BACKGROUND
mannans are complex polysaccharides representing one of the major components of hemicellulose, consisting of four types: linear mannan, glucomannan, galactomannan, and galactoglucomanan  <cit> .
each of these polysaccharides has a β- <dig> -linked backbone units including mannose or a combination of glucose and mannose residues, with the presence of α- <dig> -linked side-chain substitutions  <cit> .
it was reported that the hydrolysis of these polysaccharides requires several mannan-degrading enzymes, primarily including β-mannanase , β-mannosidase  and β-glucosidase .
other enzymes such as α-galactosidase and mannan esterase are required to remove α-galactosyl and o-acetyl side-chain substituent.
among these enzymes, two types of mannan-degrading enzymes are necessary  <cit> .
one endotype, β-mannanase, is responsible for the cleavage of β- <dig> -linked mannose residues backbone randomly to generate mannooligosaccharides.
another exotype, β-mannosidase, hydrlyses the nonreducing end of mannooligosaccharides to release mannoses  <cit> .
it is known that β-mannosidase is produced from plants, bacterial, fungi, invertebrates as well as some mammalian species  <cit> .
based on amino acid similarity and multi-domains, β-mannosidases have been mainly classified into glycoside hydrolase family   <dig>   <dig> and  <dig> .
these β-mannosidases from different ghfs possess considerable industrial applications in many fields, such as food, feed and pulp/paper industries  <cit> .
in addition, β-mannosidases have important role in saccharification of hemicellulose for fuel and other chemicals production.
in human, lack of β-mannosidase can lead to β-mannosidosis  <cit> .
during the last two decades, thermostable enzymes from thermophilic or hyperthermophilic microorganisms have become the hotspots of researches in many fields  <cit> .
the amino acid sequences of β-mannosidases are abundantly available on the constantly updating databases.
however, only a few β-mannosidases especially from hyperthermophile have been cloned, purified and characterized  <cit> .
it was found that the known hyperthermophilic β-mannosidases from pyrococcus furiosus, thermotoga maritima, and thermotoga neapolitana belonged to the ghf <dig>  ghf <dig>  and ghf <dig>  respectively  <cit> .
thermotoga thermarum, isolated from continental solfataric springs at lac abbe , is an anaerobic hyperthermophilic bacteria that grows at 80°c  <cit> .
and it has many glycoside hydrolase genes based on the genomic sequence .
the biotechnology industry is essential in modern societies  <cit> , which is reflected in the production of recombinant enzymes  and their applications.
in this study, we described the cloning, expression and functional characterizations of a novel recombinant β-mannosidase  in e. coli.
RESULTS
amino acid sequence of tth man <dig> β-mannosidase
the tth man <dig> gene isolated from the t. thermarum genome was  <dig>  bp in length coding  <dig> amino acids and it was predicted as an endo-β-mannanase  available at ncbi and cazy sites  .
as shown in figure  <dig>  tth man <dig> displayed 33% identity to β-mannosidase from sorangium cellulosum so ce <dig>  32% identity to putative β-mannosidase from actinosynnema mirum dsm  <dig> and 32% identity to the glycoside hydrolase from streptomyces flavogriseus atcc  <dig>  the results of alignments also revealed that glu <dig>  glu <dig>  glu <dig>  glu <dig> and glu <dig> were conserved amino acids among these ghf <dig> β-mannosidases.
according to the cazy database, two glutamic acids are the acid/base and the nucleophile, respectively.
against the similar catalytic domain of ghf <dig> endoglucanase  from pseudoalteromonas haloplanktis, it was presumed that active amino acids of tth man <dig> β-mannosidase were glu <dig> and glu <dig>  <cit> .
over-expression and purification of tth man <dig> β-mannosidase
when using native gene from t. thermarum for expression, the protein production was very difficult to detected .
thus, in order to increase the expression level of tth man <dig> β-mannosidase in escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence .
the mature protein without the signal peptide, allowing the insertion of a his6-tag at the c-terminus, was successfully expressed in e. coli bl <dig> , after induction with iptg for  <dig> h at 37°c.
the recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column .
finally, the purified recombinant enzyme displayed a single band on sds-page with an estimated molecular weight  of  <dig> kda , which was consistent with the predicted mw of monomer .
size exclusion chromatography was also carried out using the aktafplc™ system to compute the oligomerization state of the target protein.
it was deduced that the native protein formed 7-mer in solution with a calculated mw  <dig>  da according to the calibration curve of the gel filtration column.
athe recombinant strain was grown in lb medium  with  <dig> μg ampicillin/ml at 37°c to od <dig>  <dig> - <dig>  and was incubated further with isopropyl-β-thiogalactopyranoside  for  <dig> h. the cells were harvested by centrifugation at  <dig>  g for  <dig> min at 4°c and resuspended in  <dig> ml imidazole buffer , followed by sonication.
bthe cell extracts after sonication were heat treated at 70°c for  <dig> min, and then cooled in an ice bath, centrifuged at  <dig>  g for  <dig> min at 4°c and the supernatant was kept.
cthe obtained supernatants were loaded on to an immobilized metal affinity column , and eluted with  <dig>  m imidazole,  <dig>  m nacl, and  <dig> mm tris–hcl buffer .
biochemical characteristics of tth man <dig> β-mannosidase
the enzymatic properties of purified recombinant tth man <dig> β-mannosidase were determined and summarized in tables  <dig>   <dig> and  <dig>  substrate specificity was assayed with different substrates and tth man <dig> β-mannosidase was found to be active to p-nitrophenyl-β-d-mannopyranoside  and  <dig> -β-d-mannan.
however, no activity was detected towards konjaku powder, guar galactomannan and galactan .
these results indicated that the enzyme showed only exo-enzyme activity, which is not consistent with the prediction on its function at ncbi and cazy.
nd: not detected.
values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
afinal concentration, the former value in the table was determined at  <dig> mm.
bfinal concentration, the values in the table were determined at  <dig> mm,  <dig> %,  <dig> % and  <dig> % for edta, tween  <dig>  tris and sds, respectively.
values shown were the means of triplicate experiments.
apnpm: p-nitrophenyl-β-d-mannopyranoside.
bnd: not determined.
ccalculated by the data based on the reference.
the recombinant tth man <dig> β-mannosidase has a ph optimum of  <dig>  as shown in figure 3a.
the enzyme was relatively stable at a ph range of  <dig>  to  <dig>  and most stable at ph  <dig>  .
more than 70% of the initial enzyme activity remained at this range.
the enzyme was most active at 85°c, and it retained approximately 50% of the maximum activity at 95°c .
the thermostability data showed that it remained above 56% of its initial activity after  <dig> h of pre-incubation at temperature ranging from 75°c to 90°c .
the effects of metal ions and chemical reagents on the enzyme activity are shown in table  <dig>  in various assays, the enzyme activity was apparently stimulated by  <dig> mm mn2+, co2+ and ethylene diamine tetraacetic acid .
however, the enzyme activity was apparently inhibited by  <dig> mm cu2+ and zn2+,  <dig> % tris and  <dig> % sds.
enzyme kinetic studies in the addition of pnpm and  <dig> -β-d-mannan as the substrate at optimum temperature and ph allowed the determination of the michaelis-menten parameters .
the enzyme had an obvious km of  <dig> ± <dig>  mm , vmax of  <dig> ± <dig>  μmol min- <dig> mg- <dig> and kcat/km of  <dig> ± <dig>  mm- <dig> s- <dig> using pnpm as substrate, while km of  <dig> ± <dig>  mg ml- <dig>  vmax of  <dig> ± <dig>  μmol min- <dig> mg- <dig> and kcat/km of  <dig> ± <dig>  s- <dig> mg- <dig> ml for  <dig> -β-d-mannan.
the effect of mannose concentration on the tth man <dig> β-mannosidase activity was also investigated .
though the enzyme activity was gradually decreased with the increase of mannose concentration, the enzyme could retain 50% of its initial activity at  <dig> mm of mannose concentration, indicating tth man <dig> β-mannosidase is a mannose-tolerant β-mannosidase with a ki of  <dig> mm mannose.
hydrolysis of  <dig> -β-d-mannan by the purified tth man <dig> β-mannosidase
tth man <dig> β-mannosidase was capable of degrading not only pnpm but also polymer mannan.
the mode of action of tth man <dig> β-mannosidase was determined by analyzing the products of digestion of  <dig> -β-d-mannan .
the end products were almost the mannose after degradation for  <dig>  h and mannose concentration increased with prolonging the hydrolysis time.
the result confirmed that tth man <dig> β-mannosidase only displayed exo-enzyme activity.
phylogenetic analysis of tth man <dig> β-mannosidase
the phylogenetic trees generated from  <dig> candidate sequences were constructed to gain deeper insight into the evolutionary relationship among β-mannosidases, using the neighbor-joining  and maximum-parsimony  methods.
both trees displayed almost the same topological structures .
it revealed the presence of three well-supported clades and each clade consisting of a separated monophyletic group .
clade i, clade ii and clade iii consisted of the ghf <dig>  ghf <dig> and ghf <dig> β-mannosidases, respectively.
from the phylogenetic trees, it is obvious that there are two subclades in clade ii.
tth man <dig> β-mannosidase from t. thermarum showed an apparently distant relationship with the ghf <dig> β-mannosidases from the same genus.
therefore, it was presumed that the biochemical properties of tth man <dig> β-mannosidase might differ from the same genus β-mannosidases.
this was confirmed by the experiment results shown in table  <dig>
discussion
β-mannosidase is an important hydrolytic enzyme which attacks the non-reducing end of the β-linked mannooligosaccharides to release mannoses  <cit> .
it plays a key role in the degradation pathway of complex oligosaccharide and glycoproteins  <cit> .
to our knowledge, the hydrolytic end product by β-mannosidase, mannoses, are also fermentable sugars which can be bio-converted to bio-fuels and value-added chemicals  <cit> .
the release of the genome in database provides us an effective way to investigate the uncharacterized enzymes, which may have great potential in industrial applications.
for this study, a putative endo-β-mannanase gene  from t. thermarum was cloned and was finally defined as a β-mannosidase through the biochemical characterization.
the t. thermarum β-mannosidase is distinct from the other glycosyl hydrolases from t. thermarum.
the substrate specificity and amino acid sequence of the β-mannosidase are apparently different from those of the xylanase and β-xylosidase from t. thermarum previously reported  <cit> .
based on sequences similarity, the tth man <dig> β-mannosidase belongs to ghf <dig>  it has a homologous relationship with those from s. cellulosum , a. mirum  and s. flavogriseus  .
compared to the same genus thermotoga β-mannosidase from ghf <dig> or ghf <dig>  however, there is great difference according to phylogenetic analysis and they belong to different monophyletic groups .
this suggests that tth man <dig> β-mannosidase may have some specific properties.
like other hemicellulases, the catalytic mechanism of β-mannosidase is also a double displacement lysozyme-like reaction, involving stabilization of an oxocarbonium ion by electrostatic interaction with two glutamate acid residues at the active site  <cit> .
by the alignment with other ghf <dig> β-mannosidases and a ghf <dig> cellulase from pseudoalteromonas haloplanktis , the acid/base and the nucleophile are identified as glu <dig> and glu <dig> residues, respectively  <cit> .
it is difficult to know more details about the functional amino acid residues as lack of a suitable template for homology modeling.
therefore, a crystalline structure analysis is needed to further investigate.
the tth man <dig> β-mannosidase from t. thermarum dsm  <dig> exhibits the highest activity at temperature 85°c .
the temperature optima is higher than the β-mannosidases from homo sapiens, aplysia kurodai, bacillus licheniformis, bacillus circulans, aspergillus niger and aspergillus awamori, in which optimal temperature are ranged from 37°c to 70°c  <cit> .
it is found that the β-mannosidases from the genus thermotoga and pyrococcus furiosus exhibit the maximum activity above 80°c  <cit> .
however, ph optima for animals and fungi are near  <dig>  while for bacteria occurs at neutral environment.
as we know, the enzymes with high thermal stability are necessary in industrial progresses and thus it can minimize the enzyme consumption and enhance the catalytic efficiency.
the tth man <dig> β-mannosidase residual activity retains approximately 91% after being incubated at 75°c for  <dig> h. furthermore, the tth man <dig> β-mannosidase residual activity is still more than 50% even after being incubated at 90°c for  <dig> h. it shares similar thermostability at high temperature with the β-mannosidases from hyperthermophile.
for instance, t. neapolitana β-mannosidase was stable at 90°c for  <dig> h and t. maritima β-mannosidase was stable at 80°c for  <dig> h  <cit> .
the activity of β-mannosidase is significantly inhibited by cu2+ and zn2+ and this is similar to the β-mannosidase from homo sapiens whose activity is thoroughly decreased the activity by the addition of  <dig>  mm cu2+ or zn2+.
however, the enzyme activity is greatly stimulated by co2+ and mn2+ , very different from thermotoga maritima β-mannosidase which enzyme activity is apparently inhibited by the both ions  <cit> .
β-mannosidase is known to be a key enzyme for converting mannooligosaccharides to mannoses, the end products, which are able to inhibit the enzymatic reaction to a certain extent.
therefore, β-mannosidases with high tolerance to mannoses are beneficial to conversion of mannooligosaccharides.
however, little information about mannose-tolerant β-mannosidase is available in literatures.
as shown in figure  <dig>  tth man <dig> β-mannosidase is insensitive to mannose  whereas the ki for thermobifida fusca β-mannosidase is only  <dig>  mm  <cit> , suggesting a high tolerance to mannose inhibition.
moreover, high enzyme activity is also demanded for β-mannosidase in enzymatic hydrolysis of mannooligosaccharides.
the vmax value of tth man <dig> β-mannosidase for pnpm is  <dig> ± <dig>  μmol min- <dig> mg- <dig>  7-fold higher than thermostable β-mannosidases from t. neapolitana and p. furiosus, 38-fold higher than t. fusca β-mannosidase and 62-fold than thermoascus aurantiacus β-mannosidase  <cit> .
the kcat/km of tth man <dig> β-mannosidase for pnpm is  <dig> ± <dig>  mm- <dig> s- <dig>  approximately 2-fold higher than a. niger β-mannosidase, 11-fold higher than the p. furiosus β-mannosidase, 18-fold higher than the t. neapolitana β-mannosidase and 80-fold higher than the thermoascus aurantiacus β-mannosidase  <cit> .
generallly, it is noteworthy that tth man <dig> β-mannosidase has the highest vmax and kcat/km value .
therefore, its mannose tolerance and high catalytic efficiency are desirable features for potential industrial applications.
due to its theoretical and practical importance we investigated the hydrolysis of different substrates.
when  <dig> -β-d-mannan was degraded by tth man <dig> β-mannosidase, mannose was produced as a major product .
it states that tth man <dig> β-mannosidase is a typical β-mannosidase and acts on polymer substrate with an exolytic manner.
such an exo-type action has been found in the β-mannosidase from a marine gastropod, aplysia kurodai <cit> .
it is obvious that tth man <dig> β-mannosidase has a significant ability for the hydrolysis of  <dig> -β-d-mannan  with the vmax of  <dig> ± <dig>  μmol min- <dig> mg- <dig>  however, no activity is detected on konjaku powder and guar galactomannan which are not pretreated with any enzymes.
the results indicate that tth man <dig> β-mannosidase can act on galactomannan cooperatively with β-mannanase and α-galactosidase to produce mannose efficiently but can not act with only tth man β-mannosidase.
as tth man β-mannosidase can degrade  <dig> -β-d-mannan with high efficiency, it is deduced that it also can efficiently hydrolyze mannooligosaccharides whose degree of polymerization is less than  <dig> -β-d-mannan.
CONCLUSIONS
in this study, a novel β-mannosidase  from t. thermarum dsm  <dig> was over-expressed in e. coli with some specific features.
the results of phylogenetic analysis and biochemical properties showed that the tth man <dig> β-mannosidase was distant with the other genus thermotoga β-mannosidases.
as compared to other microorganisms, the tth man <dig> β-mannosidase possessed higher tolerance to mannose, higher catalytic efficiency and higher thermostability.
therefore, this study provides a novel and useful β-mannosidase with combined properties of high catalytic efficiency, mannose-tolerance and thermostability.
this is easily envisioned that tth man <dig> β-mannosidase exhibits a great potential for enzymatic conversion of mannans.

 mannans are complex polysaccharides representing one of the major components of hemicellulose, consisting of four types: linear mannan, glucomannan, galactomannan, and galactoglucomanan. each of these polysaccharides has a β- -linked backbone units including mannose or a combination of glucose and mannose residues, with the presence of α- -linked side-chain substitutions. it was reported that the hydrolysis of these polysaccharides requires several mannan-degrading enzymes, primarily including β-mannanase, β-mannosidase and β-glucosidase. other enzymes such as α-galactosidase and mannan esterase are required to remove α-galactosyl and o-acetyl side-chain substituent. among these enzymes, two types of mannan-degrading enzymes are necessary. one endotype, β-mannanase, is responsible for the cleavage of β- -linked mannose residues backbone randomly to generate mannooligosaccharides. another exotype, β-mannosidase, hydrlyses the nonreducing end of mannooligosaccharides to release mannoses. it is known that β-mannosidase is produced from plants, bacterial, fungi, invertebrates as well as some mammalian species. based on amino acid similarity and multi-domains, β-mannosidases have been mainly classified into glycoside hydrolase family and. these β-mannosidases from different ghfs possess considerable industrial applications in many fields, such as food, feed and pulp/paper industries. in addition, β-mannosidases have important role in saccharification of hemicellulose for fuel and other chemicals production. in human, lack of β-mannosidase can lead to β-mannosidosis. during the last two decades, thermostable enzymes from thermophilic or hyperthermophilic microorganisms have become the hotspots of researches in many fields. the amino acid sequences of β-mannosidases are abundantly available on the constantly updating databases. however, only a few β-mannosidases especially from hyperthermophile have been cloned, purified and characterized. it was found that the known hyperthermophilic β-mannosidases from pyrococcus furiosus, thermotoga maritima, and thermotoga neapolitana belonged to the ghf ghf and ghf respectively. thermotoga thermarum, isolated from continental solfataric springs at lac abbe, is an anaerobic hyperthermophilic bacteria that grows at 80°c. and it has many glycoside hydrolase genes based on the genomic sequence. the biotechnology industry is essential in modern societies, which is reflected in the production of recombinant enzymes and their applications. in this study, we described the cloning, expression and functional characterizations of a novel recombinant β-mannosidase in e. coli.  amino acid sequence of tth man β-mannosidase the tth man gene isolated from the t. thermarum genome was bp in length coding amino acids and it was predicted as an endo-β-mannanase available at ncbi and cazy sites. as shown in figure  tth man displayed 33% identity to β-mannosidase from sorangium cellulosum so ce 32% identity to putative β-mannosidase from actinosynnema mirum dsm and 32% identity to the glycoside hydrolase from streptomyces flavogriseus atcc the results of alignments also revealed that glu glu glu glu and glu were conserved amino acids among these ghf β-mannosidases. according to the cazy database, two glutamic acids are the acid/base and the nucleophile, respectively. against the similar catalytic domain of ghf endoglucanase from pseudoalteromonas haloplanktis, it was presumed that active amino acids of tth man β-mannosidase were glu and glu. over-expression and purification of tth man β-mannosidase when using native gene from t. thermarum for expression, the protein production was very difficult to detected. thus, in order to increase the expression level of tth man β-mannosidase in escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence. the mature protein without the signal peptide, allowing the insertion of a his6-tag at the c-terminus, was successfully expressed in e. coli bl, after induction with iptg for h at 37°c. the recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column. finally, the purified recombinant enzyme displayed a single band on sds-page with an estimated molecular weight of kda, which was consistent with the predicted mw of monomer. size exclusion chromatography was also carried out using the aktafplc™ system to compute the oligomerization state of the target protein. it was deduced that the native protein formed 7-mer in solution with a calculated mw da according to the calibration curve of the gel filtration column. athe recombinant strain was grown in lb medium with μg ampicillin/ml at 37°c to od - and was incubated further with isopropyl-β-thiogalactopyranoside for h. the cells were harvested by centrifugation at  g for min at 4°c and resuspended in ml imidazole buffer, followed by sonication. bthe cell extracts after sonication were heat treated at 70°c for min, and then cooled in an ice bath, centrifuged at g for min at 4°c and the supernatant was kept. cthe obtained supernatants were loaded on to an immobilized metal affinity column, and eluted with m imidazole, m nacl, and mm tris–hcl buffer. biochemical characteristics of tth man β-mannosidase the enzymatic properties of purified recombinant tth man β-mannosidase were determined and summarized in tables  and substrate specificity was assayed with different substrates and tth man β-mannosidase was found to be active to p-nitrophenyl-β-d-mannopyranoside and -β-d-mannan. however, no activity was detected towards konjaku powder, guar galactomannan and galactan. these results indicated that the enzyme showed only exo-enzyme activity, which is not consistent with the prediction on its function at ncbi and cazy. nd: not detected. values shown were the mean of triplicate experiments, and the variation about the mean was below 5%. afinal concentration, the former value in the table was determined at mm. bfinal concentration, the values in the table were determined at mm, %, % and % for edta, tween tris and sds, respectively. values shown were the means of triplicate experiments. apnpm: p-nitrophenyl-β-d-mannopyranoside. bnd: not determined. ccalculated by the data based on the reference. the recombinant tth man β-mannosidase has a ph optimum of as shown in figure 3a. the enzyme was relatively stable at a ph range of to and most stable at ph. more than 70% of the initial enzyme activity remained at this range. the enzyme was most active at 85°c, and it retained approximately 50% of the maximum activity at 95°c. the thermostability data showed that it remained above 56% of its initial activity after h of pre-incubation at temperature ranging from 75°c to 90°c. the effects of metal ions and chemical reagents on the enzyme activity are shown in table  in various assays, the enzyme activity was apparently stimulated by mm mn2+, co2+ and ethylene diamine tetraacetic acid. however, the enzyme activity was apparently inhibited by mm cu2+ and zn2+, % tris and % sds. enzyme kinetic studies in the addition of pnpm and -β-d-mannan as the substrate at optimum temperature and ph allowed the determination of the michaelis-menten parameters. the enzyme had an obvious km of ± mm, vmax of ± μmol min- mg- and kcat/km of ± mm- s- using pnpm as substrate, while km of ± mg ml- vmax of ± μmol min- mg- and kcat/km of ± s- mg- ml for -β-d-mannan. the effect of mannose concentration on the tth man β-mannosidase activity was also investigated. though the enzyme activity was gradually decreased with the increase of mannose concentration, the enzyme could retain 50% of its initial activity at mm of mannose concentration, indicating tth man β-mannosidase is a mannose-tolerant β-mannosidase with a ki of mm mannose. hydrolysis of -β-d-mannan by the purified tth man β-mannosidase tth man β-mannosidase was capable of degrading not only pnpm but also polymer mannan. the mode of action of tth man β-mannosidase was determined by analyzing the products of digestion of -β-d-mannan. the end products were almost the mannose after degradation for h and mannose concentration increased with prolonging the hydrolysis time. the result confirmed that tth man β-mannosidase only displayed exo-enzyme activity. phylogenetic analysis of tth man β-mannosidase the phylogenetic trees generated from candidate sequences were constructed to gain deeper insight into the evolutionary relationship among β-mannosidases, using the neighbor-joining and maximum-parsimony methods. both trees displayed almost the same topological structures. it revealed the presence of three well-supported clades and each clade consisting of a separated monophyletic group. clade i, clade ii and clade iii consisted of the ghf ghf and ghf β-mannosidases, respectively. from the phylogenetic trees, it is obvious that there are two subclades in clade ii. tth man β-mannosidase from t. thermarum showed an apparently distant relationship with the ghf β-mannosidases from the same genus. therefore, it was presumed that the biochemical properties of tth man β-mannosidase might differ from the same genus β-mannosidases. this was confirmed by the experiment results shown in table  discussion β-mannosidase is an important hydrolytic enzyme which attacks the non-reducing end of the β-linked mannooligosaccharides to release mannoses. it plays a key role in the degradation pathway of complex oligosaccharide and glycoproteins. to our knowledge, the hydrolytic end product by β-mannosidase, mannoses, are also fermentable sugars which can be bio-converted to bio-fuels and value-added chemicals. the release of the genome in database provides us an effective way to investigate the uncharacterized enzymes, which may have great potential in industrial applications. for this study, a putative endo-β-mannanase gene from t. thermarum was cloned and was finally defined as a β-mannosidase through the biochemical characterization. the t. thermarum β-mannosidase is distinct from the other glycosyl hydrolases from t. thermarum. the substrate specificity and amino acid sequence of the β-mannosidase are apparently different from those of the xylanase and β-xylosidase from t. thermarum previously reported. based on sequences similarity, the tth man β-mannosidase belongs to ghf it has a homologous relationship with those from s. cellulosum, a. mirum and s. flavogriseus. compared to the same genus thermotoga β-mannosidase from ghf or ghf however, there is great difference according to phylogenetic analysis and they belong to different monophyletic groups. this suggests that tth man β-mannosidase may have some specific properties. like other hemicellulases, the catalytic mechanism of β-mannosidase is also a double displacement lysozyme-like reaction, involving stabilization of an oxocarbonium ion by electrostatic interaction with two glutamate acid residues at the active site. by the alignment with other ghf β-mannosidases and a ghf cellulase from pseudoalteromonas haloplanktis, the acid/base and the nucleophile are identified as glu and glu residues, respectively. it is difficult to know more details about the functional amino acid residues as lack of a suitable template for homology modeling. therefore, a crystalline structure analysis is needed to further investigate. the tth man β-mannosidase from t. thermarum dsm exhibits the highest activity at temperature 85°c. the temperature optima is higher than the β-mannosidases from homo sapiens, aplysia kurodai, bacillus licheniformis, bacillus circulans, aspergillus niger and aspergillus awamori, in which optimal temperature are ranged from 37°c to 70°c. it is found that the β-mannosidases from the genus thermotoga and pyrococcus furiosus exhibit the maximum activity above 80°c. however, ph optima for animals and fungi are near while for bacteria occurs at neutral environment. as we know, the enzymes with high thermal stability are necessary in industrial progresses and thus it can minimize the enzyme consumption and enhance the catalytic efficiency. the tth man β-mannosidase residual activity retains approximately 91% after being incubated at 75°c for h. furthermore, the tth man β-mannosidase residual activity is still more than 50% even after being incubated at 90°c for h. it shares similar thermostability at high temperature with the β-mannosidases from hyperthermophile. for instance, t. neapolitana β-mannosidase was stable at 90°c for h and t. maritima β-mannosidase was stable at 80°c for h. the activity of β-mannosidase is significantly inhibited by cu2+ and zn2+ and this is similar to the β-mannosidase from homo sapiens whose activity is thoroughly decreased the activity by the addition of mm cu2+ or zn2+. however, the enzyme activity is greatly stimulated by co2+ and mn2+, very different from thermotoga maritima β-mannosidase which enzyme activity is apparently inhibited by the both ions. β-mannosidase is known to be a key enzyme for converting mannooligosaccharides to mannoses, the end products, which are able to inhibit the enzymatic reaction to a certain extent. therefore, β-mannosidases with high tolerance to mannoses are beneficial to conversion of mannooligosaccharides. however, little information about mannose-tolerant β-mannosidase is available in literatures. as shown in figure  tth man β-mannosidase is insensitive to mannose whereas the ki for thermobifida fusca β-mannosidase is only mm, suggesting a high tolerance to mannose inhibition. moreover, high enzyme activity is also demanded for β-mannosidase in enzymatic hydrolysis of mannooligosaccharides. the vmax value of tth man β-mannosidase for pnpm is ± μmol min- mg- 7-fold higher than thermostable β-mannosidases from t. neapolitana and p. furiosus, 38-fold higher than t. fusca β-mannosidase and 62-fold than thermoascus aurantiacus β-mannosidase. the kcat/km of tth man β-mannosidase for pnpm is ± mm- s- approximately 2-fold higher than a. niger β-mannosidase, 11-fold higher than the p. furiosus β-mannosidase, 18-fold higher than the t. neapolitana β-mannosidase and 80-fold higher than the thermoascus aurantiacus β-mannosidase. generallly, it is noteworthy that tth man β-mannosidase has the highest vmax and kcat/km value. therefore, its mannose tolerance and high catalytic efficiency are desirable features for potential industrial applications. due to its theoretical and practical importance we investigated the hydrolysis of different substrates. when -β-d-mannan was degraded by tth man β-mannosidase, mannose was produced as a major product. it states that tth man β-mannosidase is a typical β-mannosidase and acts on polymer substrate with an exolytic manner. such an exo-type action has been found in the β-mannosidase from a marine gastropod, aplysia kurodai. it is obvious that tth man β-mannosidase has a significant ability for the hydrolysis of -β-d-mannan with the vmax of ± μmol min- mg- however, no activity is detected on konjaku powder and guar galactomannan which are not pretreated with any enzymes. the results indicate that tth man β-mannosidase can act on galactomannan cooperatively with β-mannanase and α-galactosidase to produce mannose efficiently but can not act with only tth man β-mannosidase. as tth man β-mannosidase can degrade -β-d-mannan with high efficiency, it is deduced that it also can efficiently hydrolyze mannooligosaccharides whose degree of polymerization is less than -β-d-mannan.  in this study, a novel β-mannosidase from t. thermarum dsm was over-expressed in e. coli with some specific features. the results of phylogenetic analysis and biochemical properties showed that the tth man β-mannosidase was distant with the other genus thermotoga β-mannosidases. as compared to other microorganisms, the tth man β-mannosidase possessed higher tolerance to mannose, higher catalytic efficiency and higher thermostability. therefore, this study provides a novel and useful β-mannosidase with combined properties of high catalytic efficiency, mannose-tolerance and thermostability. this is easily envisioned that tth man β-mannosidase exhibits a great potential for enzymatic conversion of mannans. 
β-mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. the results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the tth man is a novel β-mannosidase of glycoside hydrolase family the optimal activity of the tth man β-mannosidase was obtained at ph and 85°c and was stable over a ph range of to and exhibited h half-life at 90°c. therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above. it displayed high tolerance to mannose, with a ki value of approximately mm. this work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.
 mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above.  a β-mannosidase gene of bp was cloned from the extremely thermophilic bacterium thermotoga thermarum dsm that encodes a protein containing amino acid residues, and was over-expressed in escherichia coli bl. the results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the tth man is a novel β-mannosidase of glycoside hydrolase family the optimal activity of the tth man β-mannosidase was obtained at ph and 85°c and was stable over a ph range of to and exhibited h half-life at 90°c. the kinetic parameters km and vmax values for p-nitrophenyl-β-d-mannopyranoside and -β-d-mannan were ± mm and ± μmol min- mg- ± mg ml- and ± μmol min- mg- respectively. the kcat/km values for p-nitrophenyl-β-d-mannopyranoside and -β-d-mannan were ± mm- s- and ± s- mg- ml, respectively. it displayed high tolerance to mannose, with a ki value of approximately mm.  this work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes. thermotoga thermarumβ-mannosidasemannose-tolerantmannanthermostabilitymannooligosaccharides 