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Inhibition of the Trichoderma viride cellulase complex by leaf litter extracts
0038-O717/87 $3.00 + 0.00 Copyright C 1987 Pergamon Journals Ltd
Soil fliol. Eiockm. Vol. 19, No. 6. pp. 719-725, 1987 Printed in Great Britain. All rights reserved
INHIBITION OF THE ~~C~~~~~~~ WUDE CELLULASE COMPLEX BY LEAF LITTER EXTRACTS ROBERTL. SINSABAIJGHand ARTHUR E. LINKINS Biotogy Department, Clarkson University, Potsdam, NY 13676. U.S.A. (Accepred 30 March
1987)
inhibition of the Trichodermauiridocellulase complex by extracts (hot water and 0.1 N NaOH) of deciduous and coniferous leaf litters in various stages of decomposition was investigated. Endocelhtlase and exocefhrlase activities were much more resistant to inhibition than /3-glucosidase. White pine extracts were more inhibitory than deciduous ones and deciduous extracts had similar inhibitory potentials ngard1es-sof litter type. Except for ~ghrcosidase inhibition by water extracts, enzyme inhibition decfined during decomposition. These rest&s indicate that direct phenohc (huntic) in~bition of celhdase components cannot account for species-specific patterns of ceiiulase activity in decomposing titter or for declining celiulase activity in the late stages of decomposition. Because /I-glucosidase, the most readily inhibited component, might potentially limit cellulolysis, it was studied further. /I-glucosidase inhibition by litter extracts was mitigated by the action of phenol oxidase and peroxidase. Complexing /3-glucosidase with water-soluble litter fractions increased pH optima and Ea values, but did not affect apparent &,. Because of the mitigating effect of oxidative enzymes and presumed differences in extraceliular localization of /?-gfucosidase refative to other cehulase components, #?-glucosidase may not limit celluto!ysis in s&u. Summary-The
iNTRODUCTION of leaf litter in forest soils can be considered a two-stage process. During the first stage. leaching and microbial utilization of labile components result in relatively rapid mass loss. In the second stage, refractory litter components, largely lignocellulose, are slowly degraded. This degradation is mediated by the activity of extracellular hydrolytic and oxidative enzymes of microbial origin. The hydrolytic enzymes catalyzing extracellular degradation of cellulose comprise the cellulase complex, which consists of three functional classes of enzymes (Reese, 1977; Eriksson. 1978). The exocellulases ~-l,~exoglucana~) cleave glucose, cellobiose, or other ~lloligo~~ha~des from the nonreducing ends of cellulose molecules. The endocellulases (B-1,4_endogelucanases) randomly hydrolyze glucosidic linkages along the interior of the polymer. Finally, I-glucosidases hydrolyze cellobiose and other soluble cellofigosaccharides into glucose Cellulases typically exhibit long-term environmental stability because they are ~ycoproteins with a large number of cystine crosslinkages (Eriksson and Wood, 1985). Consequently, these enzymes can become spatially and temporally displaced from the synthesizing organism. One implication of this displacement is that cellulose degradation may be only loosely linked to extant microbial activity. Therefore, micr~n~ronm~tal factors that directly affect ceflulase function are potentially important regulators of the decomposition process. One of these microenvironmental factors is the presence of phenolic condensates generated by the oxidative degradation of lignin. These compounds are known to inhibit the activity of several types of The decomposition
extracellular soil enzymes such as proteases (Ladd and Butler, 1969). ureases (Bundy and Bremner, 1974; Mishra et al., 1980) and phosphatases (Malcolm and Vaughan, 1979). Because cellulose and lignin are inextricably associated in litter and tend to be cometabolized (Kirk er al., 1976), cellulases must continuaIly encounter the products of lignin degradation. This interaction may influence decomposition rates. We have examined cellulase inhibition by hotwater and 0.1 N NaOH extracts of both deciduous and coniferous plant litters. We selected the ccllulosedegrading fungus Trichoderma viride as the enzyme source because its cellulase complex has been extensively studied (Eriksson and Wood, 1985). Our specific objectives were: (1) to compare the susceptibilities of exocellulase, endocelluiase and &glucosidase activities to inhibition by litter extracts; (2) to compare the inhibition potential of litter extracts of divergent chemical composition; and (3) to determine whether the inhibition potential of litter extracts changes over the course of decomposition. METHODS
Leaf litter coliection Newly senescent leaves of Cornus JIorida L. (flowering dogwood), Acer saccharum Marsh. (sugar maple), Quercur prims L. (chestnut oak) and Pinus strobus (white pine) were collected during autumnal litter fall, air dried and stored in plastic bags until used. In early April, partiallydecomposed deciduous leaf litter that had overwintered on the forest floor was collected from a woodlot adjacent to the laboratory. The litter was composed of approximately equal amounts of Ace? rtdwum L. (red maple), Be&da 719
120
ROBERT L.
SISSABAUGH and ARTHURE. LINKINS
populifolia Marsh. (gray birch) and Populus tremuloides Michx. (quaking aspen) leaves. Material was
also collected from the underlying humus. In June, white pine litter and humus were collected from a nearby monoculture stand. These litters were dried at SO’C and stored. E.wact preparation
Hot-water extracts were prepared by boiling 10 g (dry mass at SOC) of Jitter in 1.0 I of deionized water for 20min. After cooling, the extracts were filtered through 0.45 pm glass-fiber filters. The filtrate was lyophilized and stored in a desiccator until needed. The residual litter was dried at 5OC and weighed to calculate the amount of material solubilized. Basic extracts were prepared by immersing 10 g of litter in 1.0 I of 0.1 N NaOH for 3 h at room temperature. After being filtered through 0.45 pm glassfiber filters, the extracts were neutralized and deionized by eluting them through a column of Bio-Rad AG 5OW-X8 cation exchange resin. The deionized extracts were then lyophilized. Again, the residual litter was dried and weighed to calculate the quantity of material solubilized. Erryme purification
A crude cellulase preparation from T. ciride was obtained from a commercial source (Sigma, Type V). A 2.Og aliquot was dissolved in 50 ml of 50m~ acetate buffer (pH 5) and eluted through a DEAESephadex column (2.5 x 50cm) with a total bed vol of 250 ml at a rate of 3.6 ml min-‘. Acetate buffer was used as the eluant. The protein, which eluted as a single peak, was collected and lyophiiized. The lyophilate was redissolved in acetate buffer and eluted again through the regenerated column. Stock solutions of cellulase in acetate buffer were divided into small aliquots and frozen until use. Protein concentrations were determ’ned using the Bio-Rad protein assay (Bradford, 1976). Assay procedures
Each of the functional components of the celiulase complex was assayed. Exoglucanase activity was measured calorimetrically using the Nelson-Somogyi reducing sugar assay with microcrystalline cellulose as the substrate (Nelson, 1944). Endoglucanase activity was measured viscometrically using the method of Almin and Eriksson (1967). p-glucosidase activity was determined by a calorimetric assay using pnitrophenyl-b-D-glucopyranoside as the substrate (Cottingham and Mullins, 1985). Inhibition experiments
Stock solutions consisting of lyophilized litter extracts dissolved in acetate buffer to a final concentration of IOmgml-’ were prepared fresh for each assay. Dilutions containing 0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 5.0 and 8.0 mg litter extract ml-’ were prepared from the stock solution. The cellulase solution contained 4 pg protein ml-’ of acetate buffer. Equal volumes of litter extract and cellulase solutions were mixed and held for either 1 or 18 h at 20’C before assaying enzyme activity. All tests were replicated six times.
Phenol oxidase-peroxidase
inte;acrion experiments
A 0.5 ml ahquot of litter extract in 50 mM acetate buffer (pH 5) was held for I h at 20°C with 0.5 ml of mushroom tyrosinase (Sigma, EC I. 14.18.1) or horseradish peroxidase (Sigma Type II. EC 1.I I. I .7) (plus peroxide) solution, also in acetate buffer. The activity of the tyrosinase solution wa.s 40 units ml-’ with 1 unit defined as a change in otpical density at 280 nm of 0.001 min-’ at 2O’C in a mixture containing 1 ml of tyrosinase and I m! of 1 mM t.-tyrosine. The concentration of the peroxidase solution was 20 units ml-’ with 1 unit defined as the production of 3 mg purpurogallin from pyrogallol min- I at 20°C in a reaction mixture consisting of I ml of peroxidase solution and I ml of pyrogallol plus peroxidase solution. The concentration of the litter extract solutions were chosen such that, upon final dilution, the extract concentration would be near values that had been shown to result in 50% inhibition of enzyme activity. After 1 h, oxidase activity in the reaction mixtures was terminated by immersing the solutions in boiling water for 10 min. This step was necessary because of interference with the /I-glucosidase assay. After cooling, 0.5 ml of cellulase solution (4 pg protein ml-‘) in acetate buffer was added to each reaction mixture. The cellulase was allowed to react with the litter extract for I h before residual /3-glucosidase activity was assayed. Production of enzyme complexes
Soluble phenolic-humic complexes of T. uiride cellulase were prepared by dissolving 2.0 g of the crude commercial product in 100 ml of 50 mM acetate buffer (pH 5). After filtering through a 0.45 pm glass-fiber filter, the enzyme solution was combined with a litter extract solution. Both deciduous and pine complexes were prepared. The deciduous extract solution was prepared by dissolving 0.25 g each of lyophilized sugar maple and flosvering dogwood hot-water extracts in 250 ml of acetate buffer. Suspended material was removed by filtration through 0.45 pm glass-fiber filters. White pine extract solution was prepared similarly by dissolving 500 mg of lyophilized white pine hot-water extract in 250 ml of buffer. The enzyme complexes were prepared from extracts of newly senescent litters, rather than from partially decomposed ones, because these extracts had the largest effects on fi-glucosidase activity. The cellulase-litter extract solutions were held at room temperature for 2 h. By this time the enzyme complexes had spontaneously flocculated. Additional floculation was promoted by adding 50 ml of saturated ammonium su!fate solution. The suspension was centrifuged at 10,OOOg and the supematant discarded. To remove salts, the peilet was twice resuspended in 0.02 N HCI solution and centrifuged. The pellets were then dissolved in 50 ml of pH 8 modified universal buffer (MUB contains Tris-HCl, maleic acid, citric acid, boric acid and NaOH). After assaying for /I-glucosidase activity, this solution was diluted to 500ml with acetate buffer, divided into aliquots and frozen. pH profiles
Aliquots of enzyme solution were adjusted to pH values from 2.5 to 8.0 in increments of 0.5 pH units.
Cellulase inhibition by litter extracts Corresponding pH-adjusted 20 mu solutions of pnitrophenyl-fl-D-glucopyranside were prepared using stock MUB buffer. Equal 1 ml volumes of enzyme and substrate solutions were held for 1 h at 20°C. Boiled enzyme solutions served as controls. Six replicate samples were processed at each pH and the procedure was repeated to verify results. Determination of Michaelis-Menten
constants
Apparent Michaelis-Menten constants (K,,,) were determined for /3-glucosidase solutions by measuring the reaction rate every IOmin for 60 min at final p-nitrophenyl-glucoside concentrations of 0.1, 0.2, 0.3,O.j. 1.0, 1.5,2.0,2.5, 5.0 and lO.Orn~. All assays were conducted in pH 5 acetate buffer at 20°C. The entire procedure was replicated to confirm results. k;, estimates were calculated from Eadie-Hofstee plots. Determination of activation energies
/I-glucosidase activity at pH 5 was assayed at temperatures form 0 to 45°C at 5” increments. Enzyme and substrate solutions were equilibriated in the water bath before beginning the assays. Six replicate assays were conducted at each temperature. Activation energies were calculated from Arrhenius plots. RESULTS
The semi-purified Trichoderma cellulase preparation contained endocellulase, exocellulase and B-glucosidase components. These components consistently differed in their susceptability to inhibition by litter extracts (Table 1). The extract concentration (mgml-‘) resulting in a 50% reduction of enzyme activity is abbreviated as 150. 150 estimates and their SE’s were calculated by regression analysis of percentage inhibition as a function of the logarithm of the extract concentration. In some cases, when 50% inhibition was not attained, 25% inhibition values (125) were calculated. No inhibition data were obtained from 0.1 N NaOH extracts of fresh dogwood, maple or oak litters because these extracts formed insoluble tar-like residues during lyophilization. With one exception (exocellulase inhibition by water extracts of white pine surface litter), the endocellulase and exocellulase components were resistant to inhibition by extracts from partially-decomposed litters. Among the newly senescent litters, the white pine extracts were more inhibitory than the deciduous ones. For all extracts evaluated, B-glucosidase activity was the most susceptible to inhibition (Table I). The I50 values for the fresh deciduous litters were similar and approximately twice as high as the 150 estimated for the water extract of fresh pine litter. For deciduous litter, the inhibition potential of the partly decomposed surface and humified material was significantly less than that of the fresh litters, with one exception. The water extract of deciduous humus apparently contained compounds that were slowreacting, but potent inhibitors of fl-glucosidase. These results were confirmed by repeated testing. A similar result was obtained for the water extract from white pine surface litter. Inhibition of /?-glucosidase by NaOH extracts of white pine litters declined steadily with decomposition stage, but this trend was
721
not followed by the water extracts, whose inhibitory potential remained relatively stable. Overall, white pine extracts were more inhibitory to enzyme activity than corresponding deciduous ones. No differences in inhibitory potential were detected among dogwood, maple and oak litters, even though they differ markedly in composition. NaOH extracts were less inhibitory than hot-water ones. More enzyme inhibition was observed after 18 h reaction periods than after 1 h incubations. Finally, except for /?-glucosidase inhibition by white pine water extracts, enzyme inhibition generally declined with decomposition stage. Because /?-glucosidase was the most labile component of the cellulase complex and therefore might potentially limit cellulolysis, it was investigated further. In the first series of experiments, the interaction among lignin-degrading enzymes, /?-glucosidase and litter extracts was investigated. In all cases, the activity of phenol oxidase and peroxidase reduced the capacity of litter extracts to inhibit /?-glucosidase (Table 2). Two trends are apparent in the data. First, peroxidase was generally more effective than phenol oxidase in reducing the fl-glucosidase inhibition potential of litter extracts. Second, delta values were generally smaller for extracts from partially decomposed litters than for those of newly senescent litters. In addition to inhibiting or inactivating /3-glucosidase, litter extracts altered the characteristics of residual enzyme activity. The apparent optimum pH for /?-glucosidase activity shifted from 5.0 to 5.5 when the enzyme was complexed with phenolic components of deciduous and pine extracts (Fig. I). Activation energy, estimated from Arrhenius plots, increased from 50.0 kJ mol-’ for uncomplexed fl-glucosidase to 56.7 kJ mol-’ for deciduous extract complexes and 63.4 kJ mol-’ for white pine extract complexes (Fig. 2). Despite differences in pH and temperature response, K,,, values did not significantly differ, ranging between 0.09 and O.lOrn~ for both complexed and uncomplexed enzymes (Fig. 3). DISCUSSION
The interaction between phenolic substances and a number of enzymes has been investigated. However, comparisons with our data are hampered by differences in assay procedures and in the characteristics of the inhibitors used. Our results are consistent with those of Benoit and Starkey (1968) who found that compared to other exoenzymes tested, endocellulase activity was strongly resistant to inhibition by a purified tanning preparation. Goldstein and Swain (1965) found that fl-glucosidase retained about 50% of its initial activity when complexed with tannin. Many proteases are highly susceptible to humic acid inhibition, but interestingly, tyrosinase, a phenol and o-diphenol oxidase that may be involved in lignocellulose degradation, is not affected (Ladd and Butler, 1969). It appears that the extracellular enzymes involved in lignocellulose degradation may be more resistant to humic inhibition than other classes of soil enzymes, although this remains to be established. One of the trends in our data was lower enzyme inhibition by NaOH extracts than by corresponding
43
Hot-water
II 26
Hot-water
0.1 N NaOH
[
0.14 + 0.24 [I .42 + 0.081
]1.70+0.I0]
3.10+0.12
0.17 + 0.23
3.46+0.18
0.18+0.22
1.80+0.16
II
Hot-water IS
0.40 + 0. IO
0.59 + 0.09
24
0.1 N NaOH
NNaOH
I .08 + 0.22 I.21 + 0.31
I.57 + 0.32
0.26 + 0.18
0.34 + 0.14
I6
Hot-water
0. I
l.l6+0.30
NSI
NSI
NSI
NSI
NSI
36
0.1 N NaOH
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
0.21 + 0.20
2.51 + 0.08
IO
Hot-water
NSI
NSI
NSI
NSI
12.88 + O&t]
NSI
NSI
3.47 + 0.52
3.61 + 0.24
30
0.1 N NaOH
NSI
NSI
NSI
12.71 + 0.291
4.10+0X%
NSI
NSI
NSI
NSI
NSI
Il.44 + 0.261
2.84 + 0.35
NSI
NSI
NSI
NSI
NSI
NSI
2.28 + 0.22
2.70 + 0.20
I6
Hot-water
NSI
2.92 + 0.20
NSI
0.59 + 0.06
0.71 + 0.05
I8
NSI
NSI
NSI NSI
Exoccllulasc 18h ISO+SE
Exoccllulase I h ISO+SE
Hot-water
2.71’+ 0.27
2.88 + 0.59
Endocellulasc ISh ISO+SE
0.42 + 0.07
NSI
Endocellulasc I h ISO+SE
0.68 f 0.09
0.56 + 0.04
Glucosidasc 18h lSO+SE
32
0.83 + 0.06
Glucosidax ISO+SE
Ih
Hot-water
‘Ype
NSI = No Significant Inhibition. ]-125+Sc.
fresh litter Sugar maple fresh litter Chestnut oak fresh litter Mixed deciduous surface liller Mixed deciduous surface lillcr Mixed deciduous humus litter Mixed deciduous humus litter White pine fresh litter While pine fresh litter white pine surface lillcr While pine surface litter White pine humus litter While pine humus litter
mw~
Litler lype
Fraction solubilixed f%)
Extract
Table I. Inhibition of 7. oiride cellulase components by leaf litter exlracts. I50 is the extract concentration in mgml-’ that resulted in a 50% reduction in enzyme activity. Bracketed values arc erlimatcs of exlracl conanlralions causing 25% reductions in activity. Cavs where less than 20% inhibition occurred at the highest extract concentration tcstcd (5 mg ml ‘) arc abbreviated NSI (No Significant Inhibition).
Ccllulase inhibition by titter extracts
723
Table 2. Inhibition of /I-glucosidam by litter extracts modified by phenol oxidasc and peroxidasc activity. The delta value is the diffemnce between the pcrcentagc inhibition in untreated extract solutiotu and the petcentagc inhibition in modifkd extract 8olutions. Litter
Extract
type
type
Wwood fresh titter Sugar maple fresh litter Chestnut oak fresh litter Mixed deciduous surface litter Mixed deciduous humus litter Mixed deciduous fresh litter White pine fresh litter White pine surface litter White pine surface litter White pine humus litter White pine humus litter
Phenol Onidasc Lklta vahlc
Peroxidam Delta value
Hot-water
20.1
50.0
Hot-water
a.3
50.0
Hot-water
7.8
32.2
Hot-water
0.0
9.0
0.1 N NaOH
3.8
13.2
Hot-water
22.8
10.6
0.1 N NaOH
4.1
26.9
Hot-water
4.3
4.6
0.1 N NaOH
3.8
16.3
Hot-water
0.3
46.8
0.1 N NaOH
2.6
2.5
water extracts. This result could be an artifact of the extraction process. NaOH extraction solubilized a greater portion of the litter than water extractions (Table 1). The result could be a dilution of inhibitory substances by non-inhibitory ones, which would increase the apparent 150. Another factor is the modification of phenolic compounds by NaOH which could alter their inhibitory potential. Because lignin and cellulose are structurally associated, their decomposition is sustained only if both are degraded concurrently (Gressel ef al., 1983). Thus, cellulases function in an environment that exposes them to phenolic (humic) condensates produced by the oxidative degradation of lignin. However, the functional components of the Trichoderma cellulase system differ in their resistance to inactivation by these substances. According to the currently accepted model (Eriksson and Wood, 1985). the first step in hydrolytic cellulolysis is disruption of the crystalline order of native cellulose by exocellulases (cel-
lobiohydrolases). This disruption creates binding sites for endocellulases. Subsequent degradation to soluble celloligosaccharides proceeds through the synergistic interaction of both exo- and endocellulases. The final step, mediated by /I-glucosidases, is generation of glucose from soluble celloligosaccharides. This step is often spatially displaced from the sites of exo- and endocellulase activ-
I 3.2 PH
Fig. 1. pH profiles of complexed and uncomplcxcd T. uiricir b-glucosidasc
activity.
3.4
36
Temp (OK-’ x lo3 ) Fig. 2. Arrhenius plots for complexed and uncomplexed T. viride b-glucosidase activity.
ROBERT L. SINSABAUGHand ARIHURE. LINKISS
724
>, %
.
" 4. P r" 10 M
30 40 50 63 73 80 90 100
-& -aCD
mrt”
mm01 -’ x 103
Fig. 3. Eadie-Hofstee plots for complexed and uncomplexed F. uiride b-glucosidase activity. ity because fl-glucosidases, while extracellular, frequently remain associated with fungal walls, relying on diffusion for substrate from sites of polymer degradation. The relative susceptabilities of Trichoderma cellulase components to inhibition are consistent with this model. The exo- and endocellulase enzymes, which directly attack cellulose, are either unaffected or quite resistant to inhibition by hot-water and 0.1 N NaOHextractable litter fractions. In contrast, j?-glucosidase, presumably spatially displaced from the sites of lignocellulose degradation, can be markedly inhibited at moderate extract concentrations. These differences in reactivity probably reflect differences in the chemical composition of the enzymes. Most cellulases are glycoproteins with the proportion of carbohydrate ranging as high as 50% (Eriksson and Wood, 1985). Differences in carbohydrate composition may account for the differential inhibition of the Trichodefma cellulase components. Studies of cellulase dynamics on decomposing deciduous litters have shown that there are speciesspecific patterns of exocellulase and endocellulase activity and that activity declines markedly during the later stages of decomposition (Sinsabaugh er al., 1981; McClaugherty et al., 1987, submitted manuscript, unpublished data). The similarity in inhibition potential among deciduous litters of diverse composition and the lack of inhibition of exocellulase and endocellulase activity by extracts from partiallydecomposed litters indicates that direct inhibition of these enzymes does not account for these trends. The activity of phenol oxidase and peroxidase toward extracted leaf litter components reduced their capacity to inhibit T. niride fl-glucosidase. Similar patterns emerged for endocellulase and exocellulase activities (data not presented). Phenol oxidase and peroxidase enzymes exhibit both polymerizing and depolymerizing activities toward phenolic substances (Sulfita and Bollag. 1980; Leonowicz ef al., 1985). Activity toward large molecules, like lignins, results in net depolymerization and oxidation of alkyl side chains (Chen and Chang, 1985). But because these reactions proceed by free radical mechanisms (Paterson and Lundquist, 1985). reaction products condense with one another and with soluble carbohydrates, proteins and lipids to form new heterocondensates The addition of . . . . . (humic substances). phenol oxtdase and peroxidase
to fresh litter extracts
can be viewed as an acceleration of the natural decomposition process. The result was that the capacity of the modified extract to inhibit cellulase activity was sharply reduced to levels typical of those determined for partially-decomposed litters. Generally, the extracts from partially-decomposed litters were rendered only slightly less inhibitory by the action of the oxidative enzymes. Inhibition is not the only way that extractable litter components can affect enzyme activity. The functional complexes formed by the interaction of fl-glucosidase and litter extracts in solution exhibited different responses to pH and temperature, though not substrate concentration. These effects were more pronounced for white pine enzyme complexes than for deciduous ones. The functional significance of these differences is not known. However, our data suggest that in decomposing litter phenolic-humic inhibition of /I-glucosidase may be ameliorated by the presence of phenol oxidases and peroxidases. This effect, along with differences in /3-glucosidase localization, may mitigate the potential limitation of cellulolysis implied by the litter extract inhibition experiments. work was supported by the National Science Foundation. We thank Elizabeth Sechoka for her technical expertise and Holly Nachod for performing the electrophoresis. Acknowledgements-Our
REFERENCES Almin K. E. and Eriksson K-E. (1967) Enzymic degradation of polymers: I. Viscometric method for the determination of the enzymic activity. Biochimica et Biophvsica Acra . 139, 238-247. Benoit R. E. and Starkey R. L. (1968) Enzyme inactivation as a factor in the inhibition of decomoosition of oreanic matter by tannins. Soil Science 105, 203-208. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyticaf Biochemistry 72, 248-254.
Bundy L. G. and Bremner J. M. (1974) Effect of urease inhibitors on nitrilication in soils. Soil Biology & Biochemistry 6, 27-30.
Chen C-L. and Chang H-M. (1985) Chemistry of lignin biodegradation. In Biosynthesis and Biodegradarion of Wood Components (T. Higuchi. Ed.), pp. 535-555. Academic Press, New York. Cottingham C. K. and Mullins J. T. (1985) The preliminary characterization of the beta-glucosidases in Achyla ambisexualis.
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77, 38 I-389.
Eriksson K-E. (1978) Enzyme mechanisms involved in cellular hydrolysis by the rot fungus, Spororrichum pulverulenfum. 317-332.
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Cellulase inhibition by litter extracts Ladd J. N. and Butler J. H. A. (1969) Inhibition and stimulation of proteolytic enzyme activities by soil humic acids. Australian Joumal of Soil Research 7, 253-261. Leonowin A., Szklatz G. -and Wojtas-Wasilewska M. (1985) The effect of fungal lactase on fractionated lig nosulphonates (Peritan Na). Phytochemistry 24.393-396. Malcolm R. E. and Vaughan D. (1979) Comparative effects of soil organic matter-fractions on phosphatase activities in wheat roots. Plant and Soil 51. 117-126. Mishra M. M., Flaig W. and Soechtig H. (1980) The effect of quinoid and phenolic compounds on urease and dehydrogenase activity and nitrification in soil. Plant and Soil 55, 25-33. Nelson N. (1944) A photometric adaptation of the Somogyi
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method for the determination of glucose. Journal of Biological Chemistry 153, 375-380. Paterson A. and Lundauist K. (1985) Radical breakdown of lignin. Nature 316, 575-576.. ’ Reese E. T. (1977) The structure. biosvnthesis. and degradation of wood. In Recent A&nces in Phytochemirtry. Vol. 2 (F. A. Loewus and V. C. Runeckles. Eds), pp. 31 l-367. Plenum Press. New York. Sinsabaugh R. L., Bentield E. F. and Linkins A. E. (1981) Cellulase activity associated with the decomposition of leaf titter in a woodland stream. Oikos 36, 184-190. Sulfita J. M. and Bollag J-M. (1980) Gxidative coupling activity in soil extracts. Soil Biology & Biochemistry 12, 177-183.
Soil fliol. Eiockm. Vol. 19, No. 6. pp. 719-725, 1987 Printed in Great Britain. All rights reserved
INHIBITION OF THE ~~C~~~~~~~ WUDE CELLULASE COMPLEX BY LEAF LITTER EXTRACTS ROBERTL. SINSABAIJGHand ARTHUR E. LINKINS Biotogy Department, Clarkson University, Potsdam, NY 13676. U.S.A. (Accepred 30 March
1987)
inhibition of the Trichodermauiridocellulase complex by extracts (hot water and 0.1 N NaOH) of deciduous and coniferous leaf litters in various stages of decomposition was investigated. Endocelhtlase and exocefhrlase activities were much more resistant to inhibition than /3-glucosidase. White pine extracts were more inhibitory than deciduous ones and deciduous extracts had similar inhibitory potentials ngard1es-sof litter type. Except for ~ghrcosidase inhibition by water extracts, enzyme inhibition decfined during decomposition. These rest&s indicate that direct phenohc (huntic) in~bition of celhdase components cannot account for species-specific patterns of ceiiulase activity in decomposing titter or for declining celiulase activity in the late stages of decomposition. Because /I-glucosidase, the most readily inhibited component, might potentially limit cellulolysis, it was studied further. /I-glucosidase inhibition by litter extracts was mitigated by the action of phenol oxidase and peroxidase. Complexing /3-glucosidase with water-soluble litter fractions increased pH optima and Ea values, but did not affect apparent &,. Because of the mitigating effect of oxidative enzymes and presumed differences in extraceliular localization of /?-gfucosidase refative to other cehulase components, #?-glucosidase may not limit celluto!ysis in s&u. Summary-The
iNTRODUCTION of leaf litter in forest soils can be considered a two-stage process. During the first stage. leaching and microbial utilization of labile components result in relatively rapid mass loss. In the second stage, refractory litter components, largely lignocellulose, are slowly degraded. This degradation is mediated by the activity of extracellular hydrolytic and oxidative enzymes of microbial origin. The hydrolytic enzymes catalyzing extracellular degradation of cellulose comprise the cellulase complex, which consists of three functional classes of enzymes (Reese, 1977; Eriksson. 1978). The exocellulases ~-l,~exoglucana~) cleave glucose, cellobiose, or other ~lloligo~~ha~des from the nonreducing ends of cellulose molecules. The endocellulases (B-1,4_endogelucanases) randomly hydrolyze glucosidic linkages along the interior of the polymer. Finally, I-glucosidases hydrolyze cellobiose and other soluble cellofigosaccharides into glucose Cellulases typically exhibit long-term environmental stability because they are ~ycoproteins with a large number of cystine crosslinkages (Eriksson and Wood, 1985). Consequently, these enzymes can become spatially and temporally displaced from the synthesizing organism. One implication of this displacement is that cellulose degradation may be only loosely linked to extant microbial activity. Therefore, micr~n~ronm~tal factors that directly affect ceflulase function are potentially important regulators of the decomposition process. One of these microenvironmental factors is the presence of phenolic condensates generated by the oxidative degradation of lignin. These compounds are known to inhibit the activity of several types of The decomposition
extracellular soil enzymes such as proteases (Ladd and Butler, 1969). ureases (Bundy and Bremner, 1974; Mishra et al., 1980) and phosphatases (Malcolm and Vaughan, 1979). Because cellulose and lignin are inextricably associated in litter and tend to be cometabolized (Kirk er al., 1976), cellulases must continuaIly encounter the products of lignin degradation. This interaction may influence decomposition rates. We have examined cellulase inhibition by hotwater and 0.1 N NaOH extracts of both deciduous and coniferous plant litters. We selected the ccllulosedegrading fungus Trichoderma viride as the enzyme source because its cellulase complex has been extensively studied (Eriksson and Wood, 1985). Our specific objectives were: (1) to compare the susceptibilities of exocellulase, endocelluiase and &glucosidase activities to inhibition by litter extracts; (2) to compare the inhibition potential of litter extracts of divergent chemical composition; and (3) to determine whether the inhibition potential of litter extracts changes over the course of decomposition. METHODS
Leaf litter coliection Newly senescent leaves of Cornus JIorida L. (flowering dogwood), Acer saccharum Marsh. (sugar maple), Quercur prims L. (chestnut oak) and Pinus strobus (white pine) were collected during autumnal litter fall, air dried and stored in plastic bags until used. In early April, partiallydecomposed deciduous leaf litter that had overwintered on the forest floor was collected from a woodlot adjacent to the laboratory. The litter was composed of approximately equal amounts of Ace? rtdwum L. (red maple), Be&da 719
120
ROBERT L.
SISSABAUGH and ARTHURE. LINKINS
populifolia Marsh. (gray birch) and Populus tremuloides Michx. (quaking aspen) leaves. Material was
also collected from the underlying humus. In June, white pine litter and humus were collected from a nearby monoculture stand. These litters were dried at SO’C and stored. E.wact preparation
Hot-water extracts were prepared by boiling 10 g (dry mass at SOC) of Jitter in 1.0 I of deionized water for 20min. After cooling, the extracts were filtered through 0.45 pm glass-fiber filters. The filtrate was lyophilized and stored in a desiccator until needed. The residual litter was dried at 5OC and weighed to calculate the amount of material solubilized. Basic extracts were prepared by immersing 10 g of litter in 1.0 I of 0.1 N NaOH for 3 h at room temperature. After being filtered through 0.45 pm glassfiber filters, the extracts were neutralized and deionized by eluting them through a column of Bio-Rad AG 5OW-X8 cation exchange resin. The deionized extracts were then lyophilized. Again, the residual litter was dried and weighed to calculate the quantity of material solubilized. Erryme purification
A crude cellulase preparation from T. ciride was obtained from a commercial source (Sigma, Type V). A 2.Og aliquot was dissolved in 50 ml of 50m~ acetate buffer (pH 5) and eluted through a DEAESephadex column (2.5 x 50cm) with a total bed vol of 250 ml at a rate of 3.6 ml min-‘. Acetate buffer was used as the eluant. The protein, which eluted as a single peak, was collected and lyophiiized. The lyophilate was redissolved in acetate buffer and eluted again through the regenerated column. Stock solutions of cellulase in acetate buffer were divided into small aliquots and frozen until use. Protein concentrations were determ’ned using the Bio-Rad protein assay (Bradford, 1976). Assay procedures
Each of the functional components of the celiulase complex was assayed. Exoglucanase activity was measured calorimetrically using the Nelson-Somogyi reducing sugar assay with microcrystalline cellulose as the substrate (Nelson, 1944). Endoglucanase activity was measured viscometrically using the method of Almin and Eriksson (1967). p-glucosidase activity was determined by a calorimetric assay using pnitrophenyl-b-D-glucopyranoside as the substrate (Cottingham and Mullins, 1985). Inhibition experiments
Stock solutions consisting of lyophilized litter extracts dissolved in acetate buffer to a final concentration of IOmgml-’ were prepared fresh for each assay. Dilutions containing 0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 5.0 and 8.0 mg litter extract ml-’ were prepared from the stock solution. The cellulase solution contained 4 pg protein ml-’ of acetate buffer. Equal volumes of litter extract and cellulase solutions were mixed and held for either 1 or 18 h at 20’C before assaying enzyme activity. All tests were replicated six times.
Phenol oxidase-peroxidase
inte;acrion experiments
A 0.5 ml ahquot of litter extract in 50 mM acetate buffer (pH 5) was held for I h at 20°C with 0.5 ml of mushroom tyrosinase (Sigma, EC I. 14.18.1) or horseradish peroxidase (Sigma Type II. EC 1.I I. I .7) (plus peroxide) solution, also in acetate buffer. The activity of the tyrosinase solution wa.s 40 units ml-’ with 1 unit defined as a change in otpical density at 280 nm of 0.001 min-’ at 2O’C in a mixture containing 1 ml of tyrosinase and I m! of 1 mM t.-tyrosine. The concentration of the peroxidase solution was 20 units ml-’ with 1 unit defined as the production of 3 mg purpurogallin from pyrogallol min- I at 20°C in a reaction mixture consisting of I ml of peroxidase solution and I ml of pyrogallol plus peroxidase solution. The concentration of the litter extract solutions were chosen such that, upon final dilution, the extract concentration would be near values that had been shown to result in 50% inhibition of enzyme activity. After 1 h, oxidase activity in the reaction mixtures was terminated by immersing the solutions in boiling water for 10 min. This step was necessary because of interference with the /I-glucosidase assay. After cooling, 0.5 ml of cellulase solution (4 pg protein ml-‘) in acetate buffer was added to each reaction mixture. The cellulase was allowed to react with the litter extract for I h before residual /3-glucosidase activity was assayed. Production of enzyme complexes
Soluble phenolic-humic complexes of T. uiride cellulase were prepared by dissolving 2.0 g of the crude commercial product in 100 ml of 50 mM acetate buffer (pH 5). After filtering through a 0.45 pm glass-fiber filter, the enzyme solution was combined with a litter extract solution. Both deciduous and pine complexes were prepared. The deciduous extract solution was prepared by dissolving 0.25 g each of lyophilized sugar maple and flosvering dogwood hot-water extracts in 250 ml of acetate buffer. Suspended material was removed by filtration through 0.45 pm glass-fiber filters. White pine extract solution was prepared similarly by dissolving 500 mg of lyophilized white pine hot-water extract in 250 ml of buffer. The enzyme complexes were prepared from extracts of newly senescent litters, rather than from partially decomposed ones, because these extracts had the largest effects on fi-glucosidase activity. The cellulase-litter extract solutions were held at room temperature for 2 h. By this time the enzyme complexes had spontaneously flocculated. Additional floculation was promoted by adding 50 ml of saturated ammonium su!fate solution. The suspension was centrifuged at 10,OOOg and the supematant discarded. To remove salts, the peilet was twice resuspended in 0.02 N HCI solution and centrifuged. The pellets were then dissolved in 50 ml of pH 8 modified universal buffer (MUB contains Tris-HCl, maleic acid, citric acid, boric acid and NaOH). After assaying for /I-glucosidase activity, this solution was diluted to 500ml with acetate buffer, divided into aliquots and frozen. pH profiles
Aliquots of enzyme solution were adjusted to pH values from 2.5 to 8.0 in increments of 0.5 pH units.
Cellulase inhibition by litter extracts Corresponding pH-adjusted 20 mu solutions of pnitrophenyl-fl-D-glucopyranside were prepared using stock MUB buffer. Equal 1 ml volumes of enzyme and substrate solutions were held for 1 h at 20°C. Boiled enzyme solutions served as controls. Six replicate samples were processed at each pH and the procedure was repeated to verify results. Determination of Michaelis-Menten
constants
Apparent Michaelis-Menten constants (K,,,) were determined for /3-glucosidase solutions by measuring the reaction rate every IOmin for 60 min at final p-nitrophenyl-glucoside concentrations of 0.1, 0.2, 0.3,O.j. 1.0, 1.5,2.0,2.5, 5.0 and lO.Orn~. All assays were conducted in pH 5 acetate buffer at 20°C. The entire procedure was replicated to confirm results. k;, estimates were calculated from Eadie-Hofstee plots. Determination of activation energies
/I-glucosidase activity at pH 5 was assayed at temperatures form 0 to 45°C at 5” increments. Enzyme and substrate solutions were equilibriated in the water bath before beginning the assays. Six replicate assays were conducted at each temperature. Activation energies were calculated from Arrhenius plots. RESULTS
The semi-purified Trichoderma cellulase preparation contained endocellulase, exocellulase and B-glucosidase components. These components consistently differed in their susceptability to inhibition by litter extracts (Table 1). The extract concentration (mgml-‘) resulting in a 50% reduction of enzyme activity is abbreviated as 150. 150 estimates and their SE’s were calculated by regression analysis of percentage inhibition as a function of the logarithm of the extract concentration. In some cases, when 50% inhibition was not attained, 25% inhibition values (125) were calculated. No inhibition data were obtained from 0.1 N NaOH extracts of fresh dogwood, maple or oak litters because these extracts formed insoluble tar-like residues during lyophilization. With one exception (exocellulase inhibition by water extracts of white pine surface litter), the endocellulase and exocellulase components were resistant to inhibition by extracts from partially-decomposed litters. Among the newly senescent litters, the white pine extracts were more inhibitory than the deciduous ones. For all extracts evaluated, B-glucosidase activity was the most susceptible to inhibition (Table I). The I50 values for the fresh deciduous litters were similar and approximately twice as high as the 150 estimated for the water extract of fresh pine litter. For deciduous litter, the inhibition potential of the partly decomposed surface and humified material was significantly less than that of the fresh litters, with one exception. The water extract of deciduous humus apparently contained compounds that were slowreacting, but potent inhibitors of fl-glucosidase. These results were confirmed by repeated testing. A similar result was obtained for the water extract from white pine surface litter. Inhibition of /?-glucosidase by NaOH extracts of white pine litters declined steadily with decomposition stage, but this trend was
721
not followed by the water extracts, whose inhibitory potential remained relatively stable. Overall, white pine extracts were more inhibitory to enzyme activity than corresponding deciduous ones. No differences in inhibitory potential were detected among dogwood, maple and oak litters, even though they differ markedly in composition. NaOH extracts were less inhibitory than hot-water ones. More enzyme inhibition was observed after 18 h reaction periods than after 1 h incubations. Finally, except for /?-glucosidase inhibition by white pine water extracts, enzyme inhibition generally declined with decomposition stage. Because /?-glucosidase was the most labile component of the cellulase complex and therefore might potentially limit cellulolysis, it was investigated further. In the first series of experiments, the interaction among lignin-degrading enzymes, /?-glucosidase and litter extracts was investigated. In all cases, the activity of phenol oxidase and peroxidase reduced the capacity of litter extracts to inhibit /?-glucosidase (Table 2). Two trends are apparent in the data. First, peroxidase was generally more effective than phenol oxidase in reducing the fl-glucosidase inhibition potential of litter extracts. Second, delta values were generally smaller for extracts from partially decomposed litters than for those of newly senescent litters. In addition to inhibiting or inactivating /3-glucosidase, litter extracts altered the characteristics of residual enzyme activity. The apparent optimum pH for /?-glucosidase activity shifted from 5.0 to 5.5 when the enzyme was complexed with phenolic components of deciduous and pine extracts (Fig. I). Activation energy, estimated from Arrhenius plots, increased from 50.0 kJ mol-’ for uncomplexed fl-glucosidase to 56.7 kJ mol-’ for deciduous extract complexes and 63.4 kJ mol-’ for white pine extract complexes (Fig. 2). Despite differences in pH and temperature response, K,,, values did not significantly differ, ranging between 0.09 and O.lOrn~ for both complexed and uncomplexed enzymes (Fig. 3). DISCUSSION
The interaction between phenolic substances and a number of enzymes has been investigated. However, comparisons with our data are hampered by differences in assay procedures and in the characteristics of the inhibitors used. Our results are consistent with those of Benoit and Starkey (1968) who found that compared to other exoenzymes tested, endocellulase activity was strongly resistant to inhibition by a purified tanning preparation. Goldstein and Swain (1965) found that fl-glucosidase retained about 50% of its initial activity when complexed with tannin. Many proteases are highly susceptible to humic acid inhibition, but interestingly, tyrosinase, a phenol and o-diphenol oxidase that may be involved in lignocellulose degradation, is not affected (Ladd and Butler, 1969). It appears that the extracellular enzymes involved in lignocellulose degradation may be more resistant to humic inhibition than other classes of soil enzymes, although this remains to be established. One of the trends in our data was lower enzyme inhibition by NaOH extracts than by corresponding
43
Hot-water
II 26
Hot-water
0.1 N NaOH
[
0.14 + 0.24 [I .42 + 0.081
]1.70+0.I0]
3.10+0.12
0.17 + 0.23
3.46+0.18
0.18+0.22
1.80+0.16
II
Hot-water IS
0.40 + 0. IO
0.59 + 0.09
24
0.1 N NaOH
NNaOH
I .08 + 0.22 I.21 + 0.31
I.57 + 0.32
0.26 + 0.18
0.34 + 0.14
I6
Hot-water
0. I
l.l6+0.30
NSI
NSI
NSI
NSI
NSI
36
0.1 N NaOH
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
0.21 + 0.20
2.51 + 0.08
IO
Hot-water
NSI
NSI
NSI
NSI
12.88 + O&t]
NSI
NSI
3.47 + 0.52
3.61 + 0.24
30
0.1 N NaOH
NSI
NSI
NSI
12.71 + 0.291
4.10+0X%
NSI
NSI
NSI
NSI
NSI
Il.44 + 0.261
2.84 + 0.35
NSI
NSI
NSI
NSI
NSI
NSI
2.28 + 0.22
2.70 + 0.20
I6
Hot-water
NSI
2.92 + 0.20
NSI
0.59 + 0.06
0.71 + 0.05
I8
NSI
NSI
NSI NSI
Exoccllulasc 18h ISO+SE
Exoccllulase I h ISO+SE
Hot-water
2.71’+ 0.27
2.88 + 0.59
Endocellulasc ISh ISO+SE
0.42 + 0.07
NSI
Endocellulasc I h ISO+SE
0.68 f 0.09
0.56 + 0.04
Glucosidasc 18h lSO+SE
32
0.83 + 0.06
Glucosidax ISO+SE
Ih
Hot-water
‘Ype
NSI = No Significant Inhibition. ]-125+Sc.
fresh litter Sugar maple fresh litter Chestnut oak fresh litter Mixed deciduous surface liller Mixed deciduous surface lillcr Mixed deciduous humus litter Mixed deciduous humus litter White pine fresh litter While pine fresh litter white pine surface lillcr While pine surface litter White pine humus litter While pine humus litter
mw~
Litler lype
Fraction solubilixed f%)
Extract
Table I. Inhibition of 7. oiride cellulase components by leaf litter exlracts. I50 is the extract concentration in mgml-’ that resulted in a 50% reduction in enzyme activity. Bracketed values arc erlimatcs of exlracl conanlralions causing 25% reductions in activity. Cavs where less than 20% inhibition occurred at the highest extract concentration tcstcd (5 mg ml ‘) arc abbreviated NSI (No Significant Inhibition).
Ccllulase inhibition by titter extracts
723
Table 2. Inhibition of /I-glucosidam by litter extracts modified by phenol oxidasc and peroxidasc activity. The delta value is the diffemnce between the pcrcentagc inhibition in untreated extract solutiotu and the petcentagc inhibition in modifkd extract 8olutions. Litter
Extract
type
type
Wwood fresh titter Sugar maple fresh litter Chestnut oak fresh litter Mixed deciduous surface litter Mixed deciduous humus litter Mixed deciduous fresh litter White pine fresh litter White pine surface litter White pine surface litter White pine humus litter White pine humus litter
Phenol Onidasc Lklta vahlc
Peroxidam Delta value
Hot-water
20.1
50.0
Hot-water
a.3
50.0
Hot-water
7.8
32.2
Hot-water
0.0
9.0
0.1 N NaOH
3.8
13.2
Hot-water
22.8
10.6
0.1 N NaOH
4.1
26.9
Hot-water
4.3
4.6
0.1 N NaOH
3.8
16.3
Hot-water
0.3
46.8
0.1 N NaOH
2.6
2.5
water extracts. This result could be an artifact of the extraction process. NaOH extraction solubilized a greater portion of the litter than water extractions (Table 1). The result could be a dilution of inhibitory substances by non-inhibitory ones, which would increase the apparent 150. Another factor is the modification of phenolic compounds by NaOH which could alter their inhibitory potential. Because lignin and cellulose are structurally associated, their decomposition is sustained only if both are degraded concurrently (Gressel ef al., 1983). Thus, cellulases function in an environment that exposes them to phenolic (humic) condensates produced by the oxidative degradation of lignin. However, the functional components of the Trichoderma cellulase system differ in their resistance to inactivation by these substances. According to the currently accepted model (Eriksson and Wood, 1985). the first step in hydrolytic cellulolysis is disruption of the crystalline order of native cellulose by exocellulases (cel-
lobiohydrolases). This disruption creates binding sites for endocellulases. Subsequent degradation to soluble celloligosaccharides proceeds through the synergistic interaction of both exo- and endocellulases. The final step, mediated by /I-glucosidases, is generation of glucose from soluble celloligosaccharides. This step is often spatially displaced from the sites of exo- and endocellulase activ-
I 3.2 PH
Fig. 1. pH profiles of complexed and uncomplcxcd T. uiricir b-glucosidasc
activity.
3.4
36
Temp (OK-’ x lo3 ) Fig. 2. Arrhenius plots for complexed and uncomplexed T. viride b-glucosidase activity.
ROBERT L. SINSABAUGHand ARIHURE. LINKISS
724
>, %
.
" 4. P r" 10 M
30 40 50 63 73 80 90 100
-& -aCD
mrt”
mm01 -’ x 103
Fig. 3. Eadie-Hofstee plots for complexed and uncomplexed F. uiride b-glucosidase activity. ity because fl-glucosidases, while extracellular, frequently remain associated with fungal walls, relying on diffusion for substrate from sites of polymer degradation. The relative susceptabilities of Trichoderma cellulase components to inhibition are consistent with this model. The exo- and endocellulase enzymes, which directly attack cellulose, are either unaffected or quite resistant to inhibition by hot-water and 0.1 N NaOHextractable litter fractions. In contrast, j?-glucosidase, presumably spatially displaced from the sites of lignocellulose degradation, can be markedly inhibited at moderate extract concentrations. These differences in reactivity probably reflect differences in the chemical composition of the enzymes. Most cellulases are glycoproteins with the proportion of carbohydrate ranging as high as 50% (Eriksson and Wood, 1985). Differences in carbohydrate composition may account for the differential inhibition of the Trichodefma cellulase components. Studies of cellulase dynamics on decomposing deciduous litters have shown that there are speciesspecific patterns of exocellulase and endocellulase activity and that activity declines markedly during the later stages of decomposition (Sinsabaugh er al., 1981; McClaugherty et al., 1987, submitted manuscript, unpublished data). The similarity in inhibition potential among deciduous litters of diverse composition and the lack of inhibition of exocellulase and endocellulase activity by extracts from partiallydecomposed litters indicates that direct inhibition of these enzymes does not account for these trends. The activity of phenol oxidase and peroxidase toward extracted leaf litter components reduced their capacity to inhibit T. niride fl-glucosidase. Similar patterns emerged for endocellulase and exocellulase activities (data not presented). Phenol oxidase and peroxidase enzymes exhibit both polymerizing and depolymerizing activities toward phenolic substances (Sulfita and Bollag. 1980; Leonowicz ef al., 1985). Activity toward large molecules, like lignins, results in net depolymerization and oxidation of alkyl side chains (Chen and Chang, 1985). But because these reactions proceed by free radical mechanisms (Paterson and Lundquist, 1985). reaction products condense with one another and with soluble carbohydrates, proteins and lipids to form new heterocondensates The addition of . . . . . (humic substances). phenol oxtdase and peroxidase
to fresh litter extracts
can be viewed as an acceleration of the natural decomposition process. The result was that the capacity of the modified extract to inhibit cellulase activity was sharply reduced to levels typical of those determined for partially-decomposed litters. Generally, the extracts from partially-decomposed litters were rendered only slightly less inhibitory by the action of the oxidative enzymes. Inhibition is not the only way that extractable litter components can affect enzyme activity. The functional complexes formed by the interaction of fl-glucosidase and litter extracts in solution exhibited different responses to pH and temperature, though not substrate concentration. These effects were more pronounced for white pine enzyme complexes than for deciduous ones. The functional significance of these differences is not known. However, our data suggest that in decomposing litter phenolic-humic inhibition of /I-glucosidase may be ameliorated by the presence of phenol oxidases and peroxidases. This effect, along with differences in /3-glucosidase localization, may mitigate the potential limitation of cellulolysis implied by the litter extract inhibition experiments. work was supported by the National Science Foundation. We thank Elizabeth Sechoka for her technical expertise and Holly Nachod for performing the electrophoresis. Acknowledgements-Our
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Chen C-L. and Chang H-M. (1985) Chemistry of lignin biodegradation. In Biosynthesis and Biodegradarion of Wood Components (T. Higuchi. Ed.), pp. 535-555. Academic Press, New York. Cottingham C. K. and Mullins J. T. (1985) The preliminary characterization of the beta-glucosidases in Achyla ambisexualis.
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Cellulase inhibition by litter extracts Ladd J. N. and Butler J. H. A. (1969) Inhibition and stimulation of proteolytic enzyme activities by soil humic acids. Australian Joumal of Soil Research 7, 253-261. Leonowin A., Szklatz G. -and Wojtas-Wasilewska M. (1985) The effect of fungal lactase on fractionated lig nosulphonates (Peritan Na). Phytochemistry 24.393-396. Malcolm R. E. and Vaughan D. (1979) Comparative effects of soil organic matter-fractions on phosphatase activities in wheat roots. Plant and Soil 51. 117-126. Mishra M. M., Flaig W. and Soechtig H. (1980) The effect of quinoid and phenolic compounds on urease and dehydrogenase activity and nitrification in soil. Plant and Soil 55, 25-33. Nelson N. (1944) A photometric adaptation of the Somogyi
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method for the determination of glucose. Journal of Biological Chemistry 153, 375-380. Paterson A. and Lundauist K. (1985) Radical breakdown of lignin. Nature 316, 575-576.. ’ Reese E. T. (1977) The structure. biosvnthesis. and degradation of wood. In Recent A&nces in Phytochemirtry. Vol. 2 (F. A. Loewus and V. C. Runeckles. Eds), pp. 31 l-367. Plenum Press. New York. Sinsabaugh R. L., Bentield E. F. and Linkins A. E. (1981) Cellulase activity associated with the decomposition of leaf titter in a woodland stream. Oikos 36, 184-190. Sulfita J. M. and Bollag J-M. (1980) Gxidative coupling activity in soil extracts. Soil Biology & Biochemistry 12, 177-183.