Journal of General Microbiology (1989), 135, 2799-2806,
Printed in Great Britain
2799
The Role of Hydrogen Peroxide in the Degradation of Crystalline Cellulose
by Basidiomycete Fungi
By R . G . VENESS A N D C H R I S T I N E S. E V A N S *
School of Biological Sciences and Environmental Health, Thames Polytechnic, Wellington St,
London SE18 6PF, UK
(Received 20 March 1989; revised 6 June 1989; accepted 7 July 1989)
Extracellular hydrogen peroxide has in the past been implicated in the degradation of the
crystalline cellulose component of plant cell walls, particularly by brown-rot fungi. Using assays
sensitive to 1 pg peroxide ml-l no evidence could be found of extracellular hydrogen peroxide in
culture media of several basidiomycete fungi examined, although some free radicals may have
been produced on the surface of the fungal hyphae. Analysis of the products of enzymic
degradation of crystalline cellulose by gas chromatography/mass spectroscopy revealed a range
of oxidized sugars which most probably resulted from the action of peroxide free radicals
derived from hydrogen peroxide.
INTRODUCTION
Coughlan (1985) proposed a multistep process for the degradation of crystalline cellulose, the
initial step being ‘amorphogenesis’, rendering the crystalline state more susceptible to the
subsequent lytic action of the enzymes endoglucanase, exocellobiohydrolase and Q-glucosidase.
However, there is disagreement about the mechanism of this first step. Mandels & Reese (1984)
and Selby (1969) believe that amorphogenesis is brought about by the synergistic action of these
different enzymes and is not a separate process.
Hyphae of brown-rot fungi grow mainly in the cell lumen next to the S3 layer of the secondary
wood cell wall and produce degradative agents which diffuse into the S2 layer, thereby causing a
diffuse rot. The pattern of a typical white rot is different in that decay troughs are observed first,
followed by a progressive decay originating in the S3 layer and advancing towards the middle
lamella (Blanchette et al., 1985). In addition, in the early stages of crystalline cellulose
degradation by brown-rot fungi, a considerable reduction in the mechanical strength of the
wood occurs with very little weight loss; by contrast, in the case of white-rot fungi, the loss in
strength is more or less proportional to cellulose loss (Cowling, 1961).
These two factors combined led to the idea that, at least in the brown-rot fungi,
amorphogenesis was a separate process from the enzymic degradation. The apparent
diffusibility of the agent causing amorphogenesis suggested that a low-molecular-mass
compound was responsible. The action of H 2 0 2and Fe(I1) on crystalline cellulose was shown to
mimic the early stages of degradation by brown-rot fungi (Halliwell, 1965). Detection of
extracellular H 2 0 2production (Koenigs, 1970, 1972) led to the proposal that a H 2 0 2and Fe(I1)
system could be responsible for amorphogenesis of cellulose by the brown-rot fungi (Koenigs,
1974). Subsequent work (Highley, 1987) has purported to confirm extracellular H 2 0 2production
by wood-rotting fungi. However, whereas Koenigs (1974) found significantly higher levels of
H 2 0 2in cultures of brown-rot fungi compared with the white-rot fungi, Highley (1987) reported
higher levels in cultures of white-rot fungi. Some of these differences can be explained by the fact
that one method used by Koenigs (1974) to measure H202,the catalase-aminotriazole-glucose
assay, was invalid (Highley, 1981).
Abbreviation : HRP, horseradish peroxidase.
0001-5483 0 1989 SGM
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R . G . VENESS AND C . S . E V A N S
Vaheri (I 982a,b) reported the occurrence of aldonic acids following enzymic degradation of
crystalline cellulose, and showed that an oxidative reaction was involved in degradation in
addition to hydrolysis.
It was against this background that we decided to re-investigate the production of H 2 0 2 by
wood-rotting fungi.
METHODS
Fungal strains. These were as follows: Coniophora puteana FPRL (Forest Products Research Laboratory,
Madison, Wisconsin, USA) IIE. Heterobasidion annosum CMI (Commonwealth Mycological Institute, Kew, UK)
68421, CMI 104888 and FPRL 41 G, Phanerochaete chrysosporium FPRL 446, Trichoderma reesei CMI 192656ii,
Serpula lacrymans FPRL 120, Lentinus lepideus FPRL 7B, Coriolus versicolor PRL (Princes Risborough
Laboratories of the Building Research Establishment, UK) IIA ; and Armillaria mellea, Gnnoderma upplanatum,
Fibroporia vaillantii, Pleurotus sp. 'Florida', Poria contigua and Trichoderma viride from the Thames Polytechnic
culture collection. All strains were maintained on malt extract agar at room temperature after growth at 25 "C
Growth media. Fungi were grown on one or more of the following media. All concentrations are given in g 1-I
unless otherwise indicated. Glucose medium : glucose 15. L-asparagine 2.5, DL-phenylalanine 0.15 , adenine 0.0275,
thiamin. HC1 50 pg I-', KH2P04 1, Na,HPO, . 2 H 2 0 0.1, MgS04.7 H 2 0 0.5, CaC1, 0.01, FeSO,. 7 H 2 0 0.01,
MnSO, .4H@ 0.001, ZnSO, . 7 H 2 00.001, CuSO,. 5 H 2 00.002; pH 5.0. C M C medium: glucose 5 , carboxymethylcellulose (CMC) (Sigma; product no. 8758) 10, all other constituents as for glucose medium. Cellulose medium:
glucose 2.5, cellulose (macerated Whatman no. 1 filter paper) 10, all other constituents as for glucose medium.
MYGPmedium:malt extract 3 , yeast extract 3, mycological peptone 5 , glucose 10. Malt extract medium: oxoid malt
extract agar CM59 made up according to the manufacturer's directions.
For the oxygen electrode assay for peroxide, fungi were grown in 50 ml standing cultures in 250 ml flasks;
cultures on glucose medium were incubated for 7 d and cultures on CMC medium for 14-28 d. The longer
incubation times on CMC medium were used because of the lower growth rates of the fungi on this medium. For
the titanium(1V) assay for peroxide, fungi were cultured for 28 d on 50 ml CMC medium. Fungi for the catalase
spot test were cultured for 7 d on 50 ml MYGP medium; those for the quantitive catalase determinations were
cultured for 14 d on 50 ml CMC medium. Cellulose medium cultures (30 ml in 250 ml flasks) were incubated for
70 d. All cultures were incubated at 25 "C.
Oxygen electrode assay for hydrogen peroxide. A Clarke oxygen electrode (Rank Brothers, Bottisham) coupled to
a chart recorder was used. The reaction mixture contained 2-0 ml 0.05 M-potassium phospate buffer pH 6.5 and
0.5 ml filtered (0.45 pm pore size) culture fluid in a total volume of 4.0 ml and was maintained at 25 "C by
circulating water from a water bath. Nitrogen gas was bubbled through the mixture to reduce the oxygen content
to a constant level. Catalase solution (100 pl), prepared by adding 50 p1 catalase suspension (from Aspergillus niger;
Sigma product no. C3515) to 10 ml of the phosphate buffer, was added to the reaction vessel and the rise in the
oxygen level signifying the decomposition of H 2 0 2was monitored on the chart recorder. After the chart recorder
had stabilized at its new level, loop1 0.03% H 2 0 2 was added to the reaction mixture to determine if any
compounds which interfered with the assay were present. Tests on the sensitivity of this method indicated that
levels of H 2 0 2in the order of 1 pg could be detected, corresponding to levels in the culture medium of 2 pg ml-'.
Titunium(ZV)assay for H 2 0 2 .This assay is normally used to detect titanium (Kolthoff & Sandell, 1952) and,
since excess H 2 0 2 reduces the amount of colouration produced, modification of the published procedure is
necessary when the method is used in the reverse mode to detect H 2 0 2 .To overcome this, tests were made using
three different concentrations (125, 31.25 and 7.8 pg ml-I) of Ti(1V). Filtered culture fluid (1.0 ml) and 100 pl of
the Ti(1V) solution was added to 1.0 m15 M-H,SO, and the absorbance measured at 410 nm using a Cecil CE343
spectrophotometer. Since many of the culture fluids were coloured, uninoculated culture medium could not be
incorporated in the blank and the baseline was adjusted to zero using the assay mixture before addition of the
Ti(1V) solution. At the conclusion of each determination, 100 pl H 2 0 2(100 pg ml-*) was added to ensure that no
compounds were present which interfered with the assay. The tests made on the sensitivity of this assay procedure
indicated that it could be used to detect H 2 0 2 at levels of 1 pg ml-l.
Fungal extracts. After washing in 10 ml 0.05 M-potassium phosphate buffer pH 7.0, 20-25 mg of fungal
mycelium, from CMC cultures, suspended in 5 ml of the same buffer, was ground with acid-washed sand in a
pestle and mortar. The resultant homogenate was filtered through a 0.45 pm Millipore filter and stored on ice until
required. Total protein in fungal extracts was determined by the Lowry method using bovine serum albumin as
standard.
Catalase assay. A spot test for catalase activity was carried out by adding one drop of 10 vol. H 2 0 2to 5-10 mg of
whole mycelium from MYGP cultures of various fungi and observing the liberation of oxygen bubbles.
Catalase activity was determined quantitatively in reaction mixtures containing 2.9 ml H 2 0 2solution (100 p1
30% H z 0 2in 50 mlO.05 M-potassium phosphate buffer pH 7.0) and 100 pl of the fungal extract. The decrease in
absorbance at 240 nm was recorded using a Perkin Elmer 402 dual-beam spectrophotometer; catalase activity was
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Hydrogen peroxide production by basidiomycetes
2801
calculated from the time (min) for the absorbance to fall from 0.450 to 0.400. Units of activity = 3*45/time(min)
(Sigma Catalogue, 1989, p. 398).
Stability of o-diunisidine peroxiduse. Solutions of horseradish peroxidase (Sigma Type 1) (1 mg ml-I) and
o-dianisidine (1 mg ml-I) were prepared separately in 0.05 M-pOtaSSiUm phosphate buffer pH 6.0 and sterilized
by Millipore filtration (0.45 pm pore size). Portions (5-0 ml) of the same buffer in capped 16 x 150 mm test tubes
were autoclaved at 1 bar for 15 min. The peroxidase and o-dianisidine solutions were then added to the tubes
aseptically to give the desired concentrations and the tubes incubated at 25 "C. After various time intervals the
contents were transferred to 1 cm cuvettes and the absorbance at 450 nm measured. Some samples were covered
with varying amounts of sterile paraffin oil to limit the amount of available oxygen.
Difusible.free radicafproduction.Silver mirrors were prepared on the interior surface of 100 ml conical flasks by
reducing the Tollens reagent (ammoniacal silver nitrate solution) with formaldehyde solution (Cheronis &
Entrikin, 1963). Cellulose medium (30 ml) was added and the flasks sterilized by autoclaving. After inoculating
with the test fungi, flasks were incubated at 25 "C and examined periodically for signs of silver mirror removal
indicating production of free radicals. Silver removal relative to the position of the hyphae was recorded.
Treatmenf of'glucose with Fenton's reagent. Glucose (0.01 mol; 1.8 g) in 50 ml 0.2 M-H2S0, was placed in a
250 nil flask on an ice bath and rapidly stirred. Ten microlitres of H 2 0 2solution [Om01 mol(O.34 g) H t 0 2 in 1.0 ml
water] and 500 1.11 of Fe(I1) solution [0.01mol (2.78 g) FeSO,. 7Hz0 in 50 ml 0.01 M-H,SO,] were added
simultaneously and the procedure repeated 100 times over a 25 min period. The temperature remained at
approximately 10 "C throughout. A solution of 0.1 M-BaOH was then added to raise the pH to 7.1 and precipitate
Fe(II1) and SO:-. The precipitate was removed by filtration (Whatman no. 1) and the filtrate concentrated at
45 "C to 22 ml under vacuum on a rotary evaporator. The final solution was dark brown in colour.
Enzymic hydrufysis oj'Auicef.Avicel PHlOl (100 mg) (Fluka Chemicals) was suspended in 10 ml 5 mM-wdium
acetate buffer, pH 4.8, and 42 pl (about 50 mg) Celluclast 1.5L, a cellulase preparation from Trichoderma reesei
(Novo enzymes) added. The mixture was incubated for 2 h at 40 "C in a shaking water bath operated at about 75
strokes min-' then flushed with nitrogen, sealed and heated to 85 "C for 15 min to denature the enzymes. Residual
Avicel was removed by centrifugation and filtration (0.45 pm pore size) and the filtrate reduced in volume to about
0-5 ml by vacuum rotary evaporation at 45 "C. Cold ethanol (2.5 ml) was added to precipitate protein, which was
removed by centrifugation. The resultant supernatant was evaporated to dryness and the residue dissolved in
500 pl of water. A control containing about 250 mg enzyme but without Avicel was subjected to the same work-up
procedure.
Preparation of irimelhyfsilyfesters. Samples (50 pl) were placed in glass vials, together with 0.2 mg meso-inositol
as internal standard and dried in a vacuum desiccator over P,05 for 16 h. Anhydrous pyridine (500p1) and 300 p1
silylation reagent were added (Sweeley er a f . , 1963) and the resultant esters dissolved in I ml n-hexane, filtered
through a 0.45 pm membrane filter, re-evaporated and finally redissolved in 100 p1 n-hexane. Glucose, xylose,
glucuronic acid lactone, gluconic acid 1,4-lactone and glucaric acid 1+lactone (Sigma) served as sugar standards.
Gas chromatogruphy (GC) and mass spectrometry (MS).GC was performed on a Varian Vista 6000
chromatograph fitted with a 25 m x 0.22 mm BP1 wall-coated open tubular capillary column and coupled to a
Varian Vista 402 printer plotter. Injection and detector temperatures were 300 "C. The initial temperature was
125 "C held for 1.5 min followed by a temperature gradient of 8 "C min-I to 280 "C, and a final hold at this
temperature for 4 min. Samples of 0 . 1 4 5 pl were applied with a sample split of 100: 1.
GC-MS was performed using a Pye series 204 gas chromatograph fitted with an all-glass solid injector and a
0.25 mm x 25 m wall-coated open tubular column with OV1 liquid phase. The injection temperature was 260 "C,
and the operating programme 1 min at 140 "C then 3 "C min-I to 280 "C and a hold at this temperature for 10 min.
The carrier gas was helium. The chromatograph was coupled to a VG 7070H mass spectrometer operated at an
electron energy of 70 eV, and the magnet was scanned every 3.5 s. A Finnigan Incos data system was used for mass
assignment and data presentation.
Mass spectra were identified from spectra prepared from standards and described in the following references :
Anonymous (1970), Curtius et al. (1968), Kochetkov et al. (1968), Petersson (1969, 1970), and Chaplin (1986).
Statistical anu1pi.s of results. The computer program 'Amstat 1' (S. C. Coleman, Ashby-de-la-Zouch,
Leicestershire, UK) was used.
RESULTS A N D DISCUSSION
Using the oxygen electrode, no peroxide was detected in culture fluids from 14 and 28 d CMC
and 7 d glucose cultures of the following fungi : Armillaria mellea, Coriolus uersicolor, Fibroporia
vaillantii, Heterobasidion annosum, Pleurotus sp. 'Florida', Phanerochaete chrysosporium, Poria
contigua, Trichoderma reesei and T. viride. On addition of H 2 0 2 ,all tests showed an increase in
oxygen levels, indicating normal functioning of the assay system.
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R . G . VENESS A N D C. S. EVANS
0.3
7
0.2
0.1
5
15
10
20
25
Time (d)
Fig. 1. Stabilityof HRP and o-dianisidine reagents. The concentrationof o-dianisidine was 20 pg ml-l.
Concentration of HRP: a, 10 pg ml-' ; A, 20 pg ml-l; A, 40 pg ml-l.
Table 1. Catalase activity in extracts of fungal mycelium cultured for 14 d on CMC medium
Catalase activity was determined by the decrease in absorbance at 240 nm of standard H 2 0 2solution;
total protein was determined by the Lowry method, using bovine serum albumin as standard.
Fungus
Brown rots
Coniophora puteana
Fibroporia uaillantii
Lentinus lepideus
Mean
Standard deviation
White rots
Coriolus uersicolor
Ganoderma applanatum
Heterobasidium annosum 41 G
Phanerochaete chrysosporium
Pleurotus sp. 'Florida'
Poria contijpa
Mean
Standard deviation
Catalase activity
[units (Llg protein)-']
0-01
0.08
0.23
0.10
0.12
1.30
<0*01
0.08
0.02
0.03
0.24
0.28
0.5 1
Similar results were obtained with culture fluids from 28 d CMC cultures of the same fungi
when the titanium assay was used, despite the high sensitivity of this method (1 pg H 2 0 2ml-l
detectable) in our hands. In all cases, a positive colour reaction was observed when 1 pg H 2 0 2
was added at the conclusion of the assay.
A third method (Highley, 1987), in which a colour reaction occurs between o-dianisidine and
H 2 0 2in the presence of horseradish peroxidase (HRP) was investigated. In the study by Highley
(1978), liquid salts medium, containing HRP and o-dianisidine, was inoculated with the test
fungus, and the cultures were incubated at 27 "C for an unspecified time. In a repeat of his
experiment, we first examined the stability of the HRP/o-dianisidine system over a 28 d period,
judged to be the most likely growth time used in the original study (Highley, 1987). Fig. 1 shows
that despite the absence of H202,increasing amounts of HRP caused an increase in ,4450 over a
period of 2-28 d. In a preliminary experiment, old stock of HRP was used and A450 values as
high as 0.78 were obtained in only 7 d. This suggests that unless the HRP is fresh, as in the case
of the experiments reported in Fig. 1, the system is even more unstable and therefore less
reliable. Addition of paraffin oil to the reaction mixture, so diminishing oxygen availability, did
not affect colour development. Hence the results of Highley (1987) were probably not affected by
a reduction in the oxygen tension due to fungal metabolism. The reported variations may have
been produced by some fungal metabolite which further destabilized an already unstable assay
system.
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Hydrogen peroxide production by basidiomycetes
Stage 2a
Stage 1
Stage 3
Fig. 2. Typical appearance of flasks in tests for free radicals using internally silvered flasks as fungal
culture vessels. The silver mirrors are indicated by hatching, and the extent of surface growth of the
fungus (in cellulose medium) is shown.
Table 2. Time required to achieve the various stages of silver removal
Fungi were grown in 100 ml conical flasks whose interior surfaces had been coated with silver mirrors.
Flasks contained 30 ml of cellulose medium and were incubated at 25 "C.The time (days) required to
effect various stages of silver removal was recorded.
Fungus
Stage 1
Stage 2a
Stage 2b
Stage 3
Coriolus versicolor
Fibroporia vaillantii
Heterobasidium annosum 4 1 G
Lentinus lepideus
Poria contigua
Serpula lacrymans
-
19
70
40
70
-, Not observed.
-
-
70
-
70
40
t
8
t
t
70
t
t
t Not reached at conclusion of experiment.
t
t
t
19
t
Koenigs (1972) measured H 2 0 2production by four methods : catalase-aminotriazole-glucose
(C-At-G), haemoglobin agar (HA), sheep's blood agar (SBA) and the reduction of the
methaemoglobin peak at 630nm in haemoglobin broth (HB). The C-At-G system was later
shown by Highley (1981) to be invalid for the detection of H 2 0 2 because of interfering
contaminants in the culture preparations. This assay is potentially susceptible to interference
from a wide range of contaminants at each stage of its procedure, and we agree that it is
unsuitable for use with fungal cultures. The correlation coefficients (r) between all combinations
of Koenigs' (1972) results, with the exception of his C-At-G data, were determined, with the
following results : HA and HL, r = - 0.1 75 ;HA and SBA, r = 0.330; HL and SBA, r = - 0.0574.
The interpretation we place on these results is that the three methods used by Koenigs (1972)
were not all measuring the same phenomenon, and that it is impossible to be sure which, if any,
of his results represents the true levels of H 2 0 2 produced.
A qualitative spot test to detect mycelium-associated catalase after 7 and 14 d growth proved
positive with all the fungi tested. When total catalase activity was measured in mycelial extracts
of fungi cultured for 14d on CMC medium, all of the fungi tested contained a measurable
amount of the enzyme (Table 1). This catalase activity may protect the fungus against the free
radicals so readily produced from H 2 0 2 .Any H 2 0 2which did not encounter catalase would be
available to carry out amorphogenesis by a free radical reaction.
This suggested that tests should be made for free radical production by the fungi. Free radicals
were detected by the desilvering of silver mirrors in the culture flasks. The typical appearance of
the flasks at various stages of this experiment are shown in Fig. 2. The times taken to achieve
these stages are shown in Table 2. At stage I there was no appreciable change in the appearance
of the silver mirror. Fungal growth was evident but in most cases had not reached the walls of the
flask. One exception was Serpulu lacrymans, where mycelial growth extended up the walls of the
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R . G . VENESS AND C. S . EVANS
Table 3. Identification and quantities qf niotiosaccharides observed in GC
Percentage levels of the monosaccharides derived from the treatment of Avicel with an enzyme
preparation from Trichodermn reesei, and of glucose treated with peroxide free radicals generated from
Fenton's reagent.
Percentage in? :
*?
-f>
Enzyme
preparation
Fenton's
preparation
Solvent impurity
Solvent impurity
Glyceric acid
Glyceraldehyde
Solvent impurity
Ketotetronic acid
0.17
0.2 1
0.5 1
0.79
0.49
?
0.1 1
Ketotetronic acid
Solvent impurity
0.54
0.85
1-31
0.60
1.96
0.28
Peak
no.
Probable
identity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
3
?
?
3
-
ND
ND
-
0.08
ND
Glucuronic acid lactone
Glucuronic acid lactone
1-Fructopyranose
0.1 1
b-Fructofuranose
0.25
3
3
Gluconic acid 1.4-lactone
Ara binopyranose
2-Ketopentonic acid
a-Glucopyranose
2-Ketopentonic acid
2-Deoxypentonic acid
2-Deoxypentonic acid
j-Glucopyranose
2-Deoxyarabinonic acid
Ribulonic acid
N- Acetylhexosamine
?
meso-lnosito1
ND
0.43
0.24
ND
0.64
0.17
1.15
10.28
2.1 1
3.43
ND
66.43
0.80
12.17
0.1 7
ND
std
-
-
0.70
0,78
2.45
1.26
ND
1.73
ND
0.88
ND
1.09
2.32
35.68
1.08
2.23
I .27
0.4 1
0.65
40.69
ND
0.48
std
= unidentified compound.
t -,solvent impurity, percentage not included: ND. not detected. std, ntesu-inositol added as internal standard,
percentage not included.
flask without affecting the mirror. Stage 2a occurred some days after the mycelium reached the
flask walls, and a narrow band of silver (less than 5 mm wide) was removed immediately below
the surface of the medium. Stage 26 saw a widening of this band together with the removal of
silver from the centre of the flask base as the mycelial mat expanded. Stage 3 was characterized
by extensive colonization and the complete removal of all silver below the surface of the growth
medium.
The silver mirror test indicated that free radical production occurred but was only operative
over very short distances since removal of the silver mirrors was only apparent when the
mycelium was in direct contact. The free radicals detected need not necessarily have been from
H 2 0 2 ,as a number of enzymic processes are thought to proceed by a free radical mechanism.
Since the silver mirror test indicated that free radicals were produced by fungal cultures, free
radical involvement in the degradation of Avicel was examined by comparing the oxidation
products after enzymic hydrolysis with the products obtained following treatment of glucose
with Fenton's reagent [H20,/Fe(Il)]. Table 3 shows the remarkable similarity of the
monosaccharides produced by the treatment of glucose with the free radicals generated from
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Hydrogen peroxide production by basidiornjJcete.7
Fenton's reagent, and those produced from the treatment of Avicel with an enzyme preparation
from Trichoderma reesei.
The correlation between the retention times of clearly identifiable peaks on the GC and GCMS was better than 0-99,allowing us to quantify and identify the various components. A number
of the peaks were assigned the same probable identity. This can be accounted for as follows :
ketotetronic acids (peaks 6 and lo), 2-ketopentonic acids (peaks 22 and 24) and 2-deoxypentonic
acids (peaks 25 and 26) are all most probably erythro and threo forms of these acids; the
glucuronic acid lactone (peaks 14 and 15) correspond to the cc and p anomers.
Major differences can be accounted for as follows. The p anomers of sugars are generally
oxidized more rapidly than the a anomers (Green, 1980). When treated with Fenton's reagent,
glucose first forms glucosone. The concentration of Fenton's reagent determines how glucosone
is further oxidized (Green, 1980): in concentrated solutions the major product is ribulonic acid,
whilst in dilute solutions an alternative degradative pathway operates, yielding fructosonic acid
and 2-ketopentonic acid as two early products. Hence, a high level of ribulonic acid together with
a low level of /.?-glucosewould be expected in the glucose solutions treated with Fenton's reagent.
The detection of a compound tentatively identified as an N-acetylhexosamine in the enzymetreated preparations suggests that some cell wall material, or a degradation product, is present in
the crude enzyme preparation.
The presence of both pyranose and furanose forms of fructose in the enzyme-treated
preparations and their apparent absence from the Fenton's-treated glucose suggests that a
glucose isomerase is also present in the enzyme preparation. The degradative pathway resulting
from treatment with Fenton's reagent would not account for the presence of these sugars.
The generally lower levels of more extensively degraded sugars in the enzymic preparation
are consistent with the production of lower levels of free radicals. Since most of the
monosaccharides found in the enzyme-treated preparation were also found in glucose samples
treated with peroxide-derived free radicals, and since many of these monosaccharides would
result from a simple degradation scheme involving such free radicals, this strongly indicates that
peroxide-derived free radicals participated in the degradation of Avicel.
In order for Fenton's reagent to operate in the degradation of crystalline cellulose, the iron
must be present as Fe(I1). Normally, iron is found as Fe(III), and any available Fe(I1) reacting
with H 2 0 2would be converted to Fe(II1) and therefore require regeneration in order for
production of peroxide-derived free radicals to continue. Green (1980) states that Fe(II1) reacts
with H 2 0 2to produce the hydroperoxyl radical and regenerate Fe(I1) according to the following
equation :
+
Fe3+ HOOH
-, Fe2++ H+ + 'OOH
The hydroperoxyl radical reacts with aldonic acids to produce the aldose sugar with one less
carbon and regenerates hydrogen peroxide (Green, 1980):
R--CH(OH)-COOH
+ 'OOH
-+
R-CHO
+ Cot + HOOH i- 'H
The hydrogen free radical produced would be available to abstract hydroxyl groups from sugars,
thus accounting for the formation of the deoxy sugars:
HOOC-CH(0HkR
+ 'H + HOOH
--*
HOOC-CH2-R
+ 'OOH
*
We believe that, whilst previous workers have attempted to measure and/or demonstrate the
presence of H 2 0 2 in fungal culture fluids, none have done so conclusively. Our data show that
H 2 0 2does not accumulate to detectable levels in culture fluids. We believe that, at least in the
case of Trichodermareesei, H 2 0 2is produced, but that combined consumption by the free radical
oxidation process and by catalase activity results in concentrations below the limits of sensitivity
of the assay system used. It is probable, based on the results of silver mirror tests, that peroxide is
formed by some of the fungi listed in Table 2.
The similarity in the types of oxidized sugars in an enzymically digested sample of cellulose
and in glucose solutions treated with peroxide-derived free radicals constitutes, we believe, the
best evidence yet that H 2 0 2 is involved in the degradation of crystalline cellulose.
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R . G . VENESS A N D C . S . E V A N S
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