Lipase/Esterase Activity

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

Fig. 4.4 Phenotype of 4-hydroxybutyrate dehydrogenase-positive (A) and lipase/esterase-positive (B) E. coli clones. A, 4-Hydroxy-butyrate dehydrogenase-positive clones are marked by arrows. Tetrazolium indicator plates [37] containing 4-hydroxybutyrate as test substrate were employed for the screening procedure [9], Positive clones were identified by formation of a deep-red formazan inside the colonies. B, Lipase/esterase activity of the clones was detected on LB agar [32] containing tributyrin as test substrate [14]. Zones of clearance around the colonies were indicative for lipase/ esterase activity.

Optically Active Acids and Esters. Enantioselective hydrolysis of esters of simple alcohols is a common method for the production of pure enantiomers of esters or the corresponding acids. Several representative examples are summarized ia Table 4. Lipases, esterases, and proteases accept a wide variety of esters and convert them to the corresponding acids, often ia a highly enantioselective manner. For example, the hydrolysis of (R)-methyl hydratropate [34083-55-1] (40) catalyzed by Hpase P from Amano results ia the corresponding acid ia 50% yield and 95% ee (56). Various substituents on the a-carbon (41—44) are readily tolerated by both Upases and proteases without reduction ia selectivity (57—60). The enantioselectivity of many Upases is not significantly affected by changes ia the alcohol component. As a result, activated esters may be used as a means of enhancing the reaction rate. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Several enzymes like lipases, esterases, and dehydrogenases have been active in hydrophobic environments. Thermodynamic water activity is a good predictor of the optimal hydration conditions for catalytic activity [51]. Enzyme preparation can be equilibrated at a specific water activity before the reaction [52]. When water concentration is very low, enzyme is suspended in the solid state in the water-immiscible organic solvent [46]. Enzymes are easily recovered after the reaction by the method of filtration. 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Warded, J.M., Wright, A.J., Bardsley, W.G. and D Souza, S.W. (1984) Bile salt-stimulated lipase and esterase activity in human milk after collection, storage, and heating nutritional implications. Pediatr. Res. 18, 382-386. 

Irradiation by ionizing radiation and its effect on milk lipase activity have also been studied (Tsugo and Hayashi 1962). Irradiation doses of 6.6 x 104 rads destroyed 70% of the activity. The udders of lactating cows, when exposed to 60 Co gamma rays, gave milk with decreased lipase and esterase activity (Luick and Mazrimas 1966). 

Miscellaneous. A manometric technique utilizing a Warburg apparatus has been used to follow esterase activity. The carbon dioxide liberated from sodium bicarbonate by the fatty acids is measured (Willart and Sjostrom 1959). An agar diffusion procedure has been utilized for screening microorganisms for lipolyptic enzymes. The presence of lipase is indicated by clear zones in the turbid media (Lawrence et aL 1967). 

Luick, J. R. and Mazrimas, J, A. 1966. Biological effects of ionizing radiation on milk synthesis. III. Effects on milk lipase, esterase, alkaline phosphatase, and lactoper-oxidase activities. J. Dairy Sci. 49, 1500-1504. 

Optically Active Acids ami Esters. Enannoselcctive hydrolysis of esters of simple alcohols is a common method for the production of pure enantiomers of esters or Ihe corresponding acids. Lipases, esterases, and proteases accept a wide variety of esters and convert them to the corresponding acids, often in a highly cnantiosclectivc manner. Lipasc-cutulyzed kinetic resolutions are often practical for the preparation of optically active pharmaceuticals. 

The serine hydrolase family is one of the largest and most diverse classes of enzymes. They include proteases, peptidases, lipases, esterases, and amidases and play important roles in numerous physiological and pathological process including inflammation [53], angiogenesis [54], cancer [55], and diabetes [56]. This enzyme family catalyzes the hydrolysis of ester, thioester, and amide bonds in a variety of protein and nonprotein substrates. This hydrolysis chemistry is accomplished by the activation of a conserved serine residue, which then attacks the substrate carbonyl. The resulting covalent adduct is then cleaved by a water molecule, restoring the serine to its active state [57] (Scheme 1). 

Lipases are enzymes that hydrolyse triglycerides in fats and phospholipases, as the name indicates, hydrolyse phospholipids. Lipases remove long-chain fatty acids from triglycerides, and they are also frequently described as having esterase activity. There are also specific esterases described in the GI tract, for example, carboxylesterase that is secreted by the pancreas. These enzymes are included in the discussion because their activity may be relevant to the use of macromolecular materials in novel formulations, particularly for oral peptide and nucleic acid delivery. 

Moore et al. [419] used surface-enhanced resonance Raman scattering to detect the activity of hydrolases at ultralow levels. The method was used to rapidly screen the relative activities and enantioselectivities of 14 enzymes including lipases, esterases and proteases. In the current format, the sensitivity of this technique was sufficient to detect 500 enzyme molecules, thus offering the potential to... 

Carboxylesterase activity is elevated in mastitic milk and colostrum (Fitz-Gerald et al., 1981) and may correspond to that of the reported lipases from somatic cells (Gaffney and Harper, 1965 Azzara and Dimick, 1985a) and colostrum (Driessen, 1976), respectively. The retinyl esterase activity that co-purifies with, but can be separated from, LPL may also be due to a carboxylesterase (Goldberg et al., 1986). It is of interest that the BSSL in human milk that has been shown to be identical with pancreatic carboxylesterase, has retinyl esterase activity (O Connor and Cleverly, 1989). 

Compared with the total lipase activity on emulsified milk fat or tributyrin (0.25-2.5 pmol/ml/min), the esterase activity (on soluble tribu-tyrin) is quite low, about one tenth (Downey, 1974). This may not be so for some abnormal milks where esterase levels are markedly elevated [10-12 times (Marquardt and Forster, 1962) and up to 37 times (Deeth, 1978)]. The significance of these esterases in cows milk and their relationship to each other, to LPL, and to esterases of other tissues remain to be determined. 

The natural substrates for lipases are triglycerides but, because of the complexity of these and the fact that they seldom contain a chromophore or other label to enable ready detection of the products, several synthetic substrates have been developed. These enable different detection techniques such as spectrophotometry, fluorimetry, chromatography, or radiometry to be used. It is important to note that, by definition, true lipases are active only on water-insoluble esters while esterases cleave only water-soluble esters (Jaeger et al., 1994). Thus, it is important that methods used for milk and milk products use substrates, which detect true lipase but not esterases as lipases play a major role in the hydrolysis of milk fat, while the role of esterases is considered insignificant (McKay et al., 1995). 

Lactase (Neutral) (P-Galactosidase) Activity, 801 Lipase Activity, 803 Lipase/Esterase (Forestomach) Activity, 804... 

Fig. 16. Electrophoretic separation of the lipase and esterase activities of porcine pancreas (146, 147). Starch columns equilibrated with 0.025 M acetate buffer, pH 5.25. The activities of the fractions have been determined (o) on emulsions of triolein and tributyrin (black circles), methyl oleate, methyl laurate, and p-nitro-phenyllaurate (black triangles), (b) On solutions of methyl butyrate and p-nitro-phenylacetate (crosses). White circles and dotted line, protein background. Figures along the first peak give the specific activity (lipase) of some fractions, determined against triolein emulsion. Ordinates and abscissas are the same as in Fig. 14.

The jS-eSer-a motif is likely to be present in other enzymes with esterase or thioesterase activity, although their structure may be different from that of the a/jS hydrolases. Some of these proteins have already been crystallized for X-ray studies [e.g.. Vibrio harveyi acyltransferase (Swenson et al., 1992)], and their cryst structures should throw some more light on the structural and evolutionary relationships within the lipase/esterase superfamily. 

The inhibition of hLAL by boronic acids and diethyl p-nitrophenyl phosphate (Sando and Rosenbaum, 1985 G. N. Sando and H. L. Brockman, unpublished, cited by Anderson and Sando, 1991) indicates that hLAL is a serine hydrolase. Two lipase/esterase consensus pentapep-tides, G-X-S-X-G, are found, but only one of them appears to be consistent with the packing requirements of the )8-eSer-a nucleophilic motif (see above). Susceptibility of the enzyme to sulfhydryl reagents, and the requirement of thiols for the stability of purified hLAL, prompted Anderson and Sando (1991) to propose that a cysteine residue, or rather a Cys/Ser couple, may be involved in an internal transacylation reaction. It must be pointed out, however, that hLAL has all three cysteines of the gastric enzyme (as well as six additional ones), and so the inhibitory Cys is also there. The same argument proposed herein with respect to hGL, i.e., that a free cysteine is topologically close to the active site, also holds for hLAL. 

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