Ester Hydrolases

Lipase. Lipase acts on esters at the interface between water and oil (52); the substrate is a liquid vegetable oil (olive oil, soy oil) purified by percolation over a column of activated alumina or silica gel. It is emulsified with the help of vegetable gum or bile salt. The release of fatty acid is monitored by addition of base to keep the pH constant (continuous titrimetry, or pH stat, method) or by extraction and colorimetric measurement of the copper soap (53). For the latter assay, make substrate emulsion by adding 2 mL vegetable oil solution (1% in absolute EtOH) to 100 mL 0.025 M Tris buffer, pH 8.8, containing 0.6% sodium de-oxycholate. To 5 mL substrate, add 0.1 mL enzyme, incubate 5 min, add 1 mL 1N HCI and 10 mL isooctane, shake, and allow phase separation. To 5 mL of the upper (organic solvent) layer, add 1 mL copper reagent (5% cupric acetate in water, pH adjusted to 6.1 with pyridine), shake vigorously for 90 s, and allow the phases to separate. Read the absorbance at 715 nm of the upper phase.

Esterase. The best assay for esterase activity is the pH stat method. The substrate in water or salt solution is adjusted to the desired pH, enzyme is added, and base is continually added to keep the pH constant. The plot of base consumption versus time gives the rate of the reaction. For a spectrophotometric assay the system is lightly buffered at the desired pH, an indicator dye is included, and the change in absorption is measured. An assay for pectin methylesterase (54) uses 0.5% citrus pectin in water adjusted to pH 7.5, an indicator/buffer system of 0.01% brom-thymol blue in 3 mM phosphate buffer, pH 7.5, and enzyme in the same buffer. To 2 mL of pectin solution, add 0.15 mL indicator/buffer and 0.83 mL water. Record absorbance at 620 nm briefly to establish a baseline, then add 20 fiL enzyme and record the rate of absorbance change due to es-teratic action. The system (without enzyme) is titrated with galacturonic acid to establish the correspondence between absorbance change and acid concentration.

Phosphatase. Phosphatase activity is monitored by colorimetric quantitation of P; (inorganic phosphate) formed (55). An example is the assay for acid phosphatase using glucose-6-phosphate (G-6-P) (56). Substrate is 0.2 M maleate buffer, pH 6.7, containing 60 mM G-6-P, 8 mM KF, and 8 mM EDTA. To 1 mL, add 1 mL enzyme extract, incubate 20 min at 37°C, and add 1 mL cold 10% TCA to stop. To 1 mL, add 2 mL P; reagent, hold 20 min at 45°C, and read absorbance at 820 nm. Reagent A: 10% (w/v) ascorbic acid in water. Reagent B: 4.2 g ammonium molyb-date tetrahydrate in 1 L 1 N H2S04. Pi reagent: 1 part A plus 6 parts B, made fresh daily, and kept in an ice bath until used.

Oxidases

Polyphenoloxidase. Polyphenoloxidase enzymes (eg, tyrosinase) catalyze two reactions: (1) oxygenation of a phenol to o-diphenol (cresolase) and (2) oxidation of a di-phenol to o-quinone (catecholase). Substrates commonly used for assays are p-cresol (4-methylphenol), L-tyrosine (4-hydroxy phenylalanine), and p-coumaric acid (4-hydroxycinnamic acid). The corresponding diphenols are 4-methylcatechol, L-Dopa, and caffeic acid. Rates are monitored by absorbance changes at specific wavelengths as oxygenation or oxidation occurs. An assay system that differentiates the two kinds of activities is the following (57).

For cresolase activity, mix 1.5 mL 0.05 M acetate, pH 4.8, 0.4 mL 1 mM p-cresol, 0.1 mL 1 mM 4-methylcatechol and 10 fiL enzyme. Record increase in absorbance at 291 nm. The change is due only to oxygenation; oxidation of di-phenol to quinone produces no absorbance change at 291 nm. For catecholase activity, mix 1.6 mL buffer, 0.4 mL 1 mM 4-methylcatechol, and 10 fiL enzyme, recording the decrease in absorbance at 280 nm.

Lipoxygenase. Native lipoxygenase requires activation by hydroperoxydecadienoic acid, the product of its reaction with linoleic acid and oxygen; there is a lag period in the reaction if enzyme is simply mixed with a solution of substrate (58). The delay is removed by including a small amount of product in the stock substrate solution (168 mg linoleic acid and 14 mg hydroperoxylinoleic acid in 100 mL ethanol) (59). For assay, to 2.9 mL 0.2 M borate buffer, pH 9.0, add 50 fiL substrate stock, followed by 50 fiL enzyme solution. Mix and record the increase in absorbance at 235 nm.

Ascorbic Acid Oxidase (AAO). A simple spectrophotometric assay for AAO is based on the loss of absorbance at 265 nm when ascorbate is oxidized to dehydroascorbate (60,61). To 2.8 mL buffer (0.025 M citrate, 0.05 M phosphate, pH 5.6), add 100 fiL substrate solution (0.05% ascorbate, 1% NA2EDTA, neutralized), 50 fiL 1% bovine serum albumin solution, and finally 50 fiL enzyme solution. Record the rate of disappearance of absorbance at 265 nm.

Catalase, Peroxidase. For peroxidase, the reaction mixture is 0.01 M phosphate, pH 7.0, containing 25 mg/L H202 and 2.5 g/L pyrogallol (62). After adding enzyme, record the increase at 430 nm due to the formation of purpuro-gallin. The reaction of catalase with H202 is a first-order reaction (63). Hydrogen peroxide has a broad absorption band in the far ultraviolet; molar absorbance is 120 at 200 nm and 30 at 250 nm. To 2 mL assay solution (0.05 M phosphate, pH 7.0, containing 2 g/L H202), add 1 mL diluted catalase solution and record the ultraviolet absorbance. Plot log absorbance versus time; the slope divided by 2.303 equals the first-order rate constant, which is directly proportional to the amount of catalase in the assay (or use a computer program to calculate the rate constant).

Other Enzymes

Lysozyme. Hydrolysis of bacterial cell walls by lyso-zyme causes the cells to lyse; a turbid suspension of cells will slowly clear. The turbidimetric assay (64) uses a suspension of Micrococcus lysodeikticus (0.5 g/L) in 0.06 M phosphate, pH 6.2. To 1.5 mL, add 0.5 mL 0.3 M NaCl and 1 mL enzyme, place the cuvette in a colorimeter, and record % transmittance (not absorbance) at 540 nm. A plot of %T versus time is linear from about 10 to 40%, and the rate %T/min is proportional to enzyme in the range of 0 to 10 fig.

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