APPI.IED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p. 3592-35960099-2240/94/$04.00 + 0Copyright © 1994, American Society for Microbiology

A Sensitive Method Using 4-Methylumbelliferyl-3-Cellobioseas a Substrate To Measure (1,4)-3-Glucanase

Activity in SedimentsH. T. S. BOSCHKER* AND T. E. CAPPENBERG

Centre for Limnology, Netherlands Institute of Ecology, 3631 AC Nieiuwersluis, The Netherlands

Received 1 February 1994/Accepted 19 July 1994

A sensitive method to measure (1,4)-13-glucanase activity in organic matter-rich sediments, using 4-methyl-umbelliferyl4--cellobiose as a substrate, is described. ,-Glucosidases, which were also able to hydrolyze thissubstrate, were inhibited with D-glucono-8-lactone. The produced 4-methylumbelliferone was recoveredquantitatively out of the sediment by an extraction with 80% ethanol. An inhibition experiment with knownsubstrates or inhibitors suggested that at least 59% of the measured activity could be explained by enzymes ofthe exo-(1,4)-P-glucanase type and that the contribution of endo-(1,4)-p-glucanases was minor. Results of theinhibition experiment also suggested that the measured activity was of bacterial origin in the sediment used.First results of field measurements are given for the sediment from the reed bed of Lake Gooimeer.

The major input of organic matter (OM) to the sediment oflakes is macrophyte- or alga-derived material. This material ismainly polymeric and needs to be degraded extracellularly tosmaller units before it can be taken up by microorganisms. Thepolymeric material is hydrolyzed by enzymes released by or on

the outside of microorganisms, and this process is generallyregarded as the rate-limiting step in the mineralization ofcomplex OM (3, 9). Many studies that address the importanceof extracellular enzymes in the decomposition of OM can befound (4, 17).

Cellulose is by mass the most important polymeric com-

pound in macrophytes and is degraded by a complex ofenzymes. In the cellulase system, three types of enzymes whichcooperate in the degradation of cellulose to cellobiose andglucose are distinguished: endo-(1,4)-3-glucanases, which ran-

domly split cellulose; exo-(1,4)-3-glucanases, which split offcellobiose or sometimes glucose from the end of a cellulosechain; and ,-glucosidases, which split cellobiose into twoglucose molecules (10, 22).

Fluorogenic substrates have been used for some time toassay extracellular enzymes in aquatic ecosystems (8a, 19a). Asensitive method to measure 3-glucosidase activity directly insediments that uses an artificial substrate has been described:4-methylumbelliferyl-,-glucose (MUF-Glu) (9, 12). MUF-Gluis split by the ,B-glucosidases present in the sediment sampleinto glucose and 4-methylumbelliferone (MUF). The producedMUF is measured fluorometrically down to nanomolar con-

centrations, which results in a sensitive method with shortincubation times. To our knowledge, no sensitive method forsediments has been described yet to measure the activity of thetwo enzyme types that act directly on the polymeric cellulose:endo-(1,4)-,B-glucanases and exo-(1,4)-,B-glucanases. Such a

method might be of interest because the measured activitieswould be more closely associated with the presence of cellulosethan is the case with I-glucosidases.Here we describe the development and testing of a method

to measure (1,4)-p-glucanase activity in sediment samples by

* Corresponding author. Mailing address: Centre for Limnology,Netherlands Institute of Ecology, Rijksstraatweg 6, 3631 AC Nieuw-ersluis, The Netherlands. Phone: (31)-(0)2943-3599. Fax: (31)-(0)2943-2224.

using MUF-3-cellobiose (MUF-cel) as a substrate. The prin-ciples of the method have been published by Desphande et al.(6, 7), who use p-nitrophenyl-,B-cellobiose to assay exo-(1,4)-,B-glucanases in purified cellulases. 1-Glucosidases, which alsoshowed activity towards p-nitrophenyl-3-cellobiose, were spe-cifically inhibited with D-glucono-8-lactone (GLN). We alsopresent a general experimental procedure for measuring enzy-

matic activities in sediment with MUF-labelled substrates. Inthis procedure the produced MUF is quantitatively extractedfrom the sediment with 80% ethanol.

MATERIALS AND METHODS

Sampling sites. Sediments used in this study were collectedat different sites inside and at 10 m outside the 80-m-wide reedbed on the southern shore of Lake Gooimeer. Lake Gooimeeris very shallow (mean depth, 3.6 m) with a sandy sediment andis situated in the center of the Netherlands. Common reed(Phragmites australis (Cav.) Trin. ex Steudel) is the dominantplant species in the bed. Inside the bed, OM is accumulating.The OM content of the sediment increases from <1% at thelake side of the bed to 30% on the land side. Because of thehigh OM content and the yearly input of fresh reed litter,containing about 40% cellulose, it is likely that cellulolyticenzymes play an important role in the decomposition of OM inthese sediments.

Sediments. For the testing of the enzyme assay, sedimentfrom the top 3 cm was collected and slurried for 30 s in a

Waring blender after the addition of some water (sediment/water [wt/wt] ratio = 5). To determine the profiles of enzymeactivities, sediment cores from inside and outside the bed were

taken by drilling a Perspex cylinder (7 cm inside diameter) witha sharpened cutting edge into the sediment by hand. In thelaboratory, cores were sliced with 1-cm intervals over the top 7cm and with 2-cm intervals below. Slices were mixed in a

Waring blender. Sediments were slurried because of thepresence of course reed litter fragments that otherwise couldnot be included in the assay. Living plant parts were removedas far as possible before slurrying.The dry matter (DM) content of the sediment was measured

after an overnight drying at 105°C, and OM content was

determined as the subsequent loss of weight after 4 h at 550°C.

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MEASURING (1,4)-P-GLUCANASE ACTIVITY IN SEDIMENTS 3593

TABLE 1. Effects of additions on the enzymatic activity againstMUF-Glu and MUF-cel+GLN'

% Activity" of control (mean [SD])Addition MUF-cel+GLN

(concn [g/litcrj) MUF-Glu Uce+L(1.5 mM) 1.5 mM 0.3 mM

Control 100 (6) 100 (7) 100 (3)Cellulose (10) 102 (2) 100 (2)CMC-medium (10) 72 (12)* 90 (10)*CMC-low (25) 48 (9)* 86 (11)* 73 (2)*MC-low (25) 100 (12) 73 (6)* 66 (5)*Cellodextrins (10) 65 (5)* 72 (2)* 41 (1)*Cellobiose (10) 75 (6)* 101 (4)Glucose (10) 59 (16)* 100 (7)

"Sediment (16% OM) was collected from the top 3 cm on 27 September 1990."*, significantly (P = 0.95) different from control.

Chemicals and solutions. All substrates and other chemicalswere purchased from Sigma, except for cellulose MN300,which was from Hachery Nagel & Co., and cellodextrinmixture I, which was from Merck. According to batch analysesby Merck, cellodextrin mixture I contained 0.9% glucose,14.3% cellobiose, 25.5% cellotriose, 30.2% cellotetraose, 16.3%cellopentaose, and 3.9% cellohexaose. To remove low-molec-ular-weight compounds, methylcellulose of low viscosity (MC-low) and carboxymethyl celluloses of medium (CMC-medium)and low (CMC-low) viscosities were repeatedly suspended in80% ethanol and centrifuged down. After this treatment, thewashed material was dried at 40°C. GLN was dissolved in a 0.1M phosphate buffer and adjusted to pH 7.0. All other com-pounds were dissolved in water.Enzyme assays. Between 0.1 and 0.3 ml of sediment slurry

was added to a 15-ml centrifuge tube. Reactions were startedby adding 1 ml of MUF-Glu or MUF-cel at the desiredconcentration (final pH, 7.0 to 7.5), and tubes were incubatedon a rotary shaker at 20°C. Incubations with MUF-Glu tookbetween 5 and 20 min, and those with MUF-cel took between0.5 and 1.5 h, depending on the sediment used and the purposeof the experiment. Reactions were stopped by the addition of5 ml of 96% ethanol, mixing thoroughly, and putting the tubeson ice. Particles were removed by centrifugation at 6,000 rpmfor 20 min (-2°C), and the supernatant was decanted intoanother tube containing 1 ml of 1 M glycine-NaOH buffer, pH11. From this point, the samples were stable for at least 2 h.To measure MUF concentration, 3 ml of the sample was

added to a UV-cuvette, and fluorescence was measured on anAminco-Bowman spectrofluorometer at an excitation wave-length of 365 nm and an emission wavelength of 455 nm. Forblanks, MUF-Glu and MUF-cel additions were made shortlyafter 5 ml of 96% ethanol was added to the centrifuge tubecontaining the sediment sample. After the blank fluorescencewas read, MUF fluorescence was calibrated by adding 200 pul of50 pFM MUF standard to the cuvette containing the blank,after which fluorescence was measured again. At the wave-length settings used, MUF-Glu and MUF-cel incubations andfree MUF showed maximum fluorescence. Blanks for bothMUF-Glu and MUF-cel incubations had low and similarfluorescence signals, which suggested that increased fluores-cence in the incubations was due to free MUF.

Characterization of enzyme activities. Inhibition experi-ments were conducted to characterize the enzyme activitiesmeasured with MUF-Glu and MUF-cel plus 500 mM GLN(MUF-cel+GLN). Seven known substrates or inhibitors ofP-glucosidases or 3-glucanases (Table 1) (14a, 22, 24) were

z-i

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80

60

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0 200 400 600 800 1000

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FIG. 1. Effects of GLN on the MUF production from MUF-Gluand MUF-cel. Above 400 mM GLN, no interference from ,B-glucosi-dases in the hydrolysis of MUF-cel was observed. Sediment (16% OM)was collected from the top 3 cm on 6 February 1990. All pointsrepresent duplicates. SDs in the lower part of the figure were smallerthan the symbols: 0, MUF-cel; El, MUF-Glu.

tested for their inhibiting capacities on enzymatic activitiesagainst MUF-Glu or MUF-cel+GLN. Additions were madeshortly before the MUF-labelled substrate was added. Multipleregression analysis with dummy variables was used to test forstatistically significant effects of the different additions.

RESULTS

Testing the application of MUF-cel+GLN in sediment. Aproblem associated with the use of MUF-labelled substrates insediments is that the produced MUF in the assay adsorbs ontothe sediment (9). Preliminary experiments revealed that whenno further measures were taken, only 9 to 15% of a standardMUF addition was recovered from a sediment slurry having an

OM content of 15%. Although it is possible to correct forrecovery by determining it in a separate experiment (9), thelow and variable recovery gave inaccurate results. Therefore,several methods were tested to extract MUF quantitativelyfrom the sediment. The extractants used were buffers of pH 2(0.2 M glycine-NaCl-HCl) or 11 (0.2 M glycine-NaOH), 1%Triton X-100 in water, 80% methanol, and 80% ethanol.Extraction with ethanol resulted in a recovery of 88% (stan-dard deviation [SD] = 5, n = 6). In these extracts, MUFfluorescence was linear up to a concentration of 10 puM.The effect of GLN, a specific inhibitor of f-glucosidases (6,

7, 24), on the MUF production from MUF-Glu and MUF-celis shown in Fig. 1. While the activity against MUF-Glu was lowand still went down at higher concentrations of GLN, activityagainst MUF-cel levelled off at about 35% with GLN concen-trations of 400 mM or higher. We concluded that at GLNconcentrations above 400 mM, ,B-glucosidases did not signifi-cantly hydrolyze MUF-cel. A concentration of 500 mM GLNwas chosen for further incubations with MUF-cel (referred toas MUF-cel+GLN). GLN precipitated when the 5 ml ofethanol was added for MUF extraction, but this had no effecton the recovery of MUF.The MUF production from MUF-Glu was linear in time for

at least 30 min, and for MUF-cel+GLN it was linear for atleast 2 h (data not shown). A single incubation time of 5 to 15min for MUF-Glu and of 0.5 to 1.5 h for MUF-cel+GLN was

used in further experiments and was long enough to produce a

MUF fluorescence signal of 4 to 30 times the blank fluores-cence. Maximum activity for both substrates was reached at a

substrate concentration of 2 mM (Fig. 2). Unless otherwisestated, further measurements were carried out at a MUF-Glu

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3594 BOSCHKER AND CAPPENBERG

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FIG. 2. Relation between the substrate concentration and theactivity against MUF-Glu and MUF-cel+GLN. Both enzyme systemswere saturated at 2 mM. Sediment (16% OM) was collected from thetop 3 cm on 6 February 1990. (Error bars are 1 SD; when the SD issmaller than the symbol, no bar is used). 0, MUF-Glu (data shown aredivided by 10); rl, MUF-cel+GLN.

and a MUF-cel concentration of 3.3 mM. MUF production wasonly linear with the amount of sediment in the assay when verysmall amounts of sediment were used (<50 mg ofDM [Fig. 3]).These figures also show that there was an inverse relationshipbetween the OM content of the sediment and the maximum

A

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sediment amount (mg DM)FIG. 3. Effects of the sediment amount used in the assay on the

measured MUF production from MUF-Glu (A) and MUF-cel (B).The sediment with <1% OM (*) (data shown are multiplied by 10) wasfrom outside the reed bed; the other two were from different locationsinside the bed. For the two sediments (6% [C1] and 16% [0] OM) fromoutside the bed, only 50 mg of DM or less could be used before activitydeviated from linearity with the amount of sediment. For the sedimentwith <1% OM, no activity against MUF-cel+GLN was detected.Sediments were collected from the top 3 cm on 1 June 1990.

0

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0 0.5 1.0 0 0.1 0.2 0.3,........... ..... .~~~..........................

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.............. . ...........,............. ...... .......

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FIG. 4. Enzyme activities in two cores, one from inside (dottedline) and one from outside (dashed line) the reed bed, sampled on 22May 1990. In the core from outside the bed, no exo-(1,4)-P-glucanaseactivity was detected. All measurements are in duplicate. The SD wasless than 10% of the measured activity.

amount of sediment that could be used before MUF produc-tion deviated from linearity with the amount of sediment in theassay. Heat-sterilized sediment (2 h at 120°C) showed nodetectable activity (<1% of fresh sediment) for both sub-strates. Therefore, it was concluded that the activities were ofa biological or enzymatical origin.

Characterization of enzyme activities. The enzyme activitiesmeasured with MUF-Glu and MUF-cel+GLN were character-ized by performing inhibition experiments using 7 knownsubstrates or inhibitors of ,-glucosidases and endo- or exoglu-canases (Table 1). At a substrate concentration of 1.5 mM,patterns of inhibition were clearly different for MUF-Glu andMUF-cel+GLN. Cellobiose and glucose were inhibiting activ-ity only against MUF-Glu, while MC-low inhibited activity onlyagainst MUF-cel+GLN. Activity against both substrates wassignificantly inhibited by CMC-low, CMC-medium, and cello-dextrin mixture. Cellulose did not inhibit activity in either case,which was not expected. However, since cellulose was the onlysolid compound tested, a longer preincubation time might beneeded before an effect is obtained. The percent inhibition byall additions was higher at a concentration of 0.3 mM MUF-cel+GLN than at 1.5 mM. This was especially true for thecellodextrin mixture, which reduced the activity to 41% of thecontrol level.Example of field measurements. The results of an applica-

tion of the method are shown in Fig. 4; enzyme activitiesagainst MUF-Glu and MUF-cel+GLN were measured insediment cores taken inside and outside the reed bed of LakeGooimeer. Inside the bed, activities for both enzymes werehighest in the top layer of the sediment, suggesting a maininput of fresh material from above, probably in the form ofdead reed litter. The secondary peak in the enzyme activitiesbetween 3 and 4 cm of depth might be caused by an input ofmaterial from the Phragmites roots that show a maximumstanding stock at this depth (data not shown). Outside the bed,,B-glucosidase activities were lower than inside and no exo-(1,4)-,B-glucanase activity could be detected.

DISCUSSION

Testing the application of MUF-cel+GLN in sediment. Anadvantage of the method described here is that the producedMUF is quantitatively recovered from the sediment, using anextraction with 80% ethanol. This means that there is no need

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MEASURING (1,4)-p-GLUCANASE ACTIVITY IN SEDIMENTS 3595

for an internal standardization as described by King (9), whoused MUF-Glu to measure P-glucosidase activity in marinesediments. We also have used this extraction procedure withsimilar results for other MUF-labelled substrates like MUF-xylose and MUF-butyrate (no data shown).The presented method was sensitive; incubation times of

only 0.5 to 1.5 h were sufficient to produce a well-measurableMUF fluorescence. No inhibition or induction of enzymeactivity occurred during this incubation period, since MUFproduction was linear with time. A limitation was that only asmall amount of sediment could be used in the assay beforehydrolysis of the substrates deviated from linearity with theamount of sediment. This small amount of sediment impliesthat the method was sensitive to the heterogeneity in thesample. For the type of sediments used in this study, whichcontained course litter fragments, the method could be appliedonly to well-mixed samples. The OM content of the sedimentswas high, which was an important factor in the amount ofsediment that could be used. For other types of sediments,MUF-labelled substrate analogs have been used with largeramounts of sediment (9) or even whole cores (12).

In this study it was found that a GLN concentration of 400mM or higher was effective in inhibiting the hydrolysis ofMUF-cel by ,B-glucosidases in sediments. A much lower con-centration of GLN (0.6 to 3.0 mM) is sufficient to inhibit a3-glucosidase from the fungus Trichoderna reesei (6, 7). It is

possible that GLN was less available in sediments because ofadsorption and therefore was a less effective inhibitor. Analternative explanation might be found in a combination of theacidic nature of GLN and of a difference in pH between ourstudy conditions (pH 7.0 to 7.5) and those of Desphande et al.(6, 7), who use a pH of 5. It is likely that the undissociated formof the acidic GLN is the inhibiting species for ,B-glucosidasesand this species is present at a 100-times-lower concentrationat pH 7 than at pH 5.

Characterization of enzyme activities. Because sedimentsprobably contain a large variety of enzyme types, we tried tocharacterize the activities against MUF-Glu and MUF-cel+GLN in inhibition experiments with known inhibitors or sub-strates of cellulolytic enzymes. MUF-Glu is a selective sub-strate for 3-glucosidases (6, 19). This is in agreement with ourfinding of the almost complete inhibition of MUF-Glu activityby GLN, a specific inhibitor of 3-glucosidases (6, 7, 24),indicating that MUF-Glu was mainly hydrolyzed by 3-glucosi-dases. Activity against MUF-cel+GLN showed an inhibitionpattern clearly different from that of activity against MUF-Glu(Table 1) and was therefore not of the ,B-glucosidase type.Of the two other types of cellulolytic enzymes, endo-(1,4)-

3-glucanases act on the glucoside linkages along the cellulosechain while exo-(1,4)-p-glucanases act on the end groups of thecellulose molecule (10, 22). We found that the activity againstMUF-cel+GLN was more sensitive for inhibition by thecellodextrin mixture then by CMC-medium, CMC-low, orMC-low. The cellodextrin mixture has far more end groups pergram of material than CMC-medium, CMC-low, and MC-low,meaning that at least 59% of the measured activity could beexplained best by an enzyme sensitive to end groups ofcellulose or similar compounds: an exo-(1,4)-p-glucanase typeof enzyme. Data in the literature on purified cellulolyticenzymes agree with these results. Although several endo-(1,4)-,B-glucanases are able to split MUF-cel (5, 20) or p-nitrophe-nyl-3-cellobiose (6, 15), a similar substrate, most of them showno detectable activity on these substrates (2, 8, 21), and ifactivities are found, they are lower than with exo-(1,4)-,-glucanases (6). Maybe MUF-labelled longer cellodextrins (2,21) than MUF-cel could be used to measure endo-(1,4)-P-

glucanase activity in sediments, but these substances are veryexpensive or not commercially available.We found that MUF-cel+GLN activity was not inhibited by

high concentrations (10 g/liter) of both glucose and cellobiose,the primary products of cellulose decomposition. Most cellu-lases of bacterial origin are resistant to inhibition by glucose(13, 14, 16), and some are even unaffected by cellobiose (13,16). On the other hand, inhibition by both products at concen-trations used in this study is described for all studied fungalcellulases (13, 14, 22, 23). This suggests that fungal cellulaseswere of minor importance compared with bacterial cellulasesin the sediment of the reed bed. Similarly, Benner et al. (1)showed, by using size fractionation and antibiotic treatments,that bacteria are predominant in the decomposition of "C-labelled lignocellulose in both freshwater and marine sedi-ments.

Application of the method. There are not many other assaysknown for measuring exo-(1,4)-p-glucanase activity directly insediments. Glucose production from microcrystalline cellulosehas been used to determine exo-(1,4)-p-glucanase activity insediments (18) and soils (11), but incubation times are verylong (18 to 24 h), and toluene is added to prevent uptake ofglucose and induction of enzyme activity during this longperiod. Although toluene is inhibitory to 3-glucosidases (9),the effect of toluene on exo-(1,4)-13-glucanases was not deter-mined in these studies. p-Nitrophenyl-3-cellobiose was used bySinsabaugh and Linkins (18) to measure exo-(1,4)-f-glucanaseactivity with incubation times of 4 to 6 h. But their assay was

probably not selective for exo-(1,4)-p-glucanases, since P-glu-cosidases are not inhibited. In this study, inhibition experi-ments with GLN showed that 65% of the activity measuredwith MUF-cel could be explained by 3-glucosidases.

In contrast to cellulose, the natural polymeric and particu-late substrate of cellulases, MUF-cel is an oligomeric anddissolved substrate. It is therefore very unlikely that theactivities measured with MUF-cel are actual, in situ rates ofcellulose hydrolysis or exo-(1,4)-,B-glucanase activities. Fur-thermore, since MUF-cel was used at a saturating concentra-tion, the measured activities should be seen as potentialactivities (9). Despite these limitations, we believe that themethod can be used to compare activities at different samplingsites (Fig. 4) and to measure changes in activity with time.Also, the effects of various additions on the cellulolytic activ-ities in sediment can be studied to characterize the enzymespresent in sediments (Fig. 2 and Table 1). These measure-ments give valuable information about the microbial contribu-tions to a particular ecosystem.The method described here will be used in our laboratory to

study extracellular enzyme activities and their roles in theinitial steps of OM decomposition in the reed bed of LakeGooimeer.

ACKNOWLEDGMENTSThis work was supported by grants from the Netherlands Integrated

Soil Research Programme and from the Institute for Inland WaterManagement and Waste Water Treatment.We thank A. J. B. Zehnder for his comments on the earlier versions

of the manuscript.

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