3. Materials and methods
3.1. Materials
There were four types of biomass in my research, the first of them was unbleached
eucalyptus kraft pulp (UEK) and the second one was bleached eucalyptus kraft pulp
(BEK). The original kraft pulps samples were produced from Australian Eucalyptus
globules chips by using an M/K digester (Peabody, MA, USA), with liquid-to-wood ratio
of 1/4. The cooking liquor consisted of NaOH and Na26ZLWKVXO¿GLW\DQG
active alkali based on chemical charge. Cooking temperature was raised from 25 to 160oC
at 1.5oC per minute, then maintained isothermally for 180 min.
Fully bleached pulps were prepared from oxygen bleached pulps by using a common
commercial DEDD bleaching sequence (Ko et al., 2010). DEDD bleaching sequence was
treated with chlorine dioxide (ClO2) and sodium hydroxide (NaOH) to remove lignin.
The dosages of chlorine dioxide were determined by active chlorine multiple and kappa
number. In the first stage (D0), there were 10% consistency pulp and chlorine dioxide
corresponding to pulp incubating for 1 hr at 70oC. In the next step, the sodium hydroxide
was used to alkali extraction to remove lignin (E). Dosage of sodium hydroxide was 1.8%
base on gram of pulp sample. Alkali extraction reaction time was 1.5 hr at 65oC. After
the alkali extraction, D1 and D2 steps of bleach sequence were carried out. The D1 and
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D2 bleach sequence used chlorine dioxide dosage of 0.35% and 0.15% base on pulp
sample for each 3.5 hr at 72oC and made the pulp brightness over than 90%.
The third biomass was eucalyptus chips hydrolyzed by 1% sulfuric acid for almost
6 days before steam explosion (ASEP) and the last one was eucalyptus chips treated by
steam explosion directly (NSEP). The steam explosion was executed by Institute of
Nuclear Energy Research. The condition of steam explosion was to put 1 kg dry treated
or not treated eucalyptus chips into reactor and the ratio of liquid/solid was 7 of each
feedstock and heated to 190oC with saturated steam for 10-20 minutes.
3.1.2. Materials Sieved
Fiber size fractionation was carried out in a BAUER-McNETT CLASSIFIER (BMC)
for 30-45 minutes classifier fitted with 28-, 50-, 100-, 200-mesh screens. The process of
manipulation followed the CNS 12428. The fraction retained by the screen was termed
RX where X refers to the mesh size. According the results of Tsai (2012), the enzymatic
hydrolysis and adsorption of R200 sieved substrate is the most efficient of all because the
specific surface area of R200 was more than other sieved substrate.
Although R200 had higher specific surface area, the result of the fiber size
distribution showed that there were more R100 pulp retained on mesh screen. Thus, in
this study R100 scale materials were used as substrates for further analysis.
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3.1.3. Enzymes
Pulpzyme HC was a commercial enzyme with xylanase activity supplied from
Novozyme. XylX produced by Paenibacillus campinanesis BL11 was found in black
liquor and its mutants (H2, L2) were constructed by further research. (Ko et al. 2007,
2010). Enzyme could be departed as catalytic domain which made enzyme hydrolyzing
a certain polymer and binding domain which made enzyme attaching on polymer stably.
The catalytic domain of XylX belonged to GH11 family so it was termed GH 11 and the
binding domain also called carbohydrate binding module was abbreviated as CBM (Wang,
2013).
3.1.3.1.Incubation
E. coli (Escherichia coli) expressing system was used to produce the enzymes. E.
coli were routinely cultured in Luria–Bertani (LB) medium. LB medium contained 10
g/L Bacto-tryptone, 5 g/L yeast extract, and 5 g/L NaCl. First, E. coli were precultured
from plate to liquid broth about 3 mL LB medium in glass tube at 150 rpm overnight.
After cultured to certain concentration, E. coli were transferred to 500 mL flask and
rotating at 150 rpm for 3-4 hours. Then, IPTG (,VRSURS\Oȕ-D-1-thiogalactopyranoside)
was added to final concentration as 0.1 mM for inducing E. coli to produce enzyme.
Moreover, the enzyme of E. coli expressing system was an endo-secreted system. E. coli
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were disrupted by ultrasonication in ice bath to break the cell wall and let enzyme
suspended in buffer. After ultrasonication, the buffer would be centrifuged at 4oC 8500
rpm for 20 minutes to separate the somatic of E. coli and enzyme. The supernatant which
had enzyme was separated through 0.45 filter as crude enzyme.
3.1.3.2.Purification
The crude enzyme was purified by Ni-NTA column whose motive phase was
Tris-HCl buffer with gradient imidazole. The purification made use of affinity between
His-tag on expressed enzyme and Ni2+. After binding, we used high concentration imidazole
to competitive with His-tag to make enzyme leave Ni-NTA and collected the target
enzyme. First, 5 mM imidazole Tris-HCl buffer (binding buffer) was used to stable the
condition of all purification system. Second, crude enzyme was added to Ni-NTA column
let protein bind with Ni2+as well as there were still some non-specific binding. Thus, 20
mM imidazole Tris-HCl buffer (washing buffer) was used to break the non-specific
binding between Ni2+ and non-specific protein because the non-specific binding was a
weak binding whose binding site would be occupied by imidazole. After washing, 100
mM imidazole Tris-HCl buufer (elute buffer) was used to elute the target enzyme and
collected it as purified enzyme. During the process of purification, the spectrophotometer
monitored the outlet liquid at 280 nm absorbance.
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3.1.3.3.SDS-PAGE
The purified enzymes used SDS-PAGE (Sodium dodecyl sulfate - polyacrylamide
gel electrophoresis) to check the degree of purity. 12% polyacrylamide gel was used as
running gel and set it on Hoefer electrophoresis equipment. The sample preparation was
adding same volume loading dye as sample and put it in boiling water for 10 min. Then,
the sample was centrifuged 5000 rpm for 30 min. After loading sample in stacking gel,
the volt of power supplement was set at 200 mV for 5 min to stack the protein. Next, the
volt of power supplement was changed to 120 mV for 120 min to make the protein
separated by the size. Finally, the SDS-PAGE was stained by comassie blue for 30 min
and destained by methanol acetate buffer overnight.