ENZYME ASSAYS

III. ENZYME ASSAYS

To determine whether an enzyme is present and the quantities in which it is present, a test called an assay is run. The activity of an enzyme is the velocity per milliliter or milligram of enzyme, where velocity is defined as the number of molecules of substrate transformed per unit of time. Activity is therefore the number of substrate molecules trans- formed per unit of time per milliliter or milligram of enzyme. Activity is expressed in units, where a unit is defined as the amount of enzyme required to transform 1 µmol of substrate per minute under the conditions of the assay. The last phrase, ‘‘under the condi- tions of the assay,’’ is a disclaimer that accounts for pH, temperature, ionic strength, presence of cosolvents, etc. The International Union of Biochemistry recommends the use of the Katal as a standard unit. A Katal of enzyme activity represents that amount of enzyme capable of transforming 1 mol of substrate per second and conforms with other SI units of measurement. Its use, however, has not been prevalent because of its tendency to generate smaller numbers than those scientist prefer to use. For example, 1 unit of enzyme, where a unit converts 1 µmol of substrate per minute, would correspond to 6 ⫻

10 ⫺7 Katal, or 0.6 µKatals. There are two main events in an enzyme reaction, and these happen simultaneously. During an enzyme reaction, the substrate concentration diminishes as it is converted to product, and the product concentration increases as the substrate is utilized. Therefore, the velocity of enzyme-catlyzed reaction can be monitored by measuring either the rate of disappearance of substrate or the rate of appearance of products. These differences may

be manifested as absorbance, fluorescence, optical rotation, pH, electrical conductivity, viscosity, or volume, any of which can form the basis of assay. Numerous recipes for the assay of enzymes have been published (Bergmeyer, 1974; Barman. 1969; Colwick and Kaplan, 1955). In addition, enzyme manufacturers provide information on assay methods. The U.S. Food and Drug Administration (FDA) generally follows procedures for assay recommended by the Food Chemical Codex and Nutrition Board of the National Research Council of the National Academy of Sciences in Washington, D.C. (NAS/NRC, 1981).

To study the action of amylase on starch, there are four different assay procedures in common use. Due to hydrolysis there is an appearance of reducing groups, decrease in viscosity, and an increase in maltose, glucose, or dextrins. The degree of hydrolysis is also determined by comparing the iodine color of a standard. When amylase hydrolyzes the α-1,4 glucosidic linkages of the polysaccharide, there is a rapid loss in viscosity and iodine color formation decreases. One dextrinizing unit (DU) is the quantity of amylase that will hydrolyze soluble starch in the presence of an excess of β-amylase at the rate of 1 g/h at 30 °C. Bacterial amylases are assayed in a similar manner but in the absence of amylase, and 1 bacterial amylase unit (BAU) is defined as the quantity of enzyme that will dextrinize 1 mg of starch per minute under the conditions of the assay.

Color measurements are done by comparing colors with standards. Note that 1 BAU is defined differently from 1 DU and the assay conditions vary with respect to the presence of β-amylase.

Malt is characterized by its diastase activity, or diastatic power (DP), and is different from assays for amylases. Amylase activity in malt is measured by hydrolysis of starch occuring in a 30-min period at pH 4.6 and 20 °C. The hydrolysis is quantitated titrimetri- cally by the reducing groups generated. One unit of diastatic power expressed as DP degrees ( °DP) is that amount of enzyme contained in 0.1 mL of a 5% solution of sample Malt is characterized by its diastase activity, or diastatic power (DP), and is different from assays for amylases. Amylase activity in malt is measured by hydrolysis of starch occuring in a 30-min period at pH 4.6 and 20 °C. The hydrolysis is quantitated titrimetri- cally by the reducing groups generated. One unit of diastatic power expressed as DP degrees ( °DP) is that amount of enzyme contained in 0.1 mL of a 5% solution of sample

Catalase reacts with chemically reactive oxygen species. It participates in two types of reactions: (1) a catalytic reaction and (2) a peroxide reaction. Catalase activity can be measured by various methods such as spectrophotometric, titrimetric, polarographic, and manometric methods.

Various assays given for commercial catalase preparations convert a rate of ab- sorbance change to a rate of concentration change at a set wavelength. Catalase activity is determined as Baker units (BU). The assay is an exhaustion method that quantitates the breakdown of hydrogen peroxide and the breakdown of catalase by the peroxide under specified conditions. One Baker unit of catalase activity is that amount of enzyme required to decompose 266 mg of hydrogen peroxide under the conditions of the assay. The assay is also performed by gas elecrode method, using phosphate buffer of pH 7.0 saturated with air and addition of hydrogen peroxide and catalase solution in a specific proportion

at 25 °C. The rate of increase in O 2 saturation in terms of molar concentration is recorded. 2H 2 O 2 →2H 2 O⫹O 2 Titrimetric activity is also calculated by reaction of hydrogen peroxide with standard

potassium permanganate solution in acid. Cellulase activity is measured in terms of reduction in viscosity of a reaction com- pound. Cellulose is converted to glucose by the action of a group of enzymes. The viscosity depends upon molecular weight of cellulose which reduces from 400 to 300 centipoise (cP) at pH 5.0 due to action of this enzyme. Manning (1981) has developed the methodology for measuring the viscosity change due to action of endocellulase on carboxymethylcellulose. One unit of cellulase activity (CA) is defined as that quantity of enzyme required to reduce the viscosity of 200 g of 5% solution of cellulose gum (Hercules Type 7-LF) from 400 to 300 cP at 35 ⫾ 0.1°C, pH 5.0, in 1 h.

β-Glucanase activity determination is based on a 15-min hydrolysis lichenin sub- strate at 40 °C and pH 6.5. As reducing groups are generated during hydrolysis, the increase in reducing power is quantitated spectrophotometrically. One β-glucanase unit (BGU) is that quantity of enzyme capable of liberating 1 mol of reducing sugar per minute under conditions of the assay. β-Glucanase assay is also described by Wood and Weisz (1987). This enzyme is mainly found in barley and oats. To 25 mL of MES buffer pH 5.5 was added 12.5 mg β-glucan and 125 mg agarose, and suspension was heated to boiling. The hot suspension was poured into petripalates and wells of 4 mm diameter were cut into gel. To each well 10 µL enzyme solution were added. Congo red was layered on petridishes and incubated for 18 h. The unreacted subtrate gave a purple red color sourrounding a clear zone of enzyme activity.

Glucoamylase or amyloglucosidase hydrolyzes starch, liberating one glucose mole- cule at a time. The enzyme sample is allowed to hydrolyze a cornstarch hydrolysate under controlled conditions of time, temperature, pH, and substrate concentration. The enzyme is assayed by measuring the reducing sugars resulting from the enzymatic hydrolysis of starch. A1-mL enzyme sample is mixed with an equal volume of soluble starch (30 g/L) in 0.1 M sodium acetate buffer (pH 4.8) and incubated at 30 °C. The amount of reducing sugar produced is determined with glucose as the standard. One unit glucoamylase of activity is defined as the amount of enzyme which liberates 1 µmol of reducing sugars per minute.

Glucose isomerase is the major enzyme used in the high-fructose corn syrup indus- try. Activity of glucose isomerase is generally determined using an immobilized enzyme preparation. The rate of conversion of glucose to fructose is measured using a plug flow or packed bed reactor. Enzyme assays are highly dependent upon the bed configuration, flow rate, and viscosity of the solutions. Glucose concentration is 45% w/w inlet pH 7.0–

8.5 at 25 °C (varying depending on the source of enzyme), reaction temperature 60°C, magnesium concentration 4 ⫻ 10 ⫺3 M. One unit of glucose isomerase with the column

method (GLcU) is defined as the amount of enzyme that converts glucose to fructose at an initial rate of 1 µmol/min under specified conditions. The conversion of glucose to fructose is monitored polarimetrically by measuring the change in specific rotation. In the automated assay, enzyme reacts with D-glucose and the dialyzed reaction mixture is mixed with acid carbazole. Carbazole gives a colored product only with D-fructose.

Glucose oxidase is an oxidoreductase whose activity is measured by an oxygen electrode based on the oxygen uptake in the presence of excess substrate, excess air, and excess catalase. One glucose oxidase unit (GOU) is defined as the amount of enzyme that

will cause an uptake of 10 mm 3 of oxygen per minute in a Warburg manometer in the presence of excess air and excess catalase and with a substrate containing 3.3% glucose monohydrate in 0.1 M phosphate buffer at pH 5.9 with 0.4% sodium dehydroascorbate (Scott, 1953). Glucose oxidase can also use O-benzoquinone as the hydrogen acceptor. 1 mL of benzoquinone (0.1% in water), 0.9 mL citrate buffer (pH 5.0), and 2 mL of glucose are mixed and kept at 25 °C; 0.05 mL of enzyme is added and absorbance at 290 nm is recorded at 2-min intervals. The increase in absorbance is due to an increase in concentra-

tion of hydroquinone, which has a molar absorptivity of 2.31 ⫻ 10 3 I.M. cm ⫺1 . Hemicellulase activity is measured by the ability of the enzyme to break interior

glycosidic bonds of a defined locust bean gum (Type D-200, Meer Corporation) substrate at pH 4.5 and 40 °C. The corresponding reduction in viscosity is measured using a viscome- ter. One hemicellulase unit (HCU) is defined as that amount of enzyme that will produce

a relative fluidity change of 1 over a period of 5 min in a locust bean substrate under specified conditions. The viscometer recommended is a Cannon–Fenske type size 100 apparatus.

Invertase is an enzyme used in invert sugar manufacture, and its activity is measured by 30-min hydrolysis of sucrose at 20 °C and pH 4.5. The degree of hydrolysis is measured by a change in optical rotation of the solution with a polarimeter. One invertase unit is defined as the quantity of enzyme that will hydrolyze 77% of the sucrose applied under the conditions of the assay.

Lactase ( β-galactosidase) activity is based on a 15 min assay involving the hydroly- sis of a synthetic substrate O-nitrophenyl– β-D-galactopyranoside (ONPG) at 37°C at a specified pH, dependent on the enzyme source. Substrate hydrolysis is monitored spectro- photometrically by measuring the intensity of yellow color at 420 nm. The yellow color is the O-nitrophenol liberated due to hydrolysis. One lactase unit (LaU) is defined as the amount of enzyme that hydrolyzes 1 µmol of ONPG per minute at 37°C under assay conditions.

Lipase hydrolyzes triacylglycerol liberating free fatty acids, which can be titrated. The assay is based on a 5-min hydrolysis of olive oil emulsion at pH 6.5 and 30 °C. The released fatty acids are titrated with standard base. One unit of lipase activity is defined as that amount of enzyme required to liberate 1 µmol of acid per minute from an emulsified substrate under conditions of the assay.

tributyrin, and the liberated acids are continuously titrated in a pH-stat. One lipase unit (LFU) is the activity that releases 1.25 µmol of butyric acid per minute under conditions of the assay.

Milk-clotting activity is often determined for enzyme preparations suitable for cheesemaking. Expression of a milk-clotting unit is complex because it is a ratio. In a general sense it is the number of milliliters of milk that can be clotted in 40 min at 35 °C by 1 mL of a coagulant.

Pepsin activity is measured as pepsin units. One pepsin unit is defined as that quan- tity of enzyme that digests 3000 times its weight of coagulated egg albumin under condi- tions of the assay.

Plant proteolytic activity depends on a 60-min hydrolysis of casein at pH 6.0 and

40 °C. Unhydrolyzed substrate is precipitated with trichloroacetic acid, and the concentra- tion of the solubilized protein is determined spectrophotometrically at 280 nm. One plant proteolytic unit is the quantity of enzyme that liberates 1 mg of tyrosine per hour under conditions of the assay.

The determination of proteolytic activity of bacterial proteases (PC) is similar to the assay for plant proteolytic activity and relies on measuring solubilized casein expressed as tyrosine. One unit of PC is the amount of enzyme that will liberate 1.5 mg of tyrosine equivalent per minute under the conditions of the assay.

Proteolytic activity of fungal enzyme is also similar to these methods, but instead of casein hemoglobin is used as the substrate, and therefore units are expressed as hemo- globin units on tyrosine basis (HUT). A 30-min hydrolysis of hemoglobin at pH 4.7 and