BACKGROUND
antimicrobial resistance based on hydrolysis of the antibiotic by β-lactamases is currently a worldwide problem.
it is one of the single most prevalent mechanisms responsible for resistance to β-lactams in clinical isolates of the enterobacteriaceae  <cit> .
among the four classes  of β-lactamases, plasmid mediated class a and c β-lactamases have been of high clinical concern in hospital as well as community acquired infections  <cit> .
promiscuous plasmids carrying β-lactamase encoding genes are described to spread drug resistance among different groups of microbes under local selection pressure imposed by the commonly used antibiotics  <cit> .
one of the most common plasmid mediated β-lactamase enzymes is closely related to tem and shv penicillinase  <cit> .
recently ctx-m and ampc type β-lactamase are being widely reported from enterobacteriaceae that are associated with nosocomial and community acquired infections  <cit> .
use of extended-spectrum β-lactam antibiotics has led to the occurrence of variants of these β-lactamases carrying amino acid substitutions that alter the enzyme's substrate specificity  <cit> .
shv- <dig> is an important plasmid mediated β-lactamase found in the chromosome of most strains of klebsiella pneumonia.
its hydrolytic spectrum of activity is similar to that of tem - <dig>  but it shows better activity against ampicillin  <cit> .
natural evolution and appearance of mutations has taken place in response to an array of different penicillin derivatives, cephamycins and fourth generation cephalosporins.
after identification of shv- <dig>  the first plasmid-mediated β-lactamase capable of hydrolyzing extended-spectrum cephalosporins, several point mutations in shv β-lactamase have been reported that altered the architecture of the active site of the enzyme  <cit> .
this modification leads to either an increase in minimum inhibitory concentration  or broadens the spectrum of the antimicrobial resistance observed.
amino acids from the region around the position  <dig> to the catalytic triad do not generally tolerate substitution in tem β-lactamase and are thought to be necessary for proper core packing and catalytic residue orientation  <cit> .
highly conserved residues on class a β-lactamases  are involved in hydrophobic core packing interactions.
likewise thr  <dig> and lys  <dig> are important for proper positioning of the catalytic residues ser  <dig> and asn  <dig>  <cit> .
however, the effect of substitutions on amino-acid residues that alter the substrate hydrolyzing property of shv enzyme is still unknown.
the shv β-lactamases identified in our study contained a single l138p change compared to wild-type enzyme shv- <dig>  since this mutation occurred naturally in shv- <dig> β-lactamases, we speculated that any changes in the substrate affinity must be attributed to this single amino acid substitution.
thus, to gain deeper insight we performed cloning, expression and enzyme kinetics of shv l138p β-lactamase.
for uniformity and comparative study we cloned a wild type blashv- <dig> gene from k. pneumoniae into the pet  <dig> cloning and expression vector.
this plasmid was used as template for creating shv- <dig> and target mutant shv alleles ) by site directed mutagenesis.
since shv- <dig> has a single amino-acid substitution in shv- <dig> and was previously identified in our study, we used these known β-lactamases as control.
the phenotypic and enzyme kinetics results were also verified by a molecular docking simulation experiment.
methods
bacterial strains
e. coli was isolated from the feces of pigs with mixed clinical signs of digestive and a respiratory disorder was identified by biochemical tests and by vitek .
once identified, the culture was stored in tryptic soy broth   mixed with 20% glycerol  at -70°c until use.
bacterial strains and antimicrobial tests
an e. coli isolated from the feces of pigs with mixed clinical signs of digestive and a respiratory disorder was tested with antibiotic susceptibility discs according to the guidelines of the clinical and laboratory standards institute   <cit> .
the active ingredients of the selected antibiotics were cefotaxime , ceftazidime , cefoxitin  and ceftiofur .
the isolate was further tested by the double disk diffusion tests using cefotaxime , ceftazidime , cefoxitin  in combination with amoxicillin/clavulanic acid   and oxoid ltd., uk)  <cit> .
the mics were determined by micro broth dilution method for the cephalosporins that showed complete or decreased inhibition zone diameter in the disk diffusion test.
performance and evaluation of the mic determinations followed the recommendation of the clsi  <cit> .
sequence analysis of the β-lactamases genes
oligonucleotide primers targeting tem and shv β-lactamases and sequencing of the pcr products was performed as described in our previous study  <cit> .
the search for the homologous sequence was conducted in the genbank database using the basic local alignment search tool  through the national center for biotechnology information  web site .
nucleotide substitutions were analyzed based on information available in http://www.lahey.org/studies/webt.htm.
site directed mutagenesis of blashv- <dig> genes
wild type blashv- <dig> gene from k. pneumoniae was cloned in pet  <dig> cloning vector.
this plasmid was used as template for generating blashv, blashv- <dig> and blashv- <dig> genes by site directed mutagenesis following the procedures described by zheng et.
al  <cit> .
description of the primers used in the study are listed in table  <dig>  all the pcr-amplified products were evaluated by agarose gel electrophoresis and the band with the expected size was extracted using qiaex® ii gel extraction kit  and further treated with  <dig> u dpni  and incubated at 37°c for  <dig> hrs.
an aliquot of  <dig> μl of this pcr product was transformed into topo  <dig> competent cells and plated on tryptic soy agar   agar plate containing  <dig> μg/ml of kanamycin.
a total of  <dig> colonies were selected and their plasmids were extracted using mini-prep.
sequences of all these β-lactamases were confirmed twice by the nucleotide sequencing using t <dig> forward and reverse primers.
a : primer used to create the mutation l138p in shv- <dig> β-lactamase identified in this study
b: primer used to create a single mutation p226s
β-lactamase expression and western blot
the expression of four different recombinant proteins encoding in the pet  <dig> expression vector carrying blashv- <dig>  blashv- <dig>  blashv- <dig> and blashv- <dig> genes was carried out in rossetta-gami  e. coli cells.
the cells pellets harvested by centrifugation were washed with pbs twice, re-suspended in lysis buffer  overnight at 4°c and lysed by sonication.
the his spin trap  were used for elution of the protein by  <dig> mm imidazol and protein concentrations of all β-lactamases were determined by bca protein assay kit  with bovine serum albumin as standard  <cit> .
proteins separated by sds-page  were transferred to hybond ecl nitrocellulose membrane , and incubated in anti his mouse igg followed by rabbit anti mouse igg.
binding was detected using an ap conjugate substrate kit  according to the manufacture's instruction.
enzyme activity assay
β-lactamase activity was determined by observing the rate of penicillin and ampicillin hydrolysis at  <dig> nm and  <dig> nm, respectively.
enzyme assay was performed at 25°c in  <dig> mm phosphate buffer   <cit> .
spectrophotometric measurements were made on analytic jena ag  using  <dig> -cm path length cuvette.
the values for km and vmax were determined using grafit  <dig> .
molecular docking simulation
the wild-type structure of shv  was used as a template for molecular modeling.
all molecular modeling simulations were performed by discovery studio  <dig>   and charmm forcefield and cff partial charge were used for all simulations.
the conformation of l138p position was optimized by the dreiding minimization and the molecular dynamics by standard dynamics cascade protocol was applied to relax the conformations of the wild-type and l138p mutant with and default parameters except that production steps was  <dig> and implicit solvent model was set to generalized born method.
among produced structures, the most stable structure with the lowest potential energy was selected as modeled structure for further docking simulation.
the docking simulations of β-lactamases were conducted by cdoker module with manually designed penicillin and ampicillin molecules.
because the active site and catalytic residues of shv and tem lactamasese are highly conserved, the structure of tem with bound penicillin g  was used as a reference structure to identify the initial binding site of penicillin and ampicillin in the wild-type and l138p lactamases.
once docking complexes of lactamases and antibiotics were predicted by cdocker module, productive docking structures of the carbonyl carbon of β-lactam ring of penicillin and ampicllin directly oriented to the oh group of catalytic s <dig> of β-lactamases  were considered further binding energy calculation .
the binding energies of the wild-type and l138p lactamases toward penicillin and ampicillin were calculated using calculate binding energies protocol with default parameters except that ligand minimization were performed to consider the flexibility of residues within binding sites and implicit solvent model was set to generalized born method.
RESULTS
antimicrobial resistance phenotype and genotype
e. coli  <dig> exhibited resistance to the commonly used antimicrobial agents on farms.
the disk diffusion test showed reduced inhibition zone diameter to cefotaxime , ceftazidime, ceftiofur  but not to cefoxitin .
this strain exhibited > <dig> mm increase in inhibition zone diameter of both cefotaxime and ceftazidime in the presence of amoxicillin/clavulanic acid  in contrast to when the antibiotics were tested alone.
rg e. coli cells carrying blashv- <dig>  blashv-, blashv- <dig> and blashv- <dig> exhibited variable zone diameter to penicillin and ampicillin in the disk diffusion test.
no decrease in zone diameter was noticed for cefotaxime , ceftazidime, ceftiofur  and cefoxitin .
the mic values for all e. coli strains are listed in table  <dig>  genotype analysis of e. coli isolate showed tem and shv β-lactamase genes showed 100% identity to blatem- <dig> and blashv- <dig> genes except at position  <dig> where leucine  to proline  polymorphism was detected.
aampicillin , penicillin , ceftiofur , cefoxitin , ceftazidime , cefotaxime
site directed mutagenesis of blashv- <dig> genes
after cloning and confirmation of blashv- <dig> genes in the pet  <dig> cloning and expression vector, reverse mutation at single point  was successfully performed by site directed mutagenesis to generate blashv-.
plasmid carrying blashv- <dig> gene was used to generate another mutation  that showed complete identity to blashv- <dig> gene.
sequence analysis also showed that the final site directed mutagenesis on the plasmid carrying blashv- <dig> gene, gave rise to the blashv- <dig>
cloning, expression and β-lactamase activity assay
all four pet  <dig> cloning and expression vectors carrying blashv- <dig>  blashv- <dig>  blashv- <dig> and blashv- <dig> genes expressed in rossetta-gami e. coli cells.
expressed proteins matched the size of  <dig>  kda in sds-page and western blot.
enzyme kinetics showed that shv β-lactamases cloned and expressed in this study exhibited variable catalytic activity of penicillin and ampicillin.
km value for both penicillin ampicillin was lowest for shv- <dig> β-lactamase followed by shv- <dig>  shv- <dig> and shv-l138p.
the description of the km, kcat and kcat/km values are given are listed in table  <dig>
molecular docking simulation of shv lactamases
the structures of the wild-type and l138p mutant were prepared by molecular dynamics.
the alpha helix of l138p mutant including  <dig> position was shorter than that of the wild-type and the orientation of the catalytic residues were slightly changed due to the proline mutation .
the productive docking structures with the lowest binding energies predicted by discovery studio  <dig>  were selected as binding structures of penicillin and ampicillin .
the wild-type showed higher binding affinity  of both penicillin  and ampicillin  than the l138p mutant, confirming that the l138p mutant had poor binding affinity  of penicillin  and ampicillin  compared to the wild-type.
the wild-type and l138p mutant had lower binding energies of penicillin  over ampicillin , consistent with experimental results that both β-lactamases preferred penicillin to ampicillin.
discussion
extensive research on β-lactam resistance has been carried among the clinical hospital isolates and majority of β-lactamases reported to date have been derived from clinical isolates of humans.
however, recent research has shown the increasing occurrence of β-lactam resistance in microbes of animal origin, especially in animal derived e. coli and salmonella, which are related to community acquired infections and food safety  <cit> .
in continuation of our study effort to address some of these issues of antimicrobial resistance in enterobacteriaceae isolated from farm animals, our study focused on the identification and understanding the dynamics of unique blashv-l138p mutation observed in e. coli isolated from swine.
phenotypic antimicrobial tests showed that the e. coli isolate was resistant to the common antimicrobial agents used in farms and also exhibited reduced sensitivity to three indicator cephalosporins included in the study.
genetic analysis showed the presence of both tem- <dig> and shv β-lactamases that differed from shv- <dig> only by a single amino acid substitution leucine to proline at position  <dig>  this mutation was of special interest as shv β-lactamses are specially related to k. pneumoniae and we wanted to see if this blashv gene with single amino-acid substitution  detected in e. coli added to its substrate hydrolyzing activity  <cit> .
all the cloned blashv genes expressed the specific protein bands that were confirmed by sds-page and western blot.
the size of the expressed shv β-lactamases was larger than reported in previous research because of the intact  <dig> amino acid pro-peptide and his tag  <cit> .
the enzyme kinetics of all the expressed β-lactamases showed differences in the affinities for penicillin and ampicillin that were included in this experiment .
the narrow spectrum β-lactamases shv- <dig> and shv- <dig> exhibited higher affinity to penicillin and ampicillin respectively, whereas shv- <dig> and shv- <dig> with only in one amino acid  mutation exhibited reduced activity for both the substrate used in study.
this indicated that leucine at position  <dig> was important for shv β-lactamase and played an important role in hydrolyzing penicillin and ampicilin.
previous experiments on shv β-lactamases have reported three natural mutations at position  <dig>   <dig> and  <dig> to be involved in conferring resistance to the inhibitors  <cit> .
proline has stronger stererochemical constraints than any other residues, with only one instead of two variable backbone angles and it lacks the normal amine backbone for hydrogen bonding.
this could have the disruptive function to regular secondary structure and decreased the length of α-helix and changed the orientation of residues of binding sites.
based on the modeled docking structures of the wild-type and l138p mutant, the wild-type had three hydrogen bonds with penicillin and ampicillin but the l138p mutant had two hydrogen bonds, indicating that these structural changes by l138p mutation may decrease the substrate binding and finally resulted in reduced activity of l138p mutant.
this result was supported by higher km value for penicillin and ampicillin of l138p mutation when inserted in shv- <dig> and shv- <dig>
CONCLUSIONS
based on our results we concluded that this mutation caused a drop in hydrolyzing penicillin and ampicillin.
under the selection pressure imposed by the use of these antimicrobials, naturally occurring l138p mutation in the conserved region of blashv gene was expected to increase substrate hydrolyzing property or widen the substrate spectrum of shv- β-lactamases.
however, adverse effect of this mutation observed on its substrate hydrolyzing properties may be a way these microbes trigger emergence or acquisition of more effective alternative mechanisms.
our speculation is in line with recent reports on ctx-m and ampc β-lactamases that have more frequently been reported than the classical tem and shv β-lactamase from farm and food materials  <cit> .
authors' contributions
nr, sbc and mks carried out cloning expression and western blot, sp contributed in enzyme kinetics, jcj did simulation docking experiment.
yjy and hsy provided guidance and helped coordination.
all authors have read and approved the final manuscript.

 antimicrobial resistance based on hydrolysis of the antibiotic by β-lactamases is currently a worldwide problem. it is one of the single most prevalent mechanisms responsible for resistance to β-lactams in clinical isolates of the enterobacteriaceae. among the four classes of β-lactamases, plasmid mediated class a and c β-lactamases have been of high clinical concern in hospital as well as community acquired infections. promiscuous plasmids carrying β-lactamase encoding genes are described to spread drug resistance among different groups of microbes under local selection pressure imposed by the commonly used antibiotics. one of the most common plasmid mediated β-lactamase enzymes is closely related to tem and shv penicillinase. recently ctx-m and ampc type β-lactamase are being widely reported from enterobacteriaceae that are associated with nosocomial and community acquired infections. use of extended-spectrum β-lactam antibiotics has led to the occurrence of variants of these β-lactamases carrying amino acid substitutions that alter the enzyme's substrate specificity. shv- is an important plasmid mediated β-lactamase found in the chromosome of most strains of klebsiella pneumonia. its hydrolytic spectrum of activity is similar to that of tem - but it shows better activity against ampicillin. natural evolution and appearance of mutations has taken place in response to an array of different penicillin derivatives, cephamycins and fourth generation cephalosporins. after identification of shv- the first plasmid-mediated β-lactamase capable of hydrolyzing extended-spectrum cephalosporins, several point mutations in shv β-lactamase have been reported that altered the architecture of the active site of the enzyme. this modification leads to either an increase in minimum inhibitory concentration or broadens the spectrum of the antimicrobial resistance observed. amino acids from the region around the position to the catalytic triad do not generally tolerate substitution in tem β-lactamase and are thought to be necessary for proper core packing and catalytic residue orientation. highly conserved residues on class a β-lactamases are involved in hydrophobic core packing interactions. likewise thr and lys are important for proper positioning of the catalytic residues ser and asn. however, the effect of substitutions on amino-acid residues that alter the substrate hydrolyzing property of shv enzyme is still unknown. the shv β-lactamases identified in our study contained a single l138p change compared to wild-type enzyme shv- since this mutation occurred naturally in shv- β-lactamases, we speculated that any changes in the substrate affinity must be attributed to this single amino acid substitution. thus, to gain deeper insight we performed cloning, expression and enzyme kinetics of shv l138p β-lactamase. for uniformity and comparative study we cloned a wild type blashv- gene from k. pneumoniae into the pet cloning and expression vector. this plasmid was used as template for creating shv- and target mutant shv alleles ) by site directed mutagenesis. since shv- has a single amino-acid substitution in shv- and was previously identified in our study, we used these known β-lactamases as control. the phenotypic and enzyme kinetics results were also verified by a molecular docking simulation experiment. methods bacterial strains e. coli was isolated from the feces of pigs with mixed clinical signs of digestive and a respiratory disorder was identified by biochemical tests and by vitek. once identified, the culture was stored in tryptic soy broth mixed with 20% glycerol at -70°c until use. bacterial strains and antimicrobial tests an e. coli isolated from the feces of pigs with mixed clinical signs of digestive and a respiratory disorder was tested with antibiotic susceptibility discs according to the guidelines of the clinical and laboratory standards institute. the active ingredients of the selected antibiotics were cefotaxime, ceftazidime, cefoxitin and ceftiofur. the isolate was further tested by the double disk diffusion tests using cefotaxime, ceftazidime, cefoxitin in combination with amoxicillin/clavulanic acid and oxoid ltd., uk). the mics were determined by micro broth dilution method for the cephalosporins that showed complete or decreased inhibition zone diameter in the disk diffusion test. performance and evaluation of the mic determinations followed the recommendation of the clsi. sequence analysis of the β-lactamases genes oligonucleotide primers targeting tem and shv β-lactamases and sequencing of the pcr products was performed as described in our previous study. the search for the homologous sequence was conducted in the genbank database using the basic local alignment search tool through the national center for biotechnology information web site. nucleotide substitutions were analyzed based on information available in http://www.lahey.org/studies/webt.htm. site directed mutagenesis of blashv- genes wild type blashv- gene from k. pneumoniae was cloned in pet cloning vector. this plasmid was used as template for generating blashv, blashv- and blashv- genes by site directed mutagenesis following the procedures described by zheng et. al. description of the primers used in the study are listed in table all the pcr-amplified products were evaluated by agarose gel electrophoresis and the band with the expected size was extracted using qiaex® ii gel extraction kit and further treated with u dpni and incubated at 37°c for hrs. an aliquot of μl of this pcr product was transformed into topo competent cells and plated on tryptic soy agar agar plate containing μg/ml of kanamycin. a total of colonies were selected and their plasmids were extracted using mini-prep. sequences of all these β-lactamases were confirmed twice by the nucleotide sequencing using t forward and reverse primers. a: primer used to create the mutation l138p in shv- β-lactamase identified in this study b: primer used to create a single mutation p226s β-lactamase expression and western blot the expression of four different recombinant proteins encoding in the pet expression vector carrying blashv- blashv- blashv- and blashv- genes was carried out in rossetta-gami e. coli cells. the cells pellets harvested by centrifugation were washed with pbs twice, re-suspended in lysis buffer overnight at 4°c and lysed by sonication. the his spin trap were used for elution of the protein by mm imidazol and protein concentrations of all β-lactamases were determined by bca protein assay kit with bovine serum albumin as standard. proteins separated by sds-page were transferred to hybond ecl nitrocellulose membrane, and incubated in anti his mouse igg followed by rabbit anti mouse igg. binding was detected using an ap conjugate substrate kit according to the manufacture's instruction. enzyme activity assay β-lactamase activity was determined by observing the rate of penicillin and ampicillin hydrolysis at nm and nm, respectively. enzyme assay was performed at 25°c in mm phosphate buffer. spectrophotometric measurements were made on analytic jena ag using -cm path length cuvette. the values for km and vmax were determined using grafit. molecular docking simulation the wild-type structure of shv was used as a template for molecular modeling. all molecular modeling simulations were performed by discovery studio and charmm forcefield and cff partial charge were used for all simulations. the conformation of l138p position was optimized by the dreiding minimization and the molecular dynamics by standard dynamics cascade protocol was applied to relax the conformations of the wild-type and l138p mutant with and default parameters except that production steps was and implicit solvent model was set to generalized born method. among produced structures, the most stable structure with the lowest potential energy was selected as modeled structure for further docking simulation. the docking simulations of β-lactamases were conducted by cdoker module with manually designed penicillin and ampicillin molecules. because the active site and catalytic residues of shv and tem lactamasese are highly conserved, the structure of tem with bound penicillin g was used as a reference structure to identify the initial binding site of penicillin and ampicillin in the wild-type and l138p lactamases. once docking complexes of lactamases and antibiotics were predicted by cdocker module, productive docking structures of the carbonyl carbon of β-lactam ring of penicillin and ampicllin directly oriented to the oh group of catalytic s of β-lactamases were considered further binding energy calculation. the binding energies of the wild-type and l138p lactamases toward penicillin and ampicillin were calculated using calculate binding energies protocol with default parameters except that ligand minimization were performed to consider the flexibility of residues within binding sites and implicit solvent model was set to generalized born method.  antimicrobial resistance phenotype and genotype e. coli exhibited resistance to the commonly used antimicrobial agents on farms. the disk diffusion test showed reduced inhibition zone diameter to cefotaxime, ceftazidime, ceftiofur but not to cefoxitin. this strain exhibited > mm increase in inhibition zone diameter of both cefotaxime and ceftazidime in the presence of amoxicillin/clavulanic acid in contrast to when the antibiotics were tested alone. rg e. coli cells carrying blashv- blashv-, blashv- and blashv- exhibited variable zone diameter to penicillin and ampicillin in the disk diffusion test. no decrease in zone diameter was noticed for cefotaxime, ceftazidime, ceftiofur and cefoxitin. the mic values for all e. coli strains are listed in table genotype analysis of e. coli isolate showed tem and shv β-lactamase genes showed 100% identity to blatem- and blashv- genes except at position where leucine to proline polymorphism was detected. aampicillin, penicillin, ceftiofur, cefoxitin, ceftazidime, cefotaxime site directed mutagenesis of blashv- genes after cloning and confirmation of blashv- genes in the pet cloning and expression vector, reverse mutation at single point was successfully performed by site directed mutagenesis to generate blashv-. plasmid carrying blashv- gene was used to generate another mutation that showed complete identity to blashv- gene. sequence analysis also showed that the final site directed mutagenesis on the plasmid carrying blashv- gene, gave rise to the blashv- cloning, expression and β-lactamase activity assay all four pet cloning and expression vectors carrying blashv- blashv- blashv- and blashv- genes expressed in rossetta-gami e. coli cells. expressed proteins matched the size of kda in sds-page and western blot. enzyme kinetics showed that shv β-lactamases cloned and expressed in this study exhibited variable catalytic activity of penicillin and ampicillin. km value for both penicillin ampicillin was lowest for shv- β-lactamase followed by shv- shv- and shv-l138p. the description of the km, kcat and kcat/km values are given are listed in table molecular docking simulation of shv lactamases the structures of the wild-type and l138p mutant were prepared by molecular dynamics. the alpha helix of l138p mutant including position was shorter than that of the wild-type and the orientation of the catalytic residues were slightly changed due to the proline mutation. the productive docking structures with the lowest binding energies predicted by discovery studio were selected as binding structures of penicillin and ampicillin. the wild-type showed higher binding affinity of both penicillin and ampicillin than the l138p mutant, confirming that the l138p mutant had poor binding affinity of penicillin and ampicillin compared to the wild-type. the wild-type and l138p mutant had lower binding energies of penicillin over ampicillin, consistent with experimental results that both β-lactamases preferred penicillin to ampicillin. discussion extensive research on β-lactam resistance has been carried among the clinical hospital isolates and majority of β-lactamases reported to date have been derived from clinical isolates of humans. however, recent research has shown the increasing occurrence of β-lactam resistance in microbes of animal origin, especially in animal derived e. coli and salmonella, which are related to community acquired infections and food safety. in continuation of our study effort to address some of these issues of antimicrobial resistance in enterobacteriaceae isolated from farm animals, our study focused on the identification and understanding the dynamics of unique blashv-l138p mutation observed in e. coli isolated from swine. phenotypic antimicrobial tests showed that the e. coli isolate was resistant to the common antimicrobial agents used in farms and also exhibited reduced sensitivity to three indicator cephalosporins included in the study. genetic analysis showed the presence of both tem- and shv β-lactamases that differed from shv- only by a single amino acid substitution leucine to proline at position this mutation was of special interest as shv β-lactamses are specially related to k. pneumoniae and we wanted to see if this blashv gene with single amino-acid substitution detected in e. coli added to its substrate hydrolyzing activity. all the cloned blashv genes expressed the specific protein bands that were confirmed by sds-page and western blot. the size of the expressed shv β-lactamases was larger than reported in previous research because of the intact amino acid pro-peptide and his tag. the enzyme kinetics of all the expressed β-lactamases showed differences in the affinities for penicillin and ampicillin that were included in this experiment. the narrow spectrum β-lactamases shv- and shv- exhibited higher affinity to penicillin and ampicillin respectively, whereas shv- and shv- with only in one amino acid mutation exhibited reduced activity for both the substrate used in study. this indicated that leucine at position was important for shv β-lactamase and played an important role in hydrolyzing penicillin and ampicilin. previous experiments on shv β-lactamases have reported three natural mutations at position and to be involved in conferring resistance to the inhibitors. proline has stronger stererochemical constraints than any other residues, with only one instead of two variable backbone angles and it lacks the normal amine backbone for hydrogen bonding. this could have the disruptive function to regular secondary structure and decreased the length of α-helix and changed the orientation of residues of binding sites. based on the modeled docking structures of the wild-type and l138p mutant, the wild-type had three hydrogen bonds with penicillin and ampicillin but the l138p mutant had two hydrogen bonds, indicating that these structural changes by l138p mutation may decrease the substrate binding and finally resulted in reduced activity of l138p mutant. this result was supported by higher km value for penicillin and ampicillin of l138p mutation when inserted in shv- and shv-  based on our results we concluded that this mutation caused a drop in hydrolyzing penicillin and ampicillin. under the selection pressure imposed by the use of these antimicrobials, naturally occurring l138p mutation in the conserved region of blashv gene was expected to increase substrate hydrolyzing property or widen the substrate spectrum of shv- β-lactamases. however, adverse effect of this mutation observed on its substrate hydrolyzing properties may be a way these microbes trigger emergence or acquisition of more effective alternative mechanisms. our speculation is in line with recent reports on ctx-m and ampc β-lactamases that have more frequently been reported than the classical tem and shv β-lactamase from farm and food materials. authors' contributions nr, sbc and mks carried out cloning expression and western blot, sp contributed in enzyme kinetics, jcj did simulation docking experiment. yjy and hsy provided guidance and helped coordination. all authors have read and approved the final manuscript. 
the aim of this study was to analyze the significance of leucine to proline substitution at position on the hydrolysis of penicillin and ampicillin that we identified in the blashv gene of clinical escherichia coli swine isolate. kinetic analysis of the mutant proteins showed that km value of the purified l138p mutant was comparatively higher than shv- shv- and shv- enzyme for penicillin and ampicillin. these naturally occurring mutations rendering reduced function of the existing protein could trigger the emergence or acquisition of more effective alternative mechanisms for β-lactam hydrolysis. our result demonstrated that l138p has a reduced role in penicillin and ampicillin hydrolyzing properties of shv β-lactamases.
 the aim of this study was to analyze the significance of leucine to proline substitution at position on the hydrolysis of penicillin and ampicillin that we identified in the blashv gene of clinical escherichia coli swine isolate.  kinetic analysis of the mutant proteins showed that km value of the purified l138p mutant was comparatively higher than shv- shv- and shv- enzyme for penicillin and ampicillin. docking simulation of the shv- and shv- enzymes also confirmed that β-lactamases preferred penicillin to ampicillin and the shv- had a higher binding affinity for antibiotics compared to the shv- and other mutants.  our result demonstrated that l138p has a reduced role in penicillin and ampicillin hydrolyzing properties of shv β-lactamases. these naturally occurring mutations rendering reduced function of the existing protein could trigger the emergence or acquisition of more effective alternative mechanisms for β-lactam hydrolysis. 