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Optimization of extraction of bulk enzymes

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ARTICLE in JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY JULY 2003
Impact Factor: 2.49 DOI: 10.1002/jctb.852

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Vikineswary Sabaratnam
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Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 78:743752 (online: 2003)

DOI: 10.1002/jctb.852

Optimization of extraction of bulk enzymes

from spent mushroom compost
Avneesh D Singh,1 Noorlidah Abdullah2 and S Vikineswary2
1 Institute
2 Institute

of Post-Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia

of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract: The profiling of ligninase, hemicellulase and cellulase of Pleurotus sajor-caju after inoculation
of spawn in bags containing sawdust was done at monthly intervals for a period of 6 months. Xylanase (EC
3.2.1.8) was produced throughout the 6 months studied with the productivity range from 5.60 to 7.51 U g1 .
Cellulase (EC 3.2.1.4) and -glucosidase (EC 3.2.1.21) productivities were highest at 4 months, producing
3.31 U g1 and 121.13 U g1 respectively. Laccase (EC 1.10.3.2) productivity was highest at 2 months with a
value of 7.59 U g1 . Lignin peroxidase (EC 1.11.1.14) productivity was highest at 5 months with a value of
206.20 U g1 . Total soluble proteins were highest at 4 months with a value of 0.139 mg cm3 . The profiling
of lignin peroxidase in 5-month-old spent mushroom compost was monitored over a period of 10 months.
It was observed that lignin peroxidase was produced throughout the period but productivity was variable.
The average lignin peroxidase productivity ranged from 30 to 110 U g1 . The activities of the enzymes
extracted in tap water at pH 8.4 were comparable to that extracted in 50 mmol sodium citrate buffer at
pH 4.8 and distilled water at pH 5.2 at 4 C using an incubator shaker at 200 rpm for 18 h. The optimum
extraction time was 1 h using an incubator shaker at 4 C. When an incubator shaker was used, there
was no significant difference in the recovery of xylanase, cellulase and laccase at different pH values at
4 C and 28 C. No significant difference was observed in the recovery of -glucosidase using an incubator
shaker at different pH values at 4 C although the enzyme recovery was slightly higher at pH 8.12, with a
value of 29.27 U g1 . The optimum extraction of -glucosidase was at pH 4 at room temperature using an
incubator shaker. For the lignin peroxidase enzyme, the optimum pH for extraction was 6 at 4 C and pH 7
at room temperature using an incubator shaker at 200 rpm for 1 h. Homogenization for 8 min at 8000 rpm
using tap water at pH 4 had an advantage over the use of the incubator shaker for the extraction as high
titers of enzymes were recovered.
2003 Society of Chemical Industry

Keywords: enzyme extraction; lignin-degrading enzymes; xylanase; cellulase; Pleurotus sajor-caju; spent
mushroom compost

1 INTRODUCTION
Mushrooms are basidiomycetes, which are unique
in possessing the growth form and the enzymatic
capability to fully degrade the plant polymers lignin,
hemicellulose and cellulose into low molecular weight
soluble compounds.1 These soluble compounds are
then absorbed by the fungal hyphae by a process
called nutritive absorption.2 The three major groups
of enzymes involved in the breakdown of lignin and
cellulosic agro-wastes are cellulases, hemicellulases
and ligninases.
On a commercial scale the extracellular enzymes
produced by the basidiomycetes find uses in the brewing, baking, dairy, starch processing, leather and textile
industries, and also in waste treatment.3 The use
of enzymes in waste treatment includes the decolorization of olive mill waste waters,4 decolorization

of an artificial textile effluent,5 polymerization of

pentachlorophenol,6,7 degradation of azo, triphenyl
and heterocyclic dyes,8,9 and upgrading corn straw for
paper pulping.10
Mushrooms utilize a variety of lignocellulosic
residues and convert them into human food.11 Mushroom cultivation represents one of the efficient ways
by which these residues can be recycled.11 Pleurotus
sajor-caju (Berk and Br) Sacc is a basidiomycete fungus cultured on a commercial scale for its spatulate,
edible fruiting bodies which have a pleasant flavor
and a characteristic biting texture.12 This fungus has
been reported to grow on a number of lignocellulosic
wastes including Morus alba and Ricinus communis
stems and leaves and also on paddy straw,11 rice straw
supplemented with oil seed cakes,12 sago hampas13
and oil palm frond parenchyma tissue (OPFPT)

Correspondence to: S Vikineswary, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
E-mail: viki@um.edu.my
Contract/grant sponsor: Ministry of Science, Technology and Environment, Malaysia; contract/grant number: 09-02-03-0675
Contract/grant sponsor: Ministry of Science, Technology and Environment, Malaysia; contract/grant number: 01-02-03-1002
(Received 10 June 2002; revised version received 21 January 2003; accepted 12 February 2003)

2003 Society of Chemical Industry. J Chem Technol Biotechnol 02682575/2003/$30.00

743

AD Singh, N Abdullah, S Vikineswary

supplemented with palm oil sludge solids (POSS).14

In Malaysia, on a commercial scale, Ple sajor-caju is
grown on rubberwood sawdust supplemented with
rice bran and calcium carbonate in polyethylene bags.
Spent mushroom compost is made up of fungal
mycelium, extracellular enzymes produced by mushrooms during growth, and unutilized lignocellulosic
substrate used for mushroom production. For every
200 g of Pleurotus spp produced in Malaysia, about
600 g of spent compost is available. An average farm
discards about 24 tons per month of spent mushroom compost. The disposal of spent mushroom
compost is a major problem for mushroom farmers. This spent mushroom compost is either discretely
burnt or discarded.15 This residue can be used to
produce value-added products. The production of
biogas,16 the production of bulk enzymes,13 bioconversion to organic fertilizer,17 use as an animal feed
supplement,18 and degradation of PCP19,20 are among
the potential uses or products available from spent
mushroom compost.
The objectives of the study were (a) to profile
xylanase, cellulase, -D-glucosidase, laccase and lignin
peroxidase produced by Ple sajor-caju during a spawn
run after inoculation of koji in bags of sawdust, (b)
to monitor lignin peroxidase levels in 5-month-old
bags over a 10-month period and (c) to maximize the
extraction of these enzymes from the spent compost.

2 MATERIALS AND METHODS

2.1 Collection and storage of spent mushroom
compost (SMC)
Ple sajor-caju bags were obtained from a farm in
Semenyih, Selangor from the time of inoculation of
the bags of sawdust for a period of 6 months. The
bags were processed immediately or after storage in a
deep freezer at a temperature of 20 C for 1 week.
There was no loss in the enzyme activities after storage
(unpublished data).
2.2 Preparation of substrate
Six bags of Ple sajor-caju spent compost were opened
and the contents of the bags were placed in a plastic
basin. The contents were manually broken up and
mixed thoroughly. Four samples were taken from this
mixture for the extraction and analysis of enzymes.
2.3 Enzyme profiling
The profiling of the enzymes xylanase (EC 3.2.1.8),
cellulase (EC 3.2.1.4), -D-glucosidase (EC 3.2.1.21),
laccase (EC 1.10.3.2), lignin peroxidase (EC
1.11.1.14) and total soluble protein of Ple sajorcaju spent compost was done at monthly intervals
for 6 months from the time of inoculation. Enzymes
present in the spent compost were extracted using
50 mmol sodium citrate buffer at pH 4.8 in an incubator shaker at a speed of 200 rpm for 18 h at 4 C
based on the method of Kumaran.21 The supernatant
744

obtained contained the fungal enzymes and was stored

in 1.5 cm3 microcentrifuge tubes at 20 C for 24 h
prior to enzyme assays.
In the preliminary profiling studies, high titers of
lignin peroxidase were observed. As lignin peroxidase
has not been reported previously in spent mushroom
compost of Ple sajor-caju, a 10-month study was done
to monitor lignin peroxidase levels. Six bags (fivemonths old) were randomly collected each month
for a period of 10 months from the same mushroom
farm. The enzyme was extracted using an homogenizer
and analyzed.
2.4 Optimization of extraction of extracellular
enzymes from SMC
The optimization of extraction of the extracellular
enzymes was done using 5-month-old Ple sajor-caju
bags. The initial method used for extraction was
based on the method of Kumaran.21 Variations of
the extraction conditions were made from this initial
method. The extraction was optimized for xylanase,
cellulase, -glucosidase, laccase and lignin peroxidase.
The extraction of enzymes in an incubator shaker and
by homogenizing using an homogenizer was optimized
by modifying various conditions.
2.4.1 Optimization of extraction of extracellular
enzymes using an incubator shaker
2.4.1.1 Effect of type of extraction medium on the
recovery of enzymes. The effect of various parameters
such as incubation conditions, type and pH of
extraction medium, extraction time and temperature
on the recovery of enzymes from SMC was studied.
Five-month-old Ple sajor-caju spent compost was
extracted with three different media: tap water (pH
8.45), distilled water (pH 5.25) and 50 mmol sodium
citrate buffer (pH 4). The tap water used was drinking
quality as supplied by the local council. The substrate
was prepared as described above and 10 g of spent
compost was taken and mixed with 100 cm3 each
of the extraction medium separately in 250 cm3
Erlenmeyer flasks. All the flasks were placed in an
incubator shaker at 200 rpm for 18 h at 4 C. The
samples were then centrifuged at 15 000 g and the
supernatant as the crude enzyme extract was assayed.
For each extraction medium studied, four replicate
samples were extracted and analyzed.
2.4.1.2 Effect of time of incubation on the recovery of
enzymes. The substrate was prepared as described
above and 10 g of spent compost was taken and
mixed with 100 cm3 of tap water to form a slurry.
This slurry was incubated in a shaker at 200 rpm for
different intervals of time of 1, 3, 5, 7 and 18 h at
4 C. For each time studied, four replicate samples
were extracted and analyzed.
2.4.1.3 Effect of pH of extraction medium and
incubation temperature on the recovery of enzymes. The
substrate was prepared as described above and 10 g
J Chem Technol Biotechnol 78:743752 (online: 2003)

Bulk enzymes from mushroom compost

of spent compost was taken and mixed with 100 cm3

of tap water in 250 cm3 Erlenmeyer flasks. The pH
in the different flasks was adjusted with 1 mol dm3
HCl or 1 mol dm3 NaOH to pH 4, 5, 6 and 7. The
control extraction medium was tap water at pH 8.12.
All the flasks were incubated in an incubator shaker at
200 rpm at 4 C or at 28 2 C (room temperature)
for 1 h. After 1 h, the samples were centrifuged as
described earlier and the supernatant as crude extract
was analyzed for enzymes. For each pH studied at 4 C
or at 28 2 C, four replicate samples were taken for
enzyme extraction.
2.4.2 Optimization of extraction of extracellular
enzymes using an homogenizer
2.4.2.1 Effect of pH of extraction medium on the recovery
of enzymes. The effect of varying pH of the extraction
medium was studied. The extraction medium was
tap water and the pH adjustment was made using
1 mol dm3 HCl or 1 mol dm3 NaOH to pH 4, 5, 6,
7 and 8. Tap water at pH 8.6 was used as a control.
The substrate was prepared as described above and
10 g of spent compost was taken. Homogenization
with an IKA Ultra-Turrax T25 was done at 8000 rpm
for 10 min. After homogenization, the samples were
centrifuged to separate the solids and the filtrate was
used as crude extract for enzyme analysis. For each
pH studied, four replicate samples were taken for
enzyme extraction.
2.4.2.2 Effect of time of homogenization on the recovery
of enzymes. The substrate was prepared as described
above and 10 g of spent compost was taken and mixed
with 100 cm3 tap water at pH 4. Homogenization was
done at different time intervals of 4, 8, 10, 12, 16
and 20 min at 8000 rpm. After homogenization, the
samples were centrifuged and analyzed. For each time
interval studied, four replicate samples were taken for
enzyme extraction.
2.4.2.3 Effect of speed of homogenization on the recovery
of enzymes. The substrate was prepared as described
above and 10 g of spent compost was taken and mixed
with 100 cm3 tap water at pH 4. Homogenization
was done at different speeds of 8000, 9500, 13 500,
20 500 and 24 000 rpm for 8 min. For each speed
studied, four replicate samples were taken for enzyme
extraction and analysis.
2.5 Enzyme assays
Laccase activity was determined by the increase in
the absorbance due to the formation of tetramethoxyazo-bis-methylenequinine resulting from the reaction
of laccase with syringaldazine.22,23 Syringaldazine
(0.1 mmol in 50% ethanol (w/v)) was used as the
substrate. One unit of enzyme activity was defined
as the amount of enzyme producing one unit
change in absorbance min1 g1 of the substrate.
Lignin peroxidase activity was measured by recording
the increase in absorbance at 310 nm due to the
J Chem Technol Biotechnol 78:743752 (online: 2003)

oxidation of veratryl alcohol to veratraldehyde by

lignin peroxidase.24 The reaction mixture consisted
of lignin peroxidase, 100 mmol sodium tartrate
buffer (pH 3.0) and 2 mmol veratryl alcohol as
substrate. The reaction was initiated by the addition
of H2 O2 at a final concentration of 0.5 mmol.
The standard used was 3,4-dimethoxybenzaldehyde
(veratraldehyde). Xylanase activity was measured
using a 1% (w/v) suspension of xylan from oat
spelts in 50 mmol sodium citrate buffer pH 4.8.25
Xylose was used as the standard. Carboxymethyl
cellulase (CMCase) activity was estimated by using
1% carboxymethyl cellulose (medium viscosity) as
the substrate.26 The liberated reducing sugars were
determined using the dinitrosalicylic (DNS) acid
reagent method as explained by Miller.27 The
standard used was glucose. -D-glucosidase activity
was determined using 0.5 mmol p-nitrophenyl--Dglucopyranoside in 50 mmol sodium citrate buffer,
pH 4.8, as the substrate.26 The standard was pnitrophenol. The enzyme activities were expressed as
international units (U) and defined as the amount of
enzyme required to produce 1 mol product min1 and
productivity was reported as U g1 of the substrate.
The extracellular soluble protein was quantified
using the Bradford28 dye-binding method with
crystalline BSA as standard. The reducing sugar
content was determined using the DNS acid reagent
method of Miller.27

3 RESULTS AND DISCUSSION

3.1 Enzyme profiling
Like other basidiomycetes Pleurotus sajor-caju exhibits
two main phases during its life cycle. The first phase
is the complete colonization of the substrate followed
by a second phase of fruiting. In the local mushroom
farms, Ple sajor caju is cultivated in plastic bags and
it takes about 2 months for complete colonization of
the substrate by its mycelium. After colonization, Ple
sajor caju starts the fruiting phase which lasts for
23 days followed by a rest period of 1015 days
before the second cycle of fruiting starts. A total of
four to five cycles of fruiting are exhibited by Ple sajor
caju before the bags are finally discarded at 6 months.
During the different phases of the life cycle, Ple sajor
caju has different patterns of production of hydrolytic
and oxidizing enzymes. To better understand the
hydrolytic and oxidizing enzymes of Ple sajor caju,
the enzyme profiling during the growth of Ple sajorcaju in sawdust was performed. The profiles showed
a different pattern at different months for each of the
enzymes studied. (Table 1).
The productivity of enzymes analyzed range
from 5.60 to 7.51 U g1 of SMC for xylanase,
0.853.31 U g1 of SMC for celluase, 58.35121.13
U g1 of SMC for -glucosidase, 1.227.59 U g1 of
SMC for laccase and 92.85206.20 U g1 of SMC
for lignin peroxidase. The productivity of xylanase
ranges from 5.60 to 6.20 U g1 of SMC for the
745

AD Singh, N Abdullah, S Vikineswary

Table 1. Productivity of hemicellulolytic, cellulolytic and ligninolytic enzymes of Pleurotus sajor-caju from 1 month to 6 months after inoculation into
sterilized sawdust bags supplemented with 35% (w/v) rice bran and 1 % (w/v) calcium carbonate (the bags were inoculated with 2 weeks-old koji
of Ple sajor-caju grown in autoclaved wheat grains; the results are mean of four replicate samples)

Time interval in months (enzyme activities are reported as U g1 of SMC)

Enzyme
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

6.14 0.10
1.27 0.12
58.35 5.14
3.42 0.50
112.03 36.2

5.60 0.09
1.98 0.11
83.2 4.73
7.59 0.88
169.70 41.2

6.20 0.14
1.75 0.03
89.73 5.31
2.64 0.11
162.00 14.8

7.51 0.12
3.31 0.06
121.13 4.19
1.58 0.05
92.85 22

5.81 0.14
0.85 0.05
27.7 2.19
1.22 0.04
206.20 20.4

6.78 0.03
2.31 0.09
57.84 3.57
1.62 0.10
154.30 40.1

first 3 months. At the fourth month a productivity

of 7.51 U g1 SMC was recorded followed by a
decrease in the productivity by the fifth month
(Table 1). At 6 months the enzyme productivity again
increased to 6.78 U g1 of SMC. Further, an overall
high productivity was observed for xylanases when
compared with cellulases over the 6 months after
inoculation. Xylanase productivity of 10.1 U g1 by
Ple sajor-caju grown on sago hampas on day 9 has
been reported.14 This value however, dropped to
5.0 U g1 hampas by day 5 and finally reduced to
4.0 U g1 towards the end of fermentation period of
21 days.21 In another study, Ple sajor-caju, when grown
on OPFPT supplemented with POSS, produced only
2.85 U g1 of xylanase at day 30 of fermentation.14
However, high xylanase productivity of 6.6 U g1 on
day 5 was observed when Ple sajor-caju was grown
on sago hampas. A similar xylanase productivity was
also obtained in our study. The increased levels of
xylanases as compared with cellulases in our study can
be explained on the basis of structural difference as
the xylan is more complex than the cellulose and may
require a large number of different enzymes to elicit
efficient hydrolysis. Further, the polysaccharide does
not form a tightly packed structure and thus is more
accessible to hydrolytic enzymes.29
The highest cellulase productivity of 3.31 U g1 of
SMC was obtained at the fourth month, which was
the fruiting phase of the fungus (Table 1). For the
other months studied, cellulase productivity ranged
from 0.85 to 2.31 U g1 of SMC. A similar CMCase
productivity of 2.85 U g1 on day 7 from the time
of inoculation was reported when Ple sajor-caju was
grown on sago hampas.21 However an increased
cellulase productivity of 4.38 U g1 was obtained on
day 10 when Ple sajor-caju was grown on OPFPT
supplemented with urea.14 A cellulase productivity of
2.91 U g1 , similar to the cellulase productivity in our
study, was also obtained when Ple sajor-caju was grown
on OPFPT supplemented with POSS.14
The highest -glucosidase productivity of 121.13 U
g1 of SMC was also observed at the fourth month
(Table 1). The profiling of -glucosidase showed that
the productivity at the first month was 58.35 U g1 of
SMC, which gradually increased till the fourth month
followed by a rapid decrease at the fifth month. A
very high productivity of -glucosidase was observed
746

in our study as compared with the productivity of

this enzyme by the same fungus, Ple sajor-caju, grown
on sago hampas (0.040.27 U g1 )21 and grown on
OPFPT supplemented with POSS (0.23 U g1 )14 or
urea (0.35 U g1 ).14
The highest laccase productivity of 7.59 U g1
of SMC was obtained after 2 months followed by
a rapid decrease in the productivity (Table 1). A
high laccase productivity of 10.6 U g1 on day
9 after inoculation was reported in Ple sajor-caju
grown on sago hampas.21 This productivity remained
constant until day 9 and declined on day 17 with
the onset of primordia formation. A similar high
laccase productivity of 10.53 U g1 and 16.93 U g1
on day 10 was obtained when Ple sajor-caju was
grown on OPFPT supplemented with POSS and urea
respectively for a fermentation period of 30 days.14
The highest lignin peroxidase productivity of
206.20 U g1 was observed at the fifth month
(Table 1). This is the first report on the production
of lignin peroxidase by Ple sajor-caju grown on
rubberwood sawdust. Darah and Ibrahim30 had
reported that in Phanerochaete chrysosporium, the
lignin-degrading enzyme system was expressed during
the secondary metabolic phase of growth, the onset
of which is triggered by the limitation of carbon and
nitrogen sources. They had also suggested the role of
veratryl alcohol in the production of lignin peroxidase
by Ph chrysosporium. When the mycelial growth ceased
as a result of the depletion of one of the carbon or
nitrogen sources, Ph chrysosporium secreted secondary
metabolites, mainly veratryl alcohol, which acted as
a substrate as well as an inducer for the production
of ligninolytic enzymes. A similar mechanism may
be present in our study resulting in the production
of lignin peroxidase. However, Shi et al 31 reported
that Ple sajor-caju grown in a liquid medium with
glucose as the sole carbon and energy source produced
manganese peroxidase and laccase only while no
lignin peroxidase activity was detected. However,
Shanmugam and Yadav32 reported the production
of lignin peroxidase by Ple sajor-caju when grown in a
liquid medium containing coir dust.
In Malaysia, Ple sajor-caju bags were usually
discarded after 6 months. However, from 5 months
onwards, the yield of fruit bodies was low. Further,
from this study, it was shown that high titers of lignin
J Chem Technol Biotechnol 78:743752 (online: 2003)

Bulk enzymes from mushroom compost

peroxidase and significant levels of xylanase, cellulase

and -glucosidase were present in 5-month old bags.
Thus, 5-month old bags were selected for further
experiments to optimize the extraction of enzymes.
During the profiling of lignin peroxidase over a
period of 10 months, it was observed that lignin
peroxidase was produced throughout the period
studied but productivity was variable (Table 2). The
lignin peroxidase productivity ranged from 30 to
110 U g1 over the 10-month period. The highest
and lowest enzyme productivities observed were
179.36 U g1 and 1.54 U g1 respectively (Table 2).
This wide variation in the enzyme productivity could
be due to the farm practices which included a
difference in the substrate preparation, especially
the nitrogen levels in the mushroom bags; uneven
inoculum size from bag to bag; variation in the initial
moisture content from one batch to another; difference
in the amount of substrate per bag and possibly slow
growth of the fungus in certain bags. Mushroom
growing in Malaysia remains a cottage industry, even
in large farms and thus there is variability not only
in enzyme yields but also in fruit body yields (Kuan,
personal communications).
Further, a wide variation in the xylanase, cellulase
and -glucosidase productivity by Ple sajor-caju was
observed during the profiling of SMC from the
mushroom farm when compared with that reported
by Ling14 and Kumaran.21 This variability may be
Table 2. Productivity of lignin peroxidase of Pleurotus sajor-caju from
the samples collected from the local mushroom farm over a period of
10 months (the results are mean of six replicate samples)

Enzyme productivities (U g1 of SMC)

Average
productivity

Highest
productivity

Lowest
productivity

30.58 11.03
47.43 61.10
57.29 36.06
81.80 63.52
90.93 38.27
106.47 40.29
110.47 22.82
64.66 33.97
74.97 39.43
82.08 30.48

45.75
133.22
122.65
179.36
131.30
167.82
130.34
109.19
110.15
106.31

20.76
0.0
14.03
1.54
33.26
57.29
72.67
24.61
49.60
23.65

Month
June
July
August
September
October
November
December
January
February
March

attributed to the different substrates used as well as

the farm practices described earlier. Moreover, in the
studies done by Ling14 and Kumaran21 the results were
based on the experiments conducted on a laboratory
scale using 10 g of substrate in Erlenmeyer flasks for
a period of 2130 days under controlled parameters.
However, our study was based on the farm conditions
where 900 g of substrate made up of rubberwood
sawdust, 510% rice bran and calcium carbonate was
used in each bag. The complete colonization of the
substrate took 11.5 months, thus resulting in the
variation of the enzyme production.
3.2 Optimization of extraction of extracellular
enzymes from SMC using an incubator shaker
3.2.1 Effect of type of extraction medium on the recovery
of enzymes
There was no difference observed in the enzyme
recovery of xylanase and cellulase when the samples
were extracted with the three different extraction
media (Table 3). Sodium citrate buffer (50 mmol, pH
4.8) was the best medium for the extraction of glucosidase with a high recovery of 27.39 U g1 of
SMC in an incubator shaker, which was 41.7% higher
when compared with the other extraction media.
Further, the recovery of -glucosidase extracted
with distilled water or tap water was almost similar
(Table 3). Our results are in agreement with those
of Bisaria et al.33 They reported the successful
extraction of cellulases, xylanase and -glucosidase
using 50 mmol citrate buffer, pH 4.8, from cultures of
Ple sajor-caju grown on rice and wheat straw. Prasertsan
and34 however, extracted significant amounts of
cellulases, xylanase and -glucosidase by adding a
five-fold volume of distilled water to the cultures of
Myceliophthora thermophila IFO 31843 and Aspergillus
niger ATCC 6275 grown on palm cake and palm fiber.
Sodium citrate buffer (50 mmol pH 4.8) was
also the best medium for the extraction of laccase
with a recovery of 1.99 U g1 . The recovery of
laccase with tap water or distilled water was almost
similar (Table 3). Ganisan et al 35 reported a laccase
recovery of 13.67 U mg1 protein from the cultures of
Lentinula edodes grown on sawdust for 2 weeks using
0.15 mol dm3 sodium acetate buffer, pH 4.5, and
homogenizing it for 12 min in an ice-bath. However,
Matcham and Wood36 reported the recovery of laccase

Table 3. Effect of different extraction media on enzyme recovery during incubation in a shaker at 200 rpm for 18 h at 4 C (the results are mean of
four replicate samples)

Enzyme activities (U g1 of SMC)

Enzyme

50 mmol sodium
citrate (pH 4.8)

Tap water
(pH 8.4)

Distilled
water (pH 5.2)

Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

6.42 0.09
1.71 0.10
27.39 3.23
1.99 0.08
259.13 41.48

5.99 0.09
1.82 0.09
15.96 1.15
0.70 0.15
301.91 45.28

6.01 0.06
1.94 0.04
14.89 0.85
0.72 0.03
352.37 66.98

J Chem Technol Biotechnol 78:743752 (online: 2003)

747

AD Singh, N Abdullah, S Vikineswary

from the mushroom compost of Agaricus bisporus using

chilled deionized water. In another report, Dakshini37
reported laccase recovery of 20.75 U g1 on sago
hampas fermented by Ple. sajor-caju with tap water
at pH 5 as a medium in an incubator shaker at
200 rpm for 18 h at 4 C. Distilled water was the
best extraction medium for lignin peroxidase with
recovery of 352.37 U g1 of SMC. However, the
recovery 301.91 U g1 of SMC of lignin peroxidase
with tap water was also comparable to that of
distilled water (Table 3). The results showed that
all the enzymes could be recovered in comparable
amounts using different extraction media. Hence, tap
water was selected as an extraction medium for the
further studies.

3.2.2 Effect of time of incubation on the recovery of

enzymes
There was no difference in the recovery of xylanase or
cellulase enzymes at the different time intervals studied
(Table 4). The recovery of -glucosidase at 18 h was
approximately 1733% higher as compared with the
other time intervals when an incubator shaker was used
(Table 4). The laccase recovery of 0.44 U g1 of SMC
was optimum after 1 h when an incubator shaker was
used (Table 4). However, very low levels of enzyme
recoveries were generally observed at the time intervals
studied. The optimum time interval required for the
lignin peroxidase recovery of 73.15 U g1 of SMC
was 3 h when an incubator shaker was used. This was
1654% higher than the enzyme recovered at other
time intervals. However, lignin peroxidase recovery of
61.13 U g1 of SMC at the 1 h time interval was also
very close to the highest enzyme activity (Table 4). It
was observed that after an interval of 3 h, the recovery
of lignin peroxidase decreased from 73.15 U g1 to
33.26 U g1 (Table 4). One possible reason may be
due to the reabsorption of the enzyme by the fungal
mycelium over the long period of time due to an
osmotic gradient. Further investigation is required
to prove this. The results of this study showed that
high enzyme yields were obtained at the 1 h interval
when compared with the 18 h incubation. Thus the
extraction time can be reduced to 1 h, making the
extraction of the enzymes possible in only 1 day as
compared with the initial method used.

3.2.3 Effect of pH of extraction medium and incubation

temperature on the recovery of enzymes
There was an overall decrease in the enzyme recoveries
at room temperature when compared with that of
4 C, suggesting the denaturation of enzymes at
room temperature (Table 5). There was no difference
observed in the recovery of xylanase, cellulase and
laccase at all the pH values studied at 4 C and at
room temperature. An overall low recovery of laccase
was again observed (Table 5). The -glucosidase
recovery of 29.27 U g1 of SMC was 1136% higher
at pH 8.1 at 4 C when compared with other pH
values using an incubator shaker (Table 5). When
the extraction of -glucosidase enzyme was carried
out at room temperature and at different pH values
using an incubator shaker, the -glucosidase recovery
of 2.15 U g1 of SMC was observed only at pH
4 and this activity was 88% less when compared
with the enzyme activity at the same pH at 4 C
(Table 5). At room temperature, the enzyme probably
undergoes denaturation over the period of 1 h required
for extraction. The optimum pH for lignin peroxidase
recovery using the incubator shaker at 4 C was from
pH 6 to 8 and the enzyme activities range from
78.91 to 91.41 U g1 of SMC (Table 5). However,
at room temperature, the optimum pH was 7 with
a recovery of 55.36 U g1 of SMC. The recovery
of lignin peroxidase in an acidic and alkaline pH
decreased when compared with the optimum pH of 7
using an incubator shaker and ranged from 18.84 to
38.54 U g1 of SMC. A possible reason could be
that at room temperature, changes in pH caused
irreversible denaturation of lignin peroxidase over a
period of 1 h.
3.3 Optimization of extraction of extracellular
enzymes from SMC using an homogenizer
3.3.1 Effect of pH of extraction medium on the recovery
of enzymes
There was no marked effect of pH on the -glucosidase
enzyme recovery using the homogenizer. The enzyme
was recovered in significant quantities over the pH
range tested, with activities ranging from 100.36
to 107.58 U g1 of SMC (Table 6), thus exhibiting
higher activities recovered compared with an incubator
shaker. No difference was observed in the recovery of
xylanase, cellulase and laccase at different pH values
of the extraction medium (Table 6). The extraction

Table 4. Effect of time on enzyme recovery with tap water at pH 8.4 during incubation in a shaker at 200 rpm at 4 C (the results are mean of four
replicate samples)

Enzyme activities (U g1 of SMC) at different time intervals (in h)

Enzyme
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

748

6.46 0.12
1.73 0.09
11.00 1.31
0.44 0.18
61.13 28.19

6.43 0.03
1.81 0.11
11.38 0.94
0.32 0.04
73.15 21.20

6.33 0.08
1.87 0.08
13.70 0.43
0.39 0.04
33.26 7.85

6.54 0.09
2.15 0.06
11.57 2.84
0.21 0.05
39.99 27.66

18
6.28 0.06
2.11 0.15
16.65 0.96
0.30 0.05
36.62 65.078

J Chem Technol Biotechnol 78:743752 (online: 2003)

5.11 0.10
1.14 0.09
a
0.11 0.04
55.36 42.4

5.13 0.11
1.08 0.07
a
0.07 0.02
18.84 23.6

of lignin peroxidase was optimum at pH 4 with

the recovery of 150.52 U g1 of SMC (Table 6).
Darah et al 38 have reported the extraction of lignin
peroxidase from Pha chrysosporium grown on rice husks
by homogenizing in 0.2 mol dm3 sodium acetate
buffer pH 5. Forrester et al 39 reported the extraction
of manganese peroxidase from Lentinula edodes by
suspending the cultures in water acidified to pH 4 with
HCl and stirring for 2 h at 5 C. Hence, pH 4 was used
in all subsequent extractions using an homogenizer.
3.3.2 Effect of homogenization time on the recovery of
enzymes
There was no difference observed in the recovery of
xylanase, cellulase and laccase at different homogenization times studied (Table 7). Ortega et al 40
reported on the extraction of laccase, cellulase and
xylanase produced by Ple ostreatus and Pleurotus sp R
using 0.1 mol dm3 citrate or 0.2 mol dm3 phosphate buffer, pH 5, and homogenizing for 5 min.
In our experiment, the optimum extraction time of
-glucosidase using an homogenizer was found to
be 8 min with a recovery of 104.12 of SMC U g1
(Table 7), which was 1.117% higher than the other
time intervals studied. Eight min was also found to
be the optimum extraction time of lignin peroxidase
using an homogenizer with a recovery of 119.76 U g1
of SMC (Table 7). The recovery of lignin peroxidase
at 8 min was 87% higher than the recovery of enzyme
at 12 min. The recovery of lignin peroxidase was higher
by 3158% at 8 min as compared with other homogenization time intervals. Homogenization times longer
than 8 min showed a marked decrease in the enzyme
activities. The reabsorption of the enzymes by the fungal mycelium could not be possible in this case as
the maximum time used for homogenization was only
20 min. Darah et al 38 also reported on the extraction of
lignin peroxidase from Pha chrysosporium grown on rice
husks by homogenizing in a Waring blender for 3 min.
In our study 8 min was used for further optimization
of extraction using the homogenizer.

No activity observed for -glucosidase.

a

6.55 0.15
1.91 0.10
26.01 1.7
0.26 0.03
80.84 89.03
6.55 0.03
1.97 0.08
18.73 2.31
0.18 0.11
12.11 18.90
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

6.59 0.22
2.04 0.04
18.47 1.13
0.12 0.08
45.75 46.36

6.48 0.1
1.94 0.07
23.31 3.11
0.25 0.05
91.41 56.08

6.58 0.08
1.91 0.13
29.27 1.2
0.40 0.13
78.91 77.79

4.83 0.07
1.15 0.04
2.15 0.24
0.07 0.06
38.54 22.7

4.93 0.05
0.99 0.04
a
0.12 0.03
36.14 9.2

4.93 0.09
1.11 0.06
0.08 0.92
0.07 0.01
20.76 29.4

8.1
7
6
5
4
7
5
Enzyme

8.1

Enzyme activities (U g1 of SMC) at different pH values at 28 2 C

Enzyme activities (U g1 of SMC) at different pH values at 4 C

Table 5. Effect of pH of tap water on enzyme recovery during incubation in a shaker at 200 rpm for 1 h at 4 C and at 28 C. The results are mean of four replicate samples

Bulk enzymes from mushroom compost

J Chem Technol Biotechnol 78:743752 (online: 2003)

3.3.3 Effect of speed of homogenization on the recovery

of enzymes
Different speeds of homogenization had no effect
on the recovery of xylanase, cellulase and laccase
(Table 8). The recovery of -glucosidase with a value
of 92.6397.91 U g1 of SMC was high at lower
speeds of 800013 500 rpm using the homogenizer
(Table 8). There was an 11.6% and 41.3% decrease
in the enzyme recovery of -glucosidase at the speeds
of 20 500 and 24 000 rpm respectively. It was observed
that at high speeds of 20 500 and 24 000 rpm,
the temperature of the samples was 45 C and
56 C respectively after homogenization. Such high
temperatures can have adverse effects on the enzyme
activities and may render them inactive by denaturing
the enzymes to random coils. This explains the lower
activities of -glucosidase at high speeds. The enzyme
activity of lignin peroxidase was also affected by
749

AD Singh, N Abdullah, S Vikineswary

Table 6. Effect of pH of tap water on enzyme recovery using an homogenizer at 8000 rpm for 10 min at 28 2 C (the results are mean of four
replicate samples)

Enzyme activity (U g1 of SMC) at different pH values

Enzyme
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

8.6

5.70 0.18
3.73 0.21
101.80 4.39
0.13 0.03
150.52 71.5

5.56 0.02
3.57 0.13
101.49 5.16
0.16 0.03
101.02 105.0

5.37 0.12
3.46 0.13
103.43 4.79
0.13 0.03
68.82 27.6

7.02 0.16
2.82 0.23
104.63 1.68
0.16 0.02
49.60 56.3

7.08 0.16
2.92 0.11
107.58 2.54
0.15 0.03
74.59 31.9

6.94 0.12
2.62 0.05
100.36 2.48
0.13 0.02
30.37 37.2

Table 7. Effect of time on enzyme recovery in tap water at pH 4.0 using an homogenizer at 8000 rpm at 28 2 C (the results are mean of four
replicate samples)

Enzyme activity (U g1 of SMC) at different time intervals (in min)

Enzyme
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

10

12

16

20

6.39 0.09
2.08 0.22
99.60 3.58
0.11 0.03
50.08 36.2

6.53 0.11
2.24 0.19
104.12 3.07
0.16 0.01
119.76 40.7

6.57 0.07
1.95 0.06
102.93 3.92
0.12 0.03
54.40 38.3

6.10 0.04
2.49 0.11
87.04 3.90
0.10 0.0
14.99 48.2

6.13 0.01
2.24 0.13
86.54 2.84
0.10 0.0
81.32 41.8

5.96 0.11
2.29 0.07
95.77 3.18
0.05 0.06
82.28 42.0

Table 8. Effect of speed of homogenizer on enzyme recovery in tap water at pH 4.0 for 8 min at 28 2 C (the results are mean of four
replicate samples)

Enzyme activity (U g1 of SMC) at different speeds (in rpm)

Enzyme
Xylanase
Cellulase
-Glucosidase
Laccase
Lignin peroxidase

8000

9500

13500

20500

24000

6.96 0.06
2.99 0.14
92.63 1.76
0.05 0.02
96.21 68.5

6.88 0.11
2.86 0.23
94.45 1.25
0.03 0.01
89.01 29.5

6.86 0.15
2.68 0.20
97.91 2.86
0.07 0.01
108.71 47.8

6.86 0.07
2.59 0.24
86.48 6.65
0.09 0.01
113.52 88.7

6.65 0.04
1.52 0.21
57.47 3.37
0.07 0.04
148.60 41.7

the speed of the homogenizer. The enzyme activity

increased with the increase in speed with the maximum
recovery of 148.60 U g1 of SMC at the highest speed
of 24 000 rpm (Table 8). There was probably a release
of some intracellular enzyme due to the rupture of the
mycelium during homogenization at high speeds, thus
giving high enzyme recovery. Though it was observed
that the temperature of the samples was 45 C and
56 C respectively after homogenization at the speed
of 20 500 and 24 000 rpm, the enzyme activity was
not affected, indicating that probably the enzyme
is thermostable. Shanmugam and Yadav32 reported
the production of a thermostable lignin peroxidase
from Ple sajor-caju with an optimum temperature
between 50 C and 60 C. They reported that the
lignin peroxidase of Ple sajor-caju was relatively more
stable than lignin peroxidase of Pha chrysosporium.

4 CONCLUSIONS
When grown on rubberwood sawdust, Pleurotus sajorcaju produced hydrolytic and oxidative enzymes
during the colonization and fruiting phases. The
750

monthly enzyme profiles showed that 5-month-old

bags of Ple sajor-caju can be used as a source of
bulk enzymes. Further, a high productivity of lignin
peroxidase was observed over the growth period.
To our knowledge, this is the first report of lignin
peroxidase production from Ple sajor-caju growing in
rubberwood sawdust via solid substrate fermentation
and consistently being produced over the period of
10 months. However, the results obtained so far are
based on the spectrophotometric analysis. Further
enzyme characterization including purification will be
done to confirm the enzyme.
The extraction of enzymes using an incubator shaker
and tap water at 28 2 C (room temperature) for
1 h is an economical method for the extraction of
significant amounts of industrial enzymes. The use of
an homogenizer, however, offers an advantage over
the use of an incubator shaker by eliminating the
need for a cold environment. High titers of all the
enzymes studied could be extracted in tap water using
an homogenizer for 8 min at 28 2 C. The use of
an homogenizer thus makes the extraction of bulk
enzymes much easier and cost effective.
J Chem Technol Biotechnol 78:743752 (online: 2003)

Bulk enzymes from mushroom compost

ACKNOWLEDGEMENTS
The authors thank the Ministry of Science, Technology and Environment, Malaysia, for IRPA grants
09-02-03-0675, and 01-02-03-1002, and the University of Malaya for the generous support. We also thank
Mr John Kuan for providing Pleurotus sajor-caju bags.

18

19

REFERENCES
1 Scarse R, Cultivating mushroomsthe potential. Mycologist
9:1819 (1995).
2 Bushwell JA, Production of lignocellulolytic enzymes by edible
mushrooms and their role in substrate utilization. Paper presented at ICRO UNESCO University Malaya, Microbiology
Society Malaysia Regional Training Workshop Course on
Current Trends in Microbial Technology for a Sustainable
Environment: Exploring Diversity for Novel Processes. Kuala
Lumpur, Malaysia, pp 15 (1998).
3 Gacesa P and Hubble J, Enzyme Technology. Open University
Press, Milton Keynes, England, pp 185 (1987).
4 Sayadi S and Radhouane E, Decolorization of olive mill wastewaters by the white rot fungus Phanerochaete chrysosporium:
involvement of lignin degrading system. Appl Microbiol
Biotechnol 37:813817 (1992).
5 Kirby N, McMullan G and Marchant R, Decolorization of
an artificial textile effluent by Phanerochaete chrysosporium.
Biotechnol Lett 17:761764 (1995).
6 Jian EL and Wang HY, Degradation kinetics of pentachlorophenol by Phanerochaete chrysosporium. Biotechnol Bioeng
35:11251134 (1989).
7 Lamar TR, Larsen MJ and Kirk TK, Sensitivity to and
degradation of pentachlorophenol by Phanerochaete sp. Appl
Environ Microbiol 56:35193526 (1990).
8 Cripps C, Bumpus JA and Aust SD, Biodegradation of azo and
heterocyclic dyes by Phanerochaete chrysosporium. Appl Environ
Microbiol 56:11141118 (1990).
9 Ollikka P, Alhonmaki K, Leppanen VM, Glumoff T, Raijola T
and Suominen I, Decolorization of azo, triphenyl methane,
heterocyclic and polymeric dyes by lignin peroxidase
isoenzymes from Phanerochaete chrysosporium. Appl Environ
Microbiol 59:40104016 (1993).
10 Sermanni GG, Annibale A, Lena GD, Vitale NS, Mattia ED
and Minelli V, The production of exo-enzymes by Lentinus
edodes and Pleurotus ostreatus and their use for upgrading corn
straw. Bioresource Technology 48:173178 (1994).
11 Madan M, Vasudevan P and Sharma S, Cultivation of Pleurotus
sajor-caju on different wastes. Biological Wastes 22:214250
(1987).
12 Bano Z, Mysore NS and Rajarathnam S, Improvement of the
bioconversion and biotransformation efficiencies of the oyster
mushroom (Pleurotus sajor-caju) by supplementation of its rice
straw substrate with oil seed cakes. Enzyme Microb Technol
15:985989 (1993).
13 Kumaran S, Sastry CA and Vikineswary S, Laccase, cellulase
and xylanase activities during the growth of Pleurotus sajorcaju on sago hampas. W J Microbiol Biotechnol 13:4349
(1997).
14 Ling SK, Biochemical changes associated with growth of
Pleurotus sajor-caju on oil palm frond parenchyma tissue.
M Biotechnology thesis, Universiti Malaya, Kuala Lumpur
(1994).
15 Vimala P and Vikineswary S, Microbiological and biochemical
changes during composting of spent mushroom compost.
Paper presented at 21 National Symposium of the Malaysian
Society for Microbiology, Selangor, December (1998).
16 Bisaria R, Vasudevan P and Bisaria VS, Utilization of spent
agro-residues from mushroom cultivation for biogas production. Appl Microbiol Biotechnol 33:607609 (1990).
17 Sochtig H and Grabbe K, The production and utilization of
organic-mineral fertilizer from spent mushroom compost,

J Chem Technol Biotechnol 78:743752 (online: 2003)

20

21

22

23

24

25

26

27
28

29
30

31

32

33

34

35

36

37

38

in Science and Cultivation of Edible Fungi, ed by Elliott TJ.

Balkema, Rotterdam, pp 907915 (1995).
Vikineswary S, Kumaran S, Ling SK, Dinesh N and Shim YL,
Solid substrate fermentation of agro-residues for value added
products: the Malaysian experience, in Global Environmental
Biotechnology, ed by Wise DL. Kluwer Academic Publishers,
Great Britain (1997).
Okeke BC, Smith JE, Paterson A and Watson-Craik IA, Aerobic
metabolism of pentachlorophenol by spent sawdust culture of
Shiitake mushroom (Lentinus edodes) in soil. Biotechnol Lett
15:10771080 (1993).
Chiu SW, Ching ML, Fong KL and David M, Spent mushroom
substrate performs better than many mycelia in removing
the biocide pentacholrophenol. Mycol Res 102:15531562
(1998).
Kumaran S, Enzyme activities of Pleurotus sajor-caju during
solid substrate fermentation of sago hampas. MPhil thesis,
Universiti Malaya, Kuala Lumpur (1996).
Harkin JM and Obst JR, Syringaldazine, an effective reagent
for detecting laccase and peroxidase in fungi. Experientia
29:381387 (1973).
Leonowicz A and Grzywnowicz K, Quantitative estimation of
laccase forms in some white rot fungi using syringaldazine as
a substrate. Enzyme Microb Technol 3:5558 (1981).
Have RT, Hartmans S, Teunissen PJM and Field JA, Purification and characterization of two lignin peroxidase isoenzymes
produced by Bjerkandera sp strain BOS55. FEBS Letters
422:391394 (1998).
Bailey MJ, Bailey P and Poutanen K, Interlaboratory testing
of methods for assay of xylanase activity. J Biotechnol
23:257270 (1992).
Dong WK, Tae SK, Young KJ and Jae KL, Adsorption kinetics
and behaviors of cellulase components on microcrystalline
cellulose. J Ferment Bioeng 73:461466 (1992).
Miller GL, Use of dinitrosalicylic acid reagent for determination
of reducing sugars. Anal Chem 31:426428 (1959).
Bradford MM, A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye. Anal Chem. 72:248254 (1976).
Gilbert HJ and Hazelwood GP, Bacterial cellulases and
xylanases. J Gen Microbiol 139:187194 (1993).
Darah I and Ibrahim CO, Lignin degrading enzyme production
by entrapped cells of Phanerochaete chrysosporium in submerged culture system. Asia Pacific J Mol Biol Biotechnol
5:143154 (1997).
Shi YF, Hui-sheng Y and Bushwell JA, Effect of nutrient
nitrogen and manganese on manganese peroxidase and
laccase production by Pleurotus sajor-caju. FEMS Microbiol
Lett 147:133137 (1997).
Shanmugam V and Yadav KDS, Production of extracellular
lignin peroxidase by Pleurotus sajor-caju. Indian Journal of
Experimental Biology 34:11641165 (1996).
Bisaria R, Madan M and Vasudevan P, Utilization of agroresidues as animal feed through bioconversion. Biores Technol
59:58 (1997).
Prasertsan P and Oi S, Production of cellulolytic enzymes from
fungi and use in the saccharification of palm cake and palm
fiber. W J Microbiol Biotechnol 8:536538 (1992).
Ganisan K, Tan YH and Wahab MN, Laccase production on
supplemented rubber sawdust compared with chemically
defined liquid medium in Lentinula edodes. Paper presented at
21st Symposium of the Malaysian Society for Microbiology,
Selangor, pp 2126 (1998).
Matcham SE and Wood DA, Purification of Agaricus bisporus
extracellular laccase from mushroom compost. Biotechnol Lett
14:297300 (1992).
Dakshini H, Bioconversion of agro-residues for lignocellulotic
enzymes. BSc thesis, Universiti Malaya, Kuala Lumpur
(1999).
Darah J, Burke R and Ibrahim CO, Characteristics of a
dominant lignin peroxidase isoenzyme of Phanerochaete
chrysosporium from solid-state fermentation on rice husks.
Asia Pacific J Mol Biol Biotechnol 6:181185 (1998).
751

AD Singh, N Abdullah, S Vikineswary

39 Forrester T, Grabski AC, Mishra C, Kelley BD, Strickland WN,
Leatham G and Burgess RR, Characteristics and N-terminal
amino acid sequence of a manganese peroxidase purified
from Lentinula edodes cultures grown on a commercial wood
substrate. Appl Microbiol Biotechnol 33:359365 (1990).

752

40 Ortega GM, Martinez EO, Gonzalez PC, Betancourt D and

Otero MA, Enzyme activities and substrate degradation
during white rot fungi growth on sugar-cane straw in a
solid-state fermentation. W J Microbiol Biotechnol 9:210212
(1993).

J Chem Technol Biotechnol 78:743752 (online: 2003)