BACKGROUND
biotin  is an essential enzyme cofactor involved in multiple important metabolic pathways including fatty acids, amino acids, and carbohydrates in all three domains of life
<cit> .
this co-enzyme is synthesized de novo in microorganisms
<cit>  where enzymes catalyzing the last four steps in the biotin biosynthesis are well characterized.
these include 7-keto-8-aminopelargonic acid  synthase,  <dig>  8-diaminopelargonic acid synthase, dethiobiotin synthetase, and biotin synthase.
their encoding genes, designated biof, bioa, biod and biob, have been identified in e. coli <cit> .
more recently, two pathways, bioc-bioh and bioi, have been suggested for synthesizing pimeloyl-acp , an efficient substrate of biof
<cit> .
the bioi pathway is only present in bacillus subtilis and its close relatives, where pimeloyl-acp is to be synthesized by an o2-dependent cleavage of long chain acyl-acps
<cit> .
in the bioc-bioh pathway, bioc catalyzes s-adenosylmethionine-dependent methylation of malonyl-coa  to malonyl-coa  methyl ester that enters fatty acid synthetic pathway, where an intermediate pimeloyl-acp methyl ester was cleaved by bioh to produce pimeloyl-acp that serves as the substrate for biof to initiate biotin synthesis
<cit> .
to date, bioh genes have been cloned from e. coli, serratia marcescens, serratia sp.
and kurthia sp.
strains, and the encoded proteins exhibit carboxylesterase activity
<cit> .
esterases  catalyze the hydrolysis of esters with short-chain fatty acids.
they belong to the hydrolase superfamily with α/β protein fold
<cit> , carrying a serine nucleophile embedded in the pentapeptide motif gly-xaa-ser-xaa-gly
<cit>  and the catalytic triad ser-his-asp
<cit>  at the catalytic site.
esterases, including lipases, are very important industrial enzymes and are widely applied in detergents and degreasing formulations, paper and leather manufacture, food processing, medical diagnostics and pharmaceutical syntheses, as well as wastewater treatment
<cit> .
microorganisms comprise the main resources of hydrolases from which most commercial hydrolases are derived
<cit> .
this is mainly because microbes exhibit much great diversity than multicellular organisms.
as a result, screening for novel hydrolases from microbe-rich environments have been of a great interest from both industrial and academic standpoints.
since up to 99% of microorganisms in environments cannot be cultured by standard cultivation techniques according to taxonomic studies based on 16s rrna genes
<cit> , exploring esterase resources with any culture-dependent methodology renders those resources untapped for uncultured microbes.
recently metagenomic technology was developed to address this problem.
utilizing this culture-independent approach, several genes encoding novel industrial enzymes, including lipases and esterases, have been obtained
<cit> .
however, to our knowledge, hydrolase resources have not been explored for microbial communities in aquaculture environments, such as fishing pond sediments where diverse microorganisms thrive according to 16s rrna gene analysis .
here we report the cloning and identification of a bioh gene coding for an enzyme that is capable of hydrolysis of p-nitrophenyl  esters of fatty acids by a functional metagenomic approach.
a novel bioh, biohx, was expressed, and the recombinant protein was purified and characterized.
our results indicate that biohx is a novel carboxylesterase involved in the biotin biosynthesis, exhibiting good application potential in industry and biotechnological research.
RESULTS
identification of a tributyrin-hydrolyzing clone from a metagenomic library and sequence analysis of the cloned dna fragment
metagenomic dna was prepared from sediment samples of aquaculture ponds.
the resultant dna were mechanically sheared by sonication, yielding dna fragments of 2- <dig> kb that were used to construct a metagenomic library as described in methods.
to test the quality of the library, twenty transformants grown on lb-ampicillin agar plates were randomly picked up for plasmid preparation.
digestion of the purified plasmids with ecori indicated that they all contained an insert dna fragment of 2- <dig> kb , indicating the library was suitable for functional screening of industrial enzymes.
in an esterase screening with spirit blue agar plates containing tributyrin, one clone was found to produce a clear zone of hydrolysis on the selective plate amongst ca.
<dig>  transformants yielded from the metagenomic library.
this clone, designated pestbiohx, was used for further studies.
the complete dna sequence of the cloned fragment in pestbiohx was determined, and deposited in the genbank sequence database under the accession no.
jx <dig>  it is  <dig> -bp in length and contains six genes, designated biof, biohx, fabg, bioc, orf <dig> and sdh.
their gene organization is presented in figure
<dig>  in which the genes at the two ends were only recovered partly, i.e.
the 5´ end of biof and 3´end of sdh.
database searches revealed that the  <dig> -kb sequence displayed 91% sequence identity at the nucleotide level over its full length with a dna fragment of the stenotrophomonas maltophilia r <dig> genome , indicating that the cloned dna fragment could be derived from a s. maltophilia strain present in the fishing pond sediments.
this is consistent to the notion that s. maltophilia is ubiquitous in aqueous environments, soil and plants
<cit> .
the gene responsible for the hydrolytic activity of tributyrin, was identified as biohx positioned from  <dig> to  <dig> in the cloned dna fragment.
biohx encodes a protein of  <dig> aa, displaying high amino acid sequence similarities to putative carboxylesterase bioh identified from the complete genomes of stenotrophomonas  and xanthomonas  species in the public databases.
much lower sequence similarities were found between biohx and bioh enzymes of escherichia coli k <dig>
<cit> , escherichia coli dh5α
<cit> , serratia sp.
ses- <dig>
<cit> , serratia marcescens sr <dig>
<cit> , and kurthia sp.
538-ka <dig>
<cit> , the only bacterial bioh that have been cloned and available in the databases.
analysis of these bioh proteins by multiple sequence alignment revealed that both the pentapeptide motif gly76-trp77-ser78-met79-gly <dig> and the catalytic triad ser78-his230-asp <dig> are conserved in biohx .
as illustrated in figure
<dig>  the first  <dig> genes encoded in the cloned dna fragment could form an operon with other biotin synthetic genes.
the incomplete biof gene  is located upstream of biohx, coding for a kapa synthetase protein with an n-terminal truncation.
biof is the enzyme catalyzing the first of the four-step reactions in biotin biosynthetic pathway.
it synthesizes kapa with pimeloyl-acp and l-alanine
<cit> .
the biof displayed high sequence identities at the amino acid level to the same protein encoded in different bacteria including stenotrophomonas  and xanthomonas , as well as other bacteria, such as xylella, allochromoatium and pseudomonas .
there is a noncoding region of  <dig> bp separating biof from biohx but it does not carry any recognizable motif for a putative promoter or a ribosomal binding site.
downstream of the biohx lay fabg  and bioc  genes.
the bioc encodes a protein of  <dig> amino acids, showing a high sequence similarity to a putative biotin biosynthesis protein bioc from stenotrophomanas and close related species, while the deduced amino acid sequence of the 777-bp fabg gene exhibits a high level of sequence identity to putative 3-ketopimeloyl-acp methyl ester reductases involved in fatty acid biosynthesis in the database.
fabg reduces 3-ketopimeloyl-acp methyl ester to 3-hydroxypimeloyl-acp methyl ester in canonical fatty acid biosynthetic pathway, via which the intermediate pimeloyl-acp methyl ester was produced and hydrolyzed by bioh to form pimeloyl-acp substrate for biof
<cit> .
the intergenic regions are only for  <dig> and 15-bp starting from the second gene through the fourth with no identifiable putative promoters.
thus, biof, biohx, fabg and bioc could be co-transcripted.
several other bacteria carry the same gene organization in the bioh region as for the cloned metagenomic fragment in which bioh is a member of the biotin biosynthetic operon
<cit> .
but bioh genes in e. coli k <dig> and serratia sp.
ses- <dig>  are located elsewhere in the genomes
<cit> .
taken together, this reveals an interesting aspect of bacterial bioh diversity and evolution.
two additional genes on the sequenced dna fragment, orf <dig>  and sdh  could have different functions.
they are in the opposite orientation as for the first four genes.
orf <dig> encodes a protein of  <dig> aa, and its closest match was a protein family containing duf <dig> domain.
however, there is no clue that could lead to a possible function for this protein family in the current literature.
the n-terminal truncated sdh protein displayed a high degree of similarity with serine/threonine dehydratase, which functions in bacterial amino acid metabolism.
as there is  <dig> bp overlap between the coding sequences of orf <dig> and sdh genes, they could form an operon together with other unrecovered genes.
it is thus interesting to study if orf <dig> could have any functional connection to sdh.
based on the deduced amino acid sequences of biohx and a selected set of its homologs identified in the public databases, a phylogenetic tree was constructed by the mega <dig> .
this analysis revealed that these bioh sequences could form three families, designated i, ii, and iii .
the bioh identified from e. coli, serratia sp.
and s. marcescens, as well as kurthia sp.
<cit>  were distributed in family i and iii, respectively, whereas biohx cloned in this study fell into cluster ii, representing a novel bioh.
expression and purification of biohx gene product
the 777-bp biohx structural gene  was amplified with primers biohx-mf and biohx-mr, the purified pcr product was digested with bamhi and hindiii, and then cloned into the expression vector pet-28a at bamhi and hindiii sites.
the yielded recombinant plasmid, pet28a-biohx, was transformed into e. coli bl <dig>  kanr transformants containing pet28a-biohx were screened by colony pcr.
bamhi and hindiii digests of the pet28a-biohx verified that it carried the biohx structural gene .
a pet28a-biohx transformant was grown in lb-kanamycin medium.
the expression of biohx was induced by adding  <dig>  mm iptg to the culture, and the induction was at 21°c for  <dig> h. the cell-free extract containing the soluble target protein was analyzed by sds-page.
as presented in figure
<dig> , one band corresponded in size to the calculated molecular mass of biohx was detected.
the band was absent in control lane from the e. coli bl <dig> cells carrying only the non-recombinant pet28a vector , which was cultured and induced under the same condition as that for the e. coli bl <dig> .
the expressed recombinant protein with six his-tags at c-terminal was purified via a ni-nta his bind resin column.
figure
<dig>  shows the purified biohx with molecular mass of approximately  <dig>  kda.
purified protein was about  <dig>  mg of per  <dig> g wet biomass, and accounted for about  <dig> % of soluble total proteins in the cell-free extract.
effect of temperature and ph on biohx activity and stability
carboxylesterase activity of the purified biohx was determined for temperature range of  <dig> to 80°c using pnp-butyrate as the substrate.
as presented in figure
5a, biohx displayed the maximal enzyme activity at 30°c.
the enzyme exhibited more than 74% of the optimal specific activity for the temperature range of 20-50°c, indicating that it is moderately thermotolerant.
however, enzyme activity declined rapidly when reaction temperatures reached above 70°c.
these results indicated that biohx is a mesophilic enzyme.
to assess the effect of temperature on biohx stability, the enzyme was exposed to a broad range of temperature for over  <dig> min, and the residual activity was measured.
as shown in figure
5b, biohx showed considerable stability and retained 83-97% of its initial activity after the treatment at 10-50°c.
in addition, no loss in activity was observed after the enzyme was stored at 4°c for  <dig> h .
the effect of ph on the biohx activity was determined at 30°c, the optimal reaction temperature.
purified biohx was found to function in a range of ph  <dig> – <dig>  with more than 50% of its maximal activity, and the ph optimum was recorded as ph  <dig>  .
relative activity of biohx declined sharply at the acidic range of ph.
the enzyme stability at various ph values was measured after incubating biohx at 4°c for  <dig> h. as shown in figure
5d, the enzyme exhibited relatively strong stability at ph values ranging from neutral to alkaline conditions.
it retained the maximal activity in the ph range of  <dig>  to  <dig> , which dropped only slightly at ph  <dig>  indicating that the enzyme has a remarkable stability under alkaline conditions.
substrate specification of biohx
carboxylesterase activity of biohx on p-nitrophenyl esters with acyl chain length of up to  <dig> carbons was assayed at 30°c and ph  <dig> .
as presented in figure
<dig>  biohx displays highest activity  on pnp- butyrate , followed by 89% relative activity on pnp-c <dig>  and 39-52% on pnp-c6~c <dig>  these results are in a good agreement with those obtained with other bacterial bioh enzymes
<cit> .
at structural level, the catalytic triad of e. coli bioh  is buried between two domains and was not readily accessible for bulkier compounds
<cit> .
this serves as a good explanation as to why bioh enzymes are more active on substrates with a short side chain.
it was reported that bioh encoded in e. coli showed a low enzymatic activity of thioesterase using palmitoyl-coa as a substrate
<cit> .
given that biohx contained gly76-trp77-ser78-met79-gly <dig>  a conserved motif for acyltransferases and thioesterases, we studied the thioesterase activity of biohx.
the assay was carried out at 30°c and ph  <dig>  using palmitoyl-coa as the substrate.
biohx showed a specific activity of  <dig>  +/−  <dig>  μmol coa/min · mg protein on palmitoyl-coa, which was approximately  <dig> -times lower than its carboxylesterase activity on pnp-butyrate.
the ratio of biohx activities of hydrolyzing pnp-esters/coa-thioester was similar to that of biohs , but different from that of biohe
<cit> .
effect of metal ions on biohx stability
to test the effect of divalent metal ions on biohx activity, aliquots of the enzyme was incubated with  <dig> mm of zncl <dig>  mncl <dig>  cacl <dig>  mgcl <dig>  bacl <dig>  and cuso <dig> at 30°c for  <dig> h, and residual activity was measured at 30°c and ph  <dig>  using pnp-butyrate as the substrate.
as presented in figure
<dig>  compared with the control, ba2+ and mn2+ were found to increase enzyme activity by  <dig>  and  <dig> %, respectively, whereas ca2+ and cu2+ showed a moderate inhibition to the enzyme as the activity declined to  <dig>  and  <dig> %, respectively.
mg2+ and zn2+ did not affect the enzyme activity.
edta  showed no effect on biohx activity, suggesting that it is not a metalloenzyme.
this is consistent with the results obtained with biohe and biohs
<cit> .
effect of detergents and organic solvents on biohx stability
the effect of ionic and nonionic detergents on biohx stability including sodium dodecyl sulfate , tween  <dig> and tween  <dig> was assayed.
similar with biohe and biohs, sds strongly inhibited biohx activity, diminishing about 80% of its activity at  <dig> % concentration .
in contrast to biohe and biohs in which their activities were barely affected by  <dig> -5% of tween  <dig> and tween 80
<cit> , the non-ionic detergents greatly reduced the biohx activity, inhibiting more than 60% of its activity at a final concentration of  <dig> %, arguing for the diversity of bioh enzymes.
tolerance to organic solvents is very important for any enzymes to be explored for industrial application.
to study the stability of biohx in organic solvents, the enzyme was individually incubated with methanol, ethanol, 2-propanol, 1-butanol, acetonitrile, acetone, dimethyl sulfoxide  and dimethylformamide  at the indicated concentrations at 30°c for  <dig> h. residual activity of the treated enzymes was then determined under the standard assay condition.
as summarized in table
<dig>  biohx exhibited remarkable stability in methanol, dmso and dmf, as it retained more than 90% of the original activity after the treatment with each of the organic solvents at a final concentration of 30%.
organic solvent tolerance has been reported for biohe and biohs, from which different results have been obtained.
biohe shows strong resistance to treatments with 30% organic solvents, whereas biohs lost more than 95% activity under the same treatments
<cit> .
however, compared with biohe, biohx is more resistant to the treatments with 30% of 2-propanol or acetonitrile, as the residual activity of the treated biohx is ca.
3-fold and 7-fold of that for biohe.
furthermore, upon treatments with 50% 2-propanol or acetonitrile, biohx still exhibits  <dig>  or  <dig> % residual activity , indicating that it is very resistant to these organic solvents.
these results reinforce that biohx represents a novel bioh enzyme that is very suitable to be exploited for commercial application.
no organic solvent was included in the control samples.
the concentrations of organic solvents are indicated .
the enzyme activity was determined under the standard assay condition, and the data is represented as an average of three independent determinations.
although bioh genes are present in many microbial genomes in the public databases, very few bioh enzymes have been biochemically characterized to date.
among them, interesting diversity of bioh enzymes is emerging, such as the great differences in organic solvent tolerance revealed for biohe, biohs and biohx.
in this regard, more bioh enzymes should be investigated both for elucidating biotin biosynthetic mechanisms in microorganisms and for exploiting them for commercial application in ester hydrolysis.
there are three alternative approaches to select bioh genes for biochemical characterization:  identifying bioh genes from the public databases,  isolating microorganisms from environments for studying bioh, or  functional screening for bioh-containing clones from metagenomic libraries.
since functional screening of metagenomic libraries explore all microbes in environments where more than 90% of the resources are not represented in any culture-dependent approach and far more less resources are included in the public databases, we regard that the metagenomic approach represents the most rewarding research to explore industrial application of bioh enzymes.
CONCLUSIONS
in this study, a novel bioh gene, biohx, in a  <dig> -bp dna fragment containing a biotin biosynthetic gene cluster was cloned and identified from an environmental metagenome by a functional metagenomic approach.
the biohx gene encodes a protein of  <dig> aa with a calculated molecular mass of  <dig>  kda, displaying 24-39% amino acid sequence identity to a few characterized bacterial bioh enzymes.
biohx was expressed as a recombinant protein in escherichia coli bl <dig> using the pet expression system.
the purified biohx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain .
comparing biohx with other known bioh proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and biohx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known bioh enzymes.
the distinct bio-chemical properties positioned biohx as a novel carboxylesterase with a good application potential in industry and biotechnological research.

 biotin is an essential enzyme cofactor involved in multiple important metabolic pathways including fatty acids, amino acids, and carbohydrates in all three domains of life. this co-enzyme is synthesized de novo in microorganisms where enzymes catalyzing the last four steps in the biotin biosynthesis are well characterized. these include 7-keto-8-aminopelargonic acid synthase, 8-diaminopelargonic acid synthase, dethiobiotin synthetase, and biotin synthase. their encoding genes, designated biof, bioa, biod and biob, have been identified in e. coli. more recently, two pathways, bioc-bioh and bioi, have been suggested for synthesizing pimeloyl-acp, an efficient substrate of biof. the bioi pathway is only present in bacillus subtilis and its close relatives, where pimeloyl-acp is to be synthesized by an o2-dependent cleavage of long chain acyl-acps. in the bioc-bioh pathway, bioc catalyzes s-adenosylmethionine-dependent methylation of malonyl-coa to malonyl-coa methyl ester that enters fatty acid synthetic pathway, where an intermediate pimeloyl-acp methyl ester was cleaved by bioh to produce pimeloyl-acp that serves as the substrate for biof to initiate biotin synthesis. to date, bioh genes have been cloned from e. coli, serratia marcescens, serratia sp. and kurthia sp. strains, and the encoded proteins exhibit carboxylesterase activity. esterases catalyze the hydrolysis of esters with short-chain fatty acids. they belong to the hydrolase superfamily with α/β protein fold, carrying a serine nucleophile embedded in the pentapeptide motif gly-xaa-ser-xaa-gly and the catalytic triad ser-his-asp at the catalytic site. esterases, including lipases, are very important industrial enzymes and are widely applied in detergents and degreasing formulations, paper and leather manufacture, food processing, medical diagnostics and pharmaceutical syntheses, as well as wastewater treatment. microorganisms comprise the main resources of hydrolases from which most commercial hydrolases are derived. this is mainly because microbes exhibit much great diversity than multicellular organisms. as a result, screening for novel hydrolases from microbe-rich environments have been of a great interest from both industrial and academic standpoints. since up to 99% of microorganisms in environments cannot be cultured by standard cultivation techniques according to taxonomic studies based on 16s rrna genes, exploring esterase resources with any culture-dependent methodology renders those resources untapped for uncultured microbes. recently metagenomic technology was developed to address this problem. utilizing this culture-independent approach, several genes encoding novel industrial enzymes, including lipases and esterases, have been obtained. however, to our knowledge, hydrolase resources have not been explored for microbial communities in aquaculture environments, such as fishing pond sediments where diverse microorganisms thrive according to 16s rrna gene analysis. here we report the cloning and identification of a bioh gene coding for an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids by a functional metagenomic approach. a novel bioh, biohx, was expressed, and the recombinant protein was purified and characterized. our results indicate that biohx is a novel carboxylesterase involved in the biotin biosynthesis, exhibiting good application potential in industry and biotechnological research.  identification of a tributyrin-hydrolyzing clone from a metagenomic library and sequence analysis of the cloned dna fragment metagenomic dna was prepared from sediment samples of aquaculture ponds. the resultant dna were mechanically sheared by sonication, yielding dna fragments of 2- kb that were used to construct a metagenomic library as described in methods. to test the quality of the library, twenty transformants grown on lb-ampicillin agar plates were randomly picked up for plasmid preparation. digestion of the purified plasmids with ecori indicated that they all contained an insert dna fragment of 2- kb, indicating the library was suitable for functional screening of industrial enzymes. in an esterase screening with spirit blue agar plates containing tributyrin, one clone was found to produce a clear zone of hydrolysis on the selective plate amongst ca. transformants yielded from the metagenomic library. this clone, designated pestbiohx, was used for further studies. the complete dna sequence of the cloned fragment in pestbiohx was determined, and deposited in the genbank sequence database under the accession no. jx it is -bp in length and contains six genes, designated biof, biohx, fabg, bioc, orf and sdh. their gene organization is presented in figure  in which the genes at the two ends were only recovered partly, i.e. the 5´ end of biof and 3´end of sdh. database searches revealed that the -kb sequence displayed 91% sequence identity at the nucleotide level over its full length with a dna fragment of the stenotrophomonas maltophilia r genome, indicating that the cloned dna fragment could be derived from a s. maltophilia strain present in the fishing pond sediments. this is consistent to the notion that s. maltophilia is ubiquitous in aqueous environments, soil and plants. the gene responsible for the hydrolytic activity of tributyrin, was identified as biohx positioned from to in the cloned dna fragment. biohx encodes a protein of aa, displaying high amino acid sequence similarities to putative carboxylesterase bioh identified from the complete genomes of stenotrophomonas and xanthomonas species in the public databases. much lower sequence similarities were found between biohx and bioh enzymes of escherichia coli k, escherichia coli dh5α, serratia sp. ses-, serratia marcescens sr, and kurthia sp. 538-ka, the only bacterial bioh that have been cloned and available in the databases. analysis of these bioh proteins by multiple sequence alignment revealed that both the pentapeptide motif gly76-trp77-ser78-met79-gly and the catalytic triad ser78-his230-asp are conserved in biohx. as illustrated in figure  the first genes encoded in the cloned dna fragment could form an operon with other biotin synthetic genes. the incomplete biof gene is located upstream of biohx, coding for a kapa synthetase protein with an n-terminal truncation. biof is the enzyme catalyzing the first of the four-step reactions in biotin biosynthetic pathway. it synthesizes kapa with pimeloyl-acp and l-alanine. the biof displayed high sequence identities at the amino acid level to the same protein encoded in different bacteria including stenotrophomonas and xanthomonas, as well as other bacteria, such as xylella, allochromoatium and pseudomonas. there is a noncoding region of bp separating biof from biohx but it does not carry any recognizable motif for a putative promoter or a ribosomal binding site. downstream of the biohx lay fabg and bioc genes. the bioc encodes a protein of amino acids, showing a high sequence similarity to a putative biotin biosynthesis protein bioc from stenotrophomanas and close related species, while the deduced amino acid sequence of the 777-bp fabg gene exhibits a high level of sequence identity to putative 3-ketopimeloyl-acp methyl ester reductases involved in fatty acid biosynthesis in the database. fabg reduces 3-ketopimeloyl-acp methyl ester to 3-hydroxypimeloyl-acp methyl ester in canonical fatty acid biosynthetic pathway, via which the intermediate pimeloyl-acp methyl ester was produced and hydrolyzed by bioh to form pimeloyl-acp substrate for biof. the intergenic regions are only for and 15-bp starting from the second gene through the fourth with no identifiable putative promoters. thus, biof, biohx, fabg and bioc could be co-transcripted. several other bacteria carry the same gene organization in the bioh region as for the cloned metagenomic fragment in which bioh is a member of the biotin biosynthetic operon. but bioh genes in e. coli k and serratia sp. ses- are located elsewhere in the genomes. taken together, this reveals an interesting aspect of bacterial bioh diversity and evolution. two additional genes on the sequenced dna fragment, orf and sdh could have different functions. they are in the opposite orientation as for the first four genes. orf encodes a protein of aa, and its closest match was a protein family containing duf domain. however, there is no clue that could lead to a possible function for this protein family in the current literature. the n-terminal truncated sdh protein displayed a high degree of similarity with serine/threonine dehydratase, which functions in bacterial amino acid metabolism. as there is bp overlap between the coding sequences of orf and sdh genes, they could form an operon together with other unrecovered genes. it is thus interesting to study if orf could have any functional connection to sdh. based on the deduced amino acid sequences of biohx and a selected set of its homologs identified in the public databases, a phylogenetic tree was constructed by the mega. this analysis revealed that these bioh sequences could form three families, designated i, ii, and iii. the bioh identified from e. coli, serratia sp. and s. marcescens, as well as kurthia sp. were distributed in family i and iii, respectively, whereas biohx cloned in this study fell into cluster ii, representing a novel bioh. expression and purification of biohx gene product the 777-bp biohx structural gene was amplified with primers biohx-mf and biohx-mr, the purified pcr product was digested with bamhi and hindiii, and then cloned into the expression vector pet-28a at bamhi and hindiii sites. the yielded recombinant plasmid, pet28a-biohx, was transformed into e. coli bl kanr transformants containing pet28a-biohx were screened by colony pcr. bamhi and hindiii digests of the pet28a-biohx verified that it carried the biohx structural gene. a pet28a-biohx transformant was grown in lb-kanamycin medium. the expression of biohx was induced by adding mm iptg to the culture, and the induction was at 21°c for h. the cell-free extract containing the soluble target protein was analyzed by sds-page. as presented in figure , one band corresponded in size to the calculated molecular mass of biohx was detected. the band was absent in control lane from the e. coli bl cells carrying only the non-recombinant pet28a vector, which was cultured and induced under the same condition as that for the e. coli bl. the expressed recombinant protein with six his-tags at c-terminal was purified via a ni-nta his bind resin column. figure  shows the purified biohx with molecular mass of approximately kda. purified protein was about mg of per g wet biomass, and accounted for about % of soluble total proteins in the cell-free extract. effect of temperature and ph on biohx activity and stability carboxylesterase activity of the purified biohx was determined for temperature range of to 80°c using pnp-butyrate as the substrate. as presented in figure  5a, biohx displayed the maximal enzyme activity at 30°c. the enzyme exhibited more than 74% of the optimal specific activity for the temperature range of 20-50°c, indicating that it is moderately thermotolerant. however, enzyme activity declined rapidly when reaction temperatures reached above 70°c. these results indicated that biohx is a mesophilic enzyme. to assess the effect of temperature on biohx stability, the enzyme was exposed to a broad range of temperature for over min, and the residual activity was measured. as shown in figure  5b, biohx showed considerable stability and retained 83-97% of its initial activity after the treatment at 10-50°c. in addition, no loss in activity was observed after the enzyme was stored at 4°c for h. the effect of ph on the biohx activity was determined at 30°c, the optimal reaction temperature. purified biohx was found to function in a range of ph – with more than 50% of its maximal activity, and the ph optimum was recorded as ph. relative activity of biohx declined sharply at the acidic range of ph. the enzyme stability at various ph values was measured after incubating biohx at 4°c for h. as shown in figure  5d, the enzyme exhibited relatively strong stability at ph values ranging from neutral to alkaline conditions. it retained the maximal activity in the ph range of to, which dropped only slightly at ph indicating that the enzyme has a remarkable stability under alkaline conditions. substrate specification of biohx carboxylesterase activity of biohx on p-nitrophenyl esters with acyl chain length of up to carbons was assayed at 30°c and ph. as presented in figure  biohx displays highest activity on pnp- butyrate, followed by 89% relative activity on pnp-c and 39-52% on pnp-c6~c these results are in a good agreement with those obtained with other bacterial bioh enzymes. at structural level, the catalytic triad of e. coli bioh is buried between two domains and was not readily accessible for bulkier compounds. this serves as a good explanation as to why bioh enzymes are more active on substrates with a short side chain. it was reported that bioh encoded in e. coli showed a low enzymatic activity of thioesterase using palmitoyl-coa as a substrate. given that biohx contained gly76-trp77-ser78-met79-gly a conserved motif for acyltransferases and thioesterases, we studied the thioesterase activity of biohx. the assay was carried out at 30°c and ph using palmitoyl-coa as the substrate. biohx showed a specific activity of +/− μmol coa/min · mg protein on palmitoyl-coa, which was approximately -times lower than its carboxylesterase activity on pnp-butyrate. the ratio of biohx activities of hydrolyzing pnp-esters/coa-thioester was similar to that of biohs, but different from that of biohe. effect of metal ions on biohx stability to test the effect of divalent metal ions on biohx activity, aliquots of the enzyme was incubated with mm of zncl mncl cacl mgcl bacl and cuso at 30°c for h, and residual activity was measured at 30°c and ph using pnp-butyrate as the substrate. as presented in figure  compared with the control, ba2+ and mn2+ were found to increase enzyme activity by and %, respectively, whereas ca2+ and cu2+ showed a moderate inhibition to the enzyme as the activity declined to and %, respectively. mg2+ and zn2+ did not affect the enzyme activity. edta showed no effect on biohx activity, suggesting that it is not a metalloenzyme. this is consistent with the results obtained with biohe and biohs. effect of detergents and organic solvents on biohx stability the effect of ionic and nonionic detergents on biohx stability including sodium dodecyl sulfate, tween and tween was assayed. similar with biohe and biohs, sds strongly inhibited biohx activity, diminishing about 80% of its activity at % concentration. in contrast to biohe and biohs in which their activities were barely affected by -5% of tween and tween 80, the non-ionic detergents greatly reduced the biohx activity, inhibiting more than 60% of its activity at a final concentration of %, arguing for the diversity of bioh enzymes. tolerance to organic solvents is very important for any enzymes to be explored for industrial application. to study the stability of biohx in organic solvents, the enzyme was individually incubated with methanol, ethanol, 2-propanol, 1-butanol, acetonitrile, acetone, dimethyl sulfoxide and dimethylformamide at the indicated concentrations at 30°c for h. residual activity of the treated enzymes was then determined under the standard assay condition. as summarized in table  biohx exhibited remarkable stability in methanol, dmso and dmf, as it retained more than 90% of the original activity after the treatment with each of the organic solvents at a final concentration of 30%. organic solvent tolerance has been reported for biohe and biohs, from which different results have been obtained. biohe shows strong resistance to treatments with 30% organic solvents, whereas biohs lost more than 95% activity under the same treatments. however, compared with biohe, biohx is more resistant to the treatments with 30% of 2-propanol or acetonitrile, as the residual activity of the treated biohx is ca. 3-fold and 7-fold of that for biohe. furthermore, upon treatments with 50% 2-propanol or acetonitrile, biohx still exhibits or % residual activity, indicating that it is very resistant to these organic solvents. these results reinforce that biohx represents a novel bioh enzyme that is very suitable to be exploited for commercial application. no organic solvent was included in the control samples. the concentrations of organic solvents are indicated. the enzyme activity was determined under the standard assay condition, and the data is represented as an average of three independent determinations. although bioh genes are present in many microbial genomes in the public databases, very few bioh enzymes have been biochemically characterized to date. among them, interesting diversity of bioh enzymes is emerging, such as the great differences in organic solvent tolerance revealed for biohe, biohs and biohx. in this regard, more bioh enzymes should be investigated both for elucidating biotin biosynthetic mechanisms in microorganisms and for exploiting them for commercial application in ester hydrolysis. there are three alternative approaches to select bioh genes for biochemical characterization: identifying bioh genes from the public databases, isolating microorganisms from environments for studying bioh, or functional screening for bioh-containing clones from metagenomic libraries. since functional screening of metagenomic libraries explore all microbes in environments where more than 90% of the resources are not represented in any culture-dependent approach and far more less resources are included in the public databases, we regard that the metagenomic approach represents the most rewarding research to explore industrial application of bioh enzymes.  in this study, a novel bioh gene, biohx, in a -bp dna fragment containing a biotin biosynthetic gene cluster was cloned and identified from an environmental metagenome by a functional metagenomic approach. the biohx gene encodes a protein of aa with a calculated molecular mass of kda, displaying 24-39% amino acid sequence identity to a few characterized bacterial bioh enzymes. biohx was expressed as a recombinant protein in escherichia coli bl using the pet expression system. the purified biohx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain. comparing biohx with other known bioh proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and biohx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known bioh enzymes. the distinct bio-chemical properties positioned biohx as a novel carboxylesterase with a good application potential in industry and biotechnological research. 
the purified biohx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain. the biohx gene, encoding an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids, was expressed in escherichia coli bl using the pet expression system. the biohx gene encodes a protein of aa with a calculated molecular mass of kda, displaying 24-39% amino acid sequence identity to a few characterized bacterial bioh enzymes. the results demonstrated that biohx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioh genes from complex ecosystem. comparing biohx with other known bioh proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and biohx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known bioh enzymes. to date, very few bioh genes have been characterized. this ascribed biohx as a novel carboxylesterase with a strong potential in industrial applications. it contains a pentapeptide motif and a catalytic triad, both of which are characteristic for lipolytic enzymes. screening of an unamplified metagenomic library with a tributyrin-containing medium led to the isolation of a clone exhibiting lipolytic activity. in this study, we cloned and identified a novel bioh gene, biohx, from an environmental metagenome by a functional metagenomic approach. the biochemical property of the purified biohx protein was also investigated. this study constituted the first investigation of a novel biohx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. biohx was expressed as a recombinant protein and characterized.
 bioh is one of the key enzymes to produce the precursor pimeloyl-acp to initiate biotin biosynthesis de novo in bacteria. to date, very few bioh genes have been characterized. in this study, we cloned and identified a novel bioh gene, biohx, from an environmental metagenome by a functional metagenomic approach. the biohx gene, encoding an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids, was expressed in escherichia coli bl using the pet expression system. the biochemical property of the purified biohx protein was also investigated.  screening of an unamplified metagenomic library with a tributyrin-containing medium led to the isolation of a clone exhibiting lipolytic activity. this clone carried a -bp dna fragment encoding for six genes, designated biof, biohx, fabg, bioc, orf and sdh, four of which were implicated in the de novo biotin biosynthesis. the biohx gene encodes a protein of aa with a calculated molecular mass of kda, displaying 24-39% amino acid sequence identity to a few characterized bacterial bioh enzymes. it contains a pentapeptide motif and a catalytic triad, both of which are characteristic for lipolytic enzymes. biohx was expressed as a recombinant protein and characterized. the purified biohx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain. comparing biohx with other known bioh proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and biohx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known bioh enzymes. this ascribed biohx as a novel carboxylesterase with a strong potential in industrial applications.  this study constituted the first investigation of a novel biohx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. the biohx gene was successfully cloned, expressed and characterized. the results demonstrated that biohx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioh genes from complex ecosystem. biohbiotin biosynthetic pathwaycarboxylesterasemetagenomeaqueous environment 