A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

Article abstract of DOI:10.1016/j.procbio.2013.06.007

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 C and have an optimum temperature of 80 C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.

Full text of DOI:10.1016/j.procbio.2013.06.007

Accepted Manuscript
Title: A novel thermostable cellulase free xylanase stable in
broad range of pH from Streptomyces sp. CS428
Author: G.C. Pradeep Yun Hee Choi Yoon Seok Choi Chi
Nam Seong Seung Sik Cho Hyo Jeong Lee Jin Cheol
Yoo<ce:footnote id="fn1"><ce:note-para
id="npar0005">Both authors contributed equally to this
work.</ce:note-para></ce:footnote>
PII:
S1359-5113(13)00276-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2013.06.007
PRBI 9861
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
14-11-2012
29-5-2013
1-6-2013
Please cite this article as: Pradeep GC, Choi YH, Choi YS, Seong CN, Cho
SS, Lee HJ, Yoo JC, A novel thermostable cellulase free xylanase stable in
broad range of pH from Streptomyces sp. CS428, Process Biochemistry (2013),
http://dx.doi.org/10.1016/j.procbio.2013.06.007
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A novel thermostable cellulase free xylanase
stable in broad range of pH from Streptomyces
sp. CS428
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Pradeep G.C.#, Yun Hee Choi#, Yoon Seok Choi, Chi Nam Seonga, Seung Sik Chob, Hyo
Jeong Leec, and Jin Cheol Yoo*
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Department of Pharmacy, Chosun University, Gwangju 501-759, Korea
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aDepartment of Biology, College of Natural Sciences, Sunchon National University, Sunchon,
Jeonnam, 540-742, Korea
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bDepartment of Pharmacy, College of Pharmacy, Mokpo National University, Muan,
Jeonnam,534-729, Korea
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cDepartment of Alternative Medicine, Gwangju University, Gwangju 503-703, Republic of Korea
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#Both authors contributed equally to this work.
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*Corresponding author: Tel.: +82 62 230 6380, Fax: + 82-62-227-3963, E-mail:
jcyu@chosun.ac.kr
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Abstract
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A cellulase free thermostable xylanase from Streptomyces sp CS428 was isolated
from a Korean soil sample, purified by single-step chromatography, and biochemically
characterized. The extracellular xylanase was purified 26 fold with a 55 % yield by CM
Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428)
was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (3 to
~13.6) and to 50 ºC and have an optimum temperature of 80 ºC. Xyn428 had Km and Vmax
values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when
beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was
INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and
produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without
supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM,
and produced xylose and xylobiose as major products. It was found to degrade agro waste
materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being
simple in purification, thermo tolerant, pH stabilityin broad range and ability to
producexylooligosaccharidesshow that Xyn428 has potential applications in industries as a
biobleaching agent and for xylooligosaccharides production.
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Keywords: Xylanase, single-step purification, agro-waste, xylooligosaccharides
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1. Introduction
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Lignocellulosic biomass includes cellulose (35-50 %), hemicellulose (20-35%) and
lignin (10-25%). Xylan is the major plant hemicellulose, which constitutes 20–40% of total
plant biomass [1, 2]. Xylan consists of a backbone of β-1, 4-linked D-xylose residues having
arabinose, glucuronic acid, and/or mannose substitutes. A wide variety of microorganisms
including aerobic and anaerobic, mesophiles andthermophiles can produce enzymes that can
degrade xylan[2]. The enzymatic hydrolysis of xylanrequires the cumulative actions of endo-β-
1, 4-xylanase (EC 3.2.1.8), β- xylosidase (EC 3.2.1.37), and a series of enzymes that degrade
side chain groups. Among these, endo- β-1, 4-xylanase is the most crucial enzyme that cleaves
glycosidic bonds to produce short chain xylooligosaccharides of various lengths
[3].Xylooligosaccharidesproductions from commercial and waste xylan werewell reported
[4,5]. Industrial waste materials have become a global challengeand remained as centre of
attraction since they create pollution and unnecessarily occupy space and requires high cost for
proper management. Thus, the production of value added products, such as, bioethanol and
oligosaccharides, offers an attractive solution. Bioethanol production includes two steps, that is,
thehydrolysis of biomass to sugar and the fermentation of sugar to alcohol. Furthermore, the
alcohol produced by the hydrolysis of industrial waste materials offers an alternative option of
limited fossil fuels. In addition, to remove lignin, paper and pulp industries are using xylanase
to degrade xylan, which becomes unfriendly to chemical process[6].
In recent days xylanase production using microbes is popular because of its significant
role in food, animal feed, pulp, and paper industries [7]. In particular, xylan degrading
enzymes from Streptomyces are being increasingly reported, such as, from Streptomyces
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lividans [8], Streptomyces sp.7b [9], Streptomyces cyaneus SN32 [5], Streptomyces
rameusL2001 [4], Streptomyces thermocyaneociolaceus [10], etc. Here, we describe the
isolation and identification of a potent xylanase producer - Streptomyces sp. CS428. Further
purification by single-step column chromatography and characterization of an industrially
applicable xylanase from this strain were also performed. Apart from this we have compared
the activity and stability of xylanase from CS428 with the commercially available xylanase
from Thermomyces lanuginosus, and endo-1,4-β-xylanase from Trichoderma longibrachiatum.
2. Materials and methods
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2.1. Materials
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Xylose, Beechwood xylan, Birchwood xylan, xylanase from Thermomyces lanuginosus, and
endo-1,4-β-xylanase from Trichoderma longibrachiatum were purchased from Sigma-Aldrich
(St Louis, MO, USA). Thin layer chromatography silica gel plates were purchased from Merck
(Darmstadt, Germany). Xylobiose, Xylotriose,and Xylotetrose were from Megazyme (Ireland).
CM Trisacryl was purchased from IBF (Villeneuve-la- Garenne, France).All the reagents used
were of the highest grade available.
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2.2. Bacterial strain, growth conditions, and screening
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Ten bacterial strains collected from different Korean provinces were cultivated in xylan
medium (pH 6.5) supplemented with 1.25 % beechwood xylan, 0.5% yeast extract, 1%
tryptone, 0.75% KH2PO4, 0.15% K2HPO4, and 0.05% MgSO4. The strains were cultivated in a
250 mL Erlenmeyer flask containing 50 mL medium at 28 ºC and 130 rpm for 5 days. The
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culture broths were centrifuged at 6,000 ×g for 30 min and supernatants were tested for
enzyme activity. Strain CS428,which had good activity, was selected for further study. In
addition, comparative xylanase production was studied using agro-waste materials using corn
cob and wheat bran with and without supplements (like a nitrogen source, a carbon source, and
metal ions). The corncob and wheat bran were bought from local Nam-Gwangju market; grains
were removed, washed, dried, and then grounded for use.
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2.3. Enzyme assay and protein estimation
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Protein concentrations were determined using the Bradford method [11] using bovine
serum albumin as standard.
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Xylanase activities were assayed at 80 ºC by adding 0.1 mL enzyme (appropriately
diluted in 5mM Tris/HCl pH 7.0) sample to 0.1 mL of substrate solution containing 0.5 %
(w/v) beechwood xylan prepared in 5 mM Tris/HCl (pH 7.0) for 5 min. A control was run
simultaneously with all the reagents in ice cold water (`0ºC). The reducing sugars released
were measured using 3,5-dinitrosalicylic acid (DNS) method [12]. A standard xylose curve
was constructed to determine enzyme activities. One unit of xylanase activity was defined as
the amount of enzyme that releases 1 µmol of xylose per min under standard assay conditions.
Cellulose activities were evaluated using sigma cell cellulose, carboxymethyl cellulose, avicel
and an artificial substrate such as,paranitrophenyl D- celobioside (pNPC) and paranitrophenyl-
β- D glycopyranoside (pNPG).
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2.4.Enzyme purification
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All purification procedures were carried out at 0ºC unless stated otherwise.
Streptomyces sp. CS428 was grown for 72 h and the crude supernatant extracellular xylanase
was obtained by centrifuging the broth at 6000× for 1 h. The crude supernatant was then
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subjected to 30-75 % ammonium sulfate precipitation. Proteins were recovered by
centrifugation at 6000×g for 1 h at 4 ºC, dialyzed against 10mM citrate/phosphate (pH 7.0),
and concentrated using an ultra filtration membrane (5 kDa, Millipore Corp). The dialyzed
enzyme solution was loaded to a CM Trisacryl column (20 cm × 1.1 cm) pre-equilibrated with
10 mM citrate/phosphate buffer (pH 5.5). Proteins were eluted with 0-1 M KCl in the same
buffer at 30 mL/h (2 mL/Fraction). Xylanase active fractions were pooled, concentrated, and
analyzed for purity. Further characterizations were carried out using the pure enzyme.
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2.5. Polyacrylamide gel electrophoresis
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The purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on 12% (w/v) polyacrylamide slab gel [13]. For reference
proteins, protein size marker (MBI, Fermentas) was used. Proteins were observed by staining
with Coomassie Brilliant Blue R-250 and then destaining with a solution containing methanol:
glacial acetic acid: distilled water = (1:1:8 by vol). Molecular weight was estimated by
comparison with the relative mobility of the reference protein. In addition, xylan zymography
was performed as described [14].
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2.6. Effect of pH and temperature
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The pH and temperature optimum and stability of Xyn428 were compared with the
commercially available xylanase from Thermomyces lanuginosus, and endo-1,4-β-xylanase
from Trichoderma longibrachiatum. The optimum pH required for relative xylanase activity
were determined at 80 ºC, 70ºC, and 60 ºC respectively for xylanase from CS428,
Thermomyces lanuginosus, and Trichoderma longibrachiatum at 5 mM using 100 mM
concentrations of various pH buffers (pH values 2.0-13.6). The buffers used were;citric
acid/sodium phosphate (2-7.5), Tris/HCl (7.0-9.5), CAPS/NaOH (9-11), sodium
bicarbonate/NaOH (9.5-11), glycine/NaOH (9.5-11), and KCl/NaOH (11-13.6). Temperature
was optimized at pH 7.0 in the range 50-90ºC. To determine pH stability, enzyme samples
were incubated at 10 mMusing various pH buffers at 4 ºC for 24 hr and residual activities were
measured at 20mM under standard assay conditions. Similarly, to determine enzyme
temperature stability, enzyme samples were pre-incubated at various temperatures (30-90 ºC)
for 60 min and residual activities were measured. The assay was done under standard assay
conditions.
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2.7. Effect of salt concentration
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To determine the effect of salt concentration on the activity of Xyn428, different
concentrations (2mM, 5mM, 10mM, 20mM, 50mM, 100mM) of Tris salt at different pH
values (7.0, 7.5, 8.5, 9.5) were examined. Activities were determined under standard assay
conditions.
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2.8. Effects of metal ions
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Effects of metal ions on the activities of Xyn428 were compared with the commercially
available xylanase from Thermomyces lanuginosus, and endo-1,4-β xylanase from
Trichoderma longibrachiatum. For the metal ion effect, the pure enzyme was incubated at 1
mM concentration of Ca2+, Mg2+, Cu2+, Co2+, Zn2+, K+, Na+, Mn2+,and Fe2+. The relative
activities were determined under standard assay conditions comparing with control without
metal ions (100%).
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2.9. Effects of various organic solvents
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The enzyme samples were incubated with 10% aqueous solutions of organic solvents
that is, methanol, ethanol, iso-propanol, butanol, hexane, heptanes, octane, decane, benzene,
toluene, xylene, dichloromethane, acetonitrile, ethyl acetate, acetone, diethyl ether, glycerol,
and dimethylsulfoxide (DMSO). Enzyme activities were evaluated under standard assay
conditions. In addition, the stability of xylanase in these solvents for two hours at room
temperature was measured. Further, enzyme activities were evaluated containing different
concentrations of alcohol (0.06-2%). Residual activities were measured under standard assay
conditions.
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2.10. Effects of detergents and modulators
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Detergent and modulator effects were also examined and the activities of Xyn428 were
compared with the commercially available xylanase from Thermomyces lanuginosus, and
endo-1,4-β-xylanase from Trichoderma longibrachiatum. The effects of detergents were
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evaluated using Triton X – 100, Tween-20, Tween-80, Polyoxylethylene-4-laurylether,
Deoxycholic acid (DCA), Sodium Dodecyl Sulfate (SDS) and 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate (CHAPS) at 0.25% and the effects of modulators like
hydrogen peroxide, sodium perborate, β- mercaptoethanol, Ethylenediamine tetra-acetic acid
(EDTA), ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) were
also evaluated. In all cases, relative enzyme activities were measured under standard assay
conditions with the activity without any additive was taken control (100%).
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2.11. N- terminal amino acids
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The N-terminal amino acid sequence of Xyn428 was determined by Edman
degradation using a Procise Model 492 protein sequencer (Applied Biosystems, CA, USA).
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2.12. Substrate specificity and kinetic parameters
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To evaluate the substrate specificity of the purified enzyme, the enzyme was incubated
with 0.5% (w/v) of each substrate such as beechwood xylan, birchwood xylan, wheat bran and
corncob xylan. Enzyme activities were measured under standard assay conditions. For kinetic
parameters, five different concentrations of beechwood and birchwood xylan (1.25-20 mg/mL)
with constant enzyme concentration (0.14 µg) were prepared. Assays were performed under
standard assay conditions at optimal conditions. The Michaelis-Menten constant (Km) and
maximum velocity (Vmax) were determined from a Lineweaver-Burk plot.
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2.13. Enzymatic hydrolysis and xylooligosaccharides production
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Enzymatic hydrolysis of Xyn428 was compared with the commercially available
xylanase from Thermomyces lanuginosus and evaluated using thin layer chromatography
(TLC) [5]. Briefly, purified Xyn428 and xylanase from Thermomyces lanuginosus (2.44
mg/mL) were incubated with commercial beechwood xylan substrate (50 mg/mL) in 100 mM
Tris/HCl buffer (pH 7.0) at 50 ºC. At predetermined time 30 μl was withdrawn. The reaction
was stopped by adding 5 µl of 10% (v/v) trichloro acetic acid and samples were then spotted
on the silica gel plates 60F 254 (E. Merck, Germany). The plates were developed with solvent
system of chloroform–acetic acid–water (6:10:2, v/v/v) followed by spraying the plates with a
methanol–sulfuric acid mixture (95:5, v/v) and heated for few minutes at 150 ºC. A mixture of
xylooligosaccharides consisting of xylose (X1), xylobiose (X2), xylotriose (X3), xylotetrose
(X4) (10 mg/mL) were used as the standard.
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2.14. Xylanase for the degradation of lignocellulose biomass
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Untreated corncob and wheat bran was washed with distilled water to a neutral pH.
Both biomasses were treated with 20 U/g of purified xylanase from Streptomyces sp CS428,
Thermomyces lanuginosus and endo-1,4-β-xylanase from Trichoderma longibrachiatum at
Tris/HC pH 7.0, 50 ºC for 2 h. Degradation of biomass by Xyn428 was analyzed by electron
microscopy as previously described [4]. Biomass treated with buffer was taken as the control.
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3. Results and discussion
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3.1. Bacterial strain, growth conditions, and screening
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All ten bacterial strains collected from different Korean provinces were cultivated in xylan
medium at 28 ºC and 130 rpm for 5days. Among them, strain CS428 showed strong xylan
degrading activity and was selected for further study. Based on morphological, biochemical
and 16S rRNA sequences, the stain was identified as Streptomyces sp. CS428, as we
previously described [15]. 16S rRNA sequence of the strain showed 100% similarity with
Streptomyces rameus LMG20326 (T) (Accession no. AJ781379), Streptomyces tricolor
LMG20328 (T) (Accession no.781380) and Streptomyces bangaladeshensis AAB-4(T)
(Accession no. 750056) (Fig. not shown).
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3.2. Enzyme production and purification
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The comparative study of xylanase production from corncob and wheat bran with or
without supplements was performed shown in Fig. 4. Many reports have been issued on
xylanase production using wheat bran and corncob [4, 5]. We observed that strain CS428
produced xylanase and achieved a higher yield (4197.14 ± 1.3 U/mL in 36 h) from wheat bran
without supplements than has been previously reported. The highest xylanase activity
previously reported was 1810.9 U/mL from S. rameus L2001 [4]. Interestingly, a higher yield
of xylanase production was obtained from wheat bran without supplements than from
commercially available beechwood xylan (3329.1 ± 2.6 U/mL), which is encouraging one. In
addition, remarkable xylanase production was obtained from corncob with supplements. These
findings suggest that Xyn428 is a potent candidate in industrial application.
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Xyn428 was purified in a single-step by CM Trisacryl cation exchange
chromatography. Many xylanase from Streptomyces sp. have been purified using multiple
column chromatographic steps [4, 16-19]. Our enzyme was purified more efficiently than other
available xylanase. A summary of xylanase purification is illustrated in Table 1. Xylanase was
purified to 25.9 fold with a recovery yield of 55.1%. Furthermore, xylanase activity recovery
and purity fold were greater than those of the xylanase from Streptomyces cyaneus SN32 [5],
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Streptomyces sp. 7b [9], and from Bacillus subtilis A S H [20] that were separated from single
step chromatography.
3.3. Gel electrophoresis
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The purified enzymewas analyzed by SDS-PAGE and activity staining of the gel
(Zymography) shown in Fig. 1a and 1b respectively. The molecular mass of Xyn428was
estimated approximately at 37 kDa, and was seen on gels as a single band. In accord with
SDS-PAGE results, the pure enzyme showed a clear band by zymography,confirming that it
was a xylanase. Its molecular mass was similar to the xylanases from Streptomyces
thermocyaneociolaceu (35 kDa) [10] and Streptomyces sp TN119 (35.9 kDa) [21] shown in
Table 2.
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3.4. Effect of pH and temperature
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Enzyme activity and stability were markedly affected by pH and temperature. The effects of
pH and temperature on the activity and stability of Xyn428 and other xylanase from
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Streptomyces are shown in Table 2. The effect of pH on the activity and stability of Xyn428
were also compared with the commercially available xylanase from Thermomyces lanuginosus,
and endo-1,4-β-xylanase from Trichoderma longibrachiatum shown in Fig. 2a and 2b
respectively.The xylanase from CS428, Thermomyces lanuginosus, and Trichoderma
longibrachiatum were active from pH 6.0 to 13.0 with the highest at 7.0, 7.0, and 12.5
respectively. The enzymes were stable to a broad range of pH Fig. 2b. The xylanase from
CS428, Thermomyces lanuginosus, and Trichoderma longibrachiatum remain stable about
85%, 70% and 90 % respectively of the maximal activity at pH approximately 13.6. The
reasons behind such a stability on broad range of pH may be due to the reversible denaturation
of the protein so that there is no effect on the activity of the enzyme on incubation at different
pH. Furthermore, Xyn428 was found to be more stable in acidic media than Streptomyces
cyaneus SN32 [5], Streptomyces megasporus DSM 41476 [22], Streptomyces matensis DW67
[19], and to our knowledge the most alkaline stable xylanase reported from Streptomyces
shown in Table 2. Xylanase from CS428, Thermomyces lanuginosus, and Trichoderma
longibrachiatum exhibited a temperature optimum at 80 ºC, 70 ºC, and 60 ºC respectively Fig.
3a. When incubated for 1 h at 60 ºC, xylanase from CS428, Thermomyces lanuginosus, and
Trichoderma longibrachiatum retained almost 72%, 90%, and 67 % of the maximal activity
shown in Fig. 3b. Xyn428 was found to have the highest optimal temperature reported among
the Streptomyces, and stability is comparable to that of the xylanase from Streptomyces sp. 7b
[9], Streptomyces matensis DW67 [19] Table 2. Despite the partial stability of xylanase from
CS428, Thermomyces lanuginosus, and Trichoderma longibrachiatum at 80 ºC, Xyn428
showed highest activity at this temperature. This may be due to the protective effects of
substrate to Xyn428 for optimum time. Many industrial processes are operated at extremes of
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pH (either acidic or alikaline) and at elevated temperatures thus the enzyme must suit the
process requirements and must be capabale of withstanding such harsh conditions for
prolonged periods or at least during the process time which we can find in Xyn428. For every
10°C rise in temperature, reaction rates approximately double. Therefore, assuming the
enzyme is stable at the higher temperature, the amount of enzyme needed can be reduced or
the conversion time be shortened. There are other benefits from operating at higher
temperatures carrying out conversions at increased temperatures (above 60-65°C) markedly
reduces microbial infection of the material being processed [23]. In addition, the higher
temperatures increase the solubility of polymeric substrates such as carbohydrates, there by
improving their mechanical handling characteristics and rendering them more amenable to
enzymatic attack. Tolerance to broad range of pH values, a high optimum temperature, and its
stability makes Xyn428 a novel among xylanase previously reported from Streptomyces.
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3.5. Effect of salt concentration
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The enzyme activity of Xyn428 was remarkably affected by salt concentration. The
purified enzyme showed maximum activity at a salt concentration of 5 mM. At pH 7.0, its
activities at concentration of 2 mM, 5mM, 10 mM, 20mM, 50 mM, and 100mM were 37 U, 52
U, 51 U, 43 U, 4 U, and 4.5 U, respectively. However, its activity was substantially inhibited at
salt concentrations exceeding 20 mM (Fig. 5).
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3.6. Effects of metal ions
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The influences of metal ions on the activities of xylanases obtained from CS428,
Thermomyces lanuginosus, and endo-1,4-β-xylanase fromTrichoderma longibrachiatum were
studied (Table 3). The activity was enhanced 1.33- and 1.23-fold by Ca2+ and Co2+,
respectively, but was inhibited by Fe2+, Zn2+, Cu2+, and Mg2+, and almost unaffected by K+,
Na+, and Mn2+ similar to the commercially available xylanase Thermomyces lanuginosus and
Trichoderma longibrachiatum except Mn2+ from Thermomyces lanuginosus. Cu ions are
known to catalyze the auto-oxidation of cysteines to form intra molecular disulfide bridges or
the formation of sulphenic acid [24]. The activity was enhanced by Co2+is similar to that
reported for other xylanases [18, 25]. In addition, Fe2+suppressed the activity like the xylanase
from Streptomyces sp.SWU10 [16] but against the xylanase from Streptomyces rameus L2001
and Streptomyces matensis DW67 [4,19]. The result reveals that what kinds of metals should
be included or excluded for the industrial applications.
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3.7. Effects of organic solvents
The effects of organic solvents on the activity and stability of Xyn428 were examined
at 10% concentration (Table not shown). Xylene and ethyl acetate were found to suppress
activity, but all other solvents examined have no effect. Xyn428 was stable (2 h) in hexane,
heptane, octane and acetone where as unstable in toluene, acetonitrile, and DMSO which
suggests that organic solvents have partial or specific effects on activity and stability of
Xyn428 that have significant importance on bioindustries. Solvent polarity strongly influences
the way in which organic solvents interact with water surrounding enzyme molecules. A highly
polar solvent absorbs essential water from enzyme and inactivates it. Actually, solvents with a
log P value of <4 are toxic because their degrees of partitioning into aqueous layer are higher.
15
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327
328
329
330
331
332
333
334
335
Enzymes that are stable in organic phases may be useful for bioremediation or as biocatalysts
[26]. Xyn428 was found to have stability in solvents with a higher log P value, that is, 5.2
(decane) to 2.0 (benzene) (with the exceptions of toluene (log P=2.5) and xylene (log P=3.1))
where as instability in the solvents with lower log P value is in correlation (with the exception
of DMSO (log P=-1.37), acetonitrile (log P=-0.39) and acetone (log P=-0.21)). Furthermore,
the optimal concentration of ethyl alcohol enhances the activity of Xyn428, but higher
concentrations adversely affect its activity, as like the xylanase reported for Streptomyces
rameus L2001 [4] (Table 4). These data provide the essential data required for the application
of purified xylanase reaction in an organic phase.
336
337
3.8. Effects of detergents and modulators
338
339
340
341
342
343
344
345
346
347
The influences of detergents and modulators on the activity of xylanases obtained from
CS428, Thermomyces lanuginosus and endo-1,4-β-xylanase from Trichoderma
longibrachiatum were summarized in Table 3. In case of detergents, xylanase activity was not
affected by TritonX-100, Tween-20, Tween-80, Polyoxylethylene-4-laurylether and CHAPS
but was affected by Deooxycholic acid and SDS, which is similar to that reported for the
xylanase from Burkholderia sp. DMAX [25]. On the other hand, the modulator β-
mercaptoethanol did not affect the activity of Xyn428, but 1, 4-dithiothreitol enhanced its
activity. The inhibitions of xylanase activity by EDTA and SDS have been previously reported
[17, 27].
348
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349
3.9. N-terminal amino acids
350
351
352
353
354
355
356
357
358
The amino acid sequence of the first 20 N-terminal amino acids of Xyn428 was found
to be INRTDHNENSYLEIHNNEAR. This sequence was compared with available sequences
in the National Centre for Biotechnology Information (NCBI) protein database using BLAST
(basic local alignment search tool;http://blast.ncbi.nlm.nih.gov/Blast.cgi). The BLAST search
did not suggest homology with other Streptomyces or xylanase sequences, but showed a high
degree of homology with sequences from Streptomyces tendae TU901 and Streptomyces
clavuligerus ATCC, and low homology with sequences of the xylanases from Trichoderma sp.
SY and Caldicellulosiruptor saccharolyticus and with a putative β-1, 4-xylanase from
Cochliobolus spicifer, suggesting that Xyn428 is a β -1, 4 xylanase (Table not shown).
359
360
3.10. Substrate specificity and kinetic parameters
361
362
363
364
365
366
367
368
369
Purified Xyn428 was assayed using different natural and synthetic substrates (Table not
shown). Its activity was highest for beechwood and commercially available xylan, but least for
wheat bran xylan (5% beechwood) and agro-waste materials, which is similar to that observed
for xylanase from S. rameus L2001 [4]. Xyn428 showed no activity towards Avicel,
carboxymethyl cellulose (CMC), p-nitrophenylglucopyranoside (pNPG), and p-nitrophenyl-β-
D-celobioside (pNPC). These results show that the xylanase isolated and purified from CS428
is a cellulase free xylanase, similar to a xylanase reported earlier [4, 17, 28]. This is the major
property required for the pulp and paper industries.
17
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370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
The kinetic constants (Km) and (Vmax) of Xyn428 were determined using a Lineweaver-Burk
plotto be 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, for beechwood
xylan, and 60.5 ± 25 mg/mL and 1403.4 ± 55 mmol/min mg, respectively, for birchwood xylan.
Comparing Vmax values, Xyn428 prefered beechwood xylan than birchwood xylan. The Km and
V
max using beechwood and birchwood xylan were found 1.8 ± 0.9 mg/mL and 338 ± 66
mmol/minmg, and 18 ± 2.6 mg/mL and 1,100 ± 8 mmol/min mg, respectively [15]. For
birchwood xylan as substrate, Km and Vmax values were 2.33 mg/mL and 322.69 µmol/min mg,
respectively, for Streptomyces megasporus DSM 41476 [22]. For beechwood xylan from
Streptomyces rameus L2001, Km and Vmax values were 5.8 mg/mL and 1491 ± 33 µmol/min mg,
respectively, and for birchwood xylan, 5.3 ± 0.2 mg/mL and 1643 ± 45 µmol/min mg,
respectively [4]. Vmaxvalue comparisons suggest that Xyn428 is much more efficient than other
already reported xylanases.
3.11. Enzymatic hydrolysis and xylooligosaccharides production
Comparisons of xylooligosaccharides productions from CS428 and Thermomyces
lanuginosus were shown in Fig.6a and 6b respectively. Xylooligosaccharides were the main
oligosaccharides released initially which on further hydrolysis by Xyn428 releases xylobiose
and xylose whereas xylobiose was the main oligosaccharide from Thermomyces lanuginosus.
The mode of action of xylanase from CS428 was found to be endo type since it produced
xylobiose(X2) as the predominant end product from beechwood xylan along with higher
xylooligosaccharides as intermediates. This finding suggests that Xyn428 could be a strong
candidate used to produce xylooligosaccharides. Oligosaccharides were produced from 5 min
of incubation, which is similar to that reported for the xylanase from Actinomadura sp.
18
Page 18 of 43

393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
S14[14]. Xyn428 was assessed to evaluate whether any cellulase activity was present using
various substrates and found that cellulase was completely absent. It is similarto a xylanase
recently reported as useful in xylooligosaccharides production [29].
3.12. Xylanase for the degradation of lignocellulose biomass
Corncob and wheat bran were treated with xylanse from Streptomyces sp CS428,
Thermomyces lanuginosus and endo-1,4-β-xylanase from Trichoderma longibrachiatum at
50ºC for 2 h. At 400× magnification significant changes were observed versus the untreated
control (Fig. 7a and 7b) at 20 U/g. The amount of reducing sugars released by the xylanase
from corn cob and wheat bran werere markably increased with time and the release of
chromophores were also maximal. Thus, chromophoric material were released as a result of
the enzyme action, suggesting that there were a significant decrease in the aromaticity of the
residual lignin. Comparing to the commercial xylanase from Thermomyces lanuginosus and
endo-1,4-β-xylanase from Trichoderma longibrachiatum, Xyn428 causes significant decrease
in aromicity in corn cob and wheat barn. Xyn428 appeared to be effective at 20U/g, as has
been reported for the xylan from Streptomyces rameus L2001 [4]. In addition, an ideal
xylanase to be useful in biobleaching must be active and stable in high temperatures and
alkaline conditions and it must also have lack of cellulase activity [29, 30]. Furthermore, low
molecular weight xylanases like Xyn428 more easily penetrate into the re-precipitated xylan
on the surfaces of pulp particles [5]. This mitigates the problem of a xylan barrier on the
surface of lignin containing pulp during subsequent chemical bleaching steps [31]. Although
many microorganisms have been known to produce xylanase, CS428 produces cellulase free
xylanase which is stable over wide range of pH and temperatures. Moreover, the xylanase
19
Page 19 of 43

416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
yield from CS428 was higher than the xylanase reported. Thus, Xyn428 favors its potential
application in degradation of agro waste biomass for bio bleaching in paper and pulp industries.
4. Conclusions
A thermostable Streptomyces sp. CS428 was found to produce an endo-1, 4-β-xylanase
(Xyn428), which was purified by one-step chromatography with a yield of 55%. Activity and
stability of xylanase from CS428 was compared with the commercially available xylanase
from Thermomyces lanuginosus and endo-1,4-β-xylanase from Trichoderma longibrachiatum.
Xyn428 was found to have temperature and pH optima at 80 ºC and 7.0, respectively, and have
broad range pH stability (3 to ~13.6) (in fact, it is the most alkaline tolerant xylanase ever
reported) and thermostability (50 ºC for 1 h). Its N-terminal amino acid sequence was found to
be INRTDHNENSYLEIHNNEAR. Xyn428 activity was inhibited by Fe2+, xylene, EDTA,
EGTA, hydrogen perborate, sodium perborate etc. Xyn428 produced xylose and xylobiose as
main products, which identifies it as an endoxylanase. In addition, Xyn428 was found by
electron microscopy to potently degrade agro waste materials. The thermal and broad range pH
stability Xyn428 indicates potential applications in the biofuel, food, and textile industries and
for waste treatment.
5. Acknowledgement:
This work was supported by research fund from Chosun University, 2012
20
Page 20 of 43

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References:
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
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Kim DY, Han MK, Lee JS, Oh HW, Park DS, Shin DH, Bae KS, Son KH, Park HY.
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Klibanov AM. Improving enzymes by using them in organic solvents. Nature
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from Streptomyces halstedii JM8. Appl Environ Microbiol 1995;61:2414-2419.
Verma D, Satyanarayana T. Cloning, expression and applicability of thermo-alkali-
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Techapun C, Poosaran N, Watanabe M, Sasaki K. Thermostable and alkaline-tolerant
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22
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530
531
532
533
534
535
Figure legends:
Fig. 1: 12 % (w/v) SDS-PAGE (a) and Zymograph (b) of purified xylanase from CS428.
Lanes Mr, protein molecular weight marker (Fermentas); lane C, Crude extract; lane A.Sul,
30-75 % ammonium sulfate precipitation fraction; lane P, purified xylanase after CM Trisacryl
cation exchange chromatography.
536
537
538
539
540
541
542
543
544
Thermomyces lanuginosa
Fig. 2: (a) Optimal pH, (b) pH stability ofxylanase from CS428 ( ),
( ) and,Trichoderma longibrachiatum ( ). To determine pH optimum, the activity of
xylanases were determined at 80 ºC using different pH buffers. To examine xylanase stability,
xylanases were incubated with different pH buffers (2-13.6) at 4 ºC for 24hr and relative
xylanase activity were evaluated under standard assay conditions. Each point represents as
mean (n=3).
545
546
547
548
549
550
Fig. 3: (a) Optimal temperature, (b) thermal stability of xylanase from CS428 (),
Thermomyces lanuginosa ( ) and,Trichoderma longibrachiatum ( ). To determine optimum
temperature, the reaction was performed at different temperatures at optimum pH (7.0),
whereas to determine xylanase stability purified Xyn428 was stored at different temperatures
for one hour. Activitieswere assayed under standard assay conditions. Each point represents as
mean (n=3).
551
552
553
554
Fig. 4:The comparative study of xylanase production using agro-waste materials. Xylanase
from commercial xylan ( ), corn con in distill water (
), corn cob with supplements ( ),
wheat barn in distill water ( ), wheat barn with supplements ( ). All the activity is in U/mL.
Each point represents as mean (n=3).
23
Page 23 of 43

555
556
557
Fig. 5: Effect of Tris salt on the activity of purified Xyn428 at 80 ºC. The reaction was
performed at different salt concentrations. Activities were assayed under a standard assay
conditions. Each point represent as mean (n=3).
558
559
560
Fig. 6:Plot showing xylan degradation after treating beechwoodxylan at 50 ºC and pH 7 with
xylanase from CS428 (a), Thermomyces lanuginosus (b). M: mixture of Xylose (X1),
Xylobiose (X2), Xylotriose (X3) and Xylotetrose (X4).
561
562
563
Fig. 7: Scanning electron micrographs (× 400) of corncob (a) and wheat bran (b) after
treatment with enzyme at concentration20 U/g at 50 ºC for 2 h at pH 7. Control (A), xylanase
from CS428 (B), Thermomyces lanuginosa (C) and, Trichodermal ongibrachiatum(D).
564
565
566
567
568
569
570
571
572
573
24
Page 24 of 43

574
575
576
577
Table 1
Purification summary
Purification Step
Total protein
(mg)
Total
Specific Activity
(U/mg)
Yield
(%)
Purity Fold
Activity(U)
Culture Broth
4.7
168196.7
150448.9
35786.5
100
1
(NH4)2SO4 Fractionation
1.48
101654.7
89.4
2.8
CM Trisacryl
0.1
92610.3
926103.0
55.1
25.9
578
579
580
581
582
583
584
585
586
587
25
Page 25 of 43

588
589
Table 2
Comparison ofXyn428 with other xylanases from Streptomyces
Stain Name
Optimum
Stability
Reference
Mol. Wt
21.1
20.5
43
pH
5.3
6
Temp (ºC)
pH
Temp (ºC)
3-70
4-60
0-37
3-50
3-60
3-37
3-80
3-60
3-55
3-37
3-60
3-60
4-50
0-50
S. rameus L2001
70
60-65
60
2.2-11.3
4-9.5
-
[4]
[5]
S. cyaneus SN32
S. lividans
6
[8]
S. sp. 7b
30
6
50
6-9
[9]
S. thermocyaneociolaceus
S.sp CS802
35
5
60
4.5-10.5
7.5-13
3-9
[10]
45
12
60
[15]
S.sp. SWU10 (XynSW2A)
S.sp. SWU10 (XynSW2B)
S. matensis DW67
S. spTN119
31
6
6
60
60
[16]
44
2-9
[16]
21.2
35.9
47.6
20-45
45 & 35
37
7
65
4.5-8.0
2-11
4-11
3-9
[19]
7
50
[21]
S. megasporus DSM 41476
S. actuosus A-151
S. halstediiJM8 (Xys1L & Xys1S)
S. sp. CS428
5.5
4-6
6.3
70
[22]
60-70
60
[27]
4-10
3-13.6
[28]
7
80
Current study
590
591
592
593
594
26
Page 26 of 43

595
Table 3
596
597
598
Effects of Detergents, Modulators, and metal ions on the activities of xylanases from CS428
(a), Thermomyces lanuginosus (b), and endo-1,4-β-xylanase from Trichoderma
longibrachiatum (c).
Reagents
Concentration
Type of ion
(a) Relative
(b) Relative
(c) Relative
Activity*( % ) Activity*( % ) Activity*( % )
Triton X - 100
Tween - 20
Tween - 80
0.25%
0.25%
0.25%
0.25%
Non - ionic
Non - ionic
Non - ionic
Non - ionic
124±4.1
111 ± 4.3
114 ± 4.5
108 ± 2.5
79 ± 3
83 ± 2.7
92 ± 2.2
104 ± 0.1
179 ± 0.9
185 ± 1.1
180 ± 3.5
111 ± 3.5
Polyoxylethylene-4-
laurylether
Deooxycholic Acid
0.25%
0.25%
0.25%
5 mM
5 mM
5 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
Anionic
Anionic
74± 3
36 ± 6
95 ± 3.2
0
62 ± 1.9
45 ± 5.3
80 ± 3.7
8 ± 2.3
92 ± 4
202 ± 2
76 ± 2.8
49 ± 3.5
14 ± 5
Sodium Dodecyl Sulfate
CHAPS
Zwitterionic
Hydrogen peroxide
Sodium perborate
0
55 ± 4
β- mercaptoethanol
84 ± 3
0
87 ± 3
97 ± 2
EDTA
EGTA
Ca2+
Mg2+
Cu2+
Co2+
Zn2+
K+
7.4 ± 5
7.5 ± 6
111 ± 0.3
34 ± 3.8
2.4 ± 5
105 ± 5
32 ± 6
11 ± 1.5
5 ± 3.4
0
133 ± 0.7
55 ± 2.3
61 ± 2.5
123 ± 2
53 ± 2
103 ± 2
104 ± 1
93 ± 2
154 ± 1
53 ± 5
63 ± 3.2
235 ± 1.3
49 ± 2.7
138 ± 0.4
119 ± 1.6
101 ± 2
100 ± 6
93 ± 2
Na+
Mn2+
32 ± 6
27
Page 27 of 43

Fe2+
1 mM
-
38 ± 2.4
32 ± 2
103 ± 1.7
100 ± 0.2
None
100 ± 4.7
100 ± 2.5
599
*The results presented are the averages of three separate determinations (n=3) ±standard
deviations.
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
28
Page 28 of 43

621
622
Table 4
Effect of alcohol concentration on the activity of the purified xylanase from CS428
Final %
0.06
0.12
0.25
0.5
Alcohol
Activity (%)*
116 ± 1
122 ± 0.9
143 ± 3
140 ± 2
121 ± 3
97 ± 5
Methanol
1
2
0.06
0.12
0.25
0.5
Ethanol
112 ± 1
123 ± 2
149 ± 2
139 ± 3
110 ± 2
109 ± 1
120 ± 4
124 ± 0.95
145 ± 1
147 ± 4
111 ± 1
110 ± 2
112 ± 1
116 ± 2
150 ± 2
143 ± 1
1
2
0.06
0.12
0.25
0.5
Iso propanol
1
2
0.06
0.12
0.25
0.5
Butanol
29
Page 29 of 43