Enzyme assay lipase

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

The pronounced effects of pH on enzyme reactions emphasize the need to control this variable by means of adequate buffer solutions. Enzyme assays should be carried out at the pH of optimal activity, because the pH-activity curve has its minimum slope near this pH, and a small variation in pH will cause a minimal change in enzyme activity. The buffer system must be capable of counteracting the effect of adding the specimen (e.g, serum itself is a powerful buffer) to the assay system, and the effects of acids or bases formed during the reaction (e.g., formation of fatty acids by the action of lipase). Because buffers have their maximimi buffering capacity close to their pK values, whenever possible a buffer system should be chosen with a pK value within IpH unit of the desired pH of the assay (see Chapter 1). Interaction between buffer ions and other components of the assay system (e.g., activating metal ions) may eliminate certain buffers from consideration. 

EPTC and Butvlate Fluorescein Diacetate Assay. Spectrophotometric determinations of the hydrolysis of fluorescein diacetate have been shown to be simple, rapid, and sensitive methods for determining microbial activity in soil (18). Essentially, the hydrolytic cleavage of diacetate from fluorescein is responsible for the reaction products including fluorescein, which may be detected spectrophotometrically at 490 nm. This method is somewhat nonspecific in that it is indicative of overall activity of several enzymes (protease, lipase, esterase) rather than of a specific class of enzymes. Enzyme activity may be influenced by subtle pH changes in the sample since abiotic hydrolysis of fluorescein diacetate may occur. Also, an associated lag phase in soil hydrolytic activity must be accounted for in each assay. 

The esterification activity of immobilized lipase was measured by the consumption of oleic acid at 45°C in the esterification reaction with butanol (equimolar ratio) with 3% mim enzyme. One lipase activity unit (Ue) was defined as the amount of enzyme necessary to consume 1 pmol of oleic acid per minute under assay conditions. The enzyme activity was also evaluated in a reaction medium containing 50% v/v of hexane. 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Another method for assaying the activity and stereoselectivity of enzymes at in vitro concentrations is based on surface-enhanced resonance Raman scattering (SERRS) using silver nanoparticles (116). Turnover of a substrate leads to the release of a surface targeting dye, which is detected by SERRS. In a model study, lipase-catalyzed kinetic resolution of a dye-labeled chiral ester was investigated. It is currently unclear how precise the method is when identifying mutants which lead to E values higher than 10. The assay appears to be well suited as a pre-test for activity. 

The Bacillus subtilis lipase A (BSLA) is an unusual lipase because it lacks the so-called lid structural unit 13(3). Moreover, it is a small enzyme composed of only 181 amino acids. The initial results of an ongoing study are remarkable and illustrate the power of directed evolution 45,137). The desymmetrization of meso-, 4-diacetoxy-2-cyclopentene (15) was chosen as the model reaction, and the MS-based ee assay using an appropriately Ds-labeled substrate (Section III.C) provided a means to screen thousands of mutants. 

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. 

Groves and Teng (1992) investigated the effect of compactional pressure on biologically active proteinaceous enzymes such as a-amylase, P-glucuronidase, lipase, and urease. Assaying the activity of these enzymes before and after the compaction... 

The assays presented in this section deal with the measurement of enzyme activity, which is expected to be proportional to the amount of active enzyme present in a sample, food or otherwise, unit ci.i is an overview of the important considerations in performing activity assays unitc 1.2 illustrates how these considerations are applied to the assay of a representative food-relevant glycosyl hydrolase. Chapters C2, C3, and C4 present the first units on particular types of activity assays. In Chapter C2, two units present peptidase activity assays that use either synthetic substrates (UNITC2.1) or common, commercially available protein substrates (unit C2.2). unitC3.i presents three different assays for lipase activity. unitc4.i presents assays for diphenol oxidases, and unitc4.2 for lipoxygenase. 

Place 0.05 to 0.50 ml of 0.5 mM p-nitrophenol standard solution into ten individual 15- to 20-ml test tubes and dilute each to 5 ml with 0.1 M Tris-Cl buffer, pH 8.2. This yields a standard curve of 0.005 to 0.05 fimol p-nitrophenol/ml. 2. Measure A410 using 0.1 M Tris Cl buffer, pH 8.2, as a blank, and make a standard curve by plotting A410 versus the p-nitrophenol concentration in each tube. 3. For each lipase activity assay, place 2.5 ml of 0.1 M Tris-Cl buffer, pH 8.2, and 2.5 ml of 420 pM p-nitrophenyl laurate substrate solution into a 15- to 20-ml test tube. Prepare one extra tube for a reagent blank. 4. Add 1 ml water to the reagent blank. Lypolytic Enzymes... 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

In the event that lipase preparations are too active to allow for facile estimation of initial rates, the enzyme can be diluted and assayed again. This is illustrated using the copper soap method where the reduced level of C. rugosa lipase addition afforded a longer period of linearity to the reaction progress curve than did the more active B. cepacia lipase (Fig. C3.1.3). 

Vorderwiilbecke, T., Kieslich, K., and Erdmann, H. 1992. Comparison of lipases by different assays. Enzyme Microbiol. Technol. 14 631-639. 

Hydrolytic activities of free and immobilized lipase were assayed by the olive oil emulsion method according to the modification proposed by Soares et al. (11). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 imol of free fatty acid/min under the assay conditions (37°C, pH 7.0,150 rpm). Analyses of hydrolytic activities carried out on the lipase loading solution and immobilized preparations were used to determine the activity-coupling yield (r %), which measures the recovered enzymatic activity according to Eq. 1 ... 

Flow cytometry is well suited for the analysis of enzyme activity and kinetics at the single cell level (Watson and Dive, 1994). Flow cytometric assays for numerous enzymes including esterases, proteases, peroxidases, lipases, and oxidoreductases3 are available and are widely used in research and clinical practice. To date, flow cytometry has not been widely exploited as a screening tool for enzyme engineering purposes, but this is rapidly changing. 

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