BACKGROUND
nitrile hydratase  is a multi-subunits industrial enzyme, which can efficiently catalyze the nitrile to the corresponding amide.
according to the different metal ions in the active center, the nhase could be classified into the two types: the iron type nhase   <cit>  and the cobalt type nhase   <cit> .
due to the high-value product and high catalytic activity, nhase has attracted wide attention in the chemical industry  <cit> .
nhase is composed of α- and β-subunits  <cit> .
the molecular masses of the α-subunit and β-subunit are generally less than 30 kda, such as those in bordetella petrii dsm  <dig>  and those in bacillus pallidus rapc <dig>   <cit> .
however, an activator is necessary for the functional expression of nhase, such as those in the co-nhases from rhodococcus rhodochrous j <dig> , rhodococcus jostii rha <dig>  <cit>  and pseudomonas putida nrrl- <dig>  <cit>  as well as in the fe-nhases from pseudomonas chlororaphis b <dig>  <cit>  and rhodococcus sp.
n- <dig>  <cit> .
however, certain activators are poorly expressed , such as the activator p14k in the b. pallidus rapc <dig>  comamonas testosteroni 5-mgam-4d and p. putida nrrl- <dig>  <cit> .
until recently, the p14k in p. putida was successfully expressed through fusing a strep tag at its n-terminal, then a novel nhase maturation mechanism, self-subunit swapping, was discovered  <cit> .
however, the stability of the p14k is not yet favorable for its over-expression.
thus, to further facilitate the application of nhase, other fusion protein tags that are helpful for expression of more stable p14k are required.
additionally, all the α-subunits of nhases share a conserved characteristic motif cxlc-ser-c in the active center  <cit> , and the ser in this motif is proposed to be involved in nhase maturation  .
however, the necessity of ser was not studied by the energetic calculation and site-specific mutagenesis.fig.
<dig> the catalytic pathway involving ser <dig> during nhase maturation.
a first step: atoms of o-h and oh-co3+ participates in nhase activation .
second step: the proton of oh participates in the proton transfer pathway .
b the oxygen of the phenolate of the conserved tyr <dig> was ionized by the general base ser <dig> during nhase activation.
c the oxygen of the phenolate of the conserved tyr <dig> was ionized by the general base ser <dig> with the help of tyr <dig> during nhase activation
in this study, the nhase from b. petrii dsm  <dig> was researched.
the activator p14k was successfully expressed through fusing a his tag, and its expression and stability were further enhanced by fusing a gst tag at its n-terminus.
in addition, we found that the ser <dig> in the characteristic motif cxlc-ser-c of the active center of nhase from b. petrii is not necessary for the nhase maturation.
these results are useful for the expression of nhase activator and the understanding of the role of ser <dig> during the nhase maturation.
further, the strategy for enhancing the stability of p14k is very beneficial for other proteins with difficulty in expression.
RESULTS
expression and purification of nhase
the full-length genes of nhase  were obtained from b. petrii dsm  <dig>  in which a gene encodes α-subunit, b gene encodes β-subunit and p gene encodes p14k .
the recombinant expression vector pet-24a harboring the abp genes  was constructed and transformed into e. coli for nhase expression.
the calculated molecular mass of recombinant nhase was in accordance with that of the bands by sds-page analysis .
the nhase was successfully purified  and the specific activity was  <dig>  u/mg.
the molecular mass of nhase was nearly 106 kda detected by gel filtration chromatography, indicating that the nhase is a tetramer .fig.
<dig> sketch of plasmids construction.
pet-abp, a plasmid including nhase encoding genes a , b  and p ; pet-ab, a plasmid including nhase encoding genes a , b  and his-p ; pet-ab, a plasmid including nhase encoding genes a , b  and p-his ; pet-ab, a plasmid including nhase encoding genes a , b  and gst-p ; pet-bp, a plasmid including nhase encoding genes a* , b  and p
fig.
<dig> expression and purification of recombinant nhases.
a  <dig>  mark;  <dig>  control;  <dig>  cell extracts ;  <dig>  purified nhase ;  <dig>  cell extracts );  <dig>  purified nhase );  <dig>  cell extracts );  <dig>  purified nhase ).
b molecular mass determination on a superdex  <dig> prep grade column.
standard proteins are as follows:  thyroglobulin  ;  ferritin  ;  aldolase  ;  conalbumin  ; and  ovalbumin  .
the open circle represents the determined molecular mass.
the values represent the means ± sd for at least three independent experiments  <cit>
according to our previous work  and others’ reports  <cit>  about the nhase activators, the p14k is necessary for the functional expression of nhase in b. pallidus rapc <dig>  c. testosteroni 5-mgam-4d and p. putida nrrl- <dig>  since the amino acid sequence of nhase in b. petrii possessed a high similarity  with that in p. putida nrrl- <dig>  it is proposed that the p14k in b. petrii is also necessary for nhase activation.
here, the nhase expressed by pet-abp plasmid exhibited enough activity , indicating the successful expression of nhase and p14k, though the p14k band was not detected in sds-page .
successful expression of the activator p14k
the n-terminal residues of a protein are linked to its stability, which is conserved in bacteria  <cit> .
n-terminal lysine endows a protein short half-life, whereas glycine endows the protein long half-life   <cit> .
p14k, hardly detectable in sds-page, was successfully expressed in the nhase of p. putida through molecular modification in the light of n-end rule  <cit> .
to investigate this phenomenon in nhase activator p14k from b. petrii, a his-p14k was designed, in which a his tag was attached to the n-terminus of p14k.
since the n-terminal amino acids of p14k  were changed to the gss sequence of the designed his-p14k, the his-p14k would be more stable in terms of the n-end rule.
the plasmid pet-ab including nhase encoding genes a , b  and his-p  was transformed into e. coli and was applied for nhase expression .
as a result, the his-p14k was successfully expressed   and the enzymatic activity detected in the cell-free extracts was at the same level with that of the original nhase .
although nhase was successfully purified  and the specific activity was  <dig>  u/mg, the amount of p14k expression was still low .
to investigate the influence of the his tag on the c-terminal of p14k, a p14k-his, adding a his tag at the c-terminal of p14k, was designed.
the transformed cells carrying the pet-ab, a plasmid including nhase encoding genes a , b  and p-his , were applied for nhase expression.
as a result, the enzymatic activity detected was comparable to that of the original nhase, however, p14k-his was barely detected in sds-page .
these results indicated that the c-terminal of p14k does not play a key role in the expression of p14k.
high expression of p14k attached with a gst tag
the gst tag  encodes a highly soluble protein, which can help other proteins rapidly fold into a stable protein  <cit> .
to produce a large amount of p14k, a gst-p14k, adding a gst tag at the n-terminus of p14k, was designed.
the transformed cells carrying pet-ab, a plasmid including nhase encoding genes a , b  and gst-p , were applied for nhase expression.
as shown in fig.
<dig> lane  <dig>  a large amount of gst-p14k was detected in sds-page.
the enzymatic activity detected in cell-free extracts was at the same level with that of the original nhase.
the recombinant nhase was then successfully purified , and the specific activity was  <dig>  u/mg.
together with our previous work that the flexibility of p14k from p. putida plays an important role in nhase maturation  <cit>  and the analysis that the molecular weight mass of gst  is nearly two times larger than that of p14k , it is suggested that the p14k possesses a high flexibility indeed.fig.
<dig> stability analysis of p14k through sds-page.
<dig>  mark;  <dig>  control;  <dig>  cell extracts );  <dig>  purified nhase );  <dig>  cell extracts ) stored for 2 days;  <dig>  cell extracts ) stored for 6 days;  <dig>  cell extracts bp);  <dig>  purified nhase bp)
moreover, the protein stability of his-p14k and gst-p14k in the cell-free extracts was compared during storage at room temperature by sds-page analysis.
as shown in fig.
<dig> lane  <dig> and lane  <dig>  the his-p14k band nearly disappeared after 2 days whereas the gst-p14k band decayed to 60 % after 6 days.
the gst-p14k was far more stable than his-p14k, indicating that the thermal stability might be an important factor in the p14k expression.
a characteristic motif cxlc-ser-c has been discovered in all the α-subunits of nhases up to now  <cit> .
the ser between the two cys in this motif is proposed to be involved in the nhase maturation , by which the proton is transferred to -nh <dig>   <cit> .
these speculations indicated that the ser was necessary for the nhase maturation.
to investigate the necessity of the ser, the ser <dig> in nhase from b. petrii was researched.
an energetic difference of the proton transfer via ser <dig>  and the protein transfer not via ser <dig>   was compared through quantum chemical calculations using triton.
as shown in fig.
5b and c, the energy of the proton transferred to -nh <dig> via ser <dig>   was more than that of the direct transfer  , which indicated the type ii pathway without the involvement of the ser <dig> seemed easier to take place.
thus, a mutant nhase with the ser <dig> mutated to ala <dig> was designed to investigate the necessity of the ser <dig>  considering the structural similarity of the serine and alanine except for the -oh.
the transformed cells carrying pet-bp, a plasmid including nhase encoding genes a* , b  and p , were applied for the mutant nhase expression .
the enzymatic activity  detected in the cell-free extracts was nearly 40 % of that of original nhase.
the nhase was successfully purified , and the specific activity was  <dig>  u/mg, also nearly 40 % of that of the purified original nhase.
these findings suggested that the ser in the characteristic motif cxlc-ser-c of the active center of nhase is not necessary for the catalysis of nhase, however, it could kinetically facilitate the catalysis.fig.
<dig> energetic calculation of the proposed proton transfer pathway.
a the structure of the proposed nhase catalytic model in the active center.
the big red ball represents the cobalt ion.
the ser <dig> was shown by black dashed arrow.
the h <dig> represents the proton of substrate transition state.
the h <dig> represents the proton of ser <dig>  the type i is the proton transfer pathway: h1 → h2 → n. the type ii  is the proton transfer pathway: h1 → n. b energy calculation of proposed transfer pathway : the proton transfer pathway via the proton of oh .
c energy calculation of proposed transfer pathway : the proton direct transfer pathway )
CONCLUSIONS
the activator of nhase from b. petrii dsm  <dig> was successfully over-expressed and the ser <dig> was verified to be unnecessary for the catalysis of nhase by energetic calculation and site-specific mutagenesis.
the strategy for the over-expression of p14k adopted in the paper might be beneficial for other proteins with difficulty in expression.

 nitrile hydratase is a multi-subunits industrial enzyme, which can efficiently catalyze the nitrile to the corresponding amide. according to the different metal ions in the active center, the nhase could be classified into the two types: the iron type nhase and the cobalt type nhase. due to the high-value product and high catalytic activity, nhase has attracted wide attention in the chemical industry. nhase is composed of α- and β-subunits. the molecular masses of the α-subunit and β-subunit are generally less than 30 kda, such as those in bordetella petrii dsm and those in bacillus pallidus rapc. however, an activator is necessary for the functional expression of nhase, such as those in the co-nhases from rhodococcus rhodochrous j, rhodococcus jostii rha and pseudomonas putida nrrl- as well as in the fe-nhases from pseudomonas chlororaphis b and rhodococcus sp. n-. however, certain activators are poorly expressed, such as the activator p14k in the b. pallidus rapc comamonas testosteroni 5-mgam-4d and p. putida nrrl-. until recently, the p14k in p. putida was successfully expressed through fusing a strep tag at its n-terminal, then a novel nhase maturation mechanism, self-subunit swapping, was discovered. however, the stability of the p14k is not yet favorable for its over-expression. thus, to further facilitate the application of nhase, other fusion protein tags that are helpful for expression of more stable p14k are required. additionally, all the α-subunits of nhases share a conserved characteristic motif cxlc-ser-c in the active center, and the ser in this motif is proposed to be involved in nhase maturation. however, the necessity of ser was not studied by the energetic calculation and site-specific mutagenesis.fig. the catalytic pathway involving ser during nhase maturation. a first step: atoms of o-h and oh-co3+ participates in nhase activation. second step: the proton of oh participates in the proton transfer pathway. b the oxygen of the phenolate of the conserved tyr was ionized by the general base ser during nhase activation. c the oxygen of the phenolate of the conserved tyr was ionized by the general base ser with the help of tyr during nhase activation in this study, the nhase from b. petrii dsm was researched. the activator p14k was successfully expressed through fusing a his tag, and its expression and stability were further enhanced by fusing a gst tag at its n-terminus. in addition, we found that the ser in the characteristic motif cxlc-ser-c of the active center of nhase from b. petrii is not necessary for the nhase maturation. these results are useful for the expression of nhase activator and the understanding of the role of ser during the nhase maturation. further, the strategy for enhancing the stability of p14k is very beneficial for other proteins with difficulty in expression.  expression and purification of nhase the full-length genes of nhase were obtained from b. petrii dsm in which a gene encodes α-subunit, b gene encodes β-subunit and p gene encodes p14k. the recombinant expression vector pet-24a harboring the abp genes was constructed and transformed into e. coli for nhase expression. the calculated molecular mass of recombinant nhase was in accordance with that of the bands by sds-page analysis. the nhase was successfully purified and the specific activity was u/mg. the molecular mass of nhase was nearly 106 kda detected by gel filtration chromatography, indicating that the nhase is a tetramer.fig. sketch of plasmids construction. pet-abp, a plasmid including nhase encoding genes a, b and p; pet-ab, a plasmid including nhase encoding genes a, b and his-p; pet-ab, a plasmid including nhase encoding genes a, b and p-his; pet-ab, a plasmid including nhase encoding genes a, b and gst-p; pet-bp, a plasmid including nhase encoding genes a*, b and p fig. expression and purification of recombinant nhases. a mark; control; cell extracts; purified nhase; cell extracts ); purified nhase ); cell extracts ); purified nhase ). b molecular mass determination on a superdex prep grade column. standard proteins are as follows: thyroglobulin; ferritin; aldolase; conalbumin; and ovalbumin. the open circle represents the determined molecular mass. the values represent the means ± sd for at least three independent experiments according to our previous work and others’ reports about the nhase activators, the p14k is necessary for the functional expression of nhase in b. pallidus rapc c. testosteroni 5-mgam-4d and p. putida nrrl- since the amino acid sequence of nhase in b. petrii possessed a high similarity with that in p. putida nrrl- it is proposed that the p14k in b. petrii is also necessary for nhase activation. here, the nhase expressed by pet-abp plasmid exhibited enough activity, indicating the successful expression of nhase and p14k, though the p14k band was not detected in sds-page. successful expression of the activator p14k the n-terminal residues of a protein are linked to its stability, which is conserved in bacteria. n-terminal lysine endows a protein short half-life, whereas glycine endows the protein long half-life. p14k, hardly detectable in sds-page, was successfully expressed in the nhase of p. putida through molecular modification in the light of n-end rule. to investigate this phenomenon in nhase activator p14k from b. petrii, a his-p14k was designed, in which a his tag was attached to the n-terminus of p14k. since the n-terminal amino acids of p14k were changed to the gss sequence of the designed his-p14k, the his-p14k would be more stable in terms of the n-end rule. the plasmid pet-ab including nhase encoding genes a, b and his-p was transformed into e. coli and was applied for nhase expression. as a result, the his-p14k was successfully expressed and the enzymatic activity detected in the cell-free extracts was at the same level with that of the original nhase. although nhase was successfully purified and the specific activity was u/mg, the amount of p14k expression was still low. to investigate the influence of the his tag on the c-terminal of p14k, a p14k-his, adding a his tag at the c-terminal of p14k, was designed. the transformed cells carrying the pet-ab, a plasmid including nhase encoding genes a, b and p-his, were applied for nhase expression. as a result, the enzymatic activity detected was comparable to that of the original nhase, however, p14k-his was barely detected in sds-page. these results indicated that the c-terminal of p14k does not play a key role in the expression of p14k. high expression of p14k attached with a gst tag the gst tag encodes a highly soluble protein, which can help other proteins rapidly fold into a stable protein. to produce a large amount of p14k, a gst-p14k, adding a gst tag at the n-terminus of p14k, was designed. the transformed cells carrying pet-ab, a plasmid including nhase encoding genes a, b and gst-p, were applied for nhase expression. as shown in fig.  lane a large amount of gst-p14k was detected in sds-page. the enzymatic activity detected in cell-free extracts was at the same level with that of the original nhase. the recombinant nhase was then successfully purified, and the specific activity was u/mg. together with our previous work that the flexibility of p14k from p. putida plays an important role in nhase maturation and the analysis that the molecular weight mass of gst is nearly two times larger than that of p14k, it is suggested that the p14k possesses a high flexibility indeed.fig. stability analysis of p14k through sds-page. mark; control; cell extracts ); purified nhase ); cell extracts ) stored for 2 days; cell extracts ) stored for 6 days; cell extracts bp); purified nhase bp) moreover, the protein stability of his-p14k and gst-p14k in the cell-free extracts was compared during storage at room temperature by sds-page analysis. as shown in fig.  lane and lane the his-p14k band nearly disappeared after 2 days whereas the gst-p14k band decayed to 60 % after 6 days. the gst-p14k was far more stable than his-p14k, indicating that the thermal stability might be an important factor in the p14k expression. a characteristic motif cxlc-ser-c has been discovered in all the α-subunits of nhases up to now. the ser between the two cys in this motif is proposed to be involved in the nhase maturation, by which the proton is transferred to -nh. these speculations indicated that the ser was necessary for the nhase maturation. to investigate the necessity of the ser, the ser in nhase from b. petrii was researched. an energetic difference of the proton transfer via ser and the protein transfer not via ser was compared through quantum chemical calculations using triton. as shown in fig. 5b and c, the energy of the proton transferred to -nh via ser was more than that of the direct transfer, which indicated the type ii pathway without the involvement of the ser seemed easier to take place. thus, a mutant nhase with the ser mutated to ala was designed to investigate the necessity of the ser considering the structural similarity of the serine and alanine except for the -oh. the transformed cells carrying pet-bp, a plasmid including nhase encoding genes a*, b and p, were applied for the mutant nhase expression. the enzymatic activity detected in the cell-free extracts was nearly 40 % of that of original nhase. the nhase was successfully purified, and the specific activity was u/mg, also nearly 40 % of that of the purified original nhase. these findings suggested that the ser in the characteristic motif cxlc-ser-c of the active center of nhase is not necessary for the catalysis of nhase, however, it could kinetically facilitate the catalysis.fig. energetic calculation of the proposed proton transfer pathway. a the structure of the proposed nhase catalytic model in the active center. the big red ball represents the cobalt ion. the ser was shown by black dashed arrow. the h represents the proton of substrate transition state. the h represents the proton of ser the type i is the proton transfer pathway: h1 → h2 → n. the type ii is the proton transfer pathway: h1 → n. b energy calculation of proposed transfer pathway: the proton transfer pathway via the proton of oh. c energy calculation of proposed transfer pathway: the proton direct transfer pathway )  the activator of nhase from b. petrii dsm was successfully over-expressed and the ser was verified to be unnecessary for the catalysis of nhase by energetic calculation and site-specific mutagenesis. the strategy for the over-expression of p14k adopted in the paper might be beneficial for other proteins with difficulty in expression. 
the activator p14k is necessary for the activation of nitrile hydratase. our results are not only useful for the nhase activator expression and the understanding of the role of ser during nhase activation, but also helpful for other proteins with difficulty in heterologous expression. in addition, the ser in the characteristic motif cxlc-ser115-c of the active center of nhase was found to be unnecessary for nhase maturation. p14k was successfully expressed through fusing a his tag, and was over-expressed through fusing a gst tag at its n-terminus in the nhase of bordetella petrii dsm the stability of gst-p14k was demonstrated to be higher than that of the his-p14k.
 the activator p14k is necessary for the activation of nitrile hydratase. however, it is hard to be expressed heterogeneously. although an n-terminal strep tagged p14k could be successfully expressed from pseudomonas putida, various strategies for the over-expression of p14k are needed to facilitate further application of nhase.  p14k was successfully expressed through fusing a his tag, and was over-expressed through fusing a gst tag at its n-terminus in the nhase of bordetella petrii dsm the stability of gst-p14k was demonstrated to be higher than that of the his-p14k. in addition, the ser in the characteristic motif cxlc-ser115-c of the active center of nhase was found to be unnecessary for nhase maturation.  our results are not only useful for the nhase activator expression and the understanding of the role of ser during nhase activation, but also helpful for other proteins with difficulty in heterologous expression. keywords nhasebordetella petriip14kexpressionser115http://dx.doi.org/ /501100001809national natural science foundation of china21506172liu yi issue-copyright-statement© the author 2016 