2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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Fig. 4. Influence of water extractives in cellulase hydrolysis
A
) and
water extractives in cellulase+laccase hydrolysis
B
) of 5 mg
carboxymethylcellulose (CMC) by using 0.008 U ml
-1
cellulase
(cellulase “Onozuka R
-
10” from
Trichoderma viride
, SERVA
Electrophoresis GmbH, Heidelberg, Germany) and 0.008 U ml
-1
laccase
(Novozyme, EEC No. 420-150-4) in 5 ml 50 mM sodium acetate buffer,
pH 5.0 at an incubation temperature of 37
o
C. Water extractives were
added portions of 2.5 μl DMSO containing the amount of extractives
corresponding to 5 mg of
A. grandis
wood. To all other reactions, 2.5 μl
DMSO was added for comparison. Per treatment, each three samples
were analyzed. At 60, 90, 120 and 150 min of incubation, 150 μl per
sample were taken from the reactions and tested for glucose content
using the GAGO. An acceptable positive correlation was present with
R
2
>0.95 for each kinetic curve.
According to previous own results [23],
p
-coumaric
acid (4-hydroxy cinnamic acid), vanillic acid (3-methoxy-
4-hydroxybenzoic
acid)
and
ferulic
acid
(4-hydroxy-3-methoxy cinnamic acid) are active in
inducing a high laccase production from
Trametes
versicolor
. These phenolic compounds (
p
-coumaric acid,
vanillic acid and ferulic acid) were also found in the
highest amounts within water extractives (Fig. 3). These
phenolic compounds in pure commercial form were
therefore added in different concentrations (1 mM and
1 μM) to reactions of enzymatic CMC hydrolysis carried
out in 50 mM sodium acetate buffer, pH 5.0 (Fig. 5).
In this series of tests with 5 mg of commercial CMC
preparation, again 0.008 U ml
-1
cellulase was used and
0.5 U ml
-1
of purified laccase V from
Coprinopsis
cinerea
Okayama 7. As expected, the purified laccase
added into samples with just CMC did not lead to
recognizable conversion of cellulose into glucose like in
the samples with no added enzyme and unlike in the
samples where just cellulase was added to the CMC
(Fig. 5 till Fig. 7). Additions of the three different
phenolic compounds always lead to a reduction cellulase
activity.
PG = 0.26T
R
2
= 0.99
PG = 0.22T
R
2
= 0.99
PG = 0.22T
R
2
= 0.99
PG = 0.28T
R
2
= 0.99
0
5
10
15
20
25
30
35
40
0
30
60
90
120
Incubation time [min]
Production of glucose; PG [μg ml
-1
]
Cellulase+laccase+p-coumaric acid1mM
Cellulase+laccase+p-coumaric acid1μM
Cellulase+p-coumaric acid1mM
Cellulase+p-coumaric acid1μM
Laccase+p-coumaric acid1mM
Laccase+p-coumaric acid1μM
Cellulase+laccase
Cellulase
Laccase
Bufferwithout enzyme
Fig. 5. Influence of addition of
p
-coumaric acid on hydrolysis of 5 mg
CMC by cellulase in presence and absence of laccase. 0.008 U ml
-1
cellulase (Cellulase “Onozuka R
-
10” from
Trichoderma viride
, SERVA
Electrophoresis GmbH, Heidelberg, Germany) and 0.5 U ml
-1
of
purified laccase V of
C. cinerea
Okayama 7 in 5 ml of 50 mM sodium
acetate buffer, pH 5.0 were used.
p
-Coumaric acid was dissolved in
100 μl DMSO. To all other samples, for reasons of comparison, 100 μl
of pure DMSO was added. Per treatment, three different samples were
followed up in glucose formation over the time. At 60, 90, and 120 min
of incubation, per sample aliquots of 150 μl were taken and analyzed for
glucose
content with the GAGO kit.
The results in Fig. 5 show that
p
-coumaric acid at a
high concentration (1 mM) nearly fully blocked the
enzymatic hydrolysis of CMC whereas production of
glucose was to a certain level reduced at the lower
concentration of 1 μM of
p
-coumaric acid (Fig. 5). At the
high concentration of
p
-coumaric acid (1 mM), as in
controls without added enzymes, a neglectable rate of
production
of
glucose
much
below
than
0.03 μg ml
-1
min
-1
were obtained in comparison to a rate
of production of glucose of 0.22 μg ml
-1
min
-1
at the low
concentration of
p
-coumaric acid. When laccase was
added to samples with cellulase and 1 μM
p
-coumaric
acid, the hydrolysis activity was fully restored with a
highest rate of glucose production of about
0.28 μg ml
-1
min
-1
, respectively even increased when
compared to rates of production of glucose in samples
with cellulase alone (0.22 μg ml
-1
min
-1
). Also, some
activity was restored upon addition laccase to samples
with cellulase and 1 mM
p
-coumaric acid, although the
production rate of glucose was very low with a rate of
0.03 μg ml
-1
min
-1
(Fig. 5).
Notable is further that the combination of the
cellulase + laccase without addition of
p
-coumaric acid
PG=0.16T
R
2
=0.99
PG=0.09T
R
2
=0.96
0
5
10
15
20
25
30
35
40
Production of glucose; PG [μg ml
-1
]
Cellulase+water extractives
Cellulase
Buffer without enzyme
PG=0.16T
R
2
=0.99
PG=0.13T
R
2
=0.97
0
5
10
15
20
25
30
35
40
0
30
60
90
120
150
Incubation time; T [min]
Production of glucose; PG [μg ml
-1
]
Cellulase+laccase+water extractives
Cellulase+laccase
Buffer without enzyme
A
B
