Carbohydrolase Assays*

Transcription

1 In: Dashek, William V., ed. Methods in plant biochemistry and molecular biology. Boca Raton, FL: CRC Press: pp Chapter 25. Chapter Carbohydrolase Assays* Terry L. Highley Contents * The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin.This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright. 309

2 310 Methods in Plant Biochemistry and Molecular Biology 25.1 Introduction The principal topic of this chapter concerns methods for the detection and measurement of enzymes produced by microorganisms for the breakdown of carbohydrate polymers present in the plant cell wall. Extracellular cell wall-degrading enzymes are important to both pathogenic and saprophytic microorganisms to overcome host resistance and/or to utilize organic and inorganic materials in the environment. The main carbohydrate components of the cell wall are cellulose, hemicellulose, and, in nonwoody materials, also pectins. Enzymes must exist for their breakdown, e.g., cellulases, hemicellulases, and pectinases. Much of this organic material is woody in nature, and if it did not rot, the earth would be covered with masses of dead vegetation. The industrial and economic significance of carbohydrolases is very high. The enzymes are produced by microorganisms to deteriorate wood in service, cellulosic products, rotting of fruits and vegetables, causing great economic loss. To prevent such losses, there is interest in better understanding of how these enzymes deteriorate organic material so that their degradative activities can be stopped. These enzymes also have beneficial industrial application in that they can be used for bioconversion of agricultural and wood waste to useful products. In determining carbohydrolase specificity, substrates usually are used that are homogeneous, or nearly so. 1 This permits the classification of enzymes according to the type of sugar unit and glycosidic linkage present in the specific substrate. Such enzyme-substrate correlations emphasize the type of linkage in the oligosaccharide or polysaccharide. Thus, cellulases are described as enzymes that catalyze the hydrolysis of the β 1,4 linkages between anhydro-d-glucose units, and amylases are associated with cleavage of the β 1,4 linkages of starch and glycogen. Another means of describing the specificity of a carbohydrolase for its substrate is to refer to the glycosyl unit itself rather than to the linkage, that is. by changing the emphasis from the type of linkage to the building block of the polymer. 1 Cellulase activity can then be regarded as involving the 4-substituted β-d-glucopyranosyl unit rather than as a cleavage of the β 1,4 bond. Quantitative assays for measurement of carbohydrolase activities in culture filtrates from microorganisms are addressed first. This is followed by screening methods for detection of carbohydrolase production by living organisms on agar medium and enzyme activity in culture filtrates. Prior to discussion of an assay for an enzyme, the nature of the enzyme is briefly discussed Polysaccharidases Enzymatic hydrolysis of most polysaccharides will release reducing sugars. Various methods have been used to estimate the reducing sugars formed in the enzymatic hydrolysis of polysaccharide substrates based on the reduction of an oxidation agent by the reducing sugars. The most commonly used methods to estimate enzymatically generated reducing sugars from polysaccharides are the calorimetric Nelson Somogyi and dinitrosalicylic acid (DNS) procedures Nelson-Somogyi The procedure described here for determining polysaccharidase activity, utilizing the Nelson- Somogyi 2,3 method, is one that we have used in determining cellulase, hemicellulase, and pectinase activity in filtrates from wood-rotting basidiomycete fungi. The temperature and ph pat-meters will, of course, vary with the source of the enzyme being assayed. Briefly, 1-mL samples are combined either with 10 mg substrate and 1 ml citrate buffer (0.1 M, ph 5.0) with 1 ml substrate solubilized in 0.1 M citrate buffer (ph 5.0) at 1% (w/v) in 50-mL Folin tubes. A control for each sample is prepared with substrate and buffer. Tubes are incubated at 40 C. After incubation, 2 ml of copper reagent, consisting of four parts KNa tartrate

3 Carbohydrolase Assays 311 Na 2 CO 3 /Na 2 SO 4 /NaHCO 3 (1:2:12:1.3) and one part CuSO 4 5H 2 O/Na 2 SO 4 (1:9), are added to each tube. Both copper reagents must be prepared by boiling to completely dissolve the components; they can then be stored at room temperature. They are mixed together just prior to use. After 1 ml of sample is added to the appropriate control tubes, all tubes are boiled for 10 min in a water bath. The tubes are then cooled completely, 2 ml of arsenomolybdate reagent (25 g ammonium molybdate in 450 ml H 2 O + 21 ml H 2 SO g Na 2 HASO 4 7H 2 O dissolved in 25 ml H 2 O) is added to each tube, and the tubes are shaken thoroughly before adjusting the final volume to 25 ml with water. Individual samples are filtered through filter paper, and optical density (0. D.) is determined by transmitted light at 500 nm in a spectrophotometer. Samples should be diluted within the O.D. range to so that accurate readings can be made. A standard curve is prepared by plotting O.D. against known concentrations of reducing sugars. A unit of enzyme activity can then be expressed as that liberating 1 µmol of reducing sugar per unit of time under the specified conditions Dinitrosalicylic Acid (DNS) Heavy metals, particularly copper, interfere with the DNS method. Set up enzyme solutions and substrates with appropriate controls as previously noted. Prepare DNS reagents 4 by mixing 20 ml of 2 N NaOH containing 1 g of 3,5-dinitrosalicylic acid with 50 ml of H 2 O. Add 30 g of KNa tartrate and dilute the solution to 100 ml. Keep tightly stoppered in well-filled bottles at 4 C to protect from carbon dioxide. The reagent should be stable for 1 year. After incubation at desired temperature and time, add 2 ml of DNS reagent and shake. Place tubes in boiling water for exactly 5 min. Cool tubes in ice water and dilute to volume (25 ml). Determine O.D. at 575 nm in a spectrophotometer. A standard curve is prepared as previously noted for calculation of enzyme activity Viscosimetric Among the many techniques for measuring polysaccharidase activity, particularly endopolysaccharidase, the viscosimetric method seems to be the most sensitive because breakage of a bond near the middle of the polymer chain will significantly reduce the viscosity of the polymer. However, there are certain problems, such as the choice of the substrate, expression of enzyme activity, or the distinction between enzyme and polymer properties. The assay has been used extensively for determining cellulase and pectinase activity because of the availability of suitable substrates, carboxy - methylcellulose (CMC) for cellulase and sodium polypectate for pectinase (polygalacturonase [PG]). We use the following procedure to determine the ability of cell-free culture filtrates from wood decay fungi to reduce the viscosity of CMC (cellulase or CMCase activity). In a Waring blender, 1% (wt/v) CMC is added to 0.1 M citrate buffer (ph 5.0), mixed for 1 min, poured into a flask, and allowed to equilibrate to temperature with a water bath, controlled at the desired incubation temperature. Then 9 ml of CMC solution are placed in an 18- by 120-mm tube and 1 ml of culture filtrate is added. The reaction mixture is agitated on a Vortex mixer for about 20 s; then 8 ml of the mixture are pipetted into an Ostwald Fenskie No. 300 viscosimeter suspended in the water bath. Draw the reaction mixture above the upper line of the viscosimeter by applying suction to rubber tubing connected to the arm of the viscosimeter. Determine the efflux time of the mixture (number of seconds required for the meniscus to fall from the upper to the lower line of the viscosimeter). The first viscosity reading is taken 1 min after addition of the enzyme to the substrate. Additional readings are made periodically, depending on enzyme activity. Researchers have expressed enzyme activity using a number of methods. One method is to calculate the percentage decrease in flow time (PDFT) after each incubation (t) according to the formula

4 312 Methods in Plant Biochemistry and Molecular Biology where E is the efflux time of CMC solution containing boiled filtrate; Et is the efflux time of CMC solution containing active filtrate after incubation for time t; and E w is the efflux time of distilled water in the same viscosimeter used for the above two determinations. Plot PDFT, vs. incubation time (including PDFT t = 0 at zero incubation time), and interpolate graphically the resulting curve to determine the time in minutes required for 50% decrease in flow time (t 50 PDFT). Enzyme activity is expressed as l/t 50 enzyme multiplied by Cellulase Cellulase refers to a group of enzymes that degrade cellulose to glucose. To achieve complete hydrolysis of insoluble cellulose, the different components of the enzyme complex must be present in the right amounts under the right conditions. Components of this complex include endo-β glucanases (EC ), exe-l-4-glucanases (cellobiohydrolase; EC ), and β- 1,4-glucosidases (EC ). Two types of exoglucanases are known enzymes splitting off a glucose unit from the nonreducing end of a cellulose chain and enzymes splitting off glucose and/or cellobiose. 5 All cellulase systems probably contain enzymes (endoglucanase, CMCase) capable of degrading soluble derivatives of cellulose such as CMC, but only those that contain exoglucanase can degrade highly ordered forms of cellulose as found in cotton hairs. A good example of a partially deficient system is enzyme preparations from brown-rot wood decay fungi. These fungi extensively degrade cellulose in wood, but enzyme preparations from liquid cultures or decayed wood or cellulose appear to lack the full cellulolytic complex, because they ineffectively degrade crystalline cellulose, but do degrade cellulose that has been modified by solubilization. The requirement for an active cellulase complex acting at optimum conditions, combined with the physical heterogeneous nature of cellulose substrates on which it must act, makes assaying for cellulolytic activity a formidable problem. A variety of methods have been developed for assaying cellulolytic activity, such as the following: Loss in weight of cellulosic substrates Loss in tensile strength or mechanical properties of cellulose fibers Release of dye from dyed substrates Liberation of reducing groups or glucose Decrease in viscosity of soluble cellulose derivatives Change in turbidity of cellulose suspension Clearing of cellulose in agar Growth on cellulose agar The choice of assays employed for cellulases can be confusing, measuring the three components of the complex separately or measuring the combined activity of two or even all three components. 6 For example, weight loss of cellulose substances is the most reliable and accurate measure of exoglucanase activity; viscosity reduction of cellulose derivatives measures only endoglucanase activity; cellobiose conversion to glucose measures the glucosidase activity. Assaying reducing sugars released from crystalline cellulose measures the combined effect of all three, although glucosidase is not needed. Measurements of weight loss or tensile strength cannot be directly compared with the liberation of reducing sugars. Measuring glucose produced from cellulose is similar except that the overall activity may be only half as great, ultimately without glucosidase. Measuring glucose produced from CMC measures endoglucanase with some contribution from glucosidase, but brings in the uncertainty of the variable composition of lots of CMC. For example, the more substituent groups on CMC, the more resistant it is to enzymatic hydrolysis. In recent years, assays based on the use of chromogens (dye-labeled/substrate) have become available for the assay of many polysaccharide-degrading enzymes that offer sever-d advantages

5 Carbohydrolase Assays 313 over the more conventional assays just discussed, including specificity simplicity in use and measurement of solubilization of cellulose. Leisola and Linko 7 note that the most important activity of the cellulase complex is usually the solubilizing effect (exoglucanases required). The solubilizing effect is usually measured by determining the release of reducing sugars from insoluble cellulose produced by the combined effect of the whole cellulase complex. However, the formation of reducing sugar or glucose is not necessarily proportional to the solubilizing effect. 7 Dyed cellulose substrates permit a true measure of the solubilizing activity of acellulase complex. 7 Cellulose azure (Calbiochem, Sigma) is an acid-swollen cellulose preparation dyed with Remazol brilliant blue R (RBB). However, because it is swollen cellulose, it probably is not a true measure of the enzyme activity needed to solubilize crystalline cellulose. Avicel (FMC), dyed with RBB using the method of Leisola and Linko, 7 is the best choice of celluloses because it can be dyed easily, hydrolyzes fairly rapidly, and, because it is crystalline, measures solubilizing effect. The cellulose-solubilizing effect (exoglucanase) can be determined using RBB-cellulose, as described by Leisola and Linko. 7 Into a test tube at the appropriate incubation temperature, 2 ml of enzyme solution are pipetted and 3 ml of buffer solution containing 50 or 100 mg of substrate is added. Stir on a magnetic stirrer. After incubation, the reaction is stopped by heating and the suspensions filtered through Whatman No. 1 filter paper. Absorbance is measured at 595 nm. Soluble chromogenic substrates can also be used for determining endoglucanase activity. 8 Preparation of RBB-CM-cellulose is described by McCleary, 8 and the assay for endoglucanase recommended by McCleary is as follows. For use as substrate, 2 g of RBB-CM-cellulose is sprinkled into 80 ml of vigorously stirring hot water. On dissolution, 5 ml of 2 M sodium acetate buffer (ph 4.5) are added; the ph is adjusted to 4.5 and the volume to 100 ml. In the presence of 0.02% sodium azide, the substrate is stable for more than 12 months at 4 C. Enzyme preparation (0.1 ml) is incubated with 0.5 ml of RBB-CM-cellulose substrate solution in 0.1 M sodium acetate buffer (ph 4.5) for up to 10 min at 40 C. The reaction is terminated by the addition of 2.5 ml of a precipitant solution that contains 80% ethylene glycol monomethyl ether, 0.3 M sodium acetate buffer (ph (5), and 0.4% zinc acetate. This mixture is vortexed for 10 s, stood at room temperature for 10 min, and centrifuged at 1000 xg for 10 min. The absorbance of the supernatant is measured at 590 nm Hemicellulases Hemicelluloses constitute 20 to 30% of annual and perennial plants. Hemicelluloses make up the largest content in annual plants and have been extensively examined in agricultural crops, such as corn stalks, corn cobs, corn seed coat, wheat straw, soybean hulls, as well as wood. Defined as plant cell wall polysaccharides, other than cellulose and pectin, hemicelluloses are extractable by alkaline solutions. A few hemicelluloses are extractable in hot water, but most require mild to concentrated alkaline solution with 10% sodium hydroxide being a common extractant. The hemicellulose β 1,4-xylan is probably the most abundant polysaccharide in terrestrial plants, next to cellulose. β 1-4-Mannans are the second most common hemicellulose. It is rare for either one of these hemicelluloses to occur as purely linear or without other sugars. Hemicellulases catalyzing the hydrolysis of hemicelluloses have been found in bacteria, fungi, insects, and plant material. The most extensively studied hemicellulases are those from fungi Xylanase Xylans can be hydrolyzed by β-xylanase (EC endo-1,4,- β -D-xylan xylanohydrolase) and by the glycosidase, β-xylosidase (EC ; exe-l,4-β-d-xylan xylohydrolase). A large number of assay procedures with various xylan substrates have been used to determine endoglucanase activity, making comparison among laboratories virtually impossible. 9 Note that liberation of reducing sugars from a xylan substrate does not necessarily mean that the responsible enzyme is

6 314 Methods in Plant Biochemistry and Molecular Biology an endoxylanase. The reducing sugars in question could equally result from the action of xylosidase, arabinosidase, or glucuronidase. Thus, all routine assays should be backed up by examination of the products of hydrolysis. Because of the large interlaboratory variability in xylanase assay procedures and the consequent impossibility of drawing meaningful comparisons between results reported in the literature, the International Energy Agency set in motion an international round-robin testing of methods in This study 10 provides a standardized endoxylanase assay using 4-0-methyl glucuronoxylan as substrate and gives precise instructions for the procedures to be followed using the DNS reagent to quantify the reducing sugars released by enzyme action. The xylanase standardized assay follows: Substrate 1.0% birch wood 4-0-methyl glocuronoxylan (Roth 7500) in 0.05 M Na-citrate buffer, ph 5.3. Homogenize 1.0 g of xylan in about 80 ml buffer at 60 C (e.g., using a kitchen blender) and heat to boiling point, preferably on a heating magnetic stirrer. Cool with continued stirring, cover, and stir slowly overnight. Make up to 100 ml with buffer. Store at 4 C for a maximum of 1 week or freeze aliquots of 25 ml at 20 C. Mix well after thawing! Procedure 1. Add 1.8 ml substrate solution to a 15-mL test tube, preferably using an automatic pipette. Adjust temperate to 50 C, 2. Add 200 µl enzyme diluted appropriately in citrate buffer, mix. 3. Incubate 300 s (5 min), 50 C. 4. Add 3.0 ml DNS, mix, and remove the tube from the water bath. Boil for 5 min, cool in cold water. 5. Measure the color produced at 540 nm against the reagent blank. 6. Correct the absorbance (5) for background color in the enzyme blank. 7. Using the standard line from known xylose concentrations, convert the corrected absorbance (6) to enzyme activity units. Reagent blank 1.8 ml substrate solution 5 min, 50 C 3.0 ml DNS 0.2 ml buffer Boil, cool. Use this solution to zero the spectrophotometer. Alternative procedures for assessment of endoxylanase activity have their uses, for example, when the background reducing sugar concentration is high. 10 Procedures include measuring the decrease in viscosity of solutions of suitable xylan or CM derivatives of xylan and monitoring the decrease in turbidity of stable suspensions of suitable insoluble xylans or of the release of dye from covalently dyed soluble or insoluble xylans. Endo-acting enzymes effect large responses in each of these assays, whereas those resulting from purified xylosidases or side chain-cleaving enzymes are minimal. Each alternative assay procedure should be standardized against a reducing sugar assay for each substrate and enzyme preparation used. ) The reducing group method does not offer correct results in the presence of exo-acting glycanases. Viscometric methods are more specific, but are not suitable for analysis of insoluble enzyme sources and larger series of samples. Biely et al. 11 have eliminated some of these shortcomings by the use of soluble xylan with covalently bound RBB. Dyed xylan is prepared by dissolving RBB (0.15 to 20g) in a solution of water-soluble xylan (1 g in 30 ml of H 2 O). A solution of Na 2 SO 4 (10 mg in 10 ml of water) is added dropwise with stirring for 5 min. The mixture is then alkalized with NaOH (0.5 to 1.0 g in 10 ml of water) and stirred at room temperature for

7 Carbohydrolase Assays min. The dyed product is precipitated with two volumes of ethanol (96%), collected by filtration, and washed with a mixture of ethanol 0.05 M sodium acetate in water 2:1 (v/v) until the filtrate is colorless. The product is successively washed with ethanol/water 4:1 (v/v), ethanol, and acetone, and dried at room temperature. About 1 g of RBB-xylan is isolated in this way. To conduct the xylanase assay, 0.1 ml of enzyme in appropriate buffer is added to 0.9 ml of RBB-xylan solution at suitable incubation temperature. After incubation, the reaction is terminated by addition of 2.0 ml 96% ethanol, mixing, standing for 20 min, mixing again, and centrifuging at 2000 xg for 3 min. Absorbance of the supernatant is measured at 595 nm against a substrate blank prepared by incubating 0.1 ml buffer in place of enzyme Mannanase b- D-Mannanase (EC ; mannan endo-1-4-β-mannanase) has not received nearly the attention as xylanase. However, the enzyme is produced by a number of organisms 12 such as fungi, bacteria, higher plants (locust, coffee, almond, apricot), and in animals (mammals, snails, osyters). Like xylan, mannan substrates vary considerably, but usually have a glucose or galactose component. The assay systems for mannanase are similar to those for xylanase with substitution of a suitable mannan substrate such as carob gum or guar gum. McCleary 8 describes a dyed substrate assay for mannanase. Prior to dying with RBB, a lowviscosity carob galactomannan is prepared. In 3 L of water containing 0.2 g of cellulase preparation (Sigma C7502) at 60 C, 200 g of gum, locust bean (Sigma Chemical Co. G0753) is suspended. This is stirred with a spatula to give a thick paste. After incubation for 30 min at 40 C, the paste is homogenized in a Waring blender and then incubated at 40 C for an additional 30 min. (b- Mannanase present in the cellulase preparation causes a significant decrease in the paste viscosity during this incubation.) The slurry is again homogenized in a Waring blender, incubated in a boiling water bath until the temperature reaches 90 C (to inactivate β-mannanase), and then centrifuged at 3000 xg for 15 min. The clear supernatant solution is poured into two volumes of ethanol (95% v/v), whereupon the galactomannan precipitates as a white fibrous mass. This material is collected on a nylon screen, washed by resuspension in aqueous ethanol (60% v/v), and then dried by solvent exchange with ethanol and acetone and stored in vacuo; the yield is 60%. In 1.6 L of water at 60 C, 120 g of low-viscosity carob galactomannan is dissolved. To this is added 24 g of RBB dye and 160 g of anhydrous sodium sulfate, which is dissolved by stirring over 5 min. Then, 13 g of trisodium phosphate is added and stirring is continued for 2 h at 60 C. The solution is then cooled and dialyzed overnight against flowing tap water. On treatment of this solution with two volumes of ethanol, the dyed galactomannan, which precipitates from solution, is recovered on a nylon screen and washed with 66% (v/v) aqueous ethanol until most free dye is removed. The polymer is dissolved again in water at 60 C and reprecipitated by the addition of two volumes of ethanol. This step is repeated until all free dye is removed. The precipitated galactomannan is then washed with ethanol and acetone and dried in vacuo; the yield is 100 g. The RBB to anhydrohexose ratio is approximately 1:50. For use as substrate, 1 g of the polysaccharide is dissolved in 80 ml of water at 60 C, with vigorous stirring more than 15 min. Then 10 ml of 3 M sodium acetate buffer (ph 5.0) are added and the volume is accurately adjusted to 100 ml. In the presence of 0.02% sodium azide, the solution is stable at 4 C for at least 12 months and shows no significant tendency to settle from solution during this period. Enzyme preparation (0.5 ml) containing 0 to 0.5 units of β-mannanase activity per 0.5 ml is incubated with RBB-carob galactomannan substrate solution (1.0 ml, 1% w/v) for 5 to 20 min at 40 C. The reaction is terminated and the high-molecular weight substrate is precipitated by the addition of ethanol (3 ml). The mixture is stirred, allowed to equilibrate to room temperature for 10 min, and centrifuged at 1000 xg for 10 min. The enzyme reaction is monitored by increased absorbance (590 nm) of the supernatant solution.

8 316 Methods in Plant Biochemistry and Molecular Biology Pectinase Pectins are a group of colloidial substances with a high proportion of anhydro-galacturonic acid and have a variable and complex composition. 13 D-Galacturonic acid and its methyl ester are (1,4) linked as poly-(α-galactopyranosyl)-uronic acid in the backbone of pectin. Blocks of galacturonic acid are interspaced by (1,2) linked α-l-rhamnopyranosyl units. Nonrhamnose sugars like galactose, arabinose, glucose, mannose, and xylose also occur. Acetyl ester groups are supposedly linked to galacturonate residues in the backbone, whereas feruloyl ester groups are thought to be linked to the neutral sugar side chains, at least in sugar beet pectin. 14 Pectinases (pectate lyase [PL], polygalacturonase [PG], and pectin methylesterase [PME]) have been studied in more pathogens and in more detail than any other wall depolymerase. The original rationale for studying pectinases was that they are able to macerate tissue, the characteristic symptom of soft-rot diseases. 15 Research on pectinases has received impetus recently from the demonstration that pectinases and pectin fragments induce numerous physiological effects in plants Pectin Methylesterase Pectin methylesterase (EC 3.1.1) catalyzes the hydrolysis of methyl ester groups of pectinic acids, converting pectinic acids to pectic acids (polygalacturonic acid). Numerous assay techniques exist for measuring pectin methyesterase (PME) activity, liberation of methanol determined colorimetrically, increase in free carboxyl groups by titration, or the hydrolysis of nitrophenylacetate measured spectrophotometrically. 16 The following is a simple titration method. Substrate is prepared by mixing 0.5% citrus pectin with distilled water in a Waring blender for 1 min. The mixture is filtered through Whatman No. 1 paper on a Buchner funnel. In a 250-mL beaker containing a stirring bar, 100 ml of substrate and 3 ml of 1.0 M CaCl 2 are placed. The electrodes of a ph meter are inserted and the mixture adjusted to ph 7.5 with 1 N NaOH. Then 10 ml of enzyme preparation are added to the mixture and the ph of the enzyme substrate mixture is readjusted to 7.5 and a timer started. The ph is adjusted continually to ph 7.2 to 7.5 during the course of the assay by adding N NaOH from a burette. The total milliliters of NaOH is recorded. A control is 10 ml of boiled extract substituted for the active enzyme sample. The titration difference in milliliters of alkali required by active extract and that of the heated control multiplied by the concentration of alkali equals the milliequivalents (meq) of acid released by enzymatic action. PME activity is expressed as microequivalents (meq 1000) of carboxyl groups released per unit of time by a specified quantity of enzyme solution Pectin Lyase PL (EC 4.2.2) breaks down pectin by a trans-elimination reaction. The enzyme can be assayed by measuring the change in absorbance at 235 nm in a buffered pectin reaction mixture. 18 The assay mixture contains 10 m M MES, 0.022% pectin (86% methoxy content, Sigma), 1.5 m M CaCl 2, 75 m M NaCl, and the ph is adjusted to 6.5. The enzyme solution is added to 2 ml of the buffered pectin in a 1-cm quartz cuvette. The increase in absorption, due to the formation of uronide double bonds, is recorded at 5 min of reaction time. One unit of pectin lyase activity is defined as the amount of enzyme that causes an increase of 1.0 absorbance unit per minute at 235 nm at a specified temperature.

9 Carbohydrolase Assays Lytic Enzymes A wide variety of lytic enzymes from various microorganisms can break down fungal cell walls. The principle components of the fungal wall are chitin and β 1,3 glucan. Chitin, a 1,4 β D glucosidically linked fiber-forming polymer of N-acetyl-D-glucosamine units, also occurs in the cell wall of some algae, in the exoskeletons of arthropods including crustaceans and insects, in nematodes, in molusks, in worms, and in many other types of organisms Chitinase Assays of chitinase (EC ; b-1,4-n-acetyl glucosamine glycanhydrolase) activity include viscosimetric using soluble derivative of chitin, reduction of turbidity of a suspension of collodial chitin, estimation of reducing sugar using collodial chitin, radiochemical methods using regenerated [3H]-chitin, or methods based on insoluble or soluble dye-labeled chromogenic substrldtes. 19 Estimation of reducing sugars in the hydrolysis of purified chitin (Sigma), with N -acetylglucosamine or a reference compound, is an easy and suitable assay for chitinase. Reduction of turbidity of a suspension of colloidal chitin is also a convenient assay for measurement of chitinase activity. 20 To prepare colloidal chitin, chitin is dissolved into and reprecipitated from concentrated HCI as described by Shimahara and Takiguchi. 21 A suspension containing 1% (w/v) of moist colloidal chitin is prepared in appropriate buffer and ph. A mixture of 0.5 ml each of the chitin suspension and the enzyme to be tested is prepared and incubated for 24 h at a suitable incubation temperature. After incubation, dilute the mixture with 5 ml of water and determine O.D. at 510 nm. Activity is expressed as the percentage of reduction in turbidity relative to that of a similar suspension containing water rather than enzyme solution. Soluble dye-labeled and a colloidal dye-labeled substrate are also commercially available for a sensitive and reliable, simple, and microtiter plate-adapted calorimetric assay of chitinase activity, 19 These dyes are also applicable for rapid and selective detection of chitinolytic microorganisms in agar media (plate-clearing assay) β-1,3-glucanase β 1,3-Glucose glycanhydrolase (glucanase) (EC ) is usually assayed by the increase in reducing sugars from laminarin (Sigma) Lysozyme Another lytic enzyme is lysozyme (EC ; N-acetylmuramide glycanohydrolase), which hydrolyzes the β-1,4-glucosidic linkages in the mucopolysaccharide cell wall structure in a variety of microorganisms. Plant lysozyme is found principally in fiscus and papaya latex. Worthington 6 describes an assay method based on the lysis of Micrococcus lysodeikticus. Prepare a suspension of dried M. lysodeikticus cells (Sigma), 0.3 mg/ml in 0.1 M phosphate buffer, ph 7.0. Set spectrophotometer at 450 nm. Add 0.1 ml of enzyme solution to 2.9 ml of substrate. Record absorbance at 15-s intervals using a water blank. The first 2 min of reaction are used to calculate activity. Due to the variability of the substrate, it is advisable to assay a standard preparation of lysozyme at the same time as the unknown. One unit of enzyme activity is equal to a decrease in absorbancy, at 450 nm, of per minute at ph 7.0 and 25 C Amylase Starch and glycogen are hydrolyzed by amylases, of which there are two general types: α- and β-amylase. α-amylase (EC ) is found in nearly all plants, animals, and microorganisms. α-amylase (endoamylase) catalyzes the hydrolysis of internal α-1,4-glucan linkages in starch or

10 318 Methods in Plant Biochemistry and Molecular Biology glycogen containing three or more α-1,4-linked glucose units, yielding a mixture of maltose and glucose. β-amylase (EC ; exoamylase) occurs in resting seeds. β-amylase releases successive maltose units from the nonreducing end of a polysaccharide chain by hydrolysis of α-1,4-glucan linkage. Both types of amylases can be assayed by the reducing group assay. A calibration curve is made with maltose to convert calorimetric readings to units of activity. Commercially available starch-azure (Sigma) can be used to assay amylase activity similar to that described previously for cellulase activity using cellulose-azure. McCleary 8 describes a method using soluble dyed starch as a chromogenic substrate for determining α-amylase activity Dextranase Dextranase (EC ; 1,6-α-D-glucan 6-glucanohydrolase) hydrolyzes the bacterial polysaccharide, dextran (α-1,6-.glucan), to isomaltose residues. The reducing group assay can be used for determining enzyme activity. 6 The substrate used is 2% dextran 500 (AB Pharmacia) in 0.1 M potassium, phosphate buffer, ph 6.0, containing 0.01% merthiolate. To 1.9 ml substrate, add 0.1 ml dilute enzyme. Digest for 30 min at 37 C. At end of digestion period, use 1 ml of digest for reducing group assay. Prepare a maltose standard curve using levels 0.3 to 3.0 µm maltose. One unit of enzyme activity causes the release of 1 µm isomaltose from dextran per minute Glycosidases Glycosidases break down the oligosaccharides generated by polysaccharidases to monomer sugars. Most of these enzyme activities can be assayed by determining the liberation of p-nitrophenol from the p-nitrophenol substrate. Nitrophenol substrates are available from chemical companies such as Sigma. Some commonly assayed glycosidases and p-nitrophenol substrates are arabinosidase (EC , p-nitrophenyl-α-l-ambinofuranoside); chitobiosidase (EC , p-nitrophenyl-β-d N,N'-diacetylchitobiose); galactosamidase (EC , p-nitrophenyl N-acetyl-β-D-galactosaminide); α-galactosidase (EC ); β-galactosidase (EC ) activity using p-nitrophenyl-α-d-galactopy ranoside or p-nitrophenyl-β-d-galactopyrdnoside; β glucosidase (EC ); α-glucosidase (EC ) using p-nitrophenyl. β-d-glucopyranoside, or p-nitrophenyl-α-d-glucopyranoside; glucuronidase (EC , p-nitrophenyl-β-d-glucuronide): mannosidase (EC , p-nitrophenyl-β-mannopyranoside); or xylosidase (EC , p-nitrophenyl-β-dxylopyranoside). We use the following procedure to assay for glycosidase activity in wood decay fungi. In buffer at appropriate ph, 2 ml of 0.05% p-nitrophenyl substrate are mixed with 1 ml of enzyme solution and incubated at a suitable temperature. The reaction is terminated by the addition of 1 ml of 0.2 M NaCO 3. The resulting yellow color is immediately measured at 425 nm with a spectrophotometer. A unit of enzyme activity is defined as the amount that will liberate 1 µm of p-nitrophenol per unit of time Microplate Assay Many colorimetric assays have been modified for simple testing in microtiter plates and automated measurement in a microplate reader. These microassay systems Facilitate rapid screening of a large number of column chromatography fractions and substantially conserve time and reagents. In addition, fractions can be screened for different enzymes simultaneously on the same microplate.

11 Carbohydrolase Assays 319 Most commercial microplate readers differentiate calorimetric reactions by transmitted light (absorbance) at a specific wavelength. Either insoluble substrates, used in many assays, or the precipitates formed during an assay prevent penetration of light and render measurement by transmitted light inaccurate. We have adapted a thin-layer chromatography double-beam densitometer to accept 96-well microtiter plates for determination of reducing sugars by a modified Nelson Somogyi method. 22 Each well is read by reflectance, thus avoiding the time-consuming filtration step necessary in the standard Nelson Somogyi assay in cases when insoluble precipitate hinders reading by absorbance (transmission). In a 96-well microplate, 25 µl of sample and 25 µl of appropriate substrate solubilized in 0.1 M citrate buffer, ph 5.0, are placed in each well. The plate is covered with an acetate adhesive sheet and incubated at 40 C for 24 h. After incubation, 75 µl of Somogyi copper reagent is added and the wells are resealed with the acetate sheet. The plate is then incubated at 80 C for 30 min in a water bath. After the plate has cooled completely (15 min), 75 µl of arsenomolybdate are added, and the wells are mixed well with a Vortex mixer. Calorimetric measurements are read using reflectance at 500 nm with a dual-wavelength densitometer. A standard curve is prepared using glucose at concentrations ranging from 100 to 2000 µg/ml. The microassay has proven invaluable for rapid surveys designed to locate polysaccharidedegrading enzyme from chromatography fractions. The substantially smaller test volume reduces loss of precious enzyme. The ability to run multiple samples as quickly as single samples improves efficiency, and test reagents can be added with multichannel pipettes Screening Assays Solid Medium with Living Organism Numerous screening methods exist for detecting carbohydrolase activity in microorganisms usually grown on agar medium. A solid medium providing rapid assays is useful for the direct measurement and isolation of carbohydrolase-producing organisms from natural substrates and for the isolation of mutants. Such methods also have additional advantages over the use of culture filtrates: (1) components of an enzyme system are not denatured by preparation procedures, (2) mycelial-bound enzyme activity is measured, and (3) measurements of enzyme activity are not made at a specific harvest time which may not be optimal for all organisms. 23 Common screening techniques devised for the detection of polysaccharide-degrading enzymes by microorganisms involve plate assays where the respective polymer, or its derivative, is incorporated into the basal growth medium. The production of corresponding polysaccharide-hydrolyses is indicated by the clearing of the opaque medium as the substrate is dissolved by the enzymes produced by the growing colonies. In such procedures, the results are often difficult to interpret, because the zones of clearing are not always easy to distinguish from the unaffected medium and activity might not be detected because of the masking effect of the mycelial mass when the rate of fungal growth exceeds enzyme diffusion. Chang et al. 24 overcame this problem by inoculating a polycarbonate membrane (0.2 µm, 90 mm diameter, Nucleopore Co. ) with the test fungus. After incubation, the membrane is removed and the enzyme activity recorded. The polycarbonate membranes, which can resist penetration by fungi, are placed between moistened filter papers and sterilized by autoclaving before use. Dyed polysaccharide substrates placed over an agar medium and inoculated with an organism are also useful for screening for polysaccharide-degrading enzymes. 25 The detection of enzyme activity depends on the release of dye and its diffusion into the colorless lower layer following enzymatic hydrolysis of the dyed substrate.

12 320 Methods in Plant Biochemistry and Molecular Biology Solid Media with Cell-Free Filtrates Solid media are also used to screen cell-free extracts from microorganisms for polysaccharidedegrading enzyme activity. Generally, the enzyme solution is placed in a well (cup) cut in an agar medium containing a polysaccharide substrate. The agar surface usually needs to be flooded with a reagent to develop the zones of enzyme activity around the cup. The cup-plate assay for determining polygalacturonase activity in filtrates is a good example of this type of assay (Figure 25.1).26 In a Waring blender, 1 g of ammonium oxalate, 2 g of sodium polypectate, and 3 g of agar are mixed in 200 ml of 0.1 M acetate buffer (ph 5.0). The mixture is placed in a 500-mL flask, covered with cheesecloth and aluminum foil, and autoclave for 5 min at 15 lb/in2. Hot media is poured into a prepared plate consisting of a 10- by 10-in piece of glass with 1-in pieces around the outside edges (a petri plate can also be used). When the medium is solidified, a No. 3 cork bore is used to cut cups. Into each cup, 0.1 ml of enzyme is pipetted using a long-tipped measuring pipette. After filling the cups, the plate is topped with a single glass plate. A paper towel, saturated with water, is laid on top of the plates and the plates are placed in a sealed double plastic bag. Plates are incubated at 37 C for 14 h. Following incubation, HC1 (1:2 dilution of concentrated HC1) is poured over the plate. After approximately 3 min, the plates are rinsed with cold water. The diameter of the cleared zone is measured with calipers and recorded. Diameters are converted to enzyme activity by running a dilution of the enzyme and plotting zone diameter against the log of the enzyme concentration.

13 Carbohydrolase Assays 321