2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 41 -
present in the enzyme mixture [27]. Of these, Exo I and
Endo I for example have an outmost C-terminal 30
amino-acid long CBM domain (cellulose binding motif)
with four cysteine and four tyrosines ([28]; GenBank
accession number ACC59774), offering a good target for
ferulic-acid-mediated cross-linking through laccase
action, presenting an explanation for then here observed
reduction in cellulase activity on CMC by the presence of
laccase plus 1 μM ferulic acid (Fig. 7). In contrast, other
cellulases of
T. viride
such as Endo II (GenBank BAA
36216) and Endo III (GenBank AAQ 21383) and two
other endoglucanases with not yet assigned number
(GenBank ADJ10627 and ABQ95572) have an
N-terminal CBM domain, offering both protection from
ferulic-acid
mediated
cross-linking
by
laccase.
Accordingly,
the
low
hydrolysis
activity
(0.15 μg ml
-1
min
-1
) on cellulose in samples with added
laccase and 1 μM ferulic acid may be due to such
cellulase being less accessible from inactivation.
What are the reasons for optimizing the glucose yields
from cellulosic pellets in the presence of laccase?
Cellulases may bind to lignin and this can hinder their
enzymatic actions [29]. In the study here, by addition of
wood extractives and by addition of specific phenolic
compounds shown to be present in
A. grandis
wood
extractives to CMC and cellulase it was shown that such
compounds also hinder the cellulolytic enzymatic
activities (Fig. 4).
Water extractives,
p
-coumaric acid, vanillic acid and
ferulic acid all influenced the production of glucose from
cellulose by cellulase hydrolysis, (Fig. 4). The
concentration in water extractives of
A. grandis
wood
applied in the enzymatic test with CMC (water
extractives from 1 mg
A. grandis
wood transferred into
1 ml buffer; Fig. 4) were 0.123 mM of
p
-coumaric acid,
0.137 mM vanillic acid and 0.013 mM ferulic acid and
thus in between the two concentrations of 1 mM and
1 μM used in the test for the purified compounds (Fig. 5
to Fig. 7). From the results shown in Fig. 5 to 7, it is to be
expected that at the natural concentrations the three
compounds will also provide negative effects to cellulase
activity, in particular when considering in addition that in
the wood extractives the negative actions of the different
compounds will likely add up to a stronger effect.
Therefore, since addition of laccase to the cellulase
overcomes in concentration-dependent manner the
negative effects of the phenolic compounds, it can be
concluded that laccase has a potential to decrease
inhibitors in form of phenolic compounds in wood
extractives blocking cellulases in cellulose hydrolysis
(Fig. 4). Whilst the data show that laccase can very much
improve the enzymatic generation of glucose from
cellulosic pellets, the example of ferulic acid shows that
in certain situations their might be also some negative
effects by addition of laccase. Ferulic acid potentially
blocks upon laccase-activation a subrange of cellulase in
their activities.
In summary, however, laccase has a good potential to
improve the drawbacks of lignin and lignin-related
compounds on enzymatic cellulose degradation because
laccase is efficient in degrading of lignin and of phenolic
compound as present in wood extractives or even
somewhat of hemicellulose [30]. Thus, use of laccase in
combination of enzymatic hydrolysis can improve the
hydrolysis process and gain higher glucose yields, as
found in the study by [30] and also in this study.
Future own studies for upcoming applications will
concentrate on the predominant wood industries in
Thailand, especially in Southern Thailand, that rely
economically mainly on rubberwood and palm tree
plantations. Knowledge from this work gained by
studying
A. grandis
wood will be transferred and adapted
to such wood species playing on important role in
Thailand. As a result of the Thai wood industries, huge
amounts of wood product residues and wood sawdust
from the local tree species and fruit bunches of palm trees
accumulate. Countries in Southeast Asia including
Thailand are the biggest rubberwood producers in the
world, making use of the wood for furniture and
particleboard production. Surprisingly, only 10% of the
total wood end up as products, while about 90% of the
material represent wood residues. From this, a ratio of
54% comes from small branches, and a ratio of 32 %
wastes from wood sawmills and the 4% rest wood comes
from furniture factories. In total, there is an annual
availability of rubberwood residues of 1,100,000 tons
from rubberwood plantations and sawmills for which
optimal economical and ecological uses have to be found
[31]. Moreover, a palm oil research unit was established
in 2008 in the province of Surat Thani, where the Prince
of
Songkla University (author work’s office) is located,
by implementing the province policy to develop in the
next few years the Surat Thani province to be a “Palm
city” (
. Also this development
will lead to huge amounts of further waste materials that
should economically und ecologically be made use of.
According to the official Thai policy for generating
sustainable biomass products, producing bioethanol from
wastes sources of both tree species are highly interesting
challenges,
for Thailand in the near future’s research,
both for industry and for society.
IV.C
ONCLUSION
Laccase in combination of enzymatic hydrolysis of
cellulose by application of Onozuka R-10 cellulase in
combination or in a mixture with also xylanase improved
the glucose yields by factors of 60 to 100% as compared
to enzymatic hydrolysis reactions performed without
laccase. The conversion of cellulose to glucose reached
up to 100% when using for 1 g of
A. grandis
wood
particles 8 ml of 85% phosphoric acid for one hour
incubation at 50
o
C in a hot water bath and in subsequent
enzymatic hydrolysis of 100 mg of left cellulosic pellets
each 4 U ml
-1
of cellulase, of xylanase and of laccase, or
each 4 U ml
-1
of cellulase, of xylanase and of
β
-glucosidase, or each 4 U ml
-1
of cellulase, of xylanase,
of β
-glucosidase and of laccase, or each 4 U ml
-1
of
cellulase, of xylanase and of laccase plus 1 mM HBT, or
each 4 U ml
-1
of cellulase, of xylanase, of β
-glucosidase
and of laccase plus 1 mM HBT, respectively, in 50 mM
