BACKGROUND
lipases  catalyze the hydrolysis and synthesis of a variety of acylglycerols at the interface of lipid and water
<cit> .
owing to the properties like wide substrate specificity, high enantio- and regioselectivity, lipases have a wide range of potential applications in industry such as organic synthesis, detergent formulation, food and pharmaceutical industries
<cit> .
pseudomonas lipases display special biochemical characteristics which are not common among the lipases produced by other microorganism.
for example, it has exhibited amide hydrolyzing activity
<cit>  and high enantioselectivity towards hydrolysis of racemic esters
<cit> .
in spite of various potential applications of pseudomonas lipases, their functional overexpression requires a lipase-specific chaperone to fold into an active conformation
<cit> .
although the lipases can be produced by their homologous expression in pseudomonas hosts, many pseudomonas strains are potential pathogens and special safety regulations need to govern their cultivation.
therefore, it is necessary to set up a heterologous expression system and develop efficient refolding procedure.
recent years, many microbial lipase genes had been isolated, cloned, sequenced and overexpressed in homologous or heterologous hosts such as escherichia coli, filamentous fungi or yeast.
the expression of recombinant proteins in bacteria hosts often results in the formation of inclusion bodies which are insoluble and inactive.
many methods had been used to obtain high active expression amount of pseudomonas lipases
<cit> .
the amount of active enzyme achieved by co-expression of pseudomonas lipase and its cognate foldase in an expression vector was low because of the complexity of the gene regulatory and secretion mechanisms
<cit> .
therefore, two expression vectors had been used to solve this problem.
for example, madan b and mishra p
<cit>  put the lipa and lipb under the influence of strong t <dig> promoter in pet32xa/lic and pet29a vectors, respectively.
then e. coli bl <dig>  containing petlipa and petlipb was expressed upon induction with iptg, and the productivity attained to  <dig> u l- <dig> h- <dig>  akbari et al.
<cit>  also subcloned the lipase and lipase specific foldase into two separate expression vectors and expressed in e. coli as inactive inclusion bodies and soluble form, respectively.
then the lipase and lipase special foldase were mixed in vitro to refold active lipase.
the effect of different concentrations of various additives on the lipase refolding was investigated, and the best yield of  <dig> iu/ml was obtained.
in this work, a lipase-producing strain was screened from oil-rich soil and identified as pseudomonas aeruginosa through 16s rdna sequence analysis.
then the lipase gene lipa and the lipase special foldase gene lipb were successfully cloned and sequenced.
the expression vectors pacycduet- <dig> and petduet- <dig> containing two multiple cloning sites  were used to co-express the two target genes.
in vivo expression of the active lipase was successfully achieved.
to date, only one report by quyen et al.
<cit>  achieved the expression of in vivo functional lipase in heterologous host e. coli using a dual expression plasmid system which was derived from pet22b.
it demonstrated that a dual expression plasmid system e. coli could overproduce and secrete the active chaperone-dependent lipase  in vivo.
an improved dual expression plasmid system e. coli could be potentially applied for industrial-scale production of subfamily i.
<dig> and i.
<dig> lipases.
RESULTS
identification of the lipase-producing strain
initially, one isolate named ab from oil-rich samples enriched on rhodamine b agar plates was selected through the screening procedure.
for precise identification, 16s rrna gene sequencing of the isolate was performed and nucleotide sequence generated was aligned and analyzed for identification of bacterial species.
through the phylogenetic tree analysis, 16s rrna gene sequence of strain ab was compared with the sequences of ten pseudomonas species.
this strain had a high degree of homology  with pseudomonas aeruginosa , and thus was identified as psedomonas aeruginosa.
cloning and sequence analysis of lipase and lipase foldase genes from strain ab
genomic dna obtained from the strain ab was used to isolate the lipase and lipase specific foldase genes.
the lipase gene named lipa consisted of  <dig> nucleotides, encoding  <dig> amino acids, and the size of lipase specific foldase gene named lipb was  <dig> bp, encoding  <dig> amino acids.
both genes had 99% homology with reported sequences that from pseudomonas aeruginosa pao <dig>  or pseudomonas aeruginosa lip <dig> .
the gene lipa had nine nucleotides different from that of pseudomonas aeruginosa pao <dig>  but only one amino acid was different .
the gene lipb which encoded lipase-specific chaperone was coexpressed along with the structure gene of lipa in the same operon in p.aeruginosa.
the gene lipab which contained both lipa and lipb consisted of  <dig> nucleotides, and there were  <dig> bp between lipa and lipb.
the nucleotide sequences of lipa  and lipb  had been deposited in the genbank database under accession numbers jn <dig> and jn <dig>  respectively.
expression of the lipase using different expression plasmid systems
in this work, three expression plasmids, one single expression plasmid pet28a and two dual expression plasmids pacycduet- <dig> and petduet- <dig>  were investigated for high level expression of the active lipase.
pacycduet- <dig> and petduet- <dig> are both designed for the coexpression of two target genes.
each of the vectors contains two multiple cloning sites , and each of which is preceded by a t <dig> promoter/lac operator and ribosome binding site
<cit> .
the vector pacycduet- <dig> carries the p15a replicon, laci gene and chloramphenicol resistance gene.
while the vector petduet- <dig> carries the pbr322-derived cole <dig> replicon, laci gene and ampicillin resistance gene.
pacycduet- <dig> and petduet- <dig> can be used in combination in an appropriate host strain for the coexpression of up to  <dig> target genes.
three different expression plasmids pacycduet-lipa-lipb, petduet-lipa-lipb and pet28a-lipab contained lipa, lipb or lipab genes were successfully constructed.
the structure schematic diagram was shown in the figure
<dig>
the three recombinant plasmids were transferred into e.coli bl <dig>  and expressed under the control of t <dig> promoter, respectively.
upon induction with  <dig>  mm iptg for  <dig> h at 20°c, the cells were collected by centrifugation and disrupted by sonication.
both polypeptides lipa  and lipb  were expressed as shown in sds-page gel .
the whole cell expression level of lipa was almost the same in the three expression plasmid systems e. coli bl21/pet28a-lipab , e. coli bl21/petduet-lipalipb  and e. coli bl21/pacycduet-lipalipb  in the same condition.
however, the active lipa level in the whole cell lysate's supernatant was obviously different among the three expression plasmid systems.
the amount of active lipa expressed by the e. coli bl21/pet28a-lipab  and the e. coli bl21/petduet-lipalipb  was less than that of the e. coli bl21/pacycduet-lipalipb .
the expression level of lipb was also significantly different among the three expression plasmid systems.
the amount of the lipb expressed by the e. coli bl21/pacycduet-lipalipb  was much more than those of the other two expression plasmid systems.
the lipase activity of different recombinant e. coli cell lysate's supernatants containing active lipase was also determined.
the result showed that the activity of plasmid pet28a-lipab system was only  <dig> u/l, and the activity of plasmid petduet-lipalipb system was  <dig> u/l, but that of plasmid pacycduet-lipalipb system could up to  <dig> u/l, which was more than six times than the single expression plasmid pet28a-lipab system.
these results indicated that lipb was essential for the production of active lipa, which was agreement with many other reports.
gerritse et al.
<cit>  found that the expression level of the lipb played a key role in the expression and secretion of the active lipase when the copy number of the plasmid carrying the exogenous gene was more than  <dig>  and only when the lipb expression amount was raised, the activity of the lipase could be improved.
also, quyen et al.
<cit>  reported that the active lipase was negligible when the coexpressed chaperone lipb in the gene cluster was expressed at a negligible level in comparison to the lipa.
the ratio of lipase and chaperone was important to the lipase activity.
traub et al.
<cit>  reported that the lipase and chaperone must be present in equimolar amounts to obtain optimal refolding efficiency.
akbari et al.
<cit>  also reported that the concentration of lipase and foldase was an important parameter affecting refolding process.
in this work, the sds-page showed that the concentration of lipa and lipb was in a more equal proportion in pacycduet-lipalipb system than those of petduet-lipalipb system and pet28a-lipab system .
therefore, the lipa expressed by pacycduet-lipalipb system could be more refolded into active conformation.
this can explain the result that the lipase activity expressed by pacycduet-lipalipb system was more than those of petduet-lipalipb system and pet28a-lipab system in the same condition.
in addition, the two dual expression plasmids pacycduet- <dig> and petduet- <dig> have different replicons, so they can be recombined in the same host strain for the overexpression of target genes.
in this work, the two plasmids pacycduet-lipa-lipb and petduet-lipa-lipb were transferred into the same e. coli bl <dig> to hope further improvement of the lipa and lipb expression.
after induction with iptg, the activity of the lipase expressed by e. coli bl21/pacycduet-lipa-lipb + petduet-lipa-lipb was only  <dig> u/l, which was lower than that of e. coli bl21/petduet-lipa-lipb or e. coli bl21/pacycduet-lipa-lipb single plasmid system.
the copy number of pacycduet-lipa-lipb or/and petduet-lipa-lipb may decrease when the two plasmids existed in the same host strain.
as previously reported, the gene regulatory and secretion mechanisms were complex by coexpression of lipase and its special foldase in an expression vector.
in this work, two dual expression plasmids systems were used to enhance the expression amount of the lipb in e. coli in order to obtain more active lipase in vivo.
the result showed that the dual expression plasmids could obviously increase the expression level of active lipase.
but different dual expression plasmid showed different effect, and the plasmid pacycduet- <dig> was more suitable for the co-expression lipa and lipb than the plasmid petduet- <dig>  so far, only quyen et al.
<cit>  had chose a dual expression plasmid system to express an active lipase, but the dual expression plasmid was derived from pet22b and its construction process was complex.
the dual expression plasmids pacycduet- <dig> and petduet- <dig> were both the commercial plasmids, which were more convenient for practical application.
characterization of the lipase
to examine the properties of the lipase produced by e. coli bl21/pacycduet-lipa-lipb, the recombinant lipase was purified to homogeneity  using nickel affinity chromatography  by exploiting the histidine tag.
the optimum ph of the lipase was found to be  <dig> , and the enzyme activity was also relatively high at the ph range of 8– <dig> .
the lipase showed good ph stability, and more than 70% of the original activity could be retained at the ph range of 6– <dig> .
the optimum ph and ph stability revealed that the lipase should be alkaline in nature.
lipases from other pseudomonas sp.
such as p. mendocina pk-12cs
<cit>  and p. aeruginosa <cit>  had also been reported to possess good stability over wide ph range.
the optimal temperature of the lipase was observed to be 30°c , which showed that this lipase was a low temperature enzyme.
the thermal stability of the lipase was poor.
although the enzyme activity had almost no change with the extension of time at 30°c, it declined intensively at 40°c, and there was only less than 40% of the original activity retained at  <dig> min.
when the temperature was above 50°c, the enzyme activity lost seriously only after  <dig> min .
the lyophilized lipase catalyzed the esterification reaction between butyric acid and hexanol was first investigated.
an obvious peak appeared through gc analysis, and was identified as butyric acid hexyl ester.
this indicated that the lipase could catalyze the esterification reaction.
the optimum substrate alcohol and fatty acid for this lipase catalyzed esterification reaction were further investigated.
the result was shown in figure
<dig>  medium chain alcohols were appropriate with hexanol as the optimal alcohol .
the optimal fatty acid was octylic acid, and the  <dig> to  <dig> carbon acid was also fine, which meant that the lipase was suitable to catalyze the esterification of long chain fatty acids .
CONCLUSIONS
in this work, a lipase-producing strain identified as p. aeruginosa was isolated through the screening procedure.
the lipase gene lipa  and lipase specific foldase gene lipb  were cloned from the p.aeruginosa.
three heterologous expression systems were established for the expression of active lipase in vivo.
the plasmid pacycduet-lipa-lipb was the optimal expression plasmid among the three expression plasmids constructed in this work, and the lipase activity produced by e. coli bl21/pacycduet-lipa-lipb could be up to  <dig> u/l.
the lipase had an optimal temperature of 30°c and an optimal ph of  <dig>  the lipase showed obvious esterification activity and thus had potential biocatalytic applications.

 lipases catalyze the hydrolysis and synthesis of a variety of acylglycerols at the interface of lipid and water. owing to the properties like wide substrate specificity, high enantio- and regioselectivity, lipases have a wide range of potential applications in industry such as organic synthesis, detergent formulation, food and pharmaceutical industries. pseudomonas lipases display special biochemical characteristics which are not common among the lipases produced by other microorganism. for example, it has exhibited amide hydrolyzing activity and high enantioselectivity towards hydrolysis of racemic esters. in spite of various potential applications of pseudomonas lipases, their functional overexpression requires a lipase-specific chaperone to fold into an active conformation. although the lipases can be produced by their homologous expression in pseudomonas hosts, many pseudomonas strains are potential pathogens and special safety regulations need to govern their cultivation. therefore, it is necessary to set up a heterologous expression system and develop efficient refolding procedure. recent years, many microbial lipase genes had been isolated, cloned, sequenced and overexpressed in homologous or heterologous hosts such as escherichia coli, filamentous fungi or yeast. the expression of recombinant proteins in bacteria hosts often results in the formation of inclusion bodies which are insoluble and inactive. many methods had been used to obtain high active expression amount of pseudomonas lipases. the amount of active enzyme achieved by co-expression of pseudomonas lipase and its cognate foldase in an expression vector was low because of the complexity of the gene regulatory and secretion mechanisms. therefore, two expression vectors had been used to solve this problem. for example, madan b and mishra p put the lipa and lipb under the influence of strong t promoter in pet32xa/lic and pet29a vectors, respectively. then e. coli bl containing petlipa and petlipb was expressed upon induction with iptg, and the productivity attained to u l- h- akbari et al. also subcloned the lipase and lipase specific foldase into two separate expression vectors and expressed in e. coli as inactive inclusion bodies and soluble form, respectively. then the lipase and lipase special foldase were mixed in vitro to refold active lipase. the effect of different concentrations of various additives on the lipase refolding was investigated, and the best yield of iu/ml was obtained. in this work, a lipase-producing strain was screened from oil-rich soil and identified as pseudomonas aeruginosa through 16s rdna sequence analysis. then the lipase gene lipa and the lipase special foldase gene lipb were successfully cloned and sequenced. the expression vectors pacycduet- and petduet- containing two multiple cloning sites were used to co-express the two target genes. in vivo expression of the active lipase was successfully achieved. to date, only one report by quyen et al. achieved the expression of in vivo functional lipase in heterologous host e. coli using a dual expression plasmid system which was derived from pet22b. it demonstrated that a dual expression plasmid system e. coli could overproduce and secrete the active chaperone-dependent lipase in vivo. an improved dual expression plasmid system e. coli could be potentially applied for industrial-scale production of subfamily i. and i. lipases.  identification of the lipase-producing strain initially, one isolate named ab from oil-rich samples enriched on rhodamine b agar plates was selected through the screening procedure. for precise identification, 16s rrna gene sequencing of the isolate was performed and nucleotide sequence generated was aligned and analyzed for identification of bacterial species. through the phylogenetic tree analysis, 16s rrna gene sequence of strain ab was compared with the sequences of ten pseudomonas species. this strain had a high degree of homology with pseudomonas aeruginosa, and thus was identified as psedomonas aeruginosa. cloning and sequence analysis of lipase and lipase foldase genes from strain ab genomic dna obtained from the strain ab was used to isolate the lipase and lipase specific foldase genes. the lipase gene named lipa consisted of nucleotides, encoding amino acids, and the size of lipase specific foldase gene named lipb was bp, encoding amino acids. both genes had 99% homology with reported sequences that from pseudomonas aeruginosa pao or pseudomonas aeruginosa lip. the gene lipa had nine nucleotides different from that of pseudomonas aeruginosa pao but only one amino acid was different. the gene lipb which encoded lipase-specific chaperone was coexpressed along with the structure gene of lipa in the same operon in p.aeruginosa. the gene lipab which contained both lipa and lipb consisted of nucleotides, and there were bp between lipa and lipb. the nucleotide sequences of lipa and lipb had been deposited in the genbank database under accession numbers jn and jn respectively. expression of the lipase using different expression plasmid systems in this work, three expression plasmids, one single expression plasmid pet28a and two dual expression plasmids pacycduet- and petduet- were investigated for high level expression of the active lipase. pacycduet- and petduet- are both designed for the coexpression of two target genes. each of the vectors contains two multiple cloning sites, and each of which is preceded by a t promoter/lac operator and ribosome binding site. the vector pacycduet- carries the p15a replicon, laci gene and chloramphenicol resistance gene. while the vector petduet- carries the pbr322-derived cole replicon, laci gene and ampicillin resistance gene. pacycduet- and petduet- can be used in combination in an appropriate host strain for the coexpression of up to target genes. three different expression plasmids pacycduet-lipa-lipb, petduet-lipa-lipb and pet28a-lipab contained lipa, lipb or lipab genes were successfully constructed. the structure schematic diagram was shown in the figure the three recombinant plasmids were transferred into e.coli bl and expressed under the control of t promoter, respectively. upon induction with mm iptg for h at 20°c, the cells were collected by centrifugation and disrupted by sonication. both polypeptides lipa and lipb were expressed as shown in sds-page gel. the whole cell expression level of lipa was almost the same in the three expression plasmid systems e. coli bl21/pet28a-lipab, e. coli bl21/petduet-lipalipb and e. coli bl21/pacycduet-lipalipb in the same condition. however, the active lipa level in the whole cell lysate's supernatant was obviously different among the three expression plasmid systems. the amount of active lipa expressed by the e. coli bl21/pet28a-lipab and the e. coli bl21/petduet-lipalipb was less than that of the e. coli bl21/pacycduet-lipalipb. the expression level of lipb was also significantly different among the three expression plasmid systems. the amount of the lipb expressed by the e. coli bl21/pacycduet-lipalipb was much more than those of the other two expression plasmid systems. the lipase activity of different recombinant e. coli cell lysate's supernatants containing active lipase was also determined. the result showed that the activity of plasmid pet28a-lipab system was only u/l, and the activity of plasmid petduet-lipalipb system was u/l, but that of plasmid pacycduet-lipalipb system could up to u/l, which was more than six times than the single expression plasmid pet28a-lipab system. these results indicated that lipb was essential for the production of active lipa, which was agreement with many other reports. gerritse et al. found that the expression level of the lipb played a key role in the expression and secretion of the active lipase when the copy number of the plasmid carrying the exogenous gene was more than and only when the lipb expression amount was raised, the activity of the lipase could be improved. also, quyen et al. reported that the active lipase was negligible when the coexpressed chaperone lipb in the gene cluster was expressed at a negligible level in comparison to the lipa. the ratio of lipase and chaperone was important to the lipase activity. traub et al. reported that the lipase and chaperone must be present in equimolar amounts to obtain optimal refolding efficiency. akbari et al. also reported that the concentration of lipase and foldase was an important parameter affecting refolding process. in this work, the sds-page showed that the concentration of lipa and lipb was in a more equal proportion in pacycduet-lipalipb system than those of petduet-lipalipb system and pet28a-lipab system. therefore, the lipa expressed by pacycduet-lipalipb system could be more refolded into active conformation. this can explain the result that the lipase activity expressed by pacycduet-lipalipb system was more than those of petduet-lipalipb system and pet28a-lipab system in the same condition. in addition, the two dual expression plasmids pacycduet- and petduet- have different replicons, so they can be recombined in the same host strain for the overexpression of target genes. in this work, the two plasmids pacycduet-lipa-lipb and petduet-lipa-lipb were transferred into the same e. coli bl to hope further improvement of the lipa and lipb expression. after induction with iptg, the activity of the lipase expressed by e. coli bl21/pacycduet-lipa-lipb + petduet-lipa-lipb was only u/l, which was lower than that of e. coli bl21/petduet-lipa-lipb or e. coli bl21/pacycduet-lipa-lipb single plasmid system. the copy number of pacycduet-lipa-lipb or/and petduet-lipa-lipb may decrease when the two plasmids existed in the same host strain. as previously reported, the gene regulatory and secretion mechanisms were complex by coexpression of lipase and its special foldase in an expression vector. in this work, two dual expression plasmids systems were used to enhance the expression amount of the lipb in e. coli in order to obtain more active lipase in vivo. the result showed that the dual expression plasmids could obviously increase the expression level of active lipase. but different dual expression plasmid showed different effect, and the plasmid pacycduet- was more suitable for the co-expression lipa and lipb than the plasmid petduet- so far, only quyen et al. had chose a dual expression plasmid system to express an active lipase, but the dual expression plasmid was derived from pet22b and its construction process was complex. the dual expression plasmids pacycduet- and petduet- were both the commercial plasmids, which were more convenient for practical application. characterization of the lipase to examine the properties of the lipase produced by e. coli bl21/pacycduet-lipa-lipb, the recombinant lipase was purified to homogeneity using nickel affinity chromatography by exploiting the histidine tag. the optimum ph of the lipase was found to be, and the enzyme activity was also relatively high at the ph range of 8–. the lipase showed good ph stability, and more than 70% of the original activity could be retained at the ph range of 6–. the optimum ph and ph stability revealed that the lipase should be alkaline in nature. lipases from other pseudomonas sp. such as p. mendocina pk-12cs and p. aeruginosa had also been reported to possess good stability over wide ph range. the optimal temperature of the lipase was observed to be 30°c, which showed that this lipase was a low temperature enzyme. the thermal stability of the lipase was poor. although the enzyme activity had almost no change with the extension of time at 30°c, it declined intensively at 40°c, and there was only less than 40% of the original activity retained at min. when the temperature was above 50°c, the enzyme activity lost seriously only after min. the lyophilized lipase catalyzed the esterification reaction between butyric acid and hexanol was first investigated. an obvious peak appeared through gc analysis, and was identified as butyric acid hexyl ester. this indicated that the lipase could catalyze the esterification reaction. the optimum substrate alcohol and fatty acid for this lipase catalyzed esterification reaction were further investigated. the result was shown in figure medium chain alcohols were appropriate with hexanol as the optimal alcohol. the optimal fatty acid was octylic acid, and the to carbon acid was also fine, which meant that the lipase was suitable to catalyze the esterification of long chain fatty acids.  in this work, a lipase-producing strain identified as p. aeruginosa was isolated through the screening procedure. the lipase gene lipa and lipase specific foldase gene lipb were cloned from the p.aeruginosa. three heterologous expression systems were established for the expression of active lipase in vivo. the plasmid pacycduet-lipa-lipb was the optimal expression plasmid among the three expression plasmids constructed in this work, and the lipase activity produced by e. coli bl21/pacycduet-lipa-lipb could be up to u/l. the lipase had an optimal temperature of 30°c and an optimal ph of the lipase showed obvious esterification activity and thus had potential biocatalytic applications. 
genomic dna obtained from the strain was used to isolate the gene lipa and lipase specific foldase gene lipb. microbial lipases particularly pseudomonas lipases are widely used for biotechnological applications. the lipa showed obvious esterification activity and thus had potential biocatalytic applications. in this work, the lipase and its chaperone from a screened strain named ab which belongs to pseudomonas aeruginosa were overexpressed in e. coli with two dual expression plasmid systems to enhance the production of the active lipase lipa without in vitro refolding process. one single expression plasmid system e. coli bl21/pet28a-lipab and two dual expression plasmid systems e. coli bl21/petduet-lipa-lipb and e. coli bl21/pacycduet-lipa-lipb were successfully constructed. under the same cultured condition, the activities of the lipases expressed by e. coli bl21/pet28a-lipab and e. coli bl21/petduet-lipa-lipb were u/l and u/l, respectively, while the activity of the lipase expressed by e. coli bl21/pacycduet-lipa-lipb was up to u/l. it is a meaningful work to design experiments to obtain high-level active lipase. the active lipa could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that lipa was able to catalyze esterification reaction. the effect of different plasmid system on the active lipa expression was significantly different. pacycduet-lipa-lipb was more suitable for the expression of active lipa than pet28a-lipab and petduet-lipa-lipb. there is a limiting factor for functional overexpression of the pseudomonas lipase that a chaperone is necessary for effective folding. in this work, we screened a lipase-produced strain named ab through the screening procedure, which was identified as p. aeruginosa on the basis of 16s rdna. the most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. the lipase lipa had an optimal temperature of 30°c and an optimal ph of with a strong ph tolerance.
 microbial lipases particularly pseudomonas lipases are widely used for biotechnological applications. it is a meaningful work to design experiments to obtain high-level active lipase. there is a limiting factor for functional overexpression of the pseudomonas lipase that a chaperone is necessary for effective folding. as previously reported, several methods had been used to resolve the problem. in this work, the lipase and its chaperone from a screened strain named ab which belongs to pseudomonas aeruginosa were overexpressed in e. coli with two dual expression plasmid systems to enhance the production of the active lipase lipa without in vitro refolding process.  in this work, we screened a lipase-produced strain named ab through the screening procedure, which was identified as p. aeruginosa on the basis of 16s rdna. genomic dna obtained from the strain was used to isolate the gene lipa and lipase specific foldase gene lipb. one single expression plasmid system e. coli bl21/pet28a-lipab and two dual expression plasmid systems e. coli bl21/petduet-lipa-lipb and e. coli bl21/pacycduet-lipa-lipb were successfully constructed. the lipase activities of the three expression systems were compared to choose the optimal expression method. under the same cultured condition, the activities of the lipases expressed by e. coli bl21/pet28a-lipab and e. coli bl21/petduet-lipa-lipb were u/l and u/l, respectively, while the activity of the lipase expressed by e. coli bl21/pacycduet-lipa-lipb was up to u/l. the lipase lipa had an optimal temperature of 30°c and an optimal ph of with a strong ph tolerance. the active lipa could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that lipa was able to catalyze esterification reaction. the most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively.  the effect of different plasmid system on the active lipa expression was significantly different. pacycduet-lipa-lipb was more suitable for the expression of active lipa than pet28a-lipab and petduet-lipa-lipb. the lipa showed obvious esterification activity and thus had potential biocatalytic applications. the expression method reported here can give reference for the expression of those enzymes that require chaperones. pseudomonas aeruginosalipasechaperonedual expression plasmidesterification 