carbohydrate assays

In: Dashek, William V., ed. Methods in plant biochemistry and molecularbiology. Boca Raton, FL: CRC Press: pp. 309-321. 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 writtenand prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject tocopyright.

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25.1 Introduction

Methods in Plant Biochemistry and Molecular Biology

The principal topic of this chapter concerns methods for the detection and measurement of enzymesproduced by microorganisms for the breakdown of carbohydrate polymers present in the plant cellwall. Extracellular cell wall-degrading enzymes are important to both pathogenic and saprophyticmicroorganisms to overcome host resistance and/or to utilize organic and inorganic materials inthe 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 notrot, the earth would be covered with masses of dead vegetation.

The industrial and economic significance of carbohydrolases is very high. The enzymes areproduced by microorganisms to deteriorate wood in service, cellulosic products, rotting of fruitsand vegetables, causing great economic loss. To prevent such losses, there is interest in betterunderstanding of how these enzymes deteriorate organic material so that their degradative activitiescan be stopped. These enzymes also have beneficial industrial application in that they can be usedfor 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 andglycosidic linkage present in the specific substrate. Such enzyme-substrate correlations emphasizethe type of linkage in the oligosaccharide or polysaccharide. Thus, cellulases are described asenzymes that catalyze the hydrolysis of the β−1,4 linkages between anhydro-D-glucose units, andamylases 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 tothe glycosyl unit itself rather than to the linkage, that is. by changing the emphasis from the typeof linkage to the building block of the polymer.1 Cellulase activity can then be regarded as involvingthe 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 frommicroorganisms are addressed first. This is followed by screening methods for detection of carbo-hydrolase 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.

25.2 Polysaccharidases

Enzymatic hydrolysis of most polysaccharides will release reducing sugars. Various methods havebeen used to estimate the reducing sugars formed in the enzymatic hydrolysis of polysaccharidesubstrates based on the reduction of an oxidation agent by the reducing sugars. The most commonlyused methods to estimate enzymatically generated reducing sugars from polysaccharides are thecalorimetric Nelson–Somogyi and dinitrosalicylic acid (DNS) procedures.

25.2.1 Nelson-Somogyi

The procedure described here for determining polysaccharidase activity, utilizing the Nelson-Somogyi2,3 method, is one that we have used in determining cellulase, hemicellulase, and pectinaseactivity in filtrates from wood-rotting basidiomycete fungi. The temperature and pH pat-meterswill, 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 (0.1M, pH 5.0) with 1 mL substrate solubilized in 0.1 M citrate buffer (pH 5.0) at 1% (w/v) in 50-mLFolin tubes. A control for each sample is prepared with substrate and buffer. Tubes are incubatedat 40°C. After incubation, 2 mL of copper reagent, consisting of four parts KNa tartrate

Carbohydrolase Assays 311

Na2CO3/Na2SO4/NaHCO 3 (1:2:12:1.3) and one part CuSO4·5H2O/Na2SO4 (1:9), are added to eachtube. 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 mLof 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 molyb-date in 450 mL H2O + 21 mL H2SO4 + 3 g Na2HASO4·7H2O dissolved in 25 mL H2O) is added toeach tube, and the tubes are shaken thoroughly before adjusting the final volume to 25 mL withwater. Individual samples are filtered through filter paper, and optical density (0. D.) is determinedby transmitted light at 500 nm in a spectrophotometer. Samples should be diluted within the O.D.range 0.100 to 0.600 so that accurate readings can be made. A standard curve is prepared by plottingO.D. against known concentrations of reducing sugars. A unit of enzyme activity can then beexpressed as that liberating 1 µmol of reducing sugar per unit of time under the specified conditions.

25.2.2 Dinitrosalicylic Acid (DNS)

Heavy metals, particularly copper, interfere with the DNS method. Set up enzyme solutions andsubstrates with appropriate controls as previously noted. Prepare DNS reagents4 by mixing 20 mLof 2 N NaOH containing 1 g of 3,5-dinitrosalicylic acid with 50 mL of H2O. Add 30 g of KNatartrate and dilute the solution to 100 mL. Keep tightly stoppered in well-filled bottles at 4°C toprotect from carbon dioxide. The reagent should be stable for 1 year. After incubation at desiredtemperature and time, add 2 mL of DNS reagent and shake. Place tubes in boiling water for exactly5 min. Cool tubes in ice water and dilute to volume (25 mL). Determine O.D. at 575 nm in aspectrophotometer. A standard curve is prepared as previously noted for calculation of enzymeactivity.

25.2.3 Viscosimetric

Among the many techniques for measuring polysaccharidase activity, particularly endopolysaccha-ridase, the viscosimetric method seems to be the most sensitive because breakage of a bond nearthe 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, orthe distinction between enzyme and polymer properties. The assay has been used extensively fordetermining 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 wooddecay 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 incubationtemperature. Then 9 mL of CMC solution are placed in an 18- by 120-mm tube and 1 mL of culturefiltrate is added. The reaction mixture is agitated on a Vortex mixer for about 20 s; then 8 mL ofthe mixture are pipetted into an Ostwald–Fenskie No. 300 viscosimeter suspended in the waterbath. Draw the reaction mixture above the upper line of the viscosimeter by applying suction torubber 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 theviscosimeter). 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 haveexpressed enzyme activity using a number of methods. One method is to calculate the percentagedecrease in flow time (PDFT) after each incubation (t) according to the formula

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 CMCsolution containing active filtrate after incubation for time t; and Ew is the efflux time of distilledwater in the same viscosimeter used for the above two determinations.

Plot PDFT, vs. incubation time (including PDFTt = 0 at zero incubation time), and interpolategraphically the resulting curve to determine the time in minutes required for 50% decrease in flowtime (t50PDFT). Enzyme activity is expressed as l/t50 enzyme multiplied by 100.

25.2.4 Cellulase

Cellulase refers to a group of enzymes that degrade cellulose to glucose. To achieve completehydrolysis of insoluble cellulose, the different components of the enzyme complex must be presentin the right amounts under the right conditions. Components of this complex include endo-β -1-4-glucanases (EC 3.2.1.4), exe-l-4-glucanases (cellobiohydrolase; EC 3.2.1.91), and β-1,4-glucosidases (EC 3.2.1.21). Two types of exoglucanases are known enzymes splitting off aglucose unit from the nonreducing end of a cellulose chain and enzymes splitting off glucose and/orcellobiose. 5 All cellulase systems probably contain enzymes (endoglucanase, CMCase) capable ofdegrading soluble derivatives of cellulose such as CMC, but only those that contain exoglucanasecan degrade highly ordered forms of cellulose as found in cotton hairs. A good example of apartially deficient system is enzyme preparations from brown-rot wood decay fungi. These fungiextensively degrade cellulose in wood, but enzyme preparations from liquid cultures or decayedwood or cellulose appear to lack the full cellulolytic complex, because they ineffectively degradecrystalline cellulose, but do degrade cellulose that has been modified by solubilization. The require-ment for an active cellulase complex acting at optimum conditions, combined with the physicalheterogeneous nature of cellulose substrates on which it must act, makes assaying for cellulolyticactivity a formidable problem. A variety of methods have been developed for assaying cellulolyticactivity, 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 componentsof 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 ofexoglucanase activity; viscosity reduction of cellulose derivatives measures only endoglucanaseactivity; cellobiose conversion to glucose measures the glucosidase activity. Assaying reducingsugars released from crystalline cellulose measures the combined effect of all three, althoughglucosidase is not needed. Measurements of weight loss or tensile strength cannot be directlycompared with the liberation of reducing sugars. Measuring glucose produced from cellulose issimilar except that the overall activity may be only half as great, ultimately without glucosidase.Measuring glucose produced from CMC measures endoglucanase with some contribution fromglucosidase, 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 becomeavailable for the assay of many polysaccharide-degrading enzymes that offer sever-d advantages


over the more conventional assays just discussed, including specificity simplicity in use andmeasurement of solubilization of cellulose. Leisola and Linko7 note that the most important activityof the cellulase complex is usually the solubilizing effect (exoglucanases required). The solubilizingeffect is usually measured by determining the release of reducing sugars from insoluble celluloseproduced by the combined effect of the whole cellulase complex. However, the formation ofreducing sugar or glucose is not necessarily proportional to the solubilizing effect.7 Dyed cellulosesubstrates permit a true measure of the solubilizing activity of acellulase complex.7

Cellulose azure (Calbiochem, Sigma) is an acid-swollen cellulose preparation dyed with Rem-azol brilliant blue R (RBB). However, because it is swollen cellulose, it probably is not a truemeasure of the enzyme activity needed to solubilize crystalline cellulose. Avicel (FMC), dyed withRBB using the method of Leisola and Linko,7 is the best choice of celluloses because it can bedyed easily, hydrolyzes fairly rapidly, and, because it is crystalline, measures solubilizing effect.

The cellulose-solubilizing effect (exoglucanase) can be determined using RBB-cellulose, asdescribed by Leisola and Linko.7 Into a test tube at the appropriate incubation temperature, 2 mLof enzyme solution are pipetted and 3 mL of buffer solution containing 50 or 100 mg of substrateis added. Stir on a magnetic stirrer. After incubation, the reaction is stopped by heating and thesuspensions 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 endoglucanaserecommended by McCleary is as follows. For use as substrate, 2 g of RBB-CM-cellulose is sprinkledinto 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 aprecipitant solution that contains 80% ethylene glycol monomethyl ether, 0.3 M sodium acetatebuffer (pH (5), and 0.4% zinc acetate. This mixture is vortexed for 10 s, stood at room temperaturefor 10 min, and centrifuged at 1000 xg for 10 min. The absorbance of the supernatant is measuredat 590 nm.

25.2.5 Hemicellulases

Hemicelluloses constitute 20 to 30% of annual and perennial plants. Hemicelluloses make up thelargest content in annual plants and have been extensively examined in agricultural crops, such ascorn stalks, corn cobs, corn seed coat, wheat straw, soybean hulls, as well as wood. Defined asplant cell wall polysaccharides, other than cellulose and pectin, hemicelluloses are extractable byalkaline solutions. A few hemicelluloses are extractable in hot water, but most require mild toconcentrated alkaline solution with 10% sodium hydroxide being a common extractant. The hemi-cellulose β−1,4-xylan is probably the most abundant polysaccharide in terrestrial plants, next tocellulose. β−1-4-Mannans are the second most common hemicellulose. It is rare for either one ofthese hemicelluloses to occur as purely linear or without other sugars. Hemicellulases catalyzingthe hydrolysis of hemicelluloses have been found in bacteria, fungi, insects, and plant material.The most extensively studied hemicellulases are those from fungi.

25.2.5.1 XylanaseXylans can be hydrolyzed by β-xylanase (EC 3.2.1.8. endo-1,4,- β -D-xylan xylanohydrolase) andby the glycosidase, β-xylosidase (EC 3.2.1.37; exe-l ,4-β-D-xylan xylohydrolase). A large numberof assay procedures with various xylan substrates have been used to determine endoglucanaseactivity, making comparison among laboratories virtually impossible.9 Note that liberation ofreducing sugars from a xylan substrate does not necessarily mean that the responsible enzyme is


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 ofthe products of hydrolysis.

Because of the large interlaboratory variability in xylanase assay procedures and the consequentimpossibility of drawing meaningful comparisons between results reported in the literature, theInternational Energy Agency set in motion an international round-robin testing of methods in 1989.9

This study10 provides a standardized endoxylanase assay using 4-0-methyl glucuronoxylan assubstrate and gives precise instructions for the procedures to be followed using the DNS reagentto quantify the reducing sugars released by enzyme action. The xylanase standardized assay follows:

Substrate1.0% 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 heatto boiling point, preferably on a heating magnetic stirrer. Cool with continued stirring, cover, andstir slowly overnight. Make up to 100 mL with buffer. Store at 4°C for a maximum of 1 week orfreeze 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. Adjusttemperate 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) toenzyme 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 thedecrease in viscosity of solutions of suitable xylan or CM derivatives of xylan and monitoring thedecrease in turbidity of stable suspensions of suitable insoluble xylans or of the release of dye fromcovalently dyed soluble or insoluble xylans. Endo-acting enzymes effect large responses in eachof these assays, whereas those resulting from purified xylosidases or side chain-cleaving enzymesare minimal. Each alternative assay procedure should be standardized against a reducing sugarassay for each substrate and enzyme preparation used.”)

The reducing group method does not offer correct results in the presence of exo-actingglycanases. Viscometric methods are more specific, but are not suitable for analysis of insolubleenzyme sources and larger series of samples. Biely et al.11 have eliminated some of these short-comings by the use of soluble xylan with covalently bound RBB. Dyed xylan is prepared bydissolving RBB (0.15 to 20g) in a solution of water-soluble xylan (1 g in 30 mL of H2O). A solutionof Na2SO4 (10 mg in 10 mL of water) is added dropwise with stirring for 5 min. The mixture isthen alkalized with NaOH (0.5 to 1.0 g in 10 mL of water) and stirred at room temperature for


90 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 filtrateis 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 ofRBB-xylan solution at suitable incubation temperature. After incubation, the reaction is terminatedby addition of 2.0 mL 96% ethanol, mixing, standing for 20 min, mixing again, and centrifugingat 2000 xg for 3 min. Absorbance of the supernatant is measured at 595 nm against a substrateblank prepared by incubating 0.1 mL buffer in place of enzyme.

2 5 . 2 . 5 . 2 M a n n a n a s eb- D-Mannanase (EC 3.2. 1.78; mannan endo-1-4-β-mannanase) has not received nearly the attentionas xylanase. However, the enzyme is produced by a number of organisms12 such as fungi, bacteria,higher plants (locust, coffee, almond, apricot), and in animals (mammals, snails, osyters). Likexylan, 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 suitablemannan substrate such as carob gum or guar gum.

McCleary 8 describes a dyed substrate assay for mannanase. Prior to dying with RBB, a low-viscosity 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 pasteis 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 viscosityduring this incubation.) The slurry is again homogenized in a Waring blender, incubated in a boilingwater bath until the temperature reaches 90°C (to inactivate β-mannanase), and then centrifugedat 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 collectedon a nylon screen, washed by resuspension in aqueous ethanol (60% v/v), and then dried by solventexchange 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 thisis added 24 g of RBB dye and 160 g of anhydrous sodium sulfate, which is dissolved by stirringover 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 thissolution 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 isremoved. The polymer is dissolved again in water at 60°C and reprecipitated by the addition oftwo volumes of ethanol. This step is repeated until all free dye is removed. The precipitatedgalactomannan 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, withvigorous stirring more than 15 min. Then 10 mL of 3 M sodium acetate buffer (pH 5.0) are addedand the volume is accurately adjusted to 100 mL. In the presence of 0.02% sodium azide, thesolution is stable at 4°C for at least 12 months and shows no significant tendency to settle fromsolution during this period.

Enzyme preparation (0.5 mL) containing 0 to 0.5 units of β-mannanase activity per 0.5 mL isincubated with RBB-carob galactomannan substrate solution (1.0 mL, 1% w/v) for 5 to 20 min at40°C. The reaction is terminated and the high-molecular weight substrate is precipitated by theaddition of ethanol (3 mL). The mixture is stirred, allowed to equilibrate to room temperature for10 min, and centrifuged at 1000 xg for 10 min. The enzyme reaction is monitored by increasedabsorbance (590 nm) of the supernatant solution.


25.2.6 Pectinase

Pectins are a group of colloidial substances with a high proportion of anhydro-galacturonic acidand 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 galacturonicacid 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 togalacturonate residues in the backbone, whereas feruloyl ester groups are thought to be linked tothe neutral sugar side chains, at least in sugar beet pectin.14

Pectinases (pectate lyase [PL], polygalacturonase [PG], and pectin methylesterase [PME]) havebeen studied in more pathogens and in more detail than any other wall depolymerase. The originalrationale for studying pectinases was that they are able to macerate tissue, the characteristicsymptom of soft-rot diseases.15 Research on pectinases has received impetus recently from thedemonstration that pectinases and pectin fragments induce numerous physiological effects inplants.15

25.2.6.1 Pectin MethylesterasePectin 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 existfor measuring pectin methyesterase (PME) activity, liberation of methanol determined colorimet-rically, increase in free carboxyl groups by titration, or the hydrolysis of nitrophenylacetate mea-sured spectrophotometrically.16 The following is a simple titration method. Substrate is preparedby mixing 0.5% citrus pectin with distilled water in a Waring blender for 1 min. The mixture isfiltered through Whatman No. 1 paper on a Buchner funnel. In a 250-mL beaker containing astirring bar, 100 mL of substrate and 3 mL of 1.0 M CaCl2 are placed. The electrodes of a pH meterare inserted and the mixture adjusted to pH 7.5 with 1 N NaOH. Then 10 mL of enzyme preparationare added to the mixture and the pH of the enzyme substrate mixture is readjusted to 7.5 and atimer started. The pH is adjusted continually to pH 7.2 to 7.5 during the course of the assay byadding 0.005 N NaOH from a burette. The total milliliters of NaOH is recorded. A control is 10 mLof boiled extract substituted for the active enzyme sample. The titration difference in milliliters ofalkali required by active extract and that of the heated control multiplied by the concentration ofalkali equals the milliequivalents (meq) of acid released by enzymatic action. PME activity isexpressed as microequivalents (meq × 1000) of carboxyl groups released per unit of time by aspecified quantity of enzyme solution.

25.2.6.3 Pectin LyasePL (EC 4.2.2) breaks down pectin by a trans-elimination reaction. The enzyme can be assayed bymeasuring the change in absorbance at 235 nm in a buffered pectin reaction mixture.18 The assaymixture contains 10 m M MES, 0.022% pectin (86% methoxy content, Sigma), 1.5 m M CaCl2,75 m M NaCl, and the pH is adjusted to 6.5. The enzyme solution is added to 2 mL of the bufferedpectin in a 1-cm quartz cuvette. The increase in absorption, due to the formation of uronide doublebonds, is recorded at 5 min of reaction time. One unit of pectin lyase activity is defined as theamount of enzyme that causes an increase of 1.0 absorbance unit per minute at 235 nm at a specifiedtemperature.


25.2.7 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 gluco-sidically linked fiber-forming polymer of N-acetyl-D-glucosamine units, also occurs in the cell wallof some algae, in the exoskeletons of arthropods including crustaceans and insects, in nematodes,in molusks, in worms, and in many other types of organisms.

25.2.7.1 ChitinaseAssays of chitinase (EC 3.2.1.14; b-1,4-N-acetyl glucosamine glycanhydrolase) activity includeviscosimetric using soluble derivative of chitin, reduction of turbidity of a suspension of collodialchitin, 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 -acetylglu-cosamine or a reference compound, is an easy and suitable assay for chitinase. Reduction of turbidityof 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 asdescribed by Shimahara and Takiguchi.21 A suspension containing 1% (w/v) of moist colloidalchitin is prepared in appropriate buffer and pH. A mixture of 0.5 mL each of the chitin suspensionand 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 isexpressed as the percentage of reduction in turbidity relative to that of a similar suspensioncontaining water rather than enzyme solution.

Soluble dye-labeled and a colloidal dye-labeled substrate are also commercially available fora sensitive and reliable, simple, and microtiter plate-adapted calorimetric assay of chitinase activ-ity, 19 These dyes are also applicable for rapid and selective detection of chitinolytic microorganisms

in agar media (plate-clearing assay).

25.2.7.2 β-1,3-Glucanaseβ−1,3-Glucose glycanhydrolase (glucanase) (EC 3.2.1.21) is usually assayed by the increase inreducing sugars from laminarin (Sigma).

25.2.7.3 LysozymeAnother lytic enzyme is lysozyme (EC 3.2.1.17; N-acetylmuramide glycanohydrolase), whichhydrolyzes the β-1,4-glucosidic linkages in the mucopolysaccharide cell wall structure in a varietyof microorganisms. Plant lysozyme is found principally in fiscus and papaya latex. Worthington6

describes an assay method based on the lysis of Micrococcus lysodeikticus. Prepare a suspensionof dried M. lysodeikticus cells (Sigma), 0.3 mg/mL in 0.1 M phosphate buffer, pH 7.0. Set spec-trophotometer at 450 nm. Add 0.1 mL of enzyme solution to 2.9 mL of substrate. Record absorbanceat 15-s intervals using a water blank. The first 2 min of reaction are used to calculate activity. Dueto the variability of the substrate, it is advisable to assay a standard preparation of lysozyme at thesame time as the unknown. One unit of enzyme activity is equal to a decrease in absorbancy, at450 nm, of 0.001 per minute at pH 7.0 and 25°C.

25.2.8 Amylase

Starch and glycogen are hydrolyzed by amylases, of which there are two general types: α- andβ-amylase. α-amylase (EC 3.2.1.1) 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


glycogen containing three or more α-1,4-linked glucose units, yielding a mixture of maltose andglucose. β-Amylase (EC 3.2.1.2; exoamylase) occurs in resting seeds. β-Amylase releases succes-sive maltose units from the nonreducing end of a polysaccharide chain by hydrolysis of α-1,4-glucanlinkage.

Both types of amylases can be assayed by the reducing group assay. A calibration curve ismade with maltose to convert calorimetric readings to units of activity. Commercially availablestarch-azure (Sigma) can be used to assay amylase activity similar to that described previously forcellulase activity using cellulose-azure. McCleary8 describes a method using soluble dyed starchas a chromogenic substrate for determining α-amylase activity.

25.2.9 Dextranase

Dextranase (EC 3.2.1.11; 1,6-α-D-glucan 6-glucanohydrolase) hydrolyzes the bacterial polysaccha-ride, dextran (α-1,6-.glucan), to isomaltose residues. The reducing group assay can be used fordetermining enzyme activity.6 The substrate used is 2% dextran “500” (AB Pharmacia) in 0.1 Mpotassium, phosphate buffer, pH 6.0, containing 0.01% merthiolate. To 1.9 mL substrate, add 0.1 mLdilute enzyme. Digest for 30 min at 37°C. At end of digestion period, use 1 mL of digest forreducing group assay. Prepare a maltose standard curve using levels 0.3 to 3.0 µM maltose. Oneunit of enzyme activity causes the release of 1 µM isomaltose from dextran per minute.

25.3 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 fromthe p-nitrophenol substrate. Nitrophenol substrates are available from chemical companies such asSigma. Some commonly assayed glycosidases and p-nitrophenol substrates are arabinosidase (EC3.2.1.55, p-nitrophenyl-α-L-ambinofuranoside); chitobiosidase (EC 3.2.1.14, p-nitrophenyl-β-D

N,N'-diacetylchitobiose); galactosamidase (EC 3.2.1.30, p-nitrophenyl N-acetyl-β-D-galac-tosaminide); α-galactosidase (EC 3.2.1.22); β-galactosidase (EC 3.2.1.23) activity using p-nitro-phenyl-α-D-galactopy ranoside or p-nitrophenyl-β-D-galactopyrdnoside; β−glucosidase (EC 3.2.1.6);α-glucosidase (EC 3.2.1.20) using p-nitrophenyl. β-D-glucopyranoside, or p-nitrophenyl-α-D-glu-copyranoside; glucuronidase (EC 3.2.1.31, p-nitrophenyl-β-D-glucuronide): mannosidase (EC3.2.1.25, p-nitrophenyl-β-mannopyranoside); or xylosidase (EC 3.2.1.37, p-nitrophenyl-β-D-xylopyranoside).

We use the following procedure to assay for glycosidase activity in wood decay fungi. In bufferat appropriate pH, 2 mL of 0.05% p-nitrophenyl substrate are mixed with 1 mL of enzyme solutionand incubated at a suitable temperature. The reaction is terminated by the addition of 1 mL of0.2 M NaCO3. The resulting yellow color is immediately measured at 425 nm with a spectropho-tometer. A unit of enzyme activity is defined as the amount that will liberate 1 µM of p-nitrophenolper unit of time.

25.4 Microplate Assay

Many colorimetric assays have been modified for simple testing in microtiter plates and automatedmeasurement in a microplate reader. These microassay systems Facilitate rapid screening of a largenumber of column chromatography fractions and substantially conserve time and reagents. Inaddition, fractions can be screened for different enzymes simultaneously on the same microplate.


Most commercial microplate readers differentiate calorimetric reactions by transmitted light(absorbance) at a specific wavelength. Either insoluble substrates, used in many assays, or theprecipitates formed during an assay prevent penetration of light and render measurement bytransmitted light inaccurate.

We have adapted a thin-layer chromatography double-beam densitometer to accept 96-wellmicrotiter 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 thestandard 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 in0.1 M citrate buffer, pH 5.0, are placed in each well. The plate is covered with an acetate adhesivesheet and incubated at 40°C for 24 h. After incubation, 75 µL of Somogyi copper reagent is addedand the wells are resealed with the acetate sheet. The plate is then incubated at 80°C for 30 minin a water bath. After the plate has cooled completely (15 min), 75 µL of arsenomolybdate areadded, and the wells are mixed well with a Vortex mixer. Calorimetric measurements are read usingreflectance at 500 nm with a dual-wavelength densitometer. A standard curve is prepared usingglucose at concentrations ranging from 100 to 2000 µg/mL.

The microassay has proven invaluable for rapid surveys designed to locate polysaccharide-degrading enzyme from chromatography fractions. The substantially smaller test volume reducesloss of precious enzyme. The ability to run multiple samples as quickly as single samples improvesefficiency, and test reagents can be added with multichannel pipettes.

25.5 Screening Assays

25.5.1 Solid Medium with Living Organism

Numerous screening methods exist for detecting carbohydrolase activity in microorganisms usuallygrown on agar medium. A solid medium providing rapid assays is useful for the direct measurementand isolation of carbohydrolase-producing organisms from natural substrates and for the isolationof 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-boundenzyme activity is measured, and (3) measurements of enzyme activity are not made at a specificharvest time which may not be optimal for all organisms.23

Common screening techniques devised for the detection of polysaccharide-degrading enzymesby microorganisms involve plate assays where the respective polymer, or its derivative, is incor-porated into the basal growth medium. The production of corresponding polysaccharide-hydrolysesis indicated by the clearing of the opaque medium as the substrate is dissolved by the enzymesproduced 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 andactivity might not be detected because of the masking effect of the mycelial mass when the rateof fungal growth exceeds enzyme diffusion. Chang et al.24 overcame this problem by inoculatinga polycarbonate membrane (0.2 µm, 90 mm diameter, Nucleopore Co. ) with the test fungus. Afterincubation, the membrane is removed and the enzyme activity recorded. The polycarbonate mem-branes, which can resist penetration by fungi, are placed between moistened filter papers andsterilized by autoclaving before use.

Dyed polysaccharide substrates placed over an agar medium and inoculated with an organismare also useful for screening for polysaccharide-degrading enzymes.25 The detection of enzymeactivity depends on the release of dye and its diffusion into the colorless lower layer followingenzymatic hydrolysis of the dyed substrate.


25.5.2 Solid Media with Cell-Free Filtrates

Solid media are also used to screen cell-free extracts from microorganisms for polysaccharide-degrading enzyme activity. Generally, the enzyme solution is placed in a well (cup) cut in an agarmedium containing a polysaccharide substrate. The agar surface usually needs to be flooded witha reagent to develop the zones of enzyme activity around the cup.

The cup-plate assay for determining polygalacturonase activity in filtrates is a good exampleof this type of assay (Figure 25.1).26 In a Waring blender, 1 g of ammonium oxalate, 2 g of sodiumpolypectate, and 3 g of agar are mixed in 200 mL of 0.1 M acetate buffer (pH 5.0). The mixtureis placed in a 500-mL flask, covered with cheesecloth and aluminum foil, and autoclave for 5 minat 15 lb/in2. Hot media is poured into a prepared plate consisting of a 10- by 10-in piece of glasswith 1-in pieces around the outside edges (a petri plate can also be used). When the medium issolidified, a No. 3 cork bore is used to cut cups. Into each cup, 0.1 mL of enzyme is pipetted usinga 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 sealeddouble plastic bag. Plates are incubated at 37°C for 14 h. Following incubation, HC1 (1:2 dilutionof concentrated HC1) is poured over the plate. After approximately 3 min, the plates are rinsedwith cold water. The diameter of the cleared zone is measured with calipers and recorded. Diametersare converted to enzyme activity by running a dilution of the enzyme and plotting zone diameteragainst the log of the enzyme concentration.

carbohydrate assays

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