BACKGROUND
lignocellulosic biomass, the most abundant renewable carbon resources in the biosphere, has exhibited valuable industrial applications in many fields, such as pulp and paper, food processing, detergent, textile, synthetic biology, organic synthesis, pharmaceuticals and bioenergy production  <cit> .
arabinan, a polysaccharide constituent of hemicellulose, is composed of α- <dig> - and/or α- <dig> -linked to a α- <dig> -linked l-arabinofuranosyl backbone residues  <cit> .
arabinan mainly consists of l-arabinose residues, existing in rhamnogalacturonan regions of pectins in the cell walls of several plants  <cit> .
microbial enzymes including hydrolases have represented an important part in industry due to their various favorable properties  <cit> .
to generate l-arabinose from arabinan, two major hydrolytic enzymes, endo-arabinanase and α-l-arabinofuranosidase, are essential to the synergetic action.
endo-arabinanase  can hydrolyze the arabinan α- <dig> -linked l-arabinofuranosyl backbone to produce arabino-oligosaccharides or l-arabinoses, whereas α-l-arabinofuranosidase  is capable of thoroughly hydrolyzing arabino-oligosaccharides and arabinan side chain substitutions to produce l-arabinoses  <cit> .
in addition, exo-arabinanase  is able to release terminal arabinose  or arabino-oligosaccharides from arabinan  <cit> .
based on the amino acid sequence similarities, endo-arabinanases and exo-arabinanases have been classified into glycoside hydrolase  family  <dig> and gh <dig> family, respectively, whereas α-l-arabinofuranosidases belong to five gh families  .
these arabinan-degrading enzymes from different gh families have presented various applications in industrial processes such as food technology, nutritional medical research and organic synthesis  <cit> .
a few reports about the endo-arabinanases from bacteria and fungi have been published so far  <cit> .
among all the studies above, only a novel hyperthermophilic endo-arabinanase from thermotoga petrophila was biophysically characterized and the three-dimensional  structure was released  <cit> .
although the genome of hyperthermophilic thermotoga thermarum dsm  <dig> has been sequenced, no information is currently available on the endo-arabinanase.
in this study, a putative gh <dig> β-xylosidase from thermotoga thermarum, tth abn, was cloned and expressed.
its biochemical characterization and application in arabinan degradation were also investigated.
upon the evaluation of substrate specificity, it was interesting to find that the recombinant enzyme was an endo-arabinanase, which has not yet been reported.
RESULTS
amino acid sequence of tth abn
the tth abn gene isolated from the t. thermarum genome is  <dig>  bp in length coding for  <dig> amino acids.
there is a signal peptide sequence  in tth abn when analyzed by signalp  <dig>  .
therefore, only the dna fragment of  <dig>  bp was amplified.
amino acid sequence of tth abn in genbank is described as a putative β-xylosidase.
however, it contains putative domains of α-l-arabinofuranosidase , endo-arabinanase  and β-xylosidase  through the blast.
in this study, the provisional β-xylosidase was finally confirmed as an endo-arabinanase through the biochemical analysis.
tth abn exhibited 64% identity to the β-xylosidase from thermotoga sp.
rq <dig>  and thermotoga petrophila rku- <dig> , 59% identity to putative β-xylosidase dictyoglomus thermophilum h-6- <dig> , and 50% identity to the endo-arabinanase from b. subtilis abn <dig> h318a mutant .
to gain insights into the evolutionary relationships among endo-arabinanases, the phylogenetic trees with  <dig> candidate sequences were constructed using the nj and mp methods, respectively, which both supported the same topological structures as shown in figure  <dig>  phylogenetic analysis indicated that tth abn from t. thermarum had a close relationship with an endo-arabinanase from t. petrophila and a putative β-xylosidase from the same genus t. sp.
homology modeling revealed that tth abn had a similar 5-fold β-propeller structure as the b. subtilis endo-arabinanase abn <dig> h318a mutant  <cit>  .
both sequence comparison and homology modeling indicated that asp <dig> and glu <dig> residues were the catalytic nucleophile and proton donor, respectively  <cit> .
furthermore, there are other conserved amino acid residues adjacent to the catalytic center, his <dig>  asp <dig>  pro <dig>  trp <dig>  ala <dig> and asn <dig>  which may play some roles in catalytic activity.
expression and purification of tth abn
mature protein coding  <dig> amino acids was successfully expressed in the cytoplasmatic fraction of e. coli bl <dig> .
then, the protein in the cell-free extract was purified by more than 95% homogeneity after heat treatment and affinity chromatography .
finally, the purified fusion enzyme showed a single band on a sds-page gel with an estimated molecular weight of 36 kda .
to examine the oligomerization state of the enzyme, size exclusion chromatography was performed.
the native protein formed monomer in solution with a calculated mw  <dig>  da according to the calibration curve of the gel filtration column.
biochemical properties of tth abn
enzymatic characteristics of purified recombinant tth abn were determined and summarized in tables  <dig>   <dig> and  <dig>  substrate specificity was assayed with different substrates .
it was interesting to find that tth abn was active towards linear arabinan, debranched arabinan and sugar beet arabinan, but there was no activity towards  <dig> -β-d-mannan, galactan, p-nitrophenyl-β-d-xylopyranoside , and p-nitrophenyl-α-l-arabinofuranoside .
these results indicated that the enzyme only exhibited arabinanase activity.
nd: not detected.
values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
cations
a
/chemical reagents
b
afinal concentration, 1 mm.
bfinal concentration,  <dig> % tween  <dig> and tris,  <dig> % sds.
values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
t. thermarum
dsm  <dig> and other microorganisms
k
m
k
cat
nd: not determined.
data were carried out with linear arabinan , debranched arabinan , or sugar beet arabinan  as substrate.
values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
d: μmol mg−1 min− <dig>
effect of ph on tth abn activity was determined in 50 mm imidazole-potassium buffer ranging from ph  <dig>  to  <dig>  .
tth abn was found to exhibit an optimum activity at ph  <dig>  and was able to retain more than 95% of its initial activity at 70°c for 1 h .
as a function of temperature, the enzyme displayed the highest activity at 75°c and retained more than 90% of its maximum activity even at 85°c after 10 min incubation .
incubations at different temperatures to determine the enzyme’s thermal stability were also carried out.
as shown in the result, the enzyme kept nearly its initial activity at 75°c after 2 h incubation, and could still retain more than 80% of its original activity at 80°c for 2 h .
effects of metal ions and chemicals on tth abn activity were shown in table  <dig>  in various assays, the enzyme activity was apparently stimulated by 1 mm mn2+, ca2+, ba2+, or chemical reagents  <dig> % tween  <dig> and  <dig> % sds.
on the contrast, al3+, cu2+ and zn2+ could inhibit the enzyme activity evidently.
kinetic studies in the presence of three arabinans as the substrates at optimum temperature and ph allowed the determination of the michaelis-menten parameters as shown in table  <dig>
degradation of arabinan by tth abn
catalytic ability of tth abn on arabinan was investigated by analyzing the digestion products of linear , debranched and sugar beet arabinans .
clearly from the result, the end products for hydrolysis of linear and debranched arabinans were arabinose, arabinobiose and arabinotriose after degradation for 3 h. however, it displayed very weak catalytic ability towards the hydrolysis of sugar beet arabinan.
discussion
enzymatic hydrolysis of arabinose-linked hemicellulose in plant cell wall requires endo-arabinanase, arabinofuranosidase and arabinoxylan arabinofuranohydrolase, which are capable of releasing arabinosyl oligomers and l-arabinose from arabinose-containing polysaccharides  <cit> .
we reported an endo-arabinanase  from the hyperthermophilic bacterium t. thermarum here which was originally predicted as a β-xylosidase yet possessed the capability of arabinan depolymerization.
similar results have been published that endo-arabinanase from thermotoga petrophila rku- <dig> and b. subtilis were confirmed as endo-arabinanases through biophysical characterizations although once were predicted as β-xylosidases  <cit> .
it is well known that hydrolases from gh <dig> family utilize a single displacement mechanism, which results in an inverted anomeric configuration for hydrolysis products  <cit> .
amino acid sequence alignment and homology modeling indicated that asp <dig> and glu <dig> in tth abn were the catalytic nucleophile  and proton donor, respectively.
the result is also supported by the fact that aspartic acid and glutamic acid residues are the catalytic nucleophile and proton donor in all members of gh <dig> family .
residue asp <dig> may act as a pka modulator and maintain the correct alignment of the general acid residue relative to the substrate  <cit> .
residues asp <dig>  asp <dig> and glu <dig> located in the deep cavity at the centre of the β-propeller are also found in all members of gh <dig>   <dig>   <dig> and  <dig>  <cit> .
as the consequence of the flexibility reduction of the polypeptide chain, residue pro <dig> may contribute to the high thermal stability of the enzyme  <cit> .
phylogenetic analysis and enzymatic properties showed that tth abn was distant with the endo-arabinanases from bacillus subtilis, bacillus licheniformis and aspergillus aculeatus <cit> .
however, it revealed a close relationship with t. thermarum and t. petrophila, indicating that they share the similar properties  <cit> .
other endo-arabinanases from genus thermotoga including t. thermarum endo-arabinanase have not yet been studied.
since there is 64% amino acid sequences similarity between endo-arabinanases t. thermarum and t. petrophila and is also inferred by the experimental data shown in table  <dig>  it is confirmed that tth abn could be an endo-arabinanase with some specific properties.
tth abn exhibited good thermostability when incubated at 75° for 2 h. endo-arabinanase from gram-negative bacterium t. petrophila rku- <dig> has been reported to have a ph optimum at  <dig>  and retained 95% of initial activity at 90°c for 10 h  <cit> .
while gram-positive bacteria b. subtilis and b. licheniformis endo-arabinanases show optimum activity at near-neutral ph at 60°c and 35°c, respectively  <cit> .
endo-arabinanase aspergillus aculeatus from fungus displays optimum activity at ph  <dig>  and 50°c  <cit> .
in general, endo-arabinanases from bacteria exhibit the near-neutral ph optima, while those from fungi display the acid ph optima.
the enzyme activities regarding ph from bacteria and fungi are also consistent with their inherent properties.
cations and chemical reagents exhibited different effects on the activity of the endo-arabinanase tth abn.
with the evidence that one ca2+ ion was observed in the catalytic cavity of bacillus subtilis about 5 Å below the catalytic carboxylates and the presence of ions in an equivalent location in other arabinanases  <cit> .
compared to other endo-arabinanases, tth abn demonstrated the highest vmax value among all the known endo-arabinanases from thermophile and hyperthermophile when linear arabinan was used as the substrate.
the catalytic efficiency  of endo-arabinanase tth abn on debranched arabinanwas approximately 6-fold and 18-fold higher than b. licheniformis and caldicellulosiruptor saccharolyticus, respectively .
in addition, catalytic efficiency for linear, debranched and sugar beet arabinans existed obvious differences .
this may be caused by substrate’s composition and structure.
as we know, a typical sugar composition of linear arabinan is: arabinose: galactose: rhamnose: galacturonic acid =  <dig> :  <dig> :  <dig> :  <dig> .
for debranched and sugar beet arabinans, the contents of arabinose are lower than linear arabinan, and sugar beet arabinan has more substitutions attached to backbone while  <dig> - and  <dig> -α-l-arabinofuranosyl branch units of debranched arabinan have been all removed.
the fact that tth abn could hydrolyze linear and debranched arabinan into oligomers arabinose, arabinobiose and arabinotriose implied that the tth abn had endo-arabinanase activity but no exo activity.
only a few endo-arabinanases especially that from hyperthermophile have been reported to possess the function in generating arabino-oligosaccharides  <cit> .
although it was insensitive to the branched arabinan , tth abn could be used together with other arabinan-degrading enzymes in synergistic reaction for the hydrolysis of branched ones.
CONCLUSIONS
in this study, a useful endo-arabinanase  from t. thermarum dsm  <dig> was expressed in e. coli with desirable features, such as high thermostability, catalytic efficiency, and hydrolytic reaction at high temperature.
this is easily envisioned that tth abn endo-arabinanase exhibits a great potential for enzymatic conversion of arabinan through synergetic action with other arabinan-degrading enzymes.

 lignocellulosic biomass, the most abundant renewable carbon resources in the biosphere, has exhibited valuable industrial applications in many fields, such as pulp and paper, food processing, detergent, textile, synthetic biology, organic synthesis, pharmaceuticals and bioenergy production. arabinan, a polysaccharide constituent of hemicellulose, is composed of α- - and/or α- -linked to a α- -linked l-arabinofuranosyl backbone residues. arabinan mainly consists of l-arabinose residues, existing in rhamnogalacturonan regions of pectins in the cell walls of several plants. microbial enzymes including hydrolases have represented an important part in industry due to their various favorable properties. to generate l-arabinose from arabinan, two major hydrolytic enzymes, endo-arabinanase and α-l-arabinofuranosidase, are essential to the synergetic action. endo-arabinanase can hydrolyze the arabinan α- -linked l-arabinofuranosyl backbone to produce arabino-oligosaccharides or l-arabinoses, whereas α-l-arabinofuranosidase is capable of thoroughly hydrolyzing arabino-oligosaccharides and arabinan side chain substitutions to produce l-arabinoses. in addition, exo-arabinanase is able to release terminal arabinose or arabino-oligosaccharides from arabinan. based on the amino acid sequence similarities, endo-arabinanases and exo-arabinanases have been classified into glycoside hydrolase family and gh family, respectively, whereas α-l-arabinofuranosidases belong to five gh families. these arabinan-degrading enzymes from different gh families have presented various applications in industrial processes such as food technology, nutritional medical research and organic synthesis. a few reports about the endo-arabinanases from bacteria and fungi have been published so far. among all the studies above, only a novel hyperthermophilic endo-arabinanase from thermotoga petrophila was biophysically characterized and the three-dimensional structure was released. although the genome of hyperthermophilic thermotoga thermarum dsm has been sequenced, no information is currently available on the endo-arabinanase. in this study, a putative gh β-xylosidase from thermotoga thermarum, tth abn, was cloned and expressed. its biochemical characterization and application in arabinan degradation were also investigated. upon the evaluation of substrate specificity, it was interesting to find that the recombinant enzyme was an endo-arabinanase, which has not yet been reported.  amino acid sequence of tth abn the tth abn gene isolated from the t. thermarum genome is  bp in length coding for amino acids. there is a signal peptide sequence in tth abn when analyzed by signalp. therefore, only the dna fragment of  bp was amplified. amino acid sequence of tth abn in genbank is described as a putative β-xylosidase. however, it contains putative domains of α-l-arabinofuranosidase, endo-arabinanase and β-xylosidase through the blast. in this study, the provisional β-xylosidase was finally confirmed as an endo-arabinanase through the biochemical analysis. tth abn exhibited 64% identity to the β-xylosidase from thermotoga sp. rq and thermotoga petrophila rku-, 59% identity to putative β-xylosidase dictyoglomus thermophilum h-6-, and 50% identity to the endo-arabinanase from b. subtilis abn h318a mutant. to gain insights into the evolutionary relationships among endo-arabinanases, the phylogenetic trees with candidate sequences were constructed using the nj and mp methods, respectively, which both supported the same topological structures as shown in figure  phylogenetic analysis indicated that tth abn from t. thermarum had a close relationship with an endo-arabinanase from t. petrophila and a putative β-xylosidase from the same genus t. sp. homology modeling revealed that tth abn had a similar 5-fold β-propeller structure as the b. subtilis endo-arabinanase abn h318a mutant. both sequence comparison and homology modeling indicated that asp and glu residues were the catalytic nucleophile and proton donor, respectively. furthermore, there are other conserved amino acid residues adjacent to the catalytic center, his asp pro trp ala and asn which may play some roles in catalytic activity. expression and purification of tth abn mature protein coding amino acids was successfully expressed in the cytoplasmatic fraction of e. coli bl. then, the protein in the cell-free extract was purified by more than 95% homogeneity after heat treatment and affinity chromatography. finally, the purified fusion enzyme showed a single band on a sds-page gel with an estimated molecular weight of 36 kda. to examine the oligomerization state of the enzyme, size exclusion chromatography was performed. the native protein formed monomer in solution with a calculated mw  da according to the calibration curve of the gel filtration column. biochemical properties of tth abn enzymatic characteristics of purified recombinant tth abn were determined and summarized in tables  and substrate specificity was assayed with different substrates. it was interesting to find that tth abn was active towards linear arabinan, debranched arabinan and sugar beet arabinan, but there was no activity towards -β-d-mannan, galactan, p-nitrophenyl-β-d-xylopyranoside, and p-nitrophenyl-α-l-arabinofuranoside. these results indicated that the enzyme only exhibited arabinanase activity. nd: not detected. values shown were the mean of triplicate experiments, and the variation about the mean was below 5%. cations a /chemical reagents b afinal concentration, 1 mm. bfinal concentration, % tween and tris, % sds. values shown were the mean of triplicate experiments, and the variation about the mean was below 5%. t. thermarum dsm and other microorganisms k m k cat nd: not determined. data were carried out with linear arabinan, debranched arabinan, or sugar beet arabinan as substrate. values shown were the mean of triplicate experiments, and the variation about the mean was below 5%. d: μmol mg−1 min− effect of ph on tth abn activity was determined in 50 mm imidazole-potassium buffer ranging from ph  to. tth abn was found to exhibit an optimum activity at ph  and was able to retain more than 95% of its initial activity at 70°c for 1 h. as a function of temperature, the enzyme displayed the highest activity at 75°c and retained more than 90% of its maximum activity even at 85°c after 10 min incubation. incubations at different temperatures to determine the enzyme’s thermal stability were also carried out. as shown in the result, the enzyme kept nearly its initial activity at 75°c after 2 h incubation, and could still retain more than 80% of its original activity at 80°c for 2 h. effects of metal ions and chemicals on tth abn activity were shown in table  in various assays, the enzyme activity was apparently stimulated by 1 mm mn2+, ca2+, ba2+, or chemical reagents % tween and % sds. on the contrast, al3+, cu2+ and zn2+ could inhibit the enzyme activity evidently. kinetic studies in the presence of three arabinans as the substrates at optimum temperature and ph allowed the determination of the michaelis-menten parameters as shown in table  degradation of arabinan by tth abn catalytic ability of tth abn on arabinan was investigated by analyzing the digestion products of linear, debranched and sugar beet arabinans. clearly from the result, the end products for hydrolysis of linear and debranched arabinans were arabinose, arabinobiose and arabinotriose after degradation for 3 h. however, it displayed very weak catalytic ability towards the hydrolysis of sugar beet arabinan. discussion enzymatic hydrolysis of arabinose-linked hemicellulose in plant cell wall requires endo-arabinanase, arabinofuranosidase and arabinoxylan arabinofuranohydrolase, which are capable of releasing arabinosyl oligomers and l-arabinose from arabinose-containing polysaccharides. we reported an endo-arabinanase from the hyperthermophilic bacterium t. thermarum here which was originally predicted as a β-xylosidase yet possessed the capability of arabinan depolymerization. similar results have been published that endo-arabinanase from thermotoga petrophila rku- and b. subtilis were confirmed as endo-arabinanases through biophysical characterizations although once were predicted as β-xylosidases. it is well known that hydrolases from gh family utilize a single displacement mechanism, which results in an inverted anomeric configuration for hydrolysis products. amino acid sequence alignment and homology modeling indicated that asp and glu in tth abn were the catalytic nucleophile and proton donor, respectively. the result is also supported by the fact that aspartic acid and glutamic acid residues are the catalytic nucleophile and proton donor in all members of gh family. residue asp may act as a pka modulator and maintain the correct alignment of the general acid residue relative to the substrate. residues asp asp and glu located in the deep cavity at the centre of the β-propeller are also found in all members of gh and. as the consequence of the flexibility reduction of the polypeptide chain, residue pro may contribute to the high thermal stability of the enzyme. phylogenetic analysis and enzymatic properties showed that tth abn was distant with the endo-arabinanases from bacillus subtilis, bacillus licheniformis and aspergillus aculeatus. however, it revealed a close relationship with t. thermarum and t. petrophila, indicating that they share the similar properties. other endo-arabinanases from genus thermotoga including t. thermarum endo-arabinanase have not yet been studied. since there is 64% amino acid sequences similarity between endo-arabinanases t. thermarum and t. petrophila and is also inferred by the experimental data shown in table  it is confirmed that tth abn could be an endo-arabinanase with some specific properties. tth abn exhibited good thermostability when incubated at 75° for 2 h. endo-arabinanase from gram-negative bacterium t. petrophila rku- has been reported to have a ph optimum at and retained 95% of initial activity at 90°c for 10 h. while gram-positive bacteria b. subtilis and b. licheniformis endo-arabinanases show optimum activity at near-neutral ph at 60°c and 35°c, respectively. endo-arabinanase aspergillus aculeatus from fungus displays optimum activity at ph  and 50°c. in general, endo-arabinanases from bacteria exhibit the near-neutral ph optima, while those from fungi display the acid ph optima. the enzyme activities regarding ph from bacteria and fungi are also consistent with their inherent properties. cations and chemical reagents exhibited different effects on the activity of the endo-arabinanase tth abn. with the evidence that one ca2+ ion was observed in the catalytic cavity of bacillus subtilis about 5 Å below the catalytic carboxylates and the presence of ions in an equivalent location in other arabinanases. compared to other endo-arabinanases, tth abn demonstrated the highest vmax value among all the known endo-arabinanases from thermophile and hyperthermophile when linear arabinan was used as the substrate. the catalytic efficiency of endo-arabinanase tth abn on debranched arabinanwas approximately 6-fold and 18-fold higher than b. licheniformis and caldicellulosiruptor saccharolyticus, respectively. in addition, catalytic efficiency for linear, debranched and sugar beet arabinans existed obvious differences. this may be caused by substrate’s composition and structure. as we know, a typical sugar composition of linear arabinan is: arabinose: galactose: rhamnose: galacturonic acid = :::. for debranched and sugar beet arabinans, the contents of arabinose are lower than linear arabinan, and sugar beet arabinan has more substitutions attached to backbone while - and -α-l-arabinofuranosyl branch units of debranched arabinan have been all removed. the fact that tth abn could hydrolyze linear and debranched arabinan into oligomers arabinose, arabinobiose and arabinotriose implied that the tth abn had endo-arabinanase activity but no exo activity. only a few endo-arabinanases especially that from hyperthermophile have been reported to possess the function in generating arabino-oligosaccharides. although it was insensitive to the branched arabinan, tth abn could be used together with other arabinan-degrading enzymes in synergistic reaction for the hydrolysis of branched ones.  in this study, a useful endo-arabinanase from t. thermarum dsm was expressed in e. coli with desirable features, such as high thermostability, catalytic efficiency, and hydrolytic reaction at high temperature. this is easily envisioned that tth abn endo-arabinanase exhibits a great potential for enzymatic conversion of arabinan through synergetic action with other arabinan-degrading enzymes. 
this work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan. in this study, the characterization and application in arabinan degradation of an endo-arabinanase from thermotoga thermarum were investigated. the recombinant endo-arabinanase was expressed in escherichia coli bl and purified by heat treatment followed by purification on a nickel affinity column chromatography. when linear arabinan was used as the substrate, the apparent km and vmax values were determined to be  ±   mg ml− and.1 ±   μmol ml−1 min− respectively, and the calculated kcat value was  ±   s− the purified endo-arabinanase exhibited optimal activity at ph  and 75°c and its residual activity retained more than 80% of its initial activity after being incubated at 80°c for 2 h. the results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature.
 arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-l-arabinofuranosidase. in this study, the characterization and application in arabinan degradation of an endo-arabinanase from thermotoga thermarum were investigated.  the recombinant endo-arabinanase was expressed in escherichia coli bl and purified by heat treatment followed by purification on a nickel affinity column chromatography. the purified endo-arabinanase exhibited optimal activity at ph  and 75°c and its residual activity retained more than 80% of its initial activity after being incubated at 80°c for 2 h. the results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. when linear arabinan was used as the substrate, the apparent km and vmax values were determined to be  ±   mg ml− and.1 ±   μmol ml−1 min− respectively, and the calculated kcat value was  ±   s−  this work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan. arabinanarabinoseendo-arabinanasethermotoga thermarum 