BACKGROUND
bacillus cereus is a gram-positive, spore-forming, rod-shape bacterium that grows well in aerobic and anaerobic environments  <cit> .
it causes food poisoning by producing two different types of toxins: an emetic toxin and a diarrheal toxin  <cit> .
although the symptoms caused by b. cereus food poisoning are relatively mild, the incidence of the disease is gradually increasing, and it can develop into severe disease  <cit> .
in addition, b. cereus can survive at a wide temperature range and form spores in harsh environments, especially during food processing; therefore, measures to control b. cereus effectively in the food industry are necessary  <cit> .
recently, endolysins have been explored as promising antibacterial agents.
endolysins are phage-encoded enzymes that hydrolyze the peptidoglycan bacterial cell wall  <cit> .
endolysins are synthesized at the end of the phage replication cycle and allow liberation of progeny phage particles from the host cell  <cit> .
most endolysins lack secretory signal sequences, therefore, holins are needed for endolysins to pass through the inner membrane and reach peptidoglycan, defined as the canonical holin-endolysin lysis system  <cit> .
endolysins are expected to be more effective biocontrol agents toward gram-positive than gram-negative bacteria, because the latter have an outer membrane that blocks access of endolysins to the peptidoglycan layer, when applied exogenously  <cit> .
in addition, other advantages of endolysins as biocontrol agents include:  low chance of developing bacterial resistance;  species-specific lytic activity without affecting other bacteria; and  high enzymatic activity that enables bacterial cells lysis within minutes or even seconds  <cit> .
endolysins are successfully applied in food products, such as milk and banana juice, to prevent contamination of staphylococcus aureus or gram-negative bacteria  <cit> .
besides, many reports already have shown that endolysins have high potential as strong therapeutic agents against a number of human pathogens through animal model studies  <cit> .
to date, only three endolysins from b. cereus bacteriophages have been characterized, all of which are n-acetylmuramoyl-l-alanine amidase-type endolysins  <cit> .
moreover, only a few reported phages can infect b. cereus, although many bacillus-targeting bacteriophages have been reported  <cit> .
thus, more bacteriophages and endolysins targeting b. cereus should be isolated and characterized to provide additional candidates for b. cereus biocontrol agents.
in previous work, we isolated the bacteriophage b <dig> , which is a lytic phage infecting b. cereus, from forest mud, and its genome was sequenced and analyzed to annotate important features .
in the present study, an endolysin gene was identified in the b <dig> bacteriophage genome.
this endolysin gene was cloned and expressed in escherichia coli, and the purified endolysin was characterized for its biochemical properties.
to the best of our knowledge, lysb <dig> is the first endolysin belonging to the l-alanoyl-d-glutamate endopeptidases originating from b. cereus bacteriophages.
RESULTS
identification and expression of the lysb <dig> phage endolysin
annotation of bacteriophage b <dig> genome sequence identified a predicted open reading frame  for a putative endolysin gene .
this 789-bp-long orf was designated lysb <dig>  using the interproscan program , lysb <dig> was predicted to have the vany domain  at the n terminus and sh3_ <dig> domain  at the c terminus .
according to blastp analysis  <cit> , the n terminus of lysb <dig> had high similarity to l-alanoyl-d-glutamate peptidases of listeria monocytogenes fsl j1- <dig> , bacillus subtilis subsp.
subtilis str.
<dig> , the listeria phage a <dig>  and the bacillus phage spo <dig> , and the c terminus had high similarity to proteins belonging to b. cereus ah <dig> , bacillus phages tp21-l  and bg <dig> , and the lactobacillus phage ll-ku .
among these proteins, ply <dig> of listeria phage a <dig> needs zn2+ in its active site according to a structural analysis  <cit> .
the three zn2+-coordinating residues  characterized in plya <dig> were conserved in the amino acid sequence of lysb <dig>  <cit> , and the zn2+ binding domain  reported in enterococcus vanx was found in lysb <dig>   <cit> .
recombinant lysb <dig> was cloned and expressed in e. coli with an n-terminal his-tag followed by purification using affinity chromatography.
sds-page showed a single band between  <dig> and  <dig> kda, which was consistent with the calculated molecular mass .
only  <dig> μg of purified lysb <dig> could lyse b. cereus atcc  <dig> cells substantially in  <dig> min .
viable cell counting revealed that  <dig> μg of lysb <dig> under this reaction condition could reduce the viable cell number by  <dig> to 4-log after  <dig> min .
moreover, typical optical microscopy showed that most bacilli were ruptured and disappeared by addition of lysb <dig> within  <dig> min .
effect of ph, temperature and ionic strength
analysis of lytic activity at different ph showed that lysb <dig> had the highest lytic activity at ph  <dig> - <dig>  .
this endolysin was relatively stable under a wide range of ph values, as incubation at ph  <dig> - <dig>  for  <dig> min did not inactivate the lytic activity .
in addition, although this endolysin was active to lyse the susceptible bacteria between  <dig> and 75°c, the maximal activity was shown at 50°c .
however, lysb <dig> was inactivated when it was incubated at > 55°c for  <dig> min .
the influence of nacl on the lytic activity of lysb <dig> was determined from 0- <dig> mm nacl.
as the nacl concentrations increased, lysb <dig> lytic activity was reduced, resulting in approximately 60% decrease in the presence of  <dig> mm nacl .
effect of divalent metal ions
to examine the effects of divalent metal ions to lysb <dig> enzymatic activity, we first removed metal ions from the protein using  <dig>  mm edta.
as seen in table  <dig> incubation of endolysin with  <dig> mm edta significantly decreased the lytic activity, which suggests lysb <dig> required metal ions for its full lytic activity.
when  <dig>  mm zn2+or mn2+ was added to the edta-treated endolysin, the lytic activity of the enzyme was restored .
in the case of other divalent metal ions, such as ca2+ and mg2+, addition of higher concentration  restored lysb <dig> enzymatic activity.
however, addition of hg2+ and cu2+ did not resort activity of the edta-treated endolysin.
taken together, lysb <dig> requires divalent metal ions, particularly zn2+ or mn2+ for its enzymatic activity.
relative lytic activities were measured by comparing the lytic activity of tests with it of lysb <dig> that was not treated with edta initially .
values represent the mean ± standard deviation .
antimicrobial spectrum of lysb4
antimicrobial activity against several gram-positive and gram-negative bacteria  was examined.
six b. cereus strains, b. subtilis, and two l. monocytogenes strains were susceptible to  <dig> μg lysb <dig>  showing complete lysis in the reaction buffer within  <dig> min.
this enzyme did not show lytic activity against other gram-positive bacteria such as enterococcus faecalis, staphylococcus aureus strains, streptococcus thermophilus and lactococcus lactis.
furthermore, lysb <dig> lytic activity was not detected with gram-negative bacteria, since they have a different cell wall composition  from gram-positive bacteria.
however, when cells were washed with  <dig>  m edta to increase the cell wall permeability, lysb4-mediated cell lysis was detected for all tested gram-negative bacteria including e. coli, pseudomonas aeruginosa, cronobacter sakazakii, salmonella typhimurium strains, salmonella enteritidis, shigella flexneri, and shigella boydii.
in particular, e. coli o157:h <dig> strains were lysed efficiently by lysb <dig>
gram-negative bacteria were treated with edta.
relative lytic activity was obtained by comparing the lytic activity of each test to it toward b. cereus atcc10876; 1-40% +, 41-70% ++, 71-100% +++, 0% -
endopeptidase activity of lysb4
lysb <dig> had the vany domain at its n terminus.
the vany domain encoded an l-alanoyl-d-glutamate endopeptidase and therefore lysb <dig> was expected to have endopeptidase activity.
this was confirmed using the trinitrobenzene sulfonic acid  method that detects the liberated free amino groups from b. cereus peptidoglycan caused by hydrolysis of lysb <dig>  pre-existing amino groups were eliminated by acetylating the peptidoglycan.
we detected a high concentration of free amino groups  released from the acetylated peptidoglycan after incubation with lysb <dig>  for  <dig> h, whereas only  <dig>  mm was released from the peptidoglycan in the absence of lysb <dig>  moreover, this enzyme did not show any n-acetylmuramoyl-l-alanine amidase or glycosidase activity .
therefore, lysb <dig> belongs to the endopeptidases.
determination of the cleavage site by lysb <dig> in the peptidoglycan
the specific lysb <dig> cleavage site in the peptidoglycan was determined by reverse-phase -hplc and lc-ms .
a peak that was absent from the control reaction  and had a retention time of  <dig>  min was observed in cell wall samples digested with lysb <dig> .
this peak corresponded to a fragment ion at m/z of  <dig> , which seemed to be the - of  <dig> -dinitrophenol -d-glu .
both peaks at  <dig>  min in figure 4a and at  <dig>  min in figure 4b corresponded to dnp.
when non-acetylated and acetylated peptidoglycan substrate were hydrolyzed by  <dig> n hcl and analyzed by rp-hplc, the peak corresponding to dnp-d-diaminopimelic acid  appeared only with the non-acetylated peptidoglycan sample, which showed that free amino groups of diaminopimelic acid in non-cross-linked peptide stem were labeled with dnp in this sample .
the lack of this peak with the acetylated peptidoglycan sample indicated that all the free amino groups were successfully acetylated.
these results suggested that lysb <dig> acts as an l-alanoyl-d-glutamate endopeptidase to cut the peptide bond between the l-ala and d-glu .
discussion
in this study, lysb <dig>  a newly identified endolysin from the b. cereus-specific bacteriophage b <dig>  was expressed, purified, and characterized.
we showed that lysb <dig> was an l-alanoyl-d-glutamate endopeptidase.
these endopeptidases have been reported in l. monocytogenes phages, the e. coli bacteriophage t <dig>  and a b. subtilis strain  <cit> .
in contrast, all the characterized endolysins found in bacteriophages infecting bacillus species are amidases   <cit> .
thus, lysb <dig> is the first characterized l-alanoyl-d-glutamate endopeptidase originating from b. cereus phages.
lysb <dig> has two domains; the vany domain at the n-terminus and sh3_ <dig> domain at the c-terminus.
the majority of the endolysins have two domains connected by a short linker: the n-terminal catalytic domain is responsible for cell lytic activity and the c-terminal cell wall binding domain that recognizes and binds a specific substrate, such as carbohydrate in the cell wall of target bacteria  <cit> .
the catalytic vany domain is conserved in other l-alanoyl-d-glutamate endopeptidases, including cwlk in b. subtilis and ply <dig> in l. monocytogenes bacteriophage a <dig>  <cit>  and d-alanoyl-d-alanine carboxypeptidases  <cit> .
the sh3_ <dig> domain at the c-terminus was found in the putative lysins of bacillus bacterial strains, bacillus phages and lactobacillus phages , suggesting that this domain is the cell wall binding domain.
biochemical characterization showed that the lysb <dig> endolysin was slightly alkalophilic, because activity was optimal at ph  <dig> - <dig> .
it was also slightly thermophilic, with an optimal temperature of 50°c.
the maximal lytic activity occurred in the absence of nacl.
this enzyme required a divalent metal ion, such as zn2+ or mn2+, for full enzymatic activity.
a similar requirement for divalent cations was seen for ply <dig> in l. monocytogenes bacteriophage a <dig>  <cit> .
the other characterized l-alanoyl-d-glutamate peptidase, t <dig> endolysin requires ca2+ instead of zn2+ or mn2+  <cit> .
the requirement of zn2+ or mn2+ is supported by protein sequence analysis, because lysb <dig> has the three zn2+-coordinating residues  of ply <dig>  and the zn2+-binding domain   <cit> .
endolysins are generally known to be highly specific against particular species of bacteria.
however, lysb <dig> showed lytic activity against a broad range of bacterial species.
lysb <dig> showed similar activity toward susceptible gram-positive and gram-negative bacteria, whereas other reported l-alanoyl-d-glutamate endopeptidases have a much narrower target host range  <cit> .
lysb <dig> could lyse not only b. cereus strains but also other gram-positive bacteria such as b. subtilis and l. monocytogenes strains.
in addition, this enzyme also showed lytic activity toward gram-negative bacteria when treated with edta.
most gram-negative bacteria contain the alγ type peptidoglycan, and bacillus species and l. monocytogenes have the alγ type cell wall as well  <cit> .
thus, lysb <dig> probably targets alγ type peptidoglycan.
this relatively broad antibacterial spectrum of lysb <dig> was surprising, given the narrow host range of the bacteriophage b <dig>  bacteriophage b <dig> only targets one strain of b. cereus  of five tested b. cereus strains and other gram-positive bacterial species including l. monocytogenes strains, s. aureus, and ent.
faecalis .
this suggests that there are more bacterial species with the lysb <dig> cell wall recognition site than those containing the bacteriophage b <dig> receptor.
therefore, further studies are needed to determine the moiety targeted by the lysb <dig> cell-wall binding sh3_ <dig> domain.
CONCLUSIONS
lysb <dig> is the first characterized l-alanoyl-d-glutamate endopeptidase originating from a b. cereus bacteriophage.
although lysb <dig> has similar enzymatic and genetic properties to ply <dig> from l. monocytogenes bacteriophage, lysb <dig> has broader spectrum and can lyse both gram-positive and gram-negative bacteria, including a number of foodborne pathogens.
as this enzyme also shows strong lytic activity and stability in wide range of ph and temperature, lysb <dig> has high potential as an effective antibacterial agent to control foodborne pathogens.
in the presence of agents such as edta, which permeabilize the outer cell membrane  <cit> , lysb <dig> could be successfully applied exogenously to control gram-negative bacteria as well as gram-positive bacterial pathogens.

 bacillus cereus is a gram-positive, spore-forming, rod-shape bacterium that grows well in aerobic and anaerobic environments. it causes food poisoning by producing two different types of toxins: an emetic toxin and a diarrheal toxin. although the symptoms caused by b. cereus food poisoning are relatively mild, the incidence of the disease is gradually increasing, and it can develop into severe disease. in addition, b. cereus can survive at a wide temperature range and form spores in harsh environments, especially during food processing; therefore, measures to control b. cereus effectively in the food industry are necessary. recently, endolysins have been explored as promising antibacterial agents. endolysins are phage-encoded enzymes that hydrolyze the peptidoglycan bacterial cell wall. endolysins are synthesized at the end of the phage replication cycle and allow liberation of progeny phage particles from the host cell. most endolysins lack secretory signal sequences, therefore, holins are needed for endolysins to pass through the inner membrane and reach peptidoglycan, defined as the canonical holin-endolysin lysis system. endolysins are expected to be more effective biocontrol agents toward gram-positive than gram-negative bacteria, because the latter have an outer membrane that blocks access of endolysins to the peptidoglycan layer, when applied exogenously. in addition, other advantages of endolysins as biocontrol agents include: low chance of developing bacterial resistance; species-specific lytic activity without affecting other bacteria; and high enzymatic activity that enables bacterial cells lysis within minutes or even seconds. endolysins are successfully applied in food products, such as milk and banana juice, to prevent contamination of staphylococcus aureus or gram-negative bacteria. besides, many reports already have shown that endolysins have high potential as strong therapeutic agents against a number of human pathogens through animal model studies. to date, only three endolysins from b. cereus bacteriophages have been characterized, all of which are n-acetylmuramoyl-l-alanine amidase-type endolysins. moreover, only a few reported phages can infect b. cereus, although many bacillus-targeting bacteriophages have been reported. thus, more bacteriophages and endolysins targeting b. cereus should be isolated and characterized to provide additional candidates for b. cereus biocontrol agents. in previous work, we isolated the bacteriophage b, which is a lytic phage infecting b. cereus, from forest mud, and its genome was sequenced and analyzed to annotate important features. in the present study, an endolysin gene was identified in the b bacteriophage genome. this endolysin gene was cloned and expressed in escherichia coli, and the purified endolysin was characterized for its biochemical properties. to the best of our knowledge, lysb is the first endolysin belonging to the l-alanoyl-d-glutamate endopeptidases originating from b. cereus bacteriophages.  identification and expression of the lysb phage endolysin annotation of bacteriophage b genome sequence identified a predicted open reading frame for a putative endolysin gene. this 789-bp-long orf was designated lysb using the interproscan program, lysb was predicted to have the vany domain at the n terminus and sh3_ domain at the c terminus. according to blastp analysis, the n terminus of lysb had high similarity to l-alanoyl-d-glutamate peptidases of listeria monocytogenes fsl j1-, bacillus subtilis subsp. subtilis str., the listeria phage a and the bacillus phage spo, and the c terminus had high similarity to proteins belonging to b. cereus ah, bacillus phages tp21-l and bg, and the lactobacillus phage ll-ku. among these proteins, ply of listeria phage a needs zn2+ in its active site according to a structural analysis. the three zn2+-coordinating residues characterized in plya were conserved in the amino acid sequence of lysb, and the zn2+ binding domain reported in enterococcus vanx was found in lysb. recombinant lysb was cloned and expressed in e. coli with an n-terminal his-tag followed by purification using affinity chromatography. sds-page showed a single band between and kda, which was consistent with the calculated molecular mass. only μg of purified lysb could lyse b. cereus atcc cells substantially in min. viable cell counting revealed that μg of lysb under this reaction condition could reduce the viable cell number by to 4-log after min. moreover, typical optical microscopy showed that most bacilli were ruptured and disappeared by addition of lysb within min. effect of ph, temperature and ionic strength analysis of lytic activity at different ph showed that lysb had the highest lytic activity at ph -. this endolysin was relatively stable under a wide range of ph values, as incubation at ph - for min did not inactivate the lytic activity. in addition, although this endolysin was active to lyse the susceptible bacteria between and 75°c, the maximal activity was shown at 50°c. however, lysb was inactivated when it was incubated at > 55°c for min. the influence of nacl on the lytic activity of lysb was determined from 0- mm nacl. as the nacl concentrations increased, lysb lytic activity was reduced, resulting in approximately 60% decrease in the presence of mm nacl. effect of divalent metal ions to examine the effects of divalent metal ions to lysb enzymatic activity, we first removed metal ions from the protein using mm edta. as seen in table incubation of endolysin with mm edta significantly decreased the lytic activity, which suggests lysb required metal ions for its full lytic activity. when mm zn2+or mn2+ was added to the edta-treated endolysin, the lytic activity of the enzyme was restored. in the case of other divalent metal ions, such as ca2+ and mg2+, addition of higher concentration restored lysb enzymatic activity. however, addition of hg2+ and cu2+ did not resort activity of the edta-treated endolysin. taken together, lysb requires divalent metal ions, particularly zn2+ or mn2+ for its enzymatic activity. relative lytic activities were measured by comparing the lytic activity of tests with it of lysb that was not treated with edta initially. values represent the mean ± standard deviation. antimicrobial spectrum of lysb4 antimicrobial activity against several gram-positive and gram-negative bacteria was examined. six b. cereus strains, b. subtilis, and two l. monocytogenes strains were susceptible to μg lysb showing complete lysis in the reaction buffer within min. this enzyme did not show lytic activity against other gram-positive bacteria such as enterococcus faecalis, staphylococcus aureus strains, streptococcus thermophilus and lactococcus lactis. furthermore, lysb lytic activity was not detected with gram-negative bacteria, since they have a different cell wall composition from gram-positive bacteria. however, when cells were washed with m edta to increase the cell wall permeability, lysb4-mediated cell lysis was detected for all tested gram-negative bacteria including e. coli, pseudomonas aeruginosa, cronobacter sakazakii, salmonella typhimurium strains, salmonella enteritidis, shigella flexneri, and shigella boydii. in particular, e. coli o157:h strains were lysed efficiently by lysb gram-negative bacteria were treated with edta. relative lytic activity was obtained by comparing the lytic activity of each test to it toward b. cereus atcc10876; 1-40% +, 41-70% ++, 71-100% +++, 0% - endopeptidase activity of lysb4 lysb had the vany domain at its n terminus. the vany domain encoded an l-alanoyl-d-glutamate endopeptidase and therefore lysb was expected to have endopeptidase activity. this was confirmed using the trinitrobenzene sulfonic acid method that detects the liberated free amino groups from b. cereus peptidoglycan caused by hydrolysis of lysb pre-existing amino groups were eliminated by acetylating the peptidoglycan. we detected a high concentration of free amino groups released from the acetylated peptidoglycan after incubation with lysb for h, whereas only mm was released from the peptidoglycan in the absence of lysb moreover, this enzyme did not show any n-acetylmuramoyl-l-alanine amidase or glycosidase activity. therefore, lysb belongs to the endopeptidases. determination of the cleavage site by lysb in the peptidoglycan the specific lysb cleavage site in the peptidoglycan was determined by reverse-phase -hplc and lc-ms. a peak that was absent from the control reaction and had a retention time of min was observed in cell wall samples digested with lysb. this peak corresponded to a fragment ion at m/z of, which seemed to be the - of -dinitrophenol -d-glu. both peaks at min in figure 4a and at min in figure 4b corresponded to dnp. when non-acetylated and acetylated peptidoglycan substrate were hydrolyzed by n hcl and analyzed by rp-hplc, the peak corresponding to dnp-d-diaminopimelic acid appeared only with the non-acetylated peptidoglycan sample, which showed that free amino groups of diaminopimelic acid in non-cross-linked peptide stem were labeled with dnp in this sample. the lack of this peak with the acetylated peptidoglycan sample indicated that all the free amino groups were successfully acetylated. these results suggested that lysb acts as an l-alanoyl-d-glutamate endopeptidase to cut the peptide bond between the l-ala and d-glu. discussion in this study, lysb a newly identified endolysin from the b. cereus-specific bacteriophage b was expressed, purified, and characterized. we showed that lysb was an l-alanoyl-d-glutamate endopeptidase. these endopeptidases have been reported in l. monocytogenes phages, the e. coli bacteriophage t and a b. subtilis strain. in contrast, all the characterized endolysins found in bacteriophages infecting bacillus species are amidases. thus, lysb is the first characterized l-alanoyl-d-glutamate endopeptidase originating from b. cereus phages. lysb has two domains; the vany domain at the n-terminus and sh3_ domain at the c-terminus. the majority of the endolysins have two domains connected by a short linker: the n-terminal catalytic domain is responsible for cell lytic activity and the c-terminal cell wall binding domain that recognizes and binds a specific substrate, such as carbohydrate in the cell wall of target bacteria. the catalytic vany domain is conserved in other l-alanoyl-d-glutamate endopeptidases, including cwlk in b. subtilis and ply in l. monocytogenes bacteriophage a and d-alanoyl-d-alanine carboxypeptidases. the sh3_ domain at the c-terminus was found in the putative lysins of bacillus bacterial strains, bacillus phages and lactobacillus phages, suggesting that this domain is the cell wall binding domain. biochemical characterization showed that the lysb endolysin was slightly alkalophilic, because activity was optimal at ph -. it was also slightly thermophilic, with an optimal temperature of 50°c. the maximal lytic activity occurred in the absence of nacl. this enzyme required a divalent metal ion, such as zn2+ or mn2+, for full enzymatic activity. a similar requirement for divalent cations was seen for ply in l. monocytogenes bacteriophage a. the other characterized l-alanoyl-d-glutamate peptidase, t endolysin requires ca2+ instead of zn2+ or mn2+. the requirement of zn2+ or mn2+ is supported by protein sequence analysis, because lysb has the three zn2+-coordinating residues of ply and the zn2+-binding domain. endolysins are generally known to be highly specific against particular species of bacteria. however, lysb showed lytic activity against a broad range of bacterial species. lysb showed similar activity toward susceptible gram-positive and gram-negative bacteria, whereas other reported l-alanoyl-d-glutamate endopeptidases have a much narrower target host range. lysb could lyse not only b. cereus strains but also other gram-positive bacteria such as b. subtilis and l. monocytogenes strains. in addition, this enzyme also showed lytic activity toward gram-negative bacteria when treated with edta. most gram-negative bacteria contain the alγ type peptidoglycan, and bacillus species and l. monocytogenes have the alγ type cell wall as well. thus, lysb probably targets alγ type peptidoglycan. this relatively broad antibacterial spectrum of lysb was surprising, given the narrow host range of the bacteriophage b bacteriophage b only targets one strain of b. cereus of five tested b. cereus strains and other gram-positive bacterial species including l. monocytogenes strains, s. aureus, and ent. faecalis. this suggests that there are more bacterial species with the lysb cell wall recognition site than those containing the bacteriophage b receptor. therefore, further studies are needed to determine the moiety targeted by the lysb cell-wall binding sh3_ domain.  lysb is the first characterized l-alanoyl-d-glutamate endopeptidase originating from a b. cereus bacteriophage. although lysb has similar enzymatic and genetic properties to ply from l. monocytogenes bacteriophage, lysb has broader spectrum and can lyse both gram-positive and gram-negative bacteria, including a number of foodborne pathogens. as this enzyme also shows strong lytic activity and stability in wide range of ph and temperature, lysb has high potential as an effective antibacterial agent to control foodborne pathogens. in the presence of agents such as edta, which permeabilize the outer cell membrane, lysb could be successfully applied exogenously to control gram-negative bacteria as well as gram-positive bacterial pathogens. 
the properties of lysb showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes lysb a good candidate as a biocontrol agent against b. cereus and other pathogenic bacteria. the incidence of b. cereus food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against b. cereus need to be developed. the endolysin from b. cereus phage b designated lysb was identified and characterized. the antimicrobial spectrum was relatively broad because lysb lysed gram-positive bacteria such as b. cereus, bacillus subtilis and listeria monocytogenes and some gram-negative bacteria when treated with edta. lc-ms analysis of the cell wall cleavage products showed that lysb was an l-alanoyl-d-glutamate endopeptidase, making lysb the first characterized endopeptidase of this type to target b. cereus. lysb is believed to be the first reported l-alanoyl-d-glutamate endopeptidase from b. cereus-infecting bacteriophages. in silico analysis revealed that this endolysin had the vany domain at the n terminus as the catalytic domain, and the sh3_ domain at the c terminus that appears to be the cell wall binding domain. the lytic activity was dependent on divalent metal ions, especially zn2+. biochemical characterization of lysb enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at ph - and 50°c. bacillus cereus is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.
 bacillus cereus is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. the incidence of b. cereus food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against b. cereus need to be developed. endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.  the endolysin from b. cereus phage b designated lysb was identified and characterized. in silico analysis revealed that this endolysin had the vany domain at the n terminus as the catalytic domain, and the sh3_ domain at the c terminus that appears to be the cell wall binding domain. biochemical characterization of lysb enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at ph - and 50°c. the lytic activity was dependent on divalent metal ions, especially zn2+. the antimicrobial spectrum was relatively broad because lysb lysed gram-positive bacteria such as b. cereus, bacillus subtilis and listeria monocytogenes and some gram-negative bacteria when treated with edta. lc-ms analysis of the cell wall cleavage products showed that lysb was an l-alanoyl-d-glutamate endopeptidase, making lysb the first characterized endopeptidase of this type to target b. cereus.  lysb is believed to be the first reported l-alanoyl-d-glutamate endopeptidase from b. cereus-infecting bacteriophages. the properties of lysb showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes lysb a good candidate as a biocontrol agent against b. cereus and other pathogenic bacteria. 