil, 5% Arabic gum in 100 mmol/L of phosphate buffer, pH 7.0.) and incubating at 37°C and 200 rpm for 15 min. The reaction was stopped with the addition of a solution of ethanol and acetone (1 1). The fatty acids released in the hydrolysis were titrated with a solution of 0.04 N NaOH (5). Blank tests were performed by titration of a sample of emulsion without incubation, adding the enzyme extract sample just before the beginning of titration. One unit of lipase activity corresponds to the amount of enzyme that produces 1 pmol of fatty acids/min in the reaction conditions just described. 

Statistical analysis of the results was performed using the software Statistica 5.5 (Stat Soft). Maximum lipase activities and time to reach the maximum were calculated through fitting of kinetic curves. The maximum was estimated by derivation of the fits. Empirical models were built to fit maximum lipase activity in the function of incubation temperature (T), moisture of the cake (%M), and supplementation (%00). The experimental error estimated from the duplicates was considered in the parameter estimation. The choice of the best model to describe the influence of the variables on lipase activity was based on the correlation coefficient (r2) and on the x2 test. The model that best fits the experimental data is presented in Table 2. 

Esterification reactions were carried out in a closed reactor with 10 mL of dried n-heptane containing suitable amounts of alcohol and acid. A molecular sieve (aluminum sodium silicate, type 13X BHD Chemicals) was used to removal water. The mixture was incubated at 37°C for 24 h with continuous shaking at 150 rpm. The effects of concentration of immobilized lipase (5-50 mg/mL) molar ratio of reactants (0.5-2.0), acid chain length... 

Figure 5 Stability in function of pH of pancroalin lipase (broken line) compared with a frugal lipase (RNzppus javenfau) (solid line). The pancreatln Hpase was incubated for 3 horns at 37 C and the fting l lipase for 4 boon. After the preincubntian period the Activity was measured using the FTP procedure.

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