2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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had also a better rate of glucose production (0.26 μg ml
-1
min
-1
) than cellulase alone (0.22 μg ml
-1
min
-1
), (Fig. 5).
Addition of vanillic acid (Fig. 6) followed the same
trend in the production of glucose than previously
observed with samples with added
p
-coumaric acid
(Fig. 5). A high concentration of vanillic acid (1 mM)
resulted in a rate of production of glucose of less than
0.12 μg ml
-1
min
-1
, whereas in comparison a low
concentration of vanillic acid (1 μM) resulted in a rate of
production of glucose of 0.20 μg ml
-1
min
-1
which was
somewhat reduced as compared to the rate of production
of 0.22 μg ml
-1
min
-1
of glucose in samples with only
cellulase and CMC as substrate.
PG = 0.34T
R
2
= 0.99
PG = 0.26T
R
2
= 0.99
PG = 0.12T
R
2
= 0.97
PG = 0.22T
R
2
= 0.99
PG = 0.20T
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+vanillic acid1 mM
Cellulase+laccase+vanillic acid1μM
Cellulase+vanillic acid1 mM
Cellulase+vanillic acid1 μM
Laccase+vanillic acid1 mM
Laccase+vanillic acid1 μM
Cellulase+laccase
Cellulase
Laccase
Bufferwithout enzyme
Fig. 6. Influence of addition of vanillic 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. Vanillic 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.
Upon addition of laccase to samples with cellulase and
vanillic acid at a concentration of 1 μM vanillic acid,
highest rate of production of glucose with about
0.34 μg ml
-1
min
-1
a same trend was measured and at a
concentration of 1 mM vanillic acid, a reduced rate of
about 0.12 μg ml
-1
min
-1
. Thus, rates of production of
glucose in presence of laccase in combination with
cellulase and a low amount of a phenolic compound
(1 μM) and in presence of laccase in combination with
cellulase without any added phenolic compound
(0.26 μg ml
-1
min
-1
) were obtained that were higher than
the rate of production of glucose in samples with just
cellulase alone.
When testing effects of ferulic acid (Fig. 7), at the high
concentration of 1 mM no CMC hydrolysis by cellulase
was achieved but a nearly normal rate of production of
glucose of less than 0.21 μg ml
-1
min
-1
was observed
when the low concentration of 1 mM of ferulic acid was
added to samples. In comparison, upon addition of
laccase to samples with the low concentration of ferulic
acid (1 μM) a rate of production of glucose over
0.15 μg ml
-1
min
-1
was obtained. These results suggest
that by the actions of laccase in combination with ferulic
acid, negative actions on the hydrolysis of cellulose to
glucose via the applied cellulase occur.
PG = 0.26T
R
2
= 0.99
PG = 0.21T
R
2
= 0.99
PG = 0.22T
R
2
= 0.99
PG = 0.15T
R
2
= 0.99
PG = 0.04T
R
2
= 0.82
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+ferulic acid1mM
Cellulase+laccase+ferulic acid1 μM
Cellulase+ferulic acid1mM
Cellulase+ferulic acid1 μM
Laccase+ferulic acid1mM
Laccase+ferulic acid1 μM
Cellulase+laccase
Cellulase
Laccase
Bufferwithout enzyme
Fig. 7. Influence of addition of ferulic 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. Ferulic 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. Note that for the curve calculated
for samples with added cellulase, laccase and 1 mM ferulic acid, the R
2
was only low with 0.82, indicating a low reliability of the calculated
value for the rate of production of glucose via the applied cellulose
occur.
Laccases transfer ferulic acids into different radicals
and these may form several different ferulic acid dimers
and trimers [24, 25, 26]. Moreover, in presence of ferulic
acid, laccase may oligomerize peptides containing
cysteine and crosslink tyrosine containing peptides [24,
25]. On longer peptides, cross-linking may particularly
often occur when tyrosines are present close to the C-
terminal end [25]. The Onozuka cellulose R-10 applied in
this study is indeed a mixture of various cellulases for
T. viride
: six endoglucanases (Endo I to Endo VI), three
exoglucanases (Exo I to Exo III) and a
β
-glucosidase are
