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with a simple aqueous TCA solution (8). Quantitation may be direct (absorbance of filtrate at 275 nm) or colorimetric (Folin-Lowry [28], bicinchoninic acid [29], or TNBS [30]). An example of a casein-based assay is the following (8). To 5 mL casein (12 mg/mL in 0.03 M phosphate buffer, pH 7.5), add 1 mL enzyme solution and after 10 min reaction add 5 mL of buffered TCA. After 30 min the mixture is filtered and absorbance at 275 nm is measured. For Folin-Lowry quantitation, to 1 mL of filtrate add 1 mL alkaline buffer (1M Na2C03,0.25 M NaOH), 0.4 mL copper reagent (0.1% CuS04 • 5H20, 0.2% NaK tartrate), mix, and allow to stand 10 min. Then add 0.75 mL diluted phenol reagent (Folin-Ciocalteau reagent diluted with 3 volumes H20), mix, wait 10 min, and measure absorbance at 700 nm versus an appropriate reagent blank. A plot of log absorbance versus log protein is linear over the range 3 to 400 fig of protein (eg, bovine serum albumin standard) (31).

An assay using TNBS to measure freed amino groups (30) uses ./V,TV-dimethyl casein (20) (1 mg/mL) in buffer as substrate. To 1 mL in reaction tube, add 0.1 mL enzyme solution, incubate for the desired time, and heat the tube briefly in boiling water to stop the reaction. Add 1 mL 0.4 M phosphate buffer, pH 8.2 (containing 0.25% sodium do-decyl sulfate), 2 mL freshly made TNBS (1 mg/mL in water), and incubate in the dark 1 h at 60°C. Add 4 mL 0.1 N HC1, cool to room temperature, and measure absorbance at 340 nm. The reagent blank contains only buffer and TNBS; a complete assay with zero incubation time gives the correction for free amino groups in substrate and enzyme.

The absorbance change during the reaction of synthetic substrates with enzyme is recorded on a stripchart recorder, giving a continuous trace from which the initial velocity or the first-order reaction rate is obtained. Two assays will be described as examples.

1. TAME a substrate for trypsin (23). The reaction solution is 1.04 mM TAME (0.394 mg/mL) in 0.04 M Tris buffer, pH 8.1, 10 mM in CaCl. Tb 2.9 mL, add 0.1 mL enzyme solution and record the increase in absorbance at 244 nm.

2. FAGLA is a useful substrate for metalloproteases such as thermolysin (26). To 100 mL 0.05 M phosphate buffer, pH 7.2, add 76.9 mg FAGLA to give a 2.5 mM solution. This is well below the KM of ther molysin for this substrate, so the reaction is kineti-cally first order. In the cuvette place 1.5 mL substrate, 1.4 mL buffer, and 0.1 mL enzyme, and measure the decrease in absorbance at 345 nm from 0.96 (zero time) toward an infinite-time value of 0.56. Plot log(absorbance - 0.56) versus time; the slope divided by 2.303 gives the first-order rate for the reaction, which is directly proportional to the concentration of enzyme. Preferably, a simple Basic computer program (1) may also be used to calculate the first-order rate constant.

Peptidase Assays. Carboxypeptidase is assayed by incubation with an AT-acylated dipeptide, followed by measurement of the amino group freed (32). Substrate solution is 1 mM dipeptide (eg, 35.6 mg Z-Phe-Gly per 100 mL) in 0.025 M phosphate buffer, pH 7.2. To 200 /uL add 50 ¡uh enzyme, incubate for 1 h, stop by heating in boiling water, and measure free amino groups by reaction with TNBS as described previously. Carboxypeptidase may also be assayed continuously using Z-Gly-Phe in 0.05 M Tris buffer, pH 7.5, following the decrease in absorbance at 224 nm (27).

Aminopeptidase assays usually employ the /?-naphthyl-amide of an amino acid. After incubation the freed [i-naphthylamine is quantitated by diazotization (33). Substrate stock solution is 2 mM amino acid naphthylamide in 0.01 N HCI. Buffer is 0.025 M phosphate, pH 7.2. Color reagent is the diazonium salt Fast Garnet GBC (1 mg/mL) in 1M acetate buffer, pH 4.2, containing 10% (v/v) Tween 20. To the assay tube, add 1.6 mL buffer, 0.2 mL substrate, 0.2 mL enzyme, and incubate 3 h. Then add 1 mL color reagent, allow 5 min for color development, and read absorbance at 525 nm.

Carbohydrases

Substrates. Starch, the substrate for amylases, consists of a linear polymer, amylose, and a highly branched polymer, amylopectin. The ratio of these two components varies in starches obtained from different plant sources. They may be separated by treating a solution of gelatinized starch with thymol (34). In certain assays for «-amylase the use of one or the other fraction is preferable. If the assay involves colorimetric quantitation of freed reducing groups, the blank due to reducing ends of the substrate chain may be removed by reduction with NABH4 (5).

Soluble cellulose substrates for cellulase are the car-boxymethyl, hydroxypropyl, or hydroxyethyl derivatives (35). These are available as commercial products but should be characterized before use to ensure comparability between lots. Insoluble complexes of dyes with starch (36,37) or cellulose (38) are also available; enzyme activity is assayed by measuring the amount of dye that is solubi-lized during an incubation period.

Small synthetic substrates, usually the p-nitrophenyl-glycosides, are available for some glycosidases (39). Specific oligosaccharide derivatives have been used for assays of a-amylase and /(-amylase (40,41).

Amylase Assays. For a-amylase (5), use 1 mL of a 1% solution of reduced starch in 0.02 M acetate buffer, pH 4.7, containing 1 mM CaCl2. Add 1 mL enzyme solution, incubate for the time desired, then add 4 mL each of Reagent A and Reagent B of the neocuproine system (42). Heat in boiling water 12 min, make up to 25 mL, and read absorbance at 450 nm. Reagent A: in 600 mL water, dissolve 40 g anhydrous Na2C03, 10 g glycine, and 0.45 g CuS04 • 5H20. Make up to 1 L. Reagent B: dissolve 0.12 g neocuproine (2,9-dimethyl-l,10-phenanthroline-HCl) in 100 mL water. Store in a brown bottle.

Amylopectin forms a complex with I2 which absorbs at 570 nm. As it is hydrolyzed by a-amylase the color decreases, the basis for a fixed end-point assay frequently used in cereals-related industries (43). A continuous assay based on this phenomenon is the following (44). Suspend 1 g soluble starch in 10 mL water, then slowly add this to 50 mL boiling water. Boil gently for 2 min, cool, and make up to 100 mL volume. To 2.5 mL buffered iodine solution (32 mg I2, 330 mg KI, and 23.4 mg NaCl in 100 mL of 0.01 M phosphate buffer, pH 7.0), add 10 juL enzyme followed by 10 juL starch. Record the decrease in absorbance at 570 nm. This assay was developed for pancreatic a-amylase; for cereal a-amylases, the buffer system would be 0.02 M acetate, pH 4.7, 1 mM in CaCl2.

a-Amylase is an endo glycosidase, hydrolyzing internal bonds in a-1,4 linked glucose polymers (40). A colorimetric assay using a blocked substrate takes advantage of this fact. The substrate is maltoheptaose, with a p-nitrophenyl group attached to the reducing end and a blocking agent (45) at the nonreducing end. a-Amylase cleaves this molecule in the middle. The assay mixture also contains glu-coamylase and a-glucosidase, which combine to hydrolyze the short maltose (or maltotriose) derivative, freeing the chromogen, p-nitrophenol. The reaction is stopped by the addition of Tris buffer at pH 10, and the absorbance due to ionized p-nitrophenol is read at 410 nm.

/¡■-Amylase hydrolyzes soluble starch to yield maltose, which is quantitated by colorimetric reaction with DNS (dinitrosalicylic acid) reagent (46). A 1% soluble starch solution is prepared as already described, except that 10 mL 0.16 M acetate buffer, pH 4.8, is added before adjusting to 100 mL total volume. Mix 0.5 mL substrate with 0.5 mL enzyme solution, incubate at 25°C for 3 min, then add 1 mL DNS reagent (50). Heat in boiling water 5 min, cool, add 10 mL water, and read absorbance at 540 nm. DNS

reagent: in 500 mL water dissolve 10 g NAOH, 10 g 3,5-dinitrosalicylic acid, 2 g phenol, 0.5 g Na2S03, and 200 g NaK tartrate, then make up to 1 L volume.

A chromogenic substrate, p-nitrophenyl maltopentaose, is used to assay this enzyme (41). /?-Amylase removes two maltose units from the substrate, and a-glucosidase in the assay mixture then frees the chromogen. The reaction is stopped with alkaline buffer, and absorbance is read at 410 nm.

Cellulase Assays. Cellulase activity may be monitored using the DNS reagent (47). Dissolve 11.4 g CMC (carboxy-methylcellulose) in 700 mL water with stirring, then add 7 g citric acid monohydrate, 19.6 g sodium citrate dihy-drate, 0.1 g merthiolate, and 0.1 g glucose and make up to 1 L. To 1 mL substrate, add 1 mL enzyme solution, incubate 20 min at 50°C, add 3 mL DNS reagent, heat 15 min in boiling water, cool, and read absorbance at 540 nm. Cellulase activity may also be assayed by following the decrease in viscosity of a solution of CMC during incubation with enzyme (48,49). This requires a great deal of care and preparation and will not be discussed here.

Glycosidase. As an example of assays using nitrophenyl glycosides, that for yS-galactosidase is given (50). In the reaction tube, combine 50 fiL 0.1 M citrate buffer, pH 4.3, 50 fih 0.4% bovine serum albumin in water, 50 //L 20 mM pNP-/?-galactoside in water, and 50 juL enzyme solution. After 30 min at 37°C, add 2 mL 0.25 M glycine, pH 10, and read the absorbance at 410 nm.

Xylanase. Interest has been growing recently in the use of xylanases in the food and feed industries. Xylans are pentose polymers, present primarily in cell walls of plants (an older name for them, based on this origin, is hemicel-lulose). Both soluble and insoluble types are found. The substrate for xylanases is usually derived either from a cereal grain (rye is the most abundant source) or wood (beech or larch gums). Assays may involve reducing-sugar techniques, viscometry, or chromogenic substrates (51). The chromogenic substrates are available in both soluble and insoluble (cross-linked) versions.

Xylanases, like amylases, may have either endo or exo specificities (similar to a- and /¿-amylases). Recently the two types have been separated and shown to have different kinds of improving actions in baked foods. Unfortunately, the specific assays that differentiate them have not been published. Using a soluble xylan as substrate, the endo-xylanase would show a rapid decrease in solution viscosity with a relatively slow increase in reducing groups, while the exo-xylanase would display the opposite behavior. The comparison of results from a viscometric assay and a reducing-sugar assay would readily differentiate the two types.

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