β-xylanase from Thermomyces lanuginosus and its Biobleaching Application
Thermomyces lanuginosus is thermophilic fungus in which was isolated from widespread material. A high number of this fungus was found in composts especially mushroom composts. This fungus has been reported to produce a high level xylanase when cultivated in the medium containing xylan and corn cob as a carbon source. Various strains of T. lanuginosus produced a single xylanase with molecular masses in range of 22.0 to 29.0 kDa. Pure β-xylanase obtained from various strains of this fungus exhibited highly stability at high temperature and wide pH range. The optimal temperature and optimal pH of pure β-xylanase from various strains of T. lanuginosus have been reported in range of 60-75°C and pH 6.0-7.0, respectively. The great thermal stability was resulting from the present of hydrophilic amino acid on beta sheet of the surface of xylanase structure. Moreover, the relatedness between high and low xylanase producing strains can be distinguish by random amplification of polymorphic DNA (RAPD). Based on nucleotide sequences and T. lanuginosus xylanase gene has been classified to be a member of family 11 (formerly known as cellulase family G) glycosyl hydrolases. This enzyme was endo-type xylanase having main product are xylose and xylobiose. The expression of xylanase gene from T. lanuginosus was achieved in Escherichia coli and methylotrophic yeast Pichia pastoris. The ability of T. lanuginosus in which produced large amount of high thermos stable xylanase has made this fungus to be a source of xylanase production for biobleaching in pulp and paper process.
Received: February 19, 2010;
Accepted: April 28, 2010;
Published: June 23, 2010
Over the last few decades, there has been a growing interest in lignocellulose
bioconversion as a renewable energy source. Xylan is the major constituent of
hemicellulose and has a high potential for degradation to useful end products.
These compounds were present in the cell wall and in the middle lamella of plant
cells. They were classified according to the nature of the linkages joining
the xylose residues. β-1,3-linked xylans were found only in marine algae,
those xylans containing a mixture of β-1,3- and β-1,4-linkages only
in seaweeds and β-1,4-linked xylans occur in hardwoods, softwoods and
grasses (Barry and Dillon, 1940; Dekker
and Richards, 1976). Hetero-β-1,4-D-xylans constitute the major portion
of the hemicellulose in terrestrial plants (Whistler and Richards,
1970). Native xylans were complex polymers containing vary amounts of arabinose,
4-O-methylglucuronic acid and acetic acid groups attached to the main xylose
chain, depending on the botanical origin of the xylan (Johannson
and Samuelson, 1977; Puls and Schuseil, 1993). Microbial
xylanases are the preferred catalysts for xylan hydrolysis due to their high
specificity, mild reaction conditions and negligible substrate loss and side
product generation. Xylanases have found applications in the food, feed and
pulp and paper industries (Wong and Saddler, 1993). Xylanases
are special significance to the pulp and paper industry, where they can reduce
the amount of chlorine and chlorine dioxide used for bleaching paper pulp. Xylanase
pretreatment has been reported to lower bleaching chemical consumption and to
result in greater final brightness (Kulkarni et al., 1999).
Since pulp-bleaching processes are carried out at high temperature and under
alkaline conditions, thermostable and alkali-tolerant xylanases are well suited
for such industrial processes. The potential benefits of using these enzymes
for biotechnological processes has encouraged widespread research endeavors
towards producing desirable xylanases through protein engineering using techniques
such as site-directed mutagenesis (Wakarchuk et al., 1994;
Georis et al., 2000; Mesta et al.,
2001; Turunen et al., 2001, 2002;
Liu et al., 2002) directed evolution (Arase
et al., 1993; Chen et al., 2001; Inami
et al., 2003; Palackal et al., 2004) and DNA shuffling
(Shibuya et al., 2000; Ahsan et al., 2001; Gibbs et al., 2001). Thermomyces lanuginosus
DSM 5826 produces a high level of cellulase-free, thermostable xylanase, which
is catalytically active over a broad pH range (Singh et al., 2003). This xynA was first cloned into E. coli as a LacZ-fusion protein
(Schlacher et al., 1996) and the protein was later crystallized
to elucidate its enzyme structure and mechanism of catalysis (Gruber
et al., 1998). This served as the basis for further improvement of the enzyme
on the genetic level. Directed evolution has been used to improve the existing
properties of enzymes (Giver et al., 1998). This revolutionary
type of protein engineering technology mimics Darwinian evolution in nature
and does not require extensive knowledge of the gene of interest. It consists
steps of random mutagenesis, screening and recombination (Arnold
and Volkov, 1999). However, evolution in nature may give rise to strains
producing enzyme with different properties including their thermostability.
CHARACTERISTICS OF Thermomyces LANUGINOSUS
Thermophilic fungus Thermomyces lanuginosus (formerly known as Humicola lanuginosa)
was classified as a Deuteromycetes (imperfect fungus) that was unicellular or
septate and reproduces asexually by forming aleurioconidia. The colonies of
fungus grew rapidly at 45-50°C within 2 days. Initially, the colonies appeared
white but soon turn grey or greenish-grey and mature colonies appeared dark
brown to black. The colonies have a little various colour depend on isolated
source (Fig. 1a) (Khucharoenphaisan and
Kitpreechavanich, 2006). Immature conidial spore were colorless and smooth
walled and turned dark brown and globe as mature (Fig. 1b).
Mature aleuriospores showed reticulate sculpture with a diameter of 5.5-12 μm.
The aleuriopores were straight or curved, colorless or brown and smooth (Cooney
and Emerson, 1964).
T. lanuginosus was widely distributed (Emerson, 1968)
and has been isolated in various location and ecology (Singh
et al., 2000a; Hoq and Deckwer, 1995; Hoq
et al., 1994). Large amount of this fungus was found in mushroom compost
(Khucharoenphaisan and Kitpreechavanich, 2004). It grew
at temperature of 30°C to 60°C with an optimum growth temperature of
50°C. The optimum pH for growth of most strains was 6.5.
The genetic diversity among T. lanuginosus strains obtained from various geographical
locations was found to be low. PCR-based amplification of the nuclear ribosomal
DNA and the subsequent sequencing of these fragments pointed to a high degree
of conservation in the rDNA region of the genome of T. lanuginosus. The 5.8
S rDNA and the flanking ITS was conserved regions frequently used in phylogenetic
studies for differentiation among species and populations within species (Mitchell
et al., 1995). However, the study of Singh et al. (2000b)
indicated that this ITS region was appropriated for phylogenetic comparisons
within this species. A homology search by BLAST (National Center for Biotechnology
Information, USA) has shown that the ITS region and the 5.8 S rDNA sequences
were highly conserved in others thermophilic fungi, suggesting a possible recent
taxonomic divergence in this group of fungi.
The Deuteromyces genus Thermomyces was closely related to Humicola and has
been combined with it by several authors. However, it could be distinguish by
aleurioconidia, which have an ornamented surface and were generally supported
by distinct stalk cells. Aleurioconidia of Thermomyces mostly arising on 10-15
μm long lateral stalk cell, dark brown, thick walled, with wrinkled
surface, 6-10 μm. The genus of Thermomyces containing four species was
T. lanuginosus, T. verrucosus, T. ibadanensis and T. stellata. T. lanuginosus
has aleuriospores 6-12 μm diameter which globose and irregularly sculptured,
which were typical characteristics for identification of the species. T. verrucosus
was distinguished from T. lanuginosus by a verrucose aleurioconidia 10-17 μm
diameter. In case of T. ibadanensis has smooth walled aleurioconidia 4-8 μm
diameter. T. stellatus has aleurioconidia in which was singly on the tip of
the aleuriophore were dark brown and stellate with maturity, 5.3 μm
diameter and 7.6 μm in length.
Xylan was a complex structure of the hemicelluloses in wood. The two main enzymes
using for de-structure of the xylan backbone were β-xylanase and β-xylosidase.
β-xylanases hydrolyze randomly on the backbone of xylan to make shorter
chain oligomers as xylooligosaccharides xylobiose and monosaccharide as xylose.
β-xylosidases were essential for the complete breakdown backbone of xylan
to xylose at the non-reducing end (Poutanen and Puls, 1988).
To complete hydrolysis of xylan, debranching enzymes such as α-arabinofuranosidase,
α-glucuronidase, acetylxylan esterase and hydroxycinnamic acid esterases
that cleave side chain residues from the xylan backbone were required to release
the substituents on the xylan backbone. A total hydrolysis of xylan to monosaccharide
was achieved from this reaction (Fig. 2). All these enzymes
act cooperatively to convert xylan to its constituents (Sunna
and Antranikian, 1997).
PRODUCTION OF β-XYLANASE BY T. LANUGINOSUS
T. lanuginosus strain SSBP has been reported to be the best producer
of xylanase with an activity of 3575 U mL-1 than that of T. lanuginosus
strains DSM 5826 and ATCC 46882 with xylanase activity of 2172 and 2726 U mL-1,
respectively in shake-flask cultures (Singh et al.,
2000c; Puchart et al., 1999; Purkarthofer
et al., 1993; Bennett et al., 1998).
When T. lanuginosus was cultivated on various carbon sources, significant
differences of xylanase production were occurred (Khucharoenphaisan
et al., 2010a). Corncobs were found to be the most effective substrate
for xylanase production among various lignocellulosic substrates such as corn
leaf, wheat bran, wheat straw, barley husk and birchwood xylan (Singh
et al., 2000a, c; Gomes
et al., 1993a; Purkarthofer and Steiner, 1995;
Bennett et al., 1998). The strain of THKU-86
produced high level of xylanase in the medium containing xylan as a carbon source
whereas strain THKU-9 produced high level of xylanase in the medium containing
xylose as a carbon source (Khucharoenphaisan et al.,
2009). A shaking speed has most effect on xylanase production by this fungus.
At 120 rpm was provided the optimal conditions for enzyme formation whereas
decreased shaking speed to 100 rpm resulting in reduced dramatically enzyme
production. At high shaking speeds of 150-250 rpm, the adversely affect was
occurred on enzyme production due to greater hypha branching, mycelium fragmentation
and early sporulation (Purkarthofer et al., 1993).
The production of xylanase by T. lanuginosus THKU-49 was also studied
in shaking cultivation at 45°C for 7 days using 1% oat spelt xylan as a
carbon source. Xylanase production was rapidly increased during 4-day cultivation,
yielded 45.7 U mL-1. It then increased to some extent, 62.7 U mL-1 after 7 day
cultivation (Khucharoenphaisan et al., 2010b).
Random amplification of polymorphic DNA (RAPD) was a modification of the Polymerase
Chain Reaction (PCR) in which a single primer able to anneal and prime at multiple
locations throughout the genome can produce a spectrum of amplified products
that were characteristics of the template DNA (Welsh and
McClelland, 1991; Williams et al., 1990). This technique
has been used for molecular genetic studies as it was a simple and rapid method
for determining genetic diversity and similarity in various organisms. It also
has the advantage that prior knowledge of the genome under research was not
necessary (Yoon and Kim, 2001). Khucharoenphaisan
et al. (2009) reported that this fungus could be difference into two groups
based on their ability to produce xylanase using xylose as sole of carbon source.
The phylogenetic analysis obtained from random amplified polymorphic DNA (RAPD)
pattern using primer UBC 24 (5’-GCCCGACGCG-3’) pointed to greater
diversity of high (cluster B) medium (cluster A) and low (cluster C) xylanase
producing strains using xylose as a carbon source as shown in Fig.
3. This result be in line with the formerly study of Singh
et al. (2000b) whose examined the phylogenetic properties of eight T. lanuginosus
strains and found relationship between the RAPD pattern and levels of xylanase
||Dendogram indicating relationships of T. lanuginosus strains
obtained with the primer UBC 241 of xylanase producing strains with xylanase
activity obtained from 5-day cultivation using xylose as a carbon source
(Khucharoenphaisan et al., 2009)
It could be establish using certain primer UBC 241. Strains DSM 5826 and SSBP
that produced xylanase of 32000 and 59600 nkat mL-1, respectively, were apparently
closely related while strains ATCC 28083 and ATCC 58160 that produced xylanase
of 9000 and 6300 nkat mL-1, respectively, also showed a close relationship.
However, not all strains producing low levels of xylanase were grouped together
indicating that RAPD analysis with primer UBC 241 has result in an ambiguous
separation of strains based on their ability to produce xylanase. This observation
would assist in attempts to find other high xylanase producing strains.
REGULATION OF T. LANUGINOSUS XYLANASE SYNTHESIS
Xylanases has been shown to be inducible enzymes but rare constitutive xylanase
expression has also been reported. Addition of inducer in the medium showed
higher enzyme production than that of un-induced condition. In general, the
xylanase induction was a complex phenomenon and the level of response to an
individual inducer varied depend on the organisms. The substrate derivatives
and the enzymatic end products might often play a key positive role on the induction
of xylanases. However, they could also act as the end-product inhibitors, possibly
at much high concentrations (Kulkarni et al., 1999).
Generally, xylanases were induced in most microorganisms during growth on substrates
containing xylan (Purkarthofer and Steiner, 1995; Khucharoenphaisan
et al., 2010a; Ahmed et al., 2003). Xylan having
high molecular mass could not penetrate the cell wall. Thus, hydrolysates of
xylan such as xylose, xylobiose, xylooligosaccharides and heterodisaccharides
could play a key role in the regulation of xylanase biosynthesis (Kulkarni
et al., 1999). Xylanase produced by T. lanuginosus was shown to correspond
to an induction or repression mechanism with Aspergillus sydowii MG49 (Ghosh
and Nanda, 1994). A low level of xylanase was constitutively formed without
the presence of an inducing substance. Xylanase production of T. lanuginosus
DSM 5826 was induced by D-xylose and having the strongest effect (1225 nkat
mL-1) indicating that D-xylose was the natural inducer. The highest xylanase
activity (7100 nkat mL-1) of T. lanuginosus DSM 5826 was found in xylan-grown
culture whereas very low activity (3.5 nkat mL-1) was found in glucose-grown
culture (Purkarthofer and Steiner, 1995). This was differenced
from the report of Khucharoenphaisan et al. (2010a) who
reported that xylan was the best inducer for xylanase production in cell culture
of T. lanuginosus TISTR 3465. This indicated that these two strains might have
different induction mechanism. The induction mechanism of xylanase from T. lanuginosus
TISTR 3465 by xylose and xylan in resting cell was also studied by Khucharoenphaisan
et al. (2010a). With the sequential addition of xylan, xylanase formation
was delayed but lasted longer. The xylanase secretion showed a dependence on
the concentrations of the inducer. Therefore, the availability of an inducer
at low levels and over extended period was thought to lead to hyper-production
of enzyme in T. lanuginosus DSM 5826 (Purkarthofer and Steiner,
1995). Hoq et al. (1994) reported that 10 g L-1
birch wood xylan and 30 g L-1 corncob induced xylanase synthesis in cultured
growth of T. lanuginosus RT9 in which isolated in Bangladesh with activities
of 8725 and 7110 nkat mL-1, respectively. In contrast, Xylose (5 g L-1) repressed
xylanase synthesis of this fungus with activity of 19 nkat mL-1. Moreover, the
xylanase formation using 5 g L-1 glucose and non carbon source having 7 and
12 nkat mL-1, respectively, were similar to xylanase level using xylose as a
carbon source. Xiong et al. (2004) reported that 15
g L-1 of substrates such as xylan and xylose stimulated xylanase formation of
T. lanuginosus DSM 10635 with activities of 497 and 83.2 U mL-1, respectively,
in growing cell condition for 4 days. In contrast of those, glucose and non-carbon
source repressed xylanase formation with activities of 0.31 and 0.95 U mL-1,
In the presence of easily metabolisable substances such as glucose, fructose
or lactose, xylanase was also formed, although, the activity in the presence
of these repressors was similar to basal levels (Purkarthofer
and Steiner, 1995; Khucharoenphaisan et al., 2009;
Khucharoenphaisan et al., 2010a). Xylan had the most pronounced
effect on xylanase production by this fungus as the level of induction. D-xylose,
D-arabinose, D-ribose and L-arabinose does not occur to the same degree as xylan.
During the initial induction period, T. lanuginosus DSM 5826 only formed constitutive
levels of xylanase activity, which led to slow liberation of xylooligosaccharides
from xylan. These fragments induced xylanase production leading to a highly
final level of enzyme activity.
CHARACTERIZATION OF T. LANUGINOSUS XYLANASE
Xylanase of T. lanuginosus has been purified from a number of strains and used
for characterization of enzyme. The molecular mass of the enzyme was found to
be in the range of 22.0-29.0 kDa and pI value between 3.8 and 4.1 (Bennett
et al., 1998; Anand et al., 1990; Cesar
and Mrsa, 1996; Lin et al., 1999; Kitpreechavanich
et al., 1984; Bakalova et al., 2002; Xiong
et al., 2004; Khucharoenphaisan et al., 2010a, b).
The optimum temperature and pH of purified xylanase from various strains have
been reported to be in the range of 60-75°C and 6.0-7.0, respectively (Table
1). These values were similar to those observed in crude extracts of xylanase
(Lischnig et al., 1993; Singh et al., 2000a, c; Gomes et al.,
1993a, b; Alam et al., 1994;
Khucharoenphaisan et al., 2010b). The thermal stability
was considered as an major characters xylanase from T. lanuginosus. The xylanase
of T. lanuginosus strain SSBP was previously reported to be the most stable
(half-life = 337 min at 70°C), whereas DSM 5826 strain and other strains
showed lesser stability. The xylanase of T. lanuginosus strain SSBP retained
its full activity at temperatures up to 65°C and 45% of its activity after
30 min at 100°C (Singh et al., 2000d). Up to date,
xylanase produced by T. lanuginosus THKU-49 has the highest thermostability
with half-life of 336 min. Moreover, this enzyme was more stable in phosphate
buffer than that in citrate buffer. When the buffer concentration increased,
the half-life of the enzyme decreased significantly. The high thermostability
of this enzyme because of single substitution (V96G) with signal peptide was
occurred at outer surface of the enzyme structure as shown in Fig.
4 (Khucharoenphaisan et al., 2008a).
The xylanase activity obtained from T. lanuginosus THKU-49 was inhibited by
Mn2+, Sn2+ and EDTA. The xylanase showed high activity towards soluble oat spelt
xylan but it exhibited low activity towards insoluble oat spelt xylan. No activity
was found to carboxymethylcellulose, avicel, filter paper, locust bean gum,
cassava starch and p-nitrophenyl β-D-xylopyranoside. The apparent Km value
of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was
7.3±0.236 and 60.2±6.788 mg mL-1, respectively. Xylanase from
strains ATCC 46882 and SSBP liberated mainly xylose and xylobiose from beechwood
O-acetyl-4-O-methyl-D-glucuronoxylan (Bennett et al., 1998;
Lin et al., 1999). Similarly xylanase from strain ATCC
46882 released xylose and xylobiose from beechwood 4-O-methyl-D-glucuronoxylan
and in addition also released an acidic xylooligosaccharide from 4-O-methyl-D-glucuronoxylan.
The hydrolysis of oat spelt xylan yield mainly xylobiose and xylose as end
products. However, the xylanase could not release xylose from substrate as xylobiose.
This suggested that it was an endo-xylanase (Khucharoenphaisan
et al., 2010b).
The Central Composite Design (CCD) was a statistic method wildly used in many
application including enzyme technology (Heck et al., 2006).
The CCD had been used for optimization of culture condition (Couto
et al., 2006) and also optimal temperature and optimal pH for maximum enzyme
activity (Khucharoenphaisan et al., 2008b). The maximum
xylanase activity of T. lanuginosus THKU-49 was obtained from CCD analysis was
66°C and pH 6.3 (Khucharoenphaisan et al., 2008b).
The temperature stability of the purified xylanase from various strains differed
somewhat depending on the experimental conditions (Table 1).
Overall, the crude enzyme of T. lanuginosus strain was apparently more thermostable
than the purified xylanase. Lin et al. (1999) suggested
that some unknown factors might be present in the extract that stabilizes the
protein. The kinetic properties of purified xylanases from T. lanuginosus have
been investigated (Table 1).
Gruber et al. (1998) was found that the structures
of the xylanase from T. lanuginosus closely resemble structures of other family
11 xylanases. The two active-site glutamates were consistent with Glu117 acting
as the nucleophile and Glu209 acting as the acid-base catalyst. The fully conserved
residue of Arg122 stabilized the negative charge on Glu117. Modeling studies
of an enzyme-xyloheptaose complex indicated that only the three central sugar
units were rigidly bound. The thermostability of this xylanase was due to the
presence of an extra-disulfide bridge not observed in most mesophilic variants,
as well as to an increase in the number of ion-pair interactions.
Stephens et al. (2007) improved the thermostability
of the xylanase from T. lanuginosus DSM 5826 by directed evolution using error-prone
PCR. The amino acid sequences of xylanase from the mutants that enhanced the
thermostability differed in 3 amino acids for mutant 2B7-6 and had single mutation
for mutants 2B11-16 and 2B7-10. Only one amino acid substitution (D72G) of xylanase
from mutant 2B11-16 and substitution (Y58F) of xylanase from mutant 2B7-10 resulted
in increasing of half-life for 2-time (from 89 min to 168 min at 70°C) and
2.5-time (from 89 min to 215 min at 70°C), respectively. The single amino
substitutions of xylanase in mutant 2B7-10 were occurred on the β-sheet,
which was the hydrophilic at the outer surface of the enzyme structure. However,
the most of amino substitution for the mutants producing high thermostable xylanases
occurred within the β-sheet of enzyme in which forms the hydrophobic region
of the enzyme (Stephens et al., 2007). Evolution in
nature gives also rise to T. lanuginosus producing enzymes with different properties
including their thermostability (Khucharoenphaisan et al.,
Molecular and structure of xylanase from T. lanuginosus. The complementary
DNA (cDNA) of T. lanuginosus xylanase containing 989 bp and included open reading
frame (ORF) 615 bp was firstly reporteded by Schlacher et al. (1996). An ATG codon (starting site) was identified on 36 bp downstream
of the 5’ of the cDNA. The ORF of xylanase gene encoding for 225 amino
acids. The N-terminal of 31 amino acids represented a signal sequence (Schlacher
et al., 1996; Gruber et al., 1998). In addition,
the region around amino acid residue 32 reassembled a KEX-like protease cleavage
site resulting in a processed polypeptide starting at the amino acid glutamine
(Singh et al., 2003). The multiple alignment of amino
acid sequence of xylanase gene obtained from highly thermostable producing strain
(THKU-49) and low thermostable producing strain (THKU-9 and DSM 5826) showed
some differentiation among these strains (Fig. 5). One amino
acid differentiation was found at position 96. Valine (V) was found in both
of low thermostable producing strain while valine was replaced by glycine (G)
in highly thermostable producing strain. This may imply that they have some
modification in this fungus (Khucharoenphaisan et al., 2008a).
The phylogenetic relationship between fungal xylanase was studied. Based on
amino acid sequences, xylanase from T. lanuginosus TISTR 3465 is identical to
T. lanuginosus DSM 5826 and closely related to other thermophilic fungi especially
Humicola sp. and Scytalidium thermophilum as shown in Fig. 6.
||Alignment of amino acid sequence of xylanase genes from different
strains of T. lanuginosus strains. Alignment characters are indicated as
follows: ‘*’ indicates position with a conserved amino acid
residue; ‘.’ indicates position with a different amino acid
residue (Khucharoenphaisan et al., 2008a)
||Phylogenetic tree of amino acid sequence analysis of xylanase
of thermophile, mesophile and thermophilic fungi constructed by Neighbor-joining
method from MEGA 4 program. Scale bar shown distance values under the tree
means 0.05 substitutions per amino acid position. Bootstrap analyses were
performed with 1000 re-samplings and percent values are shown at the branching
points (Khucharoenphaisan et al., 2010a)
Xylanase produced by T. lanuginosus strain was folded into a single ellipsoidal
domain. The overall structures of xylanase were similar and have been described
as a partially closed right hand (Fig. 4). The active site
was located at the concave side of the cleft (Torronen et al., 1994). Two conserved glutamate residues were catalytically active residues
and were located on either side of the cleft. According to mutagenesis and mechanism-based
inhibitors, these residues have been identified as a nucleophile and an acid:base
catalyst (E86 and E177) in the T. lanuginosus xylanase structure, respectively
(Gruber et al., 1998).
Xylanase gene from T. lanuginosus has been expressed in Escherichia coli but
the expression level was lower than that of original host. Most expressed xylanase
was found as inclusion body in the cytoplasm of the cell. The absence of post
translational modification such as glycosylation in E. coli and intracellular
accumulation of recombinant xylanase have been suggested to be the key reason
(Singh et al., 2003). The xylanase gene incorporating
the secretion signal sequence of T. lanuginosus DSM 5826 was functionally expressed
in E. coli but extracellular enzyme activity was not reported (Schlacher
et al., 1996). Subsequently the recombinant E. coli was found to produce
up to 240 U mL-1 of intracellular xylanase when induced by 0.1 mM isopropyl
thiogalactoside (Singh et al., 2003). Xylanase gene
(xynA1) including signal peptide from T. lanuginosus DSM 5826 was synthesized
to construct the expression vector pHsh-xynA1. After optimization of mRNA secondary
structure in the translation initiation, the expression level was increased
from 1.3 to 13% of total cell protein. Maximum xylanase activity of 47.1 U mL-1
was obtained from cellular extract (Yin et al., 2008)
The expression of xylanase gene from T. lanuginosus THKU-49 in E. coli has been
compared between with and without 31 amino acid signal peptide from original
but the expression level was not different. Recently, the using the methylotrophic
yeast Pichia pastoris as a host is particular interesting in enzyme expression.
A distinct advantage of eukaryotic expression host is capacity to facilitate
the post translation modification of enzyme. Highly efficient expression of
xylanase gene from T. lanuginosus IOC-4145 was achieved in P. pastoris under
the control of the AOX1 promoter. The secretion level of recombinant XynA was
in range of 90 to 126 U mL-1 after 90 h induction. The maximum expression of
recombinant XynA was occurred after optimization by factorial design and showed
the enzyme activity of 360 U mL-1 (Monica et al., 2003).
The xylanase gene from T. lanuginosus 195 was also successfully expression in
P. pastoris. However, maximum xylanase activity of 26.8 U mL-1 was obtained
after 120 h induction of the recombinant culture without optimization of the
condition (Gaffney et al., 2009). The enhancing of recombinant
xylanase production in eukaryotic host may manipulation of codon usage. The
signal sequence peptide also has been affected on the heterologous expression
(Ghosalkar et al., 2008). It is anticipated that the
xylanase expression level will be identical to original production in thermophilic
fungus T. lanuginosus.
BIOLOGICAL APPLICATION OF XYLANASES IN PULPS
Among hemicellulytic enzymes obtained from T. lanuginosus, xylanase has widely
use in biotechnological applications especially pulp and paper industry. The
xylanase of T. lanuginosus belong to family 11 and found to be very efficient
in biobleaching. Therefore, the high thermostable xylanase of T. lanuginosus
was suitable for high temperature processes. The low molecular weight of this
enzyme has assisted in penetrating the enzyme into the interior part of the
fiber, resulting in removal of lignin compound from the pulp (Beg
et al., 2001). There were two hypotheses about the role of xylanases in
biobleaching process. In the first, the xylanases act on the xylan precipitated
on the lignin (Viikari et al., 1994). It is able to remove
xylan in which was precipitated at the end of the cooking stage. This action
would leave lignin to the compounds employed in the bleaching of cellulose pulp.
The second hypothesis was inhibited lignin to form complexes with polysaccharides
such as xylan during the kraft process (Buchert et al.,
1992). The xylanases acted also by cleaving the interaction between the
lignin and xylan resulting on open the structure of the cellulose pulp. Thus
low amount of chemical could increase brightness of pulp (Paice
et al., 1992).
Xylanase was applied as a bleaching agent to reduce amount of chlorine required
for increasing brightness of kraft and sulfite pulp which was produced from
sugar cane bagasse, eucalyptus and beech (Manimaran et al.,
2009). A diagrammatic flowchart involving xylanase in bleaching technology
was represented in Fig. 7. It showed excellent results by
enhance the extractability of lignin in pulp bleaching process (Viikari
et al., 1994). Several studies have been reported about the application
of xylanase in biobleaching of softwood and hardwood (Khucharoenphaisan
et al., 2001; Oakley et al., 2003). However, studies
on biobleaching of non-woody plant pulps such as wheat straw and bagasse pulp
also has been reported (Li et al., 2005; Manimaran
et al., 2009; Christopher et al., 2005). Significant
reduction of chemicals required to attain the desired kappa number was found
while increased brightness and viscosity was achieved (Gubitz
et al., 1997; Haarhoff et al., 1999; Madlala
et al., 2001). The brightness of bagass pulp was improved by two units with
50 U gram pulp-1 of crude xylanase obtained from T. lanuginosus SSBP (Manimaran
et al., 2009) as compared to xylanase obtained from T. lanuginosus ATCC
46882 and ATCC 36350 (Christopher et al., 2005). The
brightness of the wheat straw pretreated with xylanase of T. lanuginosus CBS
288.54 showed 7.8 points increase (Jiang et al., 2006).
Xylanase from T. lanuginosus TISTR 3465 (Humicola lanuginosa) exhibited promising
result when applied as prebleaching agent to paper mulberry pulp and eucalyptus
pulp. Each pulp was prebleached at 50°C for 3 h with T. lanuginosus TISTR
3465 β-xylanase obtained from solid state culture. In case of eucalyptus
pulp, the enzyme treatment resulted in 1.4 unit reduction in kappa number and
increase in brightness value of 5.3 points. The combination of enzyme treatment
and peroxidase extraction resulted to reduce the kappa numbers by 5.7 unit and
have brightness value of 17.4 points. In contrast, the enzymes could not increase
the reduction of kappa no. and brightness of paper mulberry pulp with or without
peroxide extraction. Therefore, the effectiveness of T. lanuginosus TISTR 3465
β-xylanase for biobleaching may be depended on the nature and quality
of pulps (Khucharoenphaisan et al., 2001). It is possible
that the different on brightness improvement from each T. lanuginosus xylanase
on pulps could be due to different amount of xylan present in each type of pulp.
Scanning electron microscope was used to determine to morphological of pulp
after pretreated with xylanase. The control pulps such as bagasse and wheat
straw were smooth surface and uniform whereas xylanase pretreated pulp showed
irregular on the peeled fibers on the surface as shown in Fig.
8a and b (Manimaran et al., 2009).
||Xylanase application processes flowchart
||(a) Scanning electron micrographs of control bagasse pulp
and (b) treated with crude xylanase at 50°C for 3 h (Manimaran
et al., 2009)
T. lanuginosus showed significant vary in xylanase productivity using corn
cob and xylose as substrate in submerge cultivation. This apparent contradiction
was found in RAPD analysis. The xylanase of T. lanuginosus has considerable
for its biotechnological potential in biobleaching of pulp. The properties of
xylanase are suitable for biobleaching of pulp due to a highly stable on high
temperature and alkaline pH. However, attempt to improve the stability especially
more pH stability is needed because strong alkaline pH was occurred in pulping
process. To remove the effect of unknown extracellular component contributed
the thermostability, the pure enzyme should be used as sample and amount of
enzyme should be reported because enzyme concentration also effected on its
thermostability. It is not clear why xylanase obtained from T. lanuginosus TISTR
3465 more induced by xylan than xylose, xylobiose and xylooligosaccharide at
various concentrations. This reports differed from T. lanuginosus DSM 5826.
This may imply that there is another factor importance for xylanase induction
in this fungus which interesting to future research.
The authors wish to thank Dr. Vichien Kitpreechavanich and Dr. Shinji Tokuyama
for their cooperation and help. This work was partially supported by the Thailand
Research Fund through the Royal Golden Jubilee Ph.D. Program and Student Exchange
Support Program (Scholarship for Short-term Study in Japan)-JASSO. The authors
also thank Phranakhon Rajabhat University where are our office.
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