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School of Forestry and Resource Conservation College of Bioresources and Agriculture
National Taiwan University Master Thesis
Еᆫᗐ䁙ᆶғ፦ނ܄፦ჹ֎ߕᆶНှຝϐቹៜ !
Impact of xylanase and biomass properties on adsorption and hydrolysis processes
ኻ
Yi-Yang Ou
ࡰᏤ௲Ǻద఼ റγ
Advisor: Chun-Han Ko, Ph.D.
ύ҇୯ 103 ԃ 7 Д
July 2014
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3125 ԃ 9 Д!
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όӕೀޑ୷፦Չ40oC Нှک֎ߕ၂ᡍǴӧ၂ᡍၸำύǴךॺၸෳۓځ
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ӧ 4oC ޑ֎ߕ၂ᡍύǴӕኬ֖ԖၨӭЕ፦નޑ୷፦ڀԖ֎ߕၨӭЕᆫᗐ䁙ޑ
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i
Abstract
Second generation biofuels produced from lignocellulosic feedstock materials and
the basic structure of all woody biomass consisted of three basic polymers: cellulose,
hemicelluloses and lignin. The hemicellulose was the second abundant carbohydrate
resource in the earth. If we made good use of it, we could confirm the energy supply
and decrease the dependence on petroleum energy.
In this study, we used 4 kinds of pretreated substrates to be hydrolyzed and
adsorbed by xylanase, its mutants, its catalytic domain and binding domain. Comparing
with 40oC adsorption and hydrolysis results, high lignin content substrates adsorbed
more xylanase but they released less reducing sugar, which decreased the efficiency of
xylanase. Although low lignin content substrates adsorbed less xylanase, they still
released reducing sugar more than those containing more lignin. The results showed
lignin content had an influence on adsorption and hydrolysis. On the other hand,
carbohydrate binding module (CBM) was one of important factors on hydrolysis. With
complete CBM, xylanase released more reducing sugar, the one whose CBM had
deficiency released less and it would release little reducing sugar without CBM.
On 4oC adsorption, high lignin content substrates also had higher maximum
adsorption and affected the affinity to enzyme because there was more unspecific
binding. The xylanase with complete CBM had higher binding strength with substrates.
ii
Keywords: Lignocellulose; Xylanase; Adsorption; Hydroylsis; Lignin; Carbohydrate
binding module
iii
Contents
α၂ہቩۓਜ ᖴᇞ!
ᄔा………i
Abstract……….……….ii
Contents………iv
Figure index………...vi
Table index……….………..vii
1. Introduction………..…...1
2. Literature review……….4
2.1. Lignocellulose……….4
2.1.1. Cellulose………4
2.1.2. Hemicellulose………4
2.1.3. Lignin……….5
2.2. Pretreatment……….5
2.3. Enzyme adsorption and hydrolysis………..7
3. Materials and methods………...13
3.1. Materials………13
3.1.1. Biomass………...13
3.1.2. Materials Sieved………..14
3.1.3. Enzymes………...15
3.1.3.1. Incubation………15
3.1.3.2. Purification………..16
3.1.3.3. SDS-PAGE………..17
3.2. Methods……….17
iv
3.2.1. 40oC hydrolysis and enzyme adsorption……….17
3.2.2. 4oC enzyme adsorption………18
4. Results and discussion………...20
4.1. Chemical composition of substrates………..20
4.2. 40oC hydrolysis and enzyme adsorption………...21
4.3. 4oC enzyme adsorption………..34
4.4. Parameters of 4oC enzyme adsorption………...41
5. Conclusions………...45
6. References……….47
v
Figure Index
Fig. 1. Generic block diagram of bioethanol production from lignocellulose biomass
(Balat, 2010)………..2
Fig. 2. Schematic of goals of pretreatment on lignocellulosic material (Balat, 2010)…..6
Fig. 3. 40oC hydrolysis of Pulpzyme HC………21
Fig. 4. 40oC adsorption of Pulpzyme HC………22
Fig. 5. 40oC hydrolysis of XylX………..23
Fig. 6. 40oC adsorption of XylX………..24
Fig. 7. 40oC hydrolysis of XylX-H2………25
Fig. 8. 40oC adsorption of XylX-H2………26
Fig. 9. 40oC hydrolysis of XylX-L2………27
Fig. 10. 40oC adsorption of XylX-L2………..28
Fig. 11. 40oC hydrolysis of CBM………29
Fig. 12. 40oC adsorption of CBM………30
Fig. 13. 40oC hydrolysis of GH 11………..31
Fig. 14. 40oC adsorption of GH 11………..32
Fig. 15. 4oC adsorption of Pulpzyme HC………35
Fig. 16. 4oC adsorption of XylX………..36
Fig. 17. 4oC adsorption of XylX-H2………37
Fig. 18. 4oC adsorption of XylX-L2………38
Fig. 19. 4oC adsorption of CBM………..39
Fig. 20. 4oC adsorption of GH 11………40
vi
Table Index
Table 1. Comparison of process conditions and performance of three hydrolysis
processes………...…8
Table 2. Chemical composition of lignocellulose substrates………..20
Table 3. Maximum enzyme adsorption of all substrates at 4oC………..41
Table 4. Equilibrium constant of enzyme adsorption at 4oC………...42
Table 5. Affinity of enzyme adsorption at 4oC……….42
Table 6. Binding strength of enzyme adsorption at 4oC………44
vii
1. Introduction
As decreasing resources of fossil carbon sources and the side effects of their usage
on natural environments were generated. People all around world paid attention close to
renewable energy such as solar energy, wind and bioenergy, etc. Comparing to others
energy, bioenergy was more sustainable and stable because the materials come from
biomass which was more predictable than other kinds of renewable energy. The first
generation bioethanol produced primarily from food crops such as corns, sugar crops and
oil seeds had been used in US, Brazil and China. But it was also limited by some issues
like competition for land and wateUXVHGIRUIRRGDQG¿EHUSURGXFtion, high production
and processing costs that require government supports in order to compete with petroleum
products and widely varying assessments of the net greenhouse gas (GHG) reductions
when land-use change was taken into account (Ralph et al., 2010).
The impacts of these various concerns had stimulated the development of second
generation biofuels produced from non-food biomass. The major components of non-food
biomass were lignocellulosic feedstock materials including by-products (corn straw,
sugar cane bagasse and forest residues), wastes (organic components of municipal solid
wastes), and dedicated feedstocks (purpose-grown vegetative grasses, short rotation
forests and other energy crops). Chemical composition of lignocellulosic materials was a
key factor DIIHFWLQJHI¿FLHQF\RIELRIXHOSroduction during conversion processes. The
1
basic structure of all woody biomass consisted of three basic polymers: cellulose,
hemicelluloses such as xylan and lignin. Cellulose and hemicellulose, which typically
made up two-thirds of cell wall dry matter, were polysaccharides that could be hydrolyzed
to sugars and then fermented to bioethanol. Generic block diagram of bioethanol
production from lignocellulose materials was given in Fig 1. The basic process steps in
producing bioethanol from lignocellulosic materials were: pretreatment, hydrolysis,
fermentation and product separation/distillation. (Balat, 2010).
Figure 1. Generic block diagram of bioethanol production from lignocellulose biomass
(Balat, 2010).
Biomass Pretreatment
Cellulose Hydrolysis
Pentose Fermentation Glucose
Fermentation
Distillation Ethanol
2
Although cellulose was the major material to produce bioethanol in lignocellulose,
hemicellulose whose backbone was xylan was secondly abundant carbon resource on
earth and also played an important role to rise the efficiency in the process of bioethanol
conversion. This study focused on enzymatic hydrolysis and adsorption of xylanase on
various pretreated biomass to understand relationship between biomass chemical
composition, enzymatic hydrolysis and adsorption.
3
2. Literature Review
2.1. Lignocellulose
Lignocellulosic materials consisted of mainly three types of polymers, namely
cellulose, hemicellulose and lignin (Fengel and Wegener, 1984).
2.1.1. Cellulose
Cellulose existed of D -glucose subunits, linked by ȕ-1, 4 glycosidic bonds (Fengel
and Wegener, 1984). The cellulose in a plant consisted of parts with a crystalline structure,
and parts with an amorphous structure. The cellulose strains were bundled together and
formed VRFDOOHGFHOOXORVH¿EULOVRUFHOOXORVHEXQGOHV7KHVHFHOOXORVH ¿brils were mostly
independent and weakly bound through hydrogen bonding (Laureano-Perez et al., 2005).
2.1.2. Hemicellulose
Hemicellulose was a complex carbohydrate structure that consisted of different
polymers like pentoses (like xylose and arabinose), hexoses (like mannose, glucose and
galactose), and sugar acids. The dominant component of hemicellulose for hardwood was
xylan and for softwood is glucomannan (Fengel and Wegener, 1984). Hemicellulose had
a lower molecular weight than cellulose and branched with short lateral chains that
consisted of different sugars, which were easy hydrolyzable polymers (Fengel and
4
Wegener, 1984). Hemicellulose served as a connection between the lignin and the
celluloVH¿EHUVDQGJDYe the whole cellulose–hemicellulose–lignin network more rigidity
(Laureano-Perez et al., 2005).
2.1.3. Lignin
Lignin was, after cellulose and hemicellulose, one of the most abundant polymers in
nature and was present in the cellular wall. It was an amorphous heteropolymer consisting
of three different phenylpropane units (p-coumaryl, coniferyl and sinapyl alcohol) that
were held together by different kind of linkages. The main purpose of lignin was to give
the plant structural support, impermeability, and resistance against microbial attack and
oxidative stress. The amorphous heteropolymer was also non-water soluble and optically
inactive; all these made the degradation of lignin very tough (Fengel and Wegener, 1984).
2.2. Pretreatment
One of the major barriers for lignocellulose to become the economical production of
bioethanol was its recalcitrance. Thus, we had to treat the materials with some physical
or chemical methods to break down the structure of biomass feedstock and remove the
barriers that made cellulose more accessible to hydrolytic enzymes for conversion to
glucose before the hydrolysis process started. The goals of pretreatment on lignocellulosic
5
material were depicted in Fig 2. Pretreatment had been regarded as one of the most
expensive pocessing steps within the conversion of biomass.
Figure 2. Schematic of goals of pretreatment on lignocellulosic material (Balat, 2010).
Taherzadeh and Karimi (2008) had summarized the goals for an ideal lignocellulosic
pretreatment. It should be (1) proGXFWLRQRIUHDFWLYHFHOOXORVLF¿EHUfor enzymatic attack,
(2) avoiding destruction of hemicelluloses and cellulose, (3) avoiding formation of
possible inhibitors for hydrolytic enzymes and fermenting microorganisms, (4)
minimizing the energy demand, (5) reducing the cost of size reduction for feedstocks, (6)
reducing the cost of material for construction of pretreatment reactors, (7) producing less
residues, and (8) consumption of little or no chemical and using a cheap chemical.
Pretreatment was crucial for ensuring good ultimate yields of sugars from both
6
polysaccharides. Hydrolysis without preceding pretreatment yielded typically <20%,
whereas yields after pretreatment often exceed 90%. There were physical (milling and
grinding), physico-chemical (steam explosion/autohydrolysis, hydrothermolysis, and wet
oxidation), chemical (alkali, dilute acid, oxidizing agents, and organic solvents) and
biological processes which had been used for pretreatment of lignocellulosic materials.
2.3. Enzyme adsorption and Hydrolysis
The carbohydrate polymers in lignocellulosic materials needed to be converted to
simple sugars before fermentation, which was called hydrolysis. The most commonly
applied methods could bH FODVVL¿HG LQ WZR JURXSV FKHPical hydrolysis (dilute and
concentrated acid hydrolysis) and enzymatic hydrolysis. The main characters of the
bioethanol were cellulose and hemicellulose and their hydrolytic products as follow
(Taherzadeh and Karimi, 2007):
Cellulose ՜ Glucan ՜ Gluose ՜ Decomposition products
Hemicelluloses ՜ Xylan ՜ Xylose ՜ Furfural
Acetyl groups ՜ Acetic acid
7
Comparing to acid hydrolysis, however, enzymatic hydrolysis had high specificity,
mild temperature, less environmental and corrosion problems. The high cost of acid
consumption and recovery were major barriers to economic success (Hamelinck et al.,
2005). But enzymatic hydrolysis of natural lignocellulosic materials was a very slow
process because cellulose hydrolysis was hindered by structural parameters of the
substrate, such as lignin and hemicellulose content, surface area, and cellulose
crystallinity.
Table 1. Comparison of process conditions and performance of three hydrolysis processes
(Hamelinck et al., 2005).
ġ Consumables Temperature (oC) Time Glucose yield (%) Dilute acid <1% H2SO4 215 3 min 50 - 70 Concentrated acid 30-70% H2SO4 40 2-6 h 90
Enzymatic Cellulase 50 1.5 days 75 ɦ 95
Yang and Wyman (2004) reported that lignin removal improved cellulose
digestibility, with mechanisms postulated to improve cellulose accessibility,
enhancement of cellulase effectiveness by removal of cross linkages to carbohydrates and
xylan removal enhance glucan digestibility. Jeoh et al (2007) showed that xylan removal
enhanced biomass digestibility by increasing cellulose accessibility.
8
Before the hydrolysis started, enzyme adsorption onto solids was the primary step
for enzymatic hydrolysis of pretreated substrate (Kumar and Wyman, 2009) and the
hydrolysis rate or yield was claimed to be directly related to the amount of adsorbed
enzyme generally (Jeoh et al., 2007). Both cellulase and hemicellulase needed to adsorb
on the solid surface prior to enzymatic hydrolysis but the complexity of the hemicellulose
structure and the array of enzymes involved would result in limited studies of
hemicelluloltyic enzymes-substrate interactions for biomass (Kumar and Wyman, 2009a).
Kumar and Wyman (2009a) also showed that cellulase adsorption was very rapid
for substrate with the maximum in the first 10 min, as adsorption continued with time,
the equilibrium will reach slowly in less than 2 hr. They also found a biomass which
content higher lignin than others has high affinity to cellulase. Furthermore, they reported
that xylanase adsorption not only takes place on xylan but also on glucan and lignin in
biomass. This result corresponded with the earlier studies by Ryu and Kim (1998) that
used purified Pulpzyme HC to adsorb on lignin and crystalline cellulose. They showed
WKDWDVLJQL¿FDQW amount of xylanase was adsorbed onto lignin in alkaline solutions. The
binding of xylanase onto lignin was assumed to be caused by a physical interaction such
as the van der Waals interaction but the adsorption of purified xylanase onto crystalline
cellulose was not significant. Tenkanen et al. (1995) who used hemicellulases to adsorb
on xylan, mannan and cellulose found that the hemicellulases
were also incompletely
9
bound on mannan and were found to bind readily on cellulose.
Thus, the hemicellulosesprobably contained not only hemicellulose binding domain but also cellulose binding
domain.
Mansfield et al. (1999) reviewed the van der Waals contacts and hydrogen bonds
were dominant forces in carbohydrate-binding proteins. The CBDs generally have a low
content of charged amino acids and a high content of hydroxyl amino acids. Aromatic
amino acid residues, tryptophan and tyrosine, were thought to pack onto the sugar rings
and increased additional specificity and stability to the enzyme-substrates complexes. The
removal of the CBD reduced the hydrolytic efficiency of the enzymes on crystalline
cellulose but not on amorphous cellulose.
Palonen et al. (2004) used two purified cellulases (cellobiohydrolase and
endoglucanase) and their catalytic domains on steam steam pretreated softwood and
lignin. They found that both cellobiohydrolase and its catalytic domain exhibited a higher
affinity to steam pretreated softwood than endoglucanase or its catalytic domain. They
also found that removal of cellulose binding domain decreased the binding efficiency.
Their results indicated that the cellulose binding domain had a significant role in the
unspecific binding of cellulases to lignin.
On the other hand, Kumar and Wyman (2009a) showed that delignification enhanced
cellulase and probably xylanase effectiveness significantly when the substrates produced
10
by high pH pretreatment. The substrate that contained a significant amount of xylan
enhances glucose release and much more xylose release. Várnai et al. (2011) found that
the cellulase remained mostly bound throughout the hydrolysis of two different types of
substrates: Avicel and steam pretreated spruce (SPS). The surface of SPS, rich in lignin,
ZDVWKXVREYLRXVO\PRUHUHSXOVLYHWRZDUGVWKHȕ-glucosidase than the crystalline Avicel
surface. Catalytically GHOLJQL¿HG VRIWZRRG FHOOXORVH &26 which was low-lignin
containing substrate had the highest ȕ-glucosidase activity retained at the end of the
hydrolysis. The reason might be the less ordered, easily accessible and hydrolysable
structure of this substrate and possibly also the altered surface characteristics due to the
oxidative pretreatment. The xylanase seemed to depend more on the lignin content than
on the xylan content of the substrates. The study showed that the SPS containing the
lowest xylan amount adsorbed the xylanase more than the highest xylan-containing COS
or the low xylan containing Avicel. The binding behavior of xylanase was obscured and
could not be explained by the lignin or xylan contents in the substrate. Heiss-Blanquet et
al. (2011) found that cellulase aGVRUSWLRQDQGVSHFL¿FDFWLYLW\ZHUHOLNHO\WREHLQÀXHQFHG
by structural and compositional characteristics of lignocellulosic substrates. Both were
indeed found to be directly proportional to the cellulose content and indirectly
proportional to Klason lignin of the substrates. The former was positive correlation and
the latter was negative correlation, suggesting that lignin was one of the factors restricting
11
enzymatic hydrolysis. Ju et al. (2013) showed that, apart from its hindrance effect, xylan
could IDFLOLWDWHFHOOXORVH¿EULOVZHOOLQJDQGWKXVFUHDWHd more accessible surface area,
which improved enzyme and substrate interactions. Surface lignin had a direct impact on
enzyme adsorption kinetics and hydrolysis rate. Higher surface lignin content, especially
from K\GURSKRELFOLJQLQOHGWRORZHUFHOOXODVHDI¿QLW\WRthe substrate and lower initial
hydrolysis rate. Guo et al. (2014) found that lignin resources affected enzyme adsorption
using structure features such as functional groups and lignin composition. Guaiacyl (G)
lignin had a higher adsorption capacity on enzymes than syringyl (S) lignin. The low S/G
ratio and high uniform lignin fragment size had good correlations with high adsorption
capacity. They found that cellobiohydrolase (CBH) and xylanase were adsorbed the most
by all lignins, endoglucanase (EG) VKRZHGOHVVLQKLELWLRQDQGȕ-glucosidase (BG) was
the least affected by lignins. The results indicated the important role of carbohydrate-
binding module (CBM) in protein adsorption.
12
3. Materials and methods
3.1. Materials 3.1.1. Biomass
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
13
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.
14
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
15
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.
16
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.
3.2. Methods
3.2.1. 40
oC enzymatic hydrolysis and adsorption
The pulps were treated with enzyme at 40oC, pH 6 and the consistency was 1%.
From the results of Tsai, the enzyme dosage used 5 mg per gram substrate was more
suitable for analysis. Pulps were putted in an incubator and incubated for 125 rpm. Each
experiment was sampled at 1, 2, 4, 8, 12, 24, 48 hr and then centrifuged 6000 rpm to
separate the substrates and supernatant. The supernatant is determined the free enzyme
17
and released reducing sugar measured by Bradford protein assay using bovine serum
albumin as standard and DNSA (dinitrosalicylic acid) respectively.
3.2.2. 4
oC enzyme adsorption
The enzyme was added to a 0.1% consistency substrate which was dispersed in pH
6 sodium acetate buffer at 4oC condition to avoid hydrolysis. The buffer was integrated
0.1 M sodium acetate, 20 mM calcium chloride. The enzyme dosages were 5, 10, 15, 20,
30, 40, 50, 60, 70 mg per gram of oven dry substrate. The mixtures were turned on a
rotator for 1 hr to be adsorption equilibrium. After equilibrium, the mixtures were
centrifuged and the supernatants were measured by the Bradford protein assay using
bovine serum albumin as standard. The adsorbed enzyme was calculated by the difference
between the amount of protein initially added and the supernatant named free enzyme.
The analysis of adsorption parameters (maximum adsorption capacity [ı] and
equilibrium constant [Kd]) were determined by non-linear regression of the adsorption
data to the following Langmuir expression using Sigmaplot software (Lynd et al., 2002;
Kumar and Wyman, 2008):
[CE] = ߪ[ܵ௧][ܧ] ܭௗ + [ܧ]
18
in which [CE] was the amount of adsorbed enzyme in mg/mL, [Ef] the free enzyme
FRQFHQWUDWLRQLQPJP/ıWKH maximum adsorption capacity in mg/mg substrate, [St] the
substrate concentration in mg/mL, and Kdthe equilibrium constant = [C][E]/[CE] in mg
of enzyme/mL, where [C] was the concentration of free binding sites on the substrates in
mg/mL and [E] and [Ef] were the enzyme concentrations not adsorbed on the substrate in
mg/mL.
19
4. Results and Discussion
4.1. Chemical composition of substrates
The chemical compositions of substrates were shown in Table 2. The Kraft process
was effective to remove the lignin of biomass to about 3% and bleach process by DEDD
making the lignin content decreasing down to zero. On the other hand, because ASEP was
pretreated by dilute sulfuric acid, the percentage of hollocellulose was less than non-acid
pretreated and even Kraft pulps. Furthermore, the lignin content of ASEP was about 50%
which was similar to hollocellulose. From the Table 2, the biomass pretreated by alkali
process (BEK and UEK) had low lignin content and above 90% content of hollocellulose.
On the other hand, the biomass pretreated by acid process (ASEP and NSEP) had high
lignin content from 21.21 to 56.32 and lower hollocellulose content than Kraft pulps.
Table 2. Chemical composition of lignocellulose substrates Chemical composition (%, w/w)
Substrates Hollocellulose Xylan Lignin Ash
BEK 96.50 8.34 0 0.87
UEK 94.82 9.88 3.75 0.77
ASEP 58.88 1.10 56.32 1.85
NSEP 76.86 1.41 21.21 1.93
20
4.2. 40 o C hydrolysis and enzyme adsorption
Fig. 3 showed the UEK, BEK, ASEP and NSEP hydrolyzed by Pulpzyme HC at
40oC for 48 hr. UEK hydrolysis by Pulpzyme HC released more reducing sugar at first
but it would be overtaken by BEK after 24 hr. The ASEP and NSEP hydrolyzed by
Pulpzyme HC released little reducing sugar for 48 hr. The results could confirm from the
results of substrate chemical composition. ASEP didn’t content xylan and NSEP had a
little bit of xylan. UEK had the most xylan than other substrates.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 3. 40oC hydrolysis of Pulpzyme HC
21
Fig. 4 showed the 40oC adsorption of Pulpzyme HC. ASEP and NSEP adsorbed
almost all Pulpzyme HC from the beginning. BEK adsorbed the least Pulpzyme HC that
was half of ASEP and NSEP in the end and the rate of adsorption was slowly. UEK
adsorbed 3 mg/g substrate Pulpzyme HC in the beginning and finally adsorbed 4 mg/g
substrate. The affinity of UEK 40oC adsorption was apparently higher than BEK.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEK UEK ASEP NSEP
Figure 4. 40oC adsorption of Pulpzyme HC
22
Fig. 5 showed the results of XylX was similar with Pulpzyme HC. The reducing
sugar of UEK and BEK hydrolysis were higher than the others and after hydrolyzing 24
hr, the reducing sugar of UEK was still higher than that of BEK. ASEP and NSEP also
release little reducing sugar for 48 hr.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 5. 40oC hydrolysis of XylX
23
Fig. 6 showed the adsorption of XylX at 40oC. UEK adsorbed enzyme was equal to
ASEP and NSEP and the adsorbed enzyme was stable during 48 hr hydrolysis. BEK
adsorption was also less than other lignin content substrates. During 48 hr, BEK didn’t
adsorbed more XylX significantly. To all substrates, it seemed that XylX reached max
adsorption and stable in 1 hr.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEKUEK ASEP NSEP
Figure 6. 40oC adsorption of XylX
ġ
ġ
ġ
ġ
24
Figure 7 showed the hydrolysis of XylX-H2. Its results were like above two enzymes
that BEK and UEK could release a large amount of reducing sugar than ASEP and NSEP.
Furthermore, BEK released more reducing sugar than UEK after hydrolyzing 24 hr.
ASEP and NSEP also released little reducing sugar.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 7. 40oC hydrolysis of XylX-H2
25
Fig. 8 showed the 40oC adsorption of XylX-H2. Though BEK still adsorbed the least
XylX-H2 in the beginning but its catch up with other substrate after hydrolyzing 12 hr.
After the adsorption of BEK caught up with UEK at 12 hr, the reducing sugar of BEK
overtook that of UEK at 24 hr. It seemed that the hydrolyzed reducing sugar made more
binding site for enzyme to adsorb. To BEK that had no lignin content, it would have no
non-specific adsorption and made it releasing more reducing sugar than UEK.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEK UEK ASEP NSEP
ġ Figure 8. 40oC adsorption of XylX-H2
26
Fig. 9 was the hydrolysis of XylX-L2. The reducing sugar of BEK and UEK were
still higher than ASEP and NSEP but their reducing sugar were less than above enzyme
a lot. UEK released about 70 μmole that was half of above enzymes and BEK also
released about 50 μmole in 48hr. ASEP and NSEP both still had little reducing sugar
released.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 9. 40oC hydrolysis of XylX-L2
27
Fig. 10 showed the 40oC adsorption of XylX-L2. All substrates had max adsorption
in 1 hr and the adsorbed enzyme were stable during hydrolyzing 48 hr. The result of
adsorption was different from above enzymes which BEK adsorbed more XylX-L2 than
other substrates. Because the binding site of XylX-L2 had been deleted, its binding
interaction between substrates had to investigate more detail to understand the
phenomena.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEK UEK ASEP NSEP
ġ Figure 10. 40oC adsorption of XylX-L2
28
Figure 11 showed 40oC hydrolysis of CBM. Since CBM just had binding domain,
it couldn’t hydrolyze any polysaccharide. Its reducing sugar showed very little released
or not detectable in most substrates.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 11. 40oC hydrolysis of CBM
29
Fig. 12 showed the 40oC adsorption of CBM. All substrates adsorbed CBM and
reached maximum adsorption in 1 hr, which showed high affinity of CBM to all
substrates. As time went on, BEK and UEK adsorbed more CBM but ASEP and NSEP
had slightly desorption of CBM. BEK and UEK might had more crystalline zone for
CBM which had more specific force to bind but ASEP and NSEP only had lignin that
adsorbed enzyme by van der Waals force.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEK UEK ASEP NSEP
Figure 12. 40oC adsorption of CBM
30
Fig. 13 showed 40oC hydrolysis of GH 11. BEK and UEK released low reducing sugar
about 12 μmole. The reducing sugar of BEK and UEK was less than Pulpzyme HC, XylX
and XylX-H2 and came close to zero. The phenomenon was similar with XylX-L2 which
binding domain had deficiency and let it released less reducing sugar. Thus, it was
reasonably because GH 11 just had catalytic domain without binding domain. ASEP and
NSEP still had little reducing sugar released.
0 10 20 30 40 50
0 10 20 30
Released reducing sugar (%)
Time (hr) BEK
UEK ASEP NSEP
Figure 13. 40oC hydrolysis of GH 11
31
Fig. 14 showed the 40oC adsorption of GH 11. All substrates had reached maximum
adsorption in 1 hr. In addition to UEK, other substrates had slight desorption and the
adsorption of BEK increased from 1 to 8 hr and decreased after that. Although all
substrates adsorbed more GH 11 than Pulpzyme HC and XylX, the binding of GH 11
without binding domain was unstable for xylanase to hydrolyze xylan.
0 10 20 30 40 50
0 1 2 3 4 5 6
Adsorbed enzyme (mg/g substrate)
Time (hr)
BEK UEK ASEP NSEP
Figure 14. 40oC adsorption of GH 11
According to above results, BEK didn’t fully adsorb Pulpzyme HC and XylX but the
reducing sugar was equal to full adsorption of UEK. In substrates chemical composition,
ASEP didn’t have xylan and NSEP just had a little bit of xylan. ASEP and NSEP both
adsorbed maximum enzymes and they didn’t release reducing sugar. There were some
non-specific of lignin which had influenced on enzyme adsorption so there was no direct
32
relationship between the amount of bound enzyme and the extent of hydrolysis. On the
other hand, all substrates adsorbed more XylX-L2 which binding domain was partly
deleted than XylX and Pulpzyme HC but the reducing sugar was released less than both
of them. Moreover, GH 11 whose structure was only catalytic domain adsorbed on all
substrates indeed and it just released a little bit reducing sugar during hydrolyzing 48 hr
even less than XylX-L2. Furthermore, they were slightly desorbed from substrates, which
might be unstable of enzyme adsorption to cause the low efficiency of enzyme hydrolysis.
The binding domain of enzyme played an important role to make enzyme stable
adsorption before hydrolysis. On 40oC hydrolysis, alkali pretreated biomass like BEK and
UEK released much more reducing sugar because the pretreatment removing lignin
increased accessibility for xylanase to hydrolyze xylan.
33
4.3. 4 o C enzyme adsorption
4oC enzyme adsorption was an experiment that enzyme would not hydrolyze
substrates where was a more stable environment for enzyme just attaching on substrates
because the activation energy was not enough to make reaction start. Fig. 15 showed that
Pulpzyme HC was adsorbed by all substrate. BEK adsorbed the least Pulpzyme HC and
the others adsorbed similarly from 5 to 20 mg/g substrate of addition enzyme. In the other
substrates, ASEP adsorption came to stable first almost at 60 mg/g substrate of addition
enzyme. Initially, UEK had an upward trend and was stable in 70 mg/g substrate of
addition enzyme. After trying higher enzyme concentration, UEK adsorption came to
stable from 70 mg/g substrate of enzyme addition. Three lignin content substrates had
adsorbed more lignin that could be explained as lignin had high affinity to xylanase.
Moreover, UEK adsorbed more xylanase than ASEP and NSEP at high enzyme addition.
It seemed that xylanase would be adsorbed by lignin first and it would adsorbed on xylan
with specific bond after saturation of lignin adsorption.
34
0.00 0.02 0.04 0.06 0.08 0.10 0
5 10 15 20 25 30 35 40
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 15. 4oC adsorption of Pulpzyme HC
Fig. 16 showed the 4oC adsorption of XylX. After enzyme addition came to 30 mg/g
substrate, it was apparently that all substrates divided into two parts. One part included
BEK and UEK which were pretreated by pulping process and another included ASEP and
NSEP which were pretreated by steam explosion. Look back to the review of enzyme
hydrolysis and adsorption, ASEP and NSEP which were high lignin content adsorbed
more XylX than BEK and UEK. The adsorption of UEK was slightly higher than BEK
but it was not so significant difference. Perhaps, UEK still had 3% lignin content which
made it adsorbing enzyme easily. Furthermore, three substrates which had lignin content
35
adsorbed xylanase faster than BEK. It could be explained as lignin had high affinity with
xylanase.
0.00 0.02 0.04 0.06 0.08 0.10
0 5 10 15 20 25 30 35 40
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 16. 4oC adsorption of XylX
Fig. 17 showed 4oC adsorption of XylX-H2. NSEP could adsorb the most XylX-H2
and it came to stable at 60 mg/g substrate of enzyme addition. The adsorption of BEK
and UEK was similar they came to stable at 12mg/g substrate. Furthermore, the affinity
of UEK to XylX-H2 was higher than BEK because the adsorption of UEK got higher
before 60 mg/g substrate of addition enzyme. This phenomenon was similar with XylX
adsorption. Although ASEP didn’t adsorbed so much like NSEP, it also could see that
adsorption of steam explosion pretreated substrates was higher than that of pulping
process.
36
0.00 0.02 0.04 0.06 0.08 0.10 0
5 10 15 20 25 30 35 40
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 17. 4oC adsorption of XylX-H2
Fig. 18 was 4oC adsorption of XylX-L2. The adsorption of ASEP and NSEP was a
part which had a significant rising before 40 mg/g substrate of addition enzyme and still
rising after that. This phenomenon showed lignin had high non-specific adsorption with
xylanase although its CBM had deficient. BEK adsorbed the least XylX-H2 and didn’t
more than 5 mg/g substrate. UEK adsorbed about 10 mg/g substrate and still higher than
BEK did. BEK and UEK both decreased the adsorption capacity of xylanase because the
CBM of XylX-L2 was deficient. Especially BEK, the adsorption of BEK was unstable
even if the addition enzyme came to high concentration.
37
0.00 0.02 0.04 0.06 0.08 0.10 0
5 10 15 20 25 30 35 40
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 18. 4oC adsorption of XylX-L2
Fig. 19 showed 4oC adsorption of CBM. UEK, ASEP and NSEP adsorbed more CBM
than BEK and their adsorption didn’t have so much difference. Most of them came to
stable after 40 mg/g substrate of addition enzyme. Although BEK adsorption was the
least of all, its adsorption was stable when the addition enzyme was high concentration.
It showed that CBM played an important role on specific adsorption from the adsorption
of BEK.
38
0.00 0.02 0.04 0.06 0.08 0.10 0
5 10 15 20 25 30 35 40
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 19. 4oC adsorption of CBM
Fig. 20 showed that 4oC adsorption of GH 11. All substrates divided into four parts
but it was clearly that ASEP and NSEP adsorption were higher than BEK and UEK. ASEP
showed a very rapid adsorption of GH11 and it seemed still rising after 70 mg/g substrate
of addition enzyme. The adsorption of ASEP and NSEP seemed that they came to stable
after high enzyme addition but that of UEK and BEK still rose linearly without a flat
condition to say it was saturation and stable.
39
0.00 0.02 0.04 0.06 0.08 0.10 0
5 10 15 20 25 30 35 40 45 50
Adsorbed enzyme (mg/g substrate)
Free enzyme (mg/mL)
BEK UEK ASEP NSEP
Figure 20. 4oC adsorption of GH 11
40
4.4. Parameters of 4 o C enzyme adsorption
Parameters of 4oC enzyme adsorption were obtained by fitting Langmuir equation
and all of them were R> 0.84.
Table 3. Maximum enzyme adsorption of all substrates at 4oC 0$; ı, mg/g substrate)
Enzymes Substrates
BEK UEK ASEP NSEP
Pulpzyme HC 11.91 26.69 22.54 36.90
XylX 24.31 20.87 30.70 26.53
XylX H2 13.24 12.39 20.50 27.52
XylX L2 3.67 11.80 33.76 36.31
CBM 17.20 30.61 27.95 31.57
GH 11 25.20 39.30 45.18 52.60
From Table 3, BEK adsorption was the lowest to most xylanase except for XylX and
XylX-H2. Although BEK maximum adsorption on XylX and XylX-H2 was higher than
UEK on them, the maximum adsorption of XylX and XylX-H2 were not significant
difference between UEK and BEK. On the other hand, all substrates could also be divided
into two parts that acid pretreated and alkali pretreated substrates. Comparing to BEK
and UEK, ASEP and NSEP both had higher maximum adsorption to most xylanase and
sometimes it even came to almost two times of BEK and UEK. However, the adsorption
of Pulpzyme HC and CBM on UEK was similar with ASEP and NSEP. Perhaps, the
content of xylan was also one of important factors in these two xylanase.
41
Table 4. Equilibrium constant of enzyme adsorption at 4oC Equilibrium constant (Kd, g/L)
Enzymes Substrates
BEK UEK ASEP NSEP
Pulpzyme HC 0.0435 0.0242 0.0280 0.0617
XylX 0.0135 0.0046 0.0033 0.0023
XylX H2 0.0110 0.0033 0.0060 0.0064
XylX L2 0.0077 0.0012 0.0007 0.0012
CBM 0.0008 0.0009 0.0017 0.0023
GH 11 0.0460 0.0220 0.0016 0.0096
Table 4 showed equilibrium constant of enzyme adsorption, which meant the affinity
of interaction between enzyme and substrate. From the Langmuir equation, the less value
Kd was, the easier enzyme was adsorbed. Besides, in order to be more convenient to
compare, we showed the reciprocal of Kd as affinity (A) in Table 5.
Table 5. Affinity of enzyme adsorption at 4oC Affinity (A, L/g)
Enzymes Substrates
BEK UEK ASEP NSEP
Pulpzyme HC 22.99 41.32 35.71 16.21
XylX 74.07 217.39 303.03 434.78
XylX H2 90.91 303.03 166.67 156.25
XylX L2 129.87 833.33 1428.57 833.33
CBM 1250.00 1111.11 588.24 434.78
GH 11 21.74 45.45 625.00 104.17
42
To Pulpzyme HC, UEK was the highest affinity and NSEP was the lowest. To XylX,
NSEP had the highest affinity and then was ASEP, UEK and BEK. To XylX-H2, the
affinity of UEK was best and that of BEK was lowest. The affinity of ASEP and NSEP
were 166.67 and 156.25 that were not significant difference. To XylX-L2, ASEP had the
highest affinity and the affinity of UEK was equal to that of NSEP. To CBM, the affinity
of BEK was 12500 which was the highest of all substrates and ASEP and NSEP were
lower than BEK and UEK. To GH 11, the affinity of ASEP was the highest and BEK was
the lowest. Besides, there was a trend that high lignin content substrates was higher
affinity to xylanase generally. The trend of CBM affinity was reverse to other enzymes
because carbohydrate binding domain had higher affinity to crystalline region. BEK was
treated by pulping and bleaching process which made most lignin and xylan removed.
Thus, the crystalline region of BEK exposed to surface easily and was attached by CBM.
Compared to the maximum adsorption, the substrates which had higher affinity didn’t
mean it could adsorbed more enzyme. On Pulpzyme HC, NSEP had the highest maximum
adsorption but its affinity was lower than other substrates that maximum adsorption were
lower than it. There was the same phenomenon on the other substrates where maximum
enzyme adsorption was not proportional to its affinity.
43
Table 6. Binding strength of enzyme adsorption at 4oC
Binding strength (ɐ × A, mL/g substrate)
Enzymes Substrates
BEK UEK ASEP NSEP
Pulpzyme HC 254.17 341.23 805.00 598.06
XylX 1800.74 4536.96 9303.03 11534.78
XylX H2 1203.64 1208.33 3416.67 4300.00
XylX L2 476.62 9833.33 48228.57 30258.33
CBM 21500.00 34011.11 16441.18 13726.09
GH 11 547.83 1786.36 28237.50 5479.17
Table 6 showed the binding strength of all enzymes. Binding strength which was the
value of maximum enzyme adsorption multiplying with the value of affinity presented
which substrate had more influence on hydrolysis (Kumar and Wyman, 2009b). In the
Table 6, the value of ASEP and NSEP were larger than BEK and UEK generally. UEK
was larger than BEK a lot and only XylX-H2 without significant difference. The binding
strength which was different from other parameters such as maximum adsorption and
affinity had a significant trend. It was apparently that acid pretreated substrates had higher
binding strength than alkali pulping process substrates.
44
5. Conclusion
The composition of alkali pretreated substrates removed most of lignin and contained
more carbohydrate in substrates. The acid steam explosion pretreated substrates removed
most of hemicellulose and contained more lignin in substrates.
From the results of 40oC adsorption and hydrolysis, high lignin content substrates
adsorbed more xylanase and released little reducing sugar. Low lignin content substrates
adsorbed less xylanase and released more reducing sugar than them. The lignin content
was an important factor on enzyme adsorption and hydrolysis. Furthermore, carbohydrate
binding domain had an influence on hydrolysis of substrates. The reducing sugar of XylX-
L2 whose carbohydrate binding domain had deficiency was less than XylX and XylX-H2
whose carbohydrate binding domain was complete. The reducing sugar of GH 11 which
only had catalytic domain was the least of all.
From 4oC adsorption, steam explosion pretreated substrates adsorbed more enzyme
than alkali pretreated substrates did. In alkali pretreated substrates, UEK maximum
adsorption was higher than BEK except that XylX and XylX-H2 had no significant
difference.
The parameters of 4oC adsorption, the maximum adsorption of xylanase didn’t
correlate with hydrolysis and affinity. Because there was unspecific binding with lignin,
steam explosion pretreated substrtaes had better adsorption and affinity of xylanases.
45
Binding strength of BEK showed that xylanase with carbohydrate binding domain had
strong binding. The other substrates showed lignin also had significant influence because
the binding strength was the value of maximum adsorption multiplying with affinity.
46
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