Gene cloning, expression and characterization of a cold-adapted lipase from

ORIGINAL PAPERGene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp.C18Ruipeng Chen •Lizhong Guo •Hongyue DangReceived:30January 2010/Accepted:4June 2010/Published online:23June 2010ÓSpringer Science+Business Media B.V.2010Abstract A psychrophilic bacterium Psychrobacter sp.C18previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin.A genomic DNA library was constructed and screened to obtain the corresponding lipase gene.The sequenced DNA fragment contains an open reading frame of 945bp,which was denoted as the lipX gene,from which a protein sequence LipX was deduced of 315amino acid res-idues with a molecular mass of 35,028Da.This protein contained the bacterial lipase GNSMG (GxSxG,x represents any amino acid residue)and HG consensus motifs.The recombinant pET28a(?)/lipX gene was overexpressed in heterologous host Escherichia coli BL21(DE3)cells to overproduce the lipase protein LipX His with a 69histidine tag at its C-terminus.Nickel affinity chromatography was used for purification of the expressed recombinant lipase.The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0with p -nitrophenyl myristate (C14)as a substrate.Thermostabil-ity assay indicated that the recombinant LipX His is a cold-adapted lipase,which was active in 10%methanol,ethanol,acetone and 30%glycol,and inhibited partially by Zn 2?,Co 2?,Mn 2?,Fe 3?and EDTA.Most non-ionic detergents,such as DMSO,Triton X-100,Tween 60and Tween 80enhanced the lipase activity but 1%SDS completelyinhibited the enzyme activity.Additionally,the highest lipolytic rate of the recombinant LipX His lipase was achieved when p -nitrophenyl myristate was used as a substrate,among all the p -nitrophenyl esters tested.Keywords Cold-adapted lipase ÁGenomic DNA library ÁRecombinant lipase ÁPsychrobacter ÁDeep-sea sedimentIntroductionLipases (triacylglycerol acyl hydrolases,E.C.3.1.1.3)cata-lyse the hydrolysis of long-chain acylglycerols and widely exist in animal tissues,plants and microorganisms,including fungi,bacteria and archaea (Jaeger et al.1999;Reetz 2002).In addition to being used in fat hydrolysis,regiospecificity and enantioselectivity make lipases highly useful as catalysts in synthetic organic chemistry and biotechnological appli-cations (Jaeger et al.1999;Schmidt-Dannert 1999;Jaeger and Reetz 2000;Bell et al.2002).Some lipases exhibit high organic solvent tolerance and stability as their activity relies on the occurrence of an oil–water interface (Verma et al.2008;Gupta and Khare 2009).Cold-adapted lipases may offer additional opportunities for novel biotechnological applications due to their high catalytic activity at low tem-peratures and unusual specificities (Joseph et al.2007,2008).These properties offer potential economic benefits in deter-gents,textile and food industries (Salameh and Wiegel 2007),and in bioremediation of polluted soils and waters (Joseph et al.2008).For example,lipase is an industrially important subgroup of the a /b hydrolase superfamily,with 1,000tons of lipase being used in detergents every year (Bell et al.2002;Joseph et al.2007).Furthermore,the diminishing petroleum reserves and environmental consequences caused by fossil fuel consumption make lipase-catalyzed biodieselR.Chen ÁL.Guo (&)College of Life Sciences,Qingdao Agricultural University,266109Qingdao,Chinae-mail:lizhguo963@R.Chen ÁH.Dang (&)State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology,China University of Petroleum (East China),266555Qingdao,China e-mail:DangHY@World J Microbiol Biotechnol (2011)27:431–441DOI 10.1007/s11274-010-0475-7fuel production an attractive strategy for renewable energy production(Ranganathan et al.2008;Parawira2009;Ro-bles-Medina et al.2009).Therefore,prospecting for novel lipase genes from psychrophilic bacteria is of great interest.PCR methods have been developed for screening lipase-encoding genes from environmental microorganisms(Bell et al.2002;Fan et al.2009).However,low homology is often observed among different lipase genes making the PCR screening method inefficient and ineffective infinding novel lipase genes(Bell et al.2002).Constructing genomic (Kumura et al.1998;Zhang et al.2007)or metagenomic (Henne et al.2000;Lee et al.2006;Ha˚rdeman and Sjo¨ling 2007;Kim et al.2009;Hu et al.2010)DNA libraries provides an alternative strategy effective forfinding novel lipase genes,as no sequence homology is needed for lipase gene screening and detection.Cold adapted lipases have been isolated from marine psychrophilic and psychrotolerant bacteria such as Aero-monas(Lee et al.,2003),Moraxella(Feller et al.,1990, 1991),Moritella(Yang et al.,2008),Photobacterium(Ryu et al.,2006),Pseudoalteromonas(Zeng et al.,2004;Lo Giudice et al.,2006;de Pascale et al.,2008),Pseudomonas (Zhang&Zeng,2008),and Psychrobacter(Arpigny et al., 1993,1995;Yumoto et al.,2003;Zeng et al.,2004;Zhang et al.,2007).However,psychrophilic or psychrotolerant bacteria usually grow slowly and their lipase production is limited.Advances in genetic engineering have enabled efficient overproduction of recombinant enzymes in an appropriate heterologous host strain such as Escherichia coli(Schmidt-Dannert,1999;Joseph et al.,2008).Deep-sea sediments provide a cold environment for isolation of psychrophilic and psychrotolerant bacteria and their cold active lipases(Kobayashi et al.2008;Dang et al. 2009).In a previous study,a psychrophilic bacterium C18 with extracellular lipolytic activity was isolated from the deep-sea sediments of the Southern Okinawa Trough (Dang et al.2009).This bacterium was identified as a Psychrobacter strain(originally named K2-4L-010)based on the phylogeny of its16S rRNA gene sequence(Dang et al.2009).In the current study,we cloned and sequenced its novel lipase gene and characterized the recombinant lipase overproduced in Escherichia coli to study its ther-mostability and optimum catalytic condition.Materials and methodsConstruction of genomic DNA library for lipase gene screeningGenomic DNA library construction methods(Kumura et al. 1998;Zhang et al.2007)were adopted for lipase gene screening.In detail,genomic DNA was extracted(Sambrook and Russell2001)from Psychrobacter sp.C18cells grown in Marine Broth2216medium at20°C for48h,and par-tially digested with restriction endonuclease Sau3A I(Ta-KaRa,Japan).The digested genomic DNA fragments with length of2–7kb were recovered from an agarose gel by an E.Z.N.A.Gel Extraction Kit(OMEGA,USA)and then ligated into a expression vector plasmid pUC19(TaKaRa, Japan),which had been previously digested with restriction endonuclease Bam H I(TaKaRa,Japan).Recombinant plasmids were transformed with the heat shock and cold treatment protocol into Escherichia coli DH5a competent cells prepared by the calcium chloride protocol(Sambrook and Russell2001),as detailed in a previous publication (Dang et al.2008).Recombinants were selected using Luria–Bertani(LB)agar indicator plates supplemented with100l g/ml ampicillin,100l M IPTG and1%(w/v) tributyrin(Sigma,USA).These plates were incubated overnight at37°C,followed by incubation for a few days at 4°C.Two colonies,named pL1and pL2respectively,were found to produce a clear zone on the plates,showing potentially the lipase activity.Sequencing and lipase gene sequence analyses Recombinant plasmids were extracted from pL1and pL2, and the DNA inserts were sequenced using an ABI3770 automatic sequencer(Applied BioSystems,USA)by San-gon Biotech Co.(Shanghai,China).The putative lipase protein sequences were deduced from the gene sequences. The NCBI Blastn and Blastx programs were used to identify similar DNA sequences that encoded lipases (Altschul et al.1997).Phylogeny of the obtained lipases was analyzed using the PHYLIP software package(version 3.68;Felsenstein1989)with an alignment of the reference lipase sequences using the Clustal X program(version2.0; Larkin et al.2007).The upstream regulatory sequence signatures of the putative lipase genes were analyzed using the online BPROM program(http://linux1.softberry. com/berry.phtml?topic=index&group=programs&subgroup= promoter),to identify the-35and-10promoter sequences and the ribosomal binding site(RBS).Signal peptide sequences of the putative lipase genes were predicted by the online SignalP3.0Server program(http://www.cbs.dtu. dk/services/SignalP/)(Nielsen et al.1997).Lipase gene cloning and expression in heterologous Escherichia coliThe lipase gene sequences from pL1and pL2were found to be identical,so only pL1was selected for further study.The lipase gene(without signal peptide sequence) was PCR amplified from the original genomic DNA of thestrain Psychrobacter sp.C18using primers lp1F50-GAT-TGCCCATGGGACTATTCAAACGCTTAGGCCTTG-30 (with Nco I restriction site underlined)and lp1R50-CAT-CCGGAATTCTTACTAATCCCCTCACGAAAC-30(with Eco R I restriction site underlined).PCR was performed with an initial denaturation at95°C for5min;followed by 30cycles of95°C for1min,60°C for90s,72°C for60s; and afinal extension at72°C for10min,using the high-fidelity pfu DNA polymerase(Promega,USA).PCR products were digested overnight at16°C with Nco I and EcoR I restriction enzymes(TaKaRa,Japan),purified with an E.Z.N.A.Gel Extraction Kit(Omega,USA),and ligated into the expression vector pET-28a(?)carrying a C-ter-minal69His-tag DNA sequence(Novagen,USA).The recombinant plasmids were transformed into Esche-richia coli BL21(DE3)competent cells(Novagen, USA).The transformed colonies were screened by colony PCR and plasmid sequencing was employed for lipase gene sequence confirmation.Optimization of recombinant lipX overexpressionin heterologous Escherichia coliThe Escherichia coli BL21(DE3)cells harboring the recombinant pET28a(?)/lipX plasmid were grown at37°C and180rpm.After the cell culture’s optical density at 600nm reached0.5,the expression of the recombinant LipX lipase with the C-terminal69His-tag was induced with 0.1mM IPTG and incubated at20°C for8h.Induced cells were harvested by centrifugation(10,0009g,10min at4°C), and the cell pellet was washed with phosphate-buffered sal-ine(PBS,pH7.4).The cells were disrupted by sonication using15bursts of18kHz each for30s alternated by1min of intermittent cooling on ice(Madan and Mishra2009).Cell debris was removed by centrifugation at10,0009g for 15min at4°C.Lipase expression was checked by12%SDS polyacrylamide gel electrophoresis(SDS-PAGE).The optimal expression conditions of the recombinant lipase in the heterologous host Escherichia coli BL21 (DE3)were checked for the optimal temperature,optimal expression hours and optimal IPTG induction.The results were confirmed by SDS-PAGE and lipase activity assay. For the optimization of IPTG concentration and induction time,Escherichia coli BL21(DE3)cells containing pET28a(?)/lipX were grown in4ml LB medium at37°C, 200rpm for10h.The inoculum(1%,v/v)was transferred into100ml LB medium and the cells were induced with different concentrations(0.1–1mM)of IPTG at20°C for different incubation time after the cell culture optical density at600nm reached0.5.For the optimization of cell density at the time of induction,cells were induced with0.1mM IPTG at different culture optical densities (0.4–1.0at600nm)and were incubated for8h.For the optimization of induction temperature,cells were induced with0.1mM IPTG at optical density of0.6at600nm and incubated at different temperatures(15–37°C)for8h.The lipase expression level was assayed by12%SDS-PAGE.Purification of the recombinant lipasePurification of the recombinant lipase was carried out by a one-step histidine-tagged protein purification method using immobilized nickel affinity chromatography(His-tag chromatography)with an A¨KTA Prime Plus purifier sys-tem(GE Pharmacia,USA).Cells were collected from1l culture of IPTG-induced Escherichia coli BL21(DE3) harboring the recombinant lipX gene and resuspended witha low imidazole buffer(20mM Na2HPO4–NaH2PO4,pH7.4,0.5M NaCl and25mM imidazole).After sonication the cell lysate was passed through a0.45l m syringe-end filter and loaded on a His-trap nickel affinity column(GE Healthcare,USA),which had already been equilibrated with the low imidazole buffer.The column was extensively washed with an intermediate imidazole buffer(20mM Na2HPO4–NaH2PO4,pH7.4,0.5M NaCl,50mM imid-azole)to remove any unspecifically bound and unbound proteins.The target lipase proteins were eluted by using a high imidazole buffer(20mM Na2HPO4–NaH2PO4,pH 7.4,0.5M NaCl,500mM imidazole).The purified lipase was checked using12%SDS-PAGE and the lipase activity was assayed by the above described method.Lipase activity and protein content assayLipase activity assay was performed using p-nitrophenyl palmitate(p-NPP)as a substrate(Winkler and Stuckmann 1979).The reaction mixture contained200l l sub-strate(0.1mM p-NPP ester in2-propanol),1,500l l buffer (100mM Tris-HCl buffer,pH7.0,with0.1%(w/v)Triton X-100and0.1%(w/v)Arabic gum powder).Lipase solu-tion(100l l)was added to the mixture after5min prew-arming at30°C.Reaction was stopped by adding1ml2% SDS(sodium dodecyl sulfonate)solution after20min incubation.Absorbance at405nm was determined.One unit of lipase activity was defined as the release of1l mol of p-nitrophenol per min.Lipase concentration was mea-sured by Bradford dye method(Bradford1976),with absorbance measured at595nm and BSA(bovine serum albumin)used as a protein standard.Characterization of the lipaseThe recombinant lipase molecular weight was deter-mined by SDS-PAGE with standard protein molecular weight markers(TaKaRa,Japan).The optimum lipase reaction temperature was determined by measuring thelipase reaction rate at different temperatures(15–60°C) under standard assay conditions.The lipase thermostability was determined by measuring the residual lipase activity after incubating the lipase at different temperatures(-20–70°C)for1h.The optimum lipase reaction pH was determined between pH3.6and pH11.0.The lipase pH stability was determined by measuring the residual lipase activity after incubating the lipase at30°C for1h in the following buffers:200mM sodium acetate(pH3.6,4.0,5.0 and5.6);PBS(pH5.7,6.0,6.5,7.2,7.4and8.0);200mM Tris-HCl(pH7.2and8.05);200mM Glycine-NaOH(pH 9.5,10.0,10.6and11).The effect of salinity on the lipase activity was determined with the measurement of the lipase activity after incubating the lipase in500mM Tris-HCl buffer(pH8.0)containing a specific NaCl concentration(0, 1,5,10,20,50,100or500mM)for20min at30°C.The effect of various metal ions(CoSO4,CuSO4,FeSO4, MgSO4,MnSO4,ZnSO4,CaCl2,FeCl3and KCl)and EDTA on the lipase activity was determined with the measurement of the lipase activity after incubating the lipase(in500mM Tris-HCl buffer,pH8.0)at30°C for20min supplemented with afinal concentration of1mM of the individual ion solution.The effect of various detergents on the lipase activity was determined with the measurement of the lipase activity after incubating the lipase in500mM Tris-HCl buffer(pH8.0)containing1%(w/v)of detergent SDS, Triton X-100,Tween60,Tween80or DMSO for20min at 30°C.The effect of various organic solvents on the lipase activity was determined by adding them in the reaction mixtures at a concentration of10,30or50%of a specific organic solvent(methanol,ethanol,glycol,acetonitrile or acetone)respectively then the enzyme activity was assayed. Substrate specificity of the lipase was determined at30°C and pH8.0on p-nitrophenyl ester substrates with variable chain length(C2to C18)by measuring the amount of p-nitrophenol released by lipase-catalyzed hydrolysis(Lee et al.1993).The detailed experiments for the effects of the various treatments on the lipase activity followed the descriptions in the above lipase activity assay section. Nucleotide sequence accession numbersThe gene sequence lipX of the Psychrobacter sp.C18 lipase was deposited in the GenBank database with accession number GU583649.ResultsLipase gene sequence analysesTwo colonies pL1and pL2showing clear halos on the tributyrin indicator plates were identified from the genomic DNA library of Psychrobacter sp.C18,indicating that these2colonies might harbor the recombinant plasmids with the lipase genes.The DNA inserts were found to be about3.5kb on the recombinant plasmids of these2col-onies.After DNA sequencing,the nucleotide sequences of the two plasmid inserts were found to be identical.The lipase gene sequence,named lipX,from colony pL1was selected for further study.The nucleotide sequence of lipX revealed an open reading frame(ORF)comprising945bp,encoding a putative protein sequence of315amino acids with a pre-dicted molecular weight of35,028Da(Fig.1).This ORF is preceded by a putative ribosome-binding site(AAGGA) at7bases upstream of the start codon ATG,the-35box (TTGTAG)and-10box(TGCTATAAA)were also iden-tified(Fig.1).The deduced LipX protein sequence has the GNSMG and HG fragments starting at residue139and75 (Fig.1);the GxSxG and HG consensus motifs are ubiq-uitous as the active sites in bacterial lipases.A sequence fragment of27amino acid residues at the N-terminus of LipX was predicted to be a signal peptide(Fig.1),indi-cating that LipX is potentially an extracellular bacterial lipase.The lipX gene had sequence identities of only80.9% with the lip gene(GenBank accession number X67712)of the Antarctic facultative psychrophile Psychrobacter immobilis B10(Arpigny et al.,1993,1995),and80.6% with the lipA1and pAL1-3genes(GenBank accession numbers AM229326and AM229327)of the Antarctic Psychrobacter sp.7195(Zhang et al.,2007).The deduced LipX protein sequence had86.3%identity with lipase Lip3 (GenBank accession number CAA37863)of the Antarctic psychrotroph Moraxella sp.TA144(Feller et al.,1991), 86.0and85.3%identities with lipases LipA1and PAL1-3 (GenBank accession numbers CAJ76164and CAJ76165) of Psychrobacter sp.7195(Zhang et al.,2007),and85.1% identity with lipase Lip(GenBank accession number CAA47949)of Psychrobacter immobilis B10(Arpigny et al.,1993,1995).Phylogenetic analysis confirmed that the LipX sequence was affiliated with known lipase sequences obtained mainly from psychrophilic bacteria, especially the Psychrobacter(Fig.2).Expression and optimization of expression of lipXThe lipX gene was amplified with no signal peptide from genomic DNA.The cloned lipX gene was expressed under the control of the strong T7RNA polymerase transcription promoter by using the pET28a(?)and E.coli BL21(DE3) expression system.The recombinant lipase protein was named LipX His(LipX?6xHis tag).Overproduction of LipX His was confirmed by SDS-PAGE and lipase activity assays,indicating that the lipX gene indeed encoded afunctional bacterial lipase.Due to the confirmed lipolytic activity of the gene product and its medium-level of sequence identities with known lipase genes,lipX might represent a novel lipase gene.The optimal expression of LipX His was achieved by induction with 0.2mM IPTG at 20°C for 8h after the E.coli BL21(DE3)cell culture optical density reached 0.6at 600nm (Fig.3a).Fig.1The nucleotide and deduced amino acidsequence of lipase LipX from the deep-sea psychrophilic bacterium Psychrobacter sp.C18Purification and characterization of the recombinant LipX His lipaseThe purification of the recombinant lipase LipX His using the His-tag chromatography achieved a 10.7-fold increase of target protein purity (Table 1).The purified LipX His showed a single band with an apparent molecular mass of about 35kDa on SDS-PAGE,consistent with thecalculated LipX His molecular weight (34,477.1Da)(Fig.3b).An active recombinant lipase with a specific activity of 623.3U/mg was obtained (Table 1).Effect of temperature on LipX His lipase activity and stabilityPurified lipase LipX His showed its maximum activity toward p -NPP (C16)at 30°C,when assayed at pH 7.4.Its lipase activity was reduced to 70%of the maximum value at 35°C and to 35%of the maximum activity at 45°C (Fig.4a).The thermostability of the recombinant LipX His was determined by preincubating the enzyme at different temperature (-20,4,20,30,40,55,65and 70°C)for 1h,then the remaining lipase activity was measured at 30°C.The result revealed that the recombinant lipase activity was very stable at temperature from -4to 30°C (Fig.4b).When the temperature reached above 40°C,the lipase activity declined very rapidly.When the lipase was incubated at 65°C for 1h,the lipase activity reduced to 30%of the initial activity,and the enzyme activity was reduced most rapidly in the first 15min.When the enzyme was incubated at 70°C for 15min,the enzyme activity was reduced by more than 65%.Taken together,LipX His possessed a typical property of cold-adaptedlipases.Fig.3a SDS-PAGE analysis of pET28a(?)/lipX induced with different ne 1:whole-cell proteins from E.coli BL21(DE3)without pET28a(?)/lipX ;Lane 2:whole-cell proteins from E.coli BL21(DE3)harboring pET28a(?)/lipX uninduced;Lane 3:induced with 0.1l M IPTG;Lane 4:induced with 0.2l M IPTG;Lane 5:induced with 0.6l M IPTG;Lane 6:protein molecular weight markers.b SDS-PAGE analysis of the purified recombinant lipase LipX His ,Lane 1the target protein eluted by high imidazole buffer,Lane 2:protein molecular weight markersTable 1Purification of LipX His from E.coli BL21(DE3)Step Total activity (U)Total protein (mg)Specific activity (U/mg)Purification (fold)Overall yield (%)Crude extract 4,0056958.04 1.0100Nickel-NTA2,368.53.8623.310.759.1Effects of pH on LipX His lipase activity and stability The purified LipX His was active in the pH range 6.0–11.0,with a maximum activity at pH 8.0(Fig.5a).LipX His was very stable at a pH range of 7.2–10.0for 24h incubation at 4°C (Fig.5b).Effect of NaCl concentration on LipX His lipase activity The purified LipX His lipase activity was increased by NaCl at 1–500mM NaCl concentrations,and the activity was the highest at 10mM NaCl.This result was related to the host strain Psychrobacter sp.C18that was isolated from a marine environment (Table 2).Effect of metal ion on LipX His lipase activityThe purified LipX His lipase activity was partially inhibited by Co 2?,Mn 2?,Fe 3?and EDTA,and strongly inhibited by Zn 2?(Table 3).Other tested metal ions had little or slight enhancement effect on the LipX His lipase activity.Effect of organic solvent on LipX His lipase activityThe organic solvent of 10%ethanol or acetone was found to enhance the purified LipX His lipase activity (Table 4).But at 30or 50%concentration,most of the organic solvents were found to reduce the LipX His lipase activity,with the only exception of 30%glycol that was found to enhance the lipase activity.Acetone,acetonitrile and ethanol at50%pH 3.6–5.6);PBS (pH 5.7–8.0);Tris-HCl (pH 7.19–9.10);glycine-NaOH (pH 8.6–10.6)concentration were found to abolish completely the LipX His lipase activity (Table 4).Effect of detergent on LipX His lipase activityThe tested non-ionic detergents DMSO,Triton X-100,Tween 60and Tween 80were found to increase the LipX His lipase activity (Table 5),while 1%SDS com-pletely inhibited the lipase activity.LipX His lipase substrate specificityThe LipX His lipase substrate specificity assay indicated that long chain fatty acids of p -nitrophenyl esters were better substrates for LipX His ,and the highest lipase activity was achieved with the C14substrate (Fig.6).DiscussionIn this study,a lipase gene,lipX ,was detected and cloned from a psychrophilic deep-sea sediment Psychrobacter sp.strain C18(Dang et al.,2009).The lipX gene was found to be novel based on its highest sequence identity (80.9%)with known GenBank lipase gene sequences.Numerous Psychrobacter strains were found previously to harbor lipase genes,most of which encode cold-adapted lipases (Arpigny et al.1993,1995;Yumoto et al.2003;Kulakova et al.2004;Zeng et al.2004;Zhang et al.2007).Our current study indicates that the recombinant lipX -encoded protein,LipX His ,is also a cold-adapted lipase,with the optimum reaction temperature at 30°C.Our results also indicate that the marine Psychrobacter strains may be an important resource for prospecting novel extreme lipases for various potential applications.The cold-adapted lipase LipX was found to have a calculated molecular weight of 35,028Da.Gene overex-pression of the recombinant LipX His in E .coli BL21(DE3)also produced a recombinant protein with similar apparentTable 3Effect of various metal ions on LipX His lipase activity Metal ions (1mM)or EDTA (1mM)Relative activity (%)None 100.0CoSO 485.3±0.8CuSO 4101.6±1.3MnSO 485.8±2.4FeCl 374.9±1.1FeSO 4103.2±0.9CaCl 2103.1±1.4KCl 109.8±0.5ZnSO 417.4±2.9MgSO 4104.7±1.5EDTA64.8±0.7Data are given as means ±SD,n =3Table 2Effect of different concentrition of NaCl on LipX His lipase activityNaCl Concentration (mM)Relative activity (%)None 100.01108.6±0.95119.3±0.910154.9±1.320120.8±2.050117.2±1.6100114.5±2.1500102.6±1.4Data are given as means ±SD,n =3Table 4Effect of organic solvents on the LipX His lipase activityData are given as means ±SD,n =3Organic solventRelative activity (%)Concentration (10%w/v)Concentration (30%w/v)Concentration (50%w/v)Control 100.0100.0100.0Methanol 101.6±1.380.1±0.833.7±1.6Ethanol 115.2±1.729.2±1.50Glycol49.8±2.3114.9±0.882.7±1.4Acetonitrile 52.5±0.927.3±0.60Acetone109.8±1.228.3±1.1Table 5Effect of various detergents on LipX His lipase activity DetergentsFinal concentrationin reaction mix (%w/v)Relative activity (%)Control –100DMSO 1107.9±1.2TritonX-1001112.8±0.6Tween 601114.8±2.1Tween 801119.5±1.1SDS1Data are given as means ±SD,n =3molecular weight about35kDa.Most of known Psych-robacter lipases have been reported to have a similar molecular weight.For example,the cold-adapted lipase LipA1from the Antarctic deep-sea psychrotrophic Psych-robacter sp.7195was35,210Da(Zhang et al.,2007),the lipase Lip1from the Antarctic facultative psychrophile Psychrobacter immobilis B10was35,288Da(Arpigny et al.,2003),and the lipase Lip3from a Psychrobacter-related strain,the Antarctic psychrotroph Moxaxella TA144,was34,662Da(Feller et al.,1991).Phylogenetic analysis indicates that these cold-adapted lipases may be closely related during gene evolution(Fig.2).Our recombinant cold-adapted lipase LipX His was more stable than the previously reported cold-adapted lipases, such as Lip3from Moraxella TA144(Feller et al.,1991), LipA from the Antarctic deep sea psychrotrophic Pseudo-monas sp.7323(Zhang&Zeng,2008),LipP from the Alaskan psychrotroph Pseudomonas sp.strain B11-1 (Choo et al.,1998),and the lipase from the facultatively psychrophilic Acinetobacter O16(Breuil&Kushner, 1975).The ability of cold-adapted enzymes to catalyze reactions at low or moderate temperature offers a great industrial and biotechnological potential(Gomes&Stei-ner,2004).The cold activity and thermolability might be a key factor to influence the successful application of the cold-adapted lipases.Thus,LipX may be very useful for various applications with the requirement of high catalytic rate at low temperature and resistance to temperature change in a certain range.It has been reported that metal ions and metal ion che-lators affect the lipase activity(Chakraborty and Paulraj 2009).Certain metal ions and EDTA showed inhibitory effect over the recombinant enzyme LipX His and reduced its lipase activity(Table3).However,LipX His also showed very active and stable properties in some of the other tested metal ions.LipX His is active and stable in some of the tested detergents and organic solvents,especially in methanol and ethanol.This indicated that LipX may be useful for biodiesel production and organic synthesis. Biodiesel,defined as monoalkyl fatty acid esters,is pres-ently evaluated as a promising alternative fuel that may have the potential to replace diesel.Currently there are only a few bacterial lipases that have been found to be effective in esterification and biodiesel production,such as the lipases from Burkholderia cepacia(Noureddini et al., 2005)and Pseudomonasfluorescens B68(Luo et al.,2006). LipX from Psychrobacter sp.C18may provide another option for this application.The various properties of LipX may also be explored to offer potential applications and economical benefits in many other microbial applications, such as bioremediation of polluted soils and waters. Acknowledgments The authors thank Hu Zhu for his assistance in the project.This work was supported by China Ocean Mineral Resources R&D Association grants DYXM-115-02-2-20and DYXM-115-02-2-6,Hi-Tech Research and Development Program of China grant2007AA091903,China National Natural Science Foundation grant40576069,Fundamental Research Funds for the Central Uni-versities of China grant09CX05005A,and Foundation of the State Key Laboratory of Heavy Oil Processing,China University of Petroleum grant SKL2010-02.ReferencesAltschul SF,Madden TL,Scha¨ffer AA,Zhang J,Zhang Z,Miller W, Lipman DJ(1997)Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res25:3389–3402Arpigny JL,Feller G,Gerday C(1993)Cloning,sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10.Biochim Biophys Acta117:331–333Arpigny JL,Feller G,Gerday C(1995)Corrigendum to‘‘Cloning, sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10’’.Bio-chim Biophys Acta1263:103Bell PJ,Sunna A,Gibbs MD,Curach NC,Nevalainen H,Bergquist PL(2002)Prospecting for novel lipase genes using PCR.Microbiology148:2283–2291Bradford MM(1976)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem72:248–254 Breuil C,Kushner DJ(1975)Partial purification and characterization of the lipase of a facultatively psychrophilic bacterium(Acine-tobacter O16).Can J Microbiol21:434–441Chakraborty K,Paulraj R(2009)Purification and biochemical characterization of an extracellular lipase from Pseudomonas fluorescens MTCC2421.J Agric Food Chem57:3859–3866。