Induction and Repression of β-Xylanase of Thermomyces lanuginosus TISTR 3465
The effect of carbon sources on the production of β-xylanase by Thermomyces lanuginosus TISTR 3465 was investigated. Xylan showed the highest inductive effect on the enzyme formation whereas xylobiose and xylooligosaccharides resulted in lesser effect. β-Xylanase was also produced at low level with xylose as well as other sugars tested. Xylan concentration at 15 g L-1 gave the maximum inductive effect on β-xylanase formation, whereas xylooligosaccharides and xylose were effective at a lower concentration of 2.5 g L-1. High concentrations of these sugars significantly repressed the enzyme formation. Crude enzyme from the supernatants, without and with other sugars produced a single xylanase band on non-denaturing PAGE gels. However, an intense xylanase activity band was observed from the supernatant of media with xylan, xylobiose and xylooligosaccharides as the carbon sources. An intense protein band of 24.9 kDa from the culture filtrate of xylan medium was observed. Xylan increased β-xylanase production by the fungus 16-fold when it was added to the xylose medium after cultivation for 3 days. In contrast, addition of xylose to the xylan medium decreased β-xylanase production 3-fold. A distinct appearance and disappearance of a 24.9 kDa protein and the activity band coincided with an increase and decrease of xylanase activity, respectively. This indicated the synthesis of xylanase by this strain was most induced by xylan. Moreover, the level of xylanase induction has no related to amino acid sequence of the enzyme.
Microbial xylan-degrading enzymes are potentially important in the biobleaching
process of pulp and paper, improving the quality of animal feed, increasing
the quality of bread and the conversion of lignocellulose to fermentable sugars
for the production of chemicals and fuels (Jäger
et al., 1992; Viikari et al., 1993; Sunna
and Antranikian, 1997; Beg et al., 2001).
β-Xylanase (Endo 1,4-β-D-xylan xylanohydrolase, EC 18.104.22.168) is one
of the important enzymes involved in xylan degradation working synergistically
with other debranching enzymes (Yasui et al., 1984;
Rapp and Wangner, 1986; Hrmova
et al., 1991). Numerous bacteria, streptomycetes and fungi can degrade
xylan by producing a range of enzymes (Abdeshahian et
al., 2009). Thermomyces lanuginosus is a potent thermophilic
strain which produces a single cellulose-free endo β-xylanase (Kitpreechavanish
et al., 1984; Anand et al., 1990;
Singh et al., 2000, 2003).
Xylan, xylose, xylobiose and non-metabolizable β-xylosides known to be
common inducers of xylanase and the xylanase produced by T. lanuginosus DSM
5826 was most stimulated by xylose (Purkarthofer and Steiner,
1995). Our early reported that strains of T. lanuginosus could be
differentiated into two groups based on their ability to produce xylanase using
xylan or xylose as sole of carbon source. One group of these strains produced
high xylanase activity either in the medium using xylan or xylose as a sole
carbon source. The xylanase production by T. lanuginosus ATCC 44008,
THKU-11 and THKU-25, which were the representative members of this group, increased
when xylose was added to the xylan medium. In contrast, there was another group
that produced high xylanase activity only in the xylan medium. Addition of xylose
to the xylan medium resulted decreasing of xylanase production in T. lanuginosus
ATCC 46882, TISTR 3465 and THKU-85 that belonged to this group (Khucharoenphaisan
et al., 2009). In this study, the induction and repression of β-xylanase
production of T. lanuginosus TISTR 3465 in liquid culture and resting
cell condition were examined. Furthermore, protein patterns and activity of
β-xylanase on gel electrophoresis were investigated during the induction
and repression of the enzyme.
MATERIALS AND METHODS
Microorganism and cultivation: A thermophilic fungus, Thermomyces
lanuginosus (formerly known as Humicola lanuginosa) (Domsch
et al., 1993), used in this study was a generous gift from Prof.
Tsutomu Morinaga. The strain is deposited in the MIRCEN Bangkok culture collection,
Thailand as T. launginosus TISTR 3465. Xylanase was produced in 50 mL
of basal medium in 250 mL Erlenmeyer flasks. The medium consisted of (per liter
of distilled water) 2.0 g KH2PO4, 0.3 g MgSO4.7H2O,
0.3 g CaCl2, 5.0 g peptone, 3.0 g yeast extract and 10 g of each
appropriate carbon sources; arabinose, cellobiose, glucose, galactose, xylose,
xylobiose, xylooligosaccharide and oat spelt xylan. Initially, pH of medium
was adjusted to 6.0 and autoclaved for 20 min at 121°C. The different mediums
were inoculated with 105-106 spores of 10 days T. lanuginosus
grown on wheat bran agar medium and then incubated at 45°C on reciprocal
shaker at 120 strokes min-1 (JEIO TECH, model BS-30). Moreover, various
concentrations of xylan, xylooligosaccharides and xylose were also used as carbon
Induction and repression on β-xylanase production: The fungus was grown in basal medium supplement with xylose or glucose as mention earlier. Oat spelt xylan suspension was added to the culture on the 3rd day of cultivation to make a final concentration of 10 g L-1 and further incubated. In parallel, the fungus grew in basal medium containing oat spelt xylan and then xylose or glucose was added on the 3rd of cultivation. The culture supernatant was collected by centrifugation and used as enzyme solution for determination of β-xylanase activity, soluble protein content, protein pattern and activity on gels by electrophoresis.
The freshly washed glutamic acid-grown mycelium were suspended in 50 mL sodium phosphate buffer (pH 6.0) and the aliquot (15 mL) to make a final concentration of mycelium was about 0.11 g dry weight. The inducers, oat spelt xylan or xylose was rapidly mixed with mycelium suspension (final concentrations are 10, 100, 500 and 1000 mg L-1). A control without any inducer was also set up. The mixtures were incubated at 50°C on a reciprocal shaker at 120 strokes min-1. Aliquots were taken at different time intervals (10 h) and filtrated supernatant was used as enzyme solution for determination of xylanase activity.
Sequencing of xylanase gene fragment: Total RNA of T. lanuginosus
TISTR 3465 was isolated and used as template for complementary DNA synthesized
by using Qiagen® One Step RT-PCR Kit. The PCR product was cloned to pGEM-T
easy vector following standard procedures. The plasmid DNA was isolated from
transformant by Quantum Prep Plasmid Miniprep Kit (Bio-Rad, USA). The PCR products
were sequenced using Big Dye® Terminator V3.1 cycle sequencing kit (Applied
Biosystems). Sequence gel electrophoresis was carried out and sequences obtained
by a 5500-5 DNA sequencer (Hitachi). The nucleotide sequences were translated
to amino acid and compared with other xylanase using the Genetyx version 5.0
program (Khucharoenphaisan et al., 2008). The
phylogenetic tree was constructed by using the neighbor-joining method in MEGA
version 4 software. The topology was evaluated by bootstrap analysis based on
1000 resamplings (Felsenstein, 1985).
Determination of dry weight: Culture broths were filtered using Whatman
filter paper No. 4 (Whatman International Ltd., USA). Mycelium retained on the
Whatman filter paper was then washed and dried at 105°C to a constant weight.
Determination of β-xylanase activity: β-xylanase was assayed
by determination of reducing sugars liberated from oat spelt xylan when incubated
with the diluted enzyme solutions at pH 6.0 and 50°C for 10 min. The reducing
sugars were measured using the DNS reagent (3,5-dinitrosalisylic acid) (Miller,
1959). One unit of β-xylanase activity was defined as the amount of
enzyme that produced 1 μmole of xylose in 1 min.
Determination of protein: Protein was determined using the method of
Lowry et al. (1951) with bovine serum albumin
as a standard.
Determination of molecular weight and xylanase activity band: Molecular
weight of proteins in supernatants was determined by Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis (SDS-PAGE) following the method of Laemmli
(1970). Protein and xylanase activity patterns were performed with Polyacrylamide
Gel Electrophoresis (PAGE) (Mishra and Maheshwari, 1996)
and stained with Coomassie brilliant blue R-250. For active zymogram analysis,
the gels were incubated in a 1% oat spelt xylan solution for 1 h at 50°C.
The gels were stained with 1% Congo red (Sigma Co., USA) for 15 min and destained
by 1 M NaCl until excess dye was removed from the xylanase activity band. The
gels were next immersed in 0.5% acetic acid and the background turned dark blue
and the xylanase activity bands appeared clear colorless areas (Kyu
et al., 1994).
Chemicals: All chemicals used were analytical grade. The xylan used for induction studies was oat spelt xylan (Sigma Co., USA). Xylooligosaccharides (Suntory Limited Osaka, Japan Lot. No. 92072511) contained 22.2% xylose, 55.4% xylobiose, 20.3% xylooligotriose, 2.1% glucose and 24.6% moisture content.
Place and during time: This study was conducted from 2008 to 2009 at Enzyme Technology Laboratory of Faculty of Science, Kasetsart University, Thailand.
RESULTS AND DISCUSSION
Effects of various sugars on β-xylanase production from T. lanuginosus TISTR 3465: The effect of the sugars such as arabinose, cellobiose, glucose, galactose, xylose, xylobiose, xylooligosaccharides and oat spelt xylan on the production of β-xylanase was investigated. Oat spelt xylan stimulated the highest level of β-xylanase production compared to xylooligosaccharides, xylobiose and xylose which had a lesser effect (Table 1). A low level of β-xylanase activity (<1U mL-1) was detected in the media containing other sugars as a carbon source and a control without addition of sugar. Furthermore, an intense band of xylanase activity was observed on zymogram of the culture supernatant containing xylan, xylobiose and xylooligosaccharides.
It is clearly indicates that oat spelt xylan is the best carbon source for
stimulate the production of extracellular xylanase by this fungus. However,
low level of β-xylanase activity was detected in the presence of other
inducing substances and also in non carbon source and the presence of easily
metabolizable substrates such as glucose, cellobiose, arabinose and galactose.
This finding leads to the conclusion that the β-xylanase was constitutively
produced at low level by T. lanuginosus TISTR 3465 and could be induced
by xylan. The results are agreement with the earlier reports where it was found
that xylanase production from T. lanuginosus DSM 5826 (Purkarthofer
and Steiner, 1995) and Trichoderma harzianum (Ahmed
et al., 2003) were induced by xylan and repressed by glucose.
activities of T. lanuginosus TISTR 3465 produced on different carbon
sources after 5-day cultivation
Effect of xylan, xylooligosaccharides and xylose concentration on β-xylanase
production: The effect of xylan concentration and its sugar derivatives
on enzyme production was investigated. It was found that β-xylanase activity
gradually increased with the increase of xylan concentrations up to 15 g L-1
but it was decreased when high concentration (Fig. 1). Low
concentrations of xylooligosaccharides and xylose also had a similar effect
on xylanase production but in less extent. Maximum β-xylanase activity
was obtained when concentration of xylooligosaccharides and xylose was at 2.5
g L-1 (Fig. 1).
It was possible that a small amount of sugars in which was released from the
hydrolysis of xylan played a role on induction but this appeared to be depend
on its concentration. High concentrations of the polymer repressed synthesis
of enzyme which might have resulted from the accumulation of hydrolysis products
resulting in feedback inhibition of enzyme production. At the beginning of the
culture, the constitutive level of xylanase activity was produced, which lead
to the liberation of xylooligomers from xylan. Xylanase formation in xylan culture
was extended, thus leading to a high level of enzyme activity. The reaction
of this effect was believed to be the longer availability of inducing molecules
that are slowly and continuously liberated. This mechanism was also suggested
by Ghosh and Nanda (1994) to explain the induction of
xylanase in Aspergillus sydowii MG49 by xylose.
of various concentrations of xylan, xylooligosaccharides and xylose on
production of β-xylanase at 5-day cultivation
These results are also similar to findings with Bacillus circulans B6
reported by Kyu et al. (1994) with the highest
xylanase production observed with 10 g L-1 xylan and repression at
high concentrations. The highest concentration of xylan (0.333 g L-1)
induced xylanase in resting cells of T. lanuginosus DSM 5826 (Purkarthofer
and Steiner, 1995). The highest xylanase activity was detected with 2.5
g L-1 of xylan in Cellulomonas flavigena (Hidalgo-Lara
and Farrés, 2005). In a similar manner, the effect of concentration
of xylooligosaccharides on xylanase production was almost identical to that
with xylose as shown Fig. 1. Xylooligosaccharides and xylose
also had an inductive effect on enzyme production at the low concentrations,
2.5 g L-1 (Fig. 1). Nevertheless, the inductive
effects were less pronounced in which were compared to xylan. This indicated
that readily utilized sugars such as xylose and xylooligosaccharides could be
inducers at low concentrations. However, there is a certain ambiguity about
whether xylooligosaccharides are direct inducers of β-xylanase. Xylooligosaccharides
are hydrolyzed to xylose by β-xylosidase during transport across the cell
membrane or within the cell and that the inducing effect of xylooligosaccharides
and xylobiose are an effect of xylose at the molecular level.
Induction and repression effect on β-xylanases production by T.
lanuginosus: A low β-xylanase activity was found when the fungus
was grown in the medium containing xylose or glucose as the carbon source. However,
addition of xylan to both media at the 3rd day of culture could effectively
increase β-xylanase formation 16-fold (Fig. 2).
of xylan addition to the xylose and glucose grown culture on the β-xylanase
production by T. lanuginosus. 1% xylan added to xylose grown culture,
1% xylan added to glucose grown culture, xylose grown culture, glucose
grown culture. Arrow indicated the addition of xylan
In contrast, the β-xylanase formation dramatically decreased about 3-fold
when xylose or glucose was added to the 3rd day xylan-grown culture (Fig.
3). The SDS-PAGE revealed that the main protein in the supernatant had a
molecular weight of 24.9 kDa. This main protein exhibited a β-xylanase
activity on zymogram in which was corresponded to main protein on SDS-PAGE and
PAGE. Addition of xylose or glucose to xylan grown cultures decreased the intensity
of the protein band on SDS-PAGE (Fig. 4a, lanes 2 and 3),
PAGE (Fig. 4b, lanes 2 and 3) and xylanase activity bands
on zymogram (Fig. 4c, lanes 2 and 3). In contrast, intensity
of main protein band was increase when xylan was added to the xylose or glucose
grown cultures, which detected on SDS-PAGE (Fig. 4a, lanes
5 and 7) and zymogram (Fig. 4c, lanes 5 and 7).
Effect of xylan and xylose concentration on β-xylanase induction in
resting cell: At various time intervals, the xylanase activity was determined
in the incubated buffer. Xylan and xylose were able to induce xylanase formation
in the T. lanuginosus TISTR 3465. Xylanase were increased when the concentration
of xylan or xylose was increased (Fig. 5a-c).
In the control of experiments showed very low xylanase activity due to the constitutive
formation of xylanase. The xylanase formation was faster in the presence of
xylose than xylan but xylan show higher pronounced on xylanase induction than
xylose (Fig. 5). The result was different from the report
of Purkarthofer and Steiner (1995) who reported that
xylose was the best inducer for xylanase production in resting cell of T.
lanuginosus DSM 5826.
of xylose or glucose addition to the xylan grown culture on the β-xylanase
production by T. lanuginosus. Xylan grown culture, 1% xylose added
to xylan grown culture, 1% glucose added to xylan grown culture. Arrow
indicated the addition of xylose or glucose
of SDS-PAGE (A) and Native-PAGE (B) after stained with coomassie brilliant
blue R-250 and zymogram for xylanase activity (C) of crude T. lanuginosus
TISTR 3465 β-xylanases produced in culture medium. Lanes: 1:
xylan grown culture, 2: 1% xylose added to xylan grown culture, 3: 1%
glucose added to xylan grown culture, 4: xylose grown culture, 5: 1% xylan
added to xylose grown culture, 6: 1% glucose added to xylose grown culture,
7: xylan added to glucose grown culture and M: Molecular mass standards
course of extracellular xylanase production by washed glutamic acid-grown
mycelium of T. lanuginosus TISTR 3465 during incubation in (a)
50 mM phosphate buffer (pH 6.0) supplied with separate oat spelt xylan
and (b) xylose at concentrations of 0 mg L-1, 10 mg L-1,
100 mg L-1, 500 mg L-1 and 1000 mg L-1.
Concentrations of fungal mycelium are in range of 0.11 g dry weight/experiment.
(c) The consumption of xylose from xylose-supplemented condition was also
This indicated that these two strains might have different induction mechanism.
However, we suggested that oat spelt xylan acted as the premier inducer of xylanase
in strains of T. lanuginosus TISTR 3465.
of amino acid sequence of xylanase from T. lanuginosus TISTR 3465
and DSM 5826. The alignment was done using the genetyx (version 5.0) alignment
program. Alignment characters are indicated as follows: *Indicates position
with a conserved amino acid residue and .: indicates position with a different
amino acid residue
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
Xylanase gene sequencing analysis: The complementary DNA of xylanase
genes from T. lanuginosus TISTR 3465 was sequenced. The nucleotide sequences
were translated to amino acid sequence and aligned with sequence of xylanase
from T. lanuginosus DSM 5826 (accession No. U35436) as shown in Fig.
6. Amino acid sequence of xylanase from strain TISTR 3465 was completely
similar to that of T. lanuginosus DSM 5826. Furthermore, the sequences
alignment revealed that the active site of xylanase of both strains of T.
lanuginosus were the position of E117 and E209 corresponding to the previous
reported of Lin et al. (1999). The phylogenetic
tree showed that xylanase from TISTR 3465 strain is closed to DSM 5826 strain
and related to each other thermophilic fungi (Fig. 7). This
concluded that amino acid sequence has no effect on xylanase induction. It appears
that other factors are dominant in induction of xylanase than that of amino
acid sequence. However, xylanase production using xylose as a carbon source
was related to RAPD pattern with UBC 241 primer (Khucharoenphaisan
et al., 2009).
Present finding was confirmed that the formation of β-xylanase by T. lanuginosus TISTR 3465 could be induction by xylan and its sugar derivatives. However, the mechanisms have to further clarify to make more understanding whether these are different from other T. lanuginosus strains.
This study was financially 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.
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