Research Article Identification, Cloning, and …downloads.hindawi.com/journals/tswj/2014/979858.pdfResearch Article Identification, Cloning, and Expression of L-Amino Acid Oxidase

Research ArticleIdentification Cloning and Expression of L-AminoAcid Oxidase from Marine Pseudoalteromonas sp B3

Zhiliang Yu Ning Zhou Hua Qiao and Juanping Qiu

College of Biological and Environmental Engineering Zhejiang University of Technology Hangzhou 310014 China

Correspondence should be addressed to Zhiliang Yu zhiliangyu76gmailcom

Received 25 August 2013 Accepted 22 October 2013 Published 9 January 2014

Academic Editors P Gyarmati K Hong and F Rodrigues

Copyright copy 2014 Zhiliang Yu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

L-amino acid oxidase (LAAO) is attractingmore attentions due to its broad and important biological functions Recently an LAAO-producing marine microorganism (strain B3) was isolated from the intertidal zone of Dinghai sea area China Physiologicalbiochemical and molecular identifications together with phylogenetic analysis congruously suggested that it belonged to thegenus Pseudoalteromonas Therefore it was designated as Pseudoalteromonas sp B3 Its capability of LAAO production was crosslyconfirmed bymeasuring the products of H

2O2 a-keto acids andNH

4

+ in oxidization reaction Two rounds of PCRwere performedto gain the entire B3-LAAO gene sequence of 1608 bps in length encoding for 535 amino acid residues This deduced amino acidsequence showed 60 kDa of the calculated molecular mass supporting the SDS-PAGE result Like most of flavoproteins B3-LAAOalso contained two conserved typical motifs GG-motif and 120573120572120573-dinucleotide-binding domainmotif On the other hand its uniquesubstrate spectra and sequence information suggested that B3-LAAO was a novel LAAO Our results revealed that it could befunctionally expressed in E coli BL21(DE3) using vectors pET28b(+) and pET20b(+) However compared with the native LAAOthe expression level of the recombinant one was relatively low most probably due to the formation of inclusion bodies Severalsolutions are currently being conducted in our lab to increase its expression level

1 Introduction

L-amino acid oxidase (LAAO EC 1432) is usually dimericflavoprotein containing a noncovalently bound FADmolecule as cofactor for each subunit It is able to catalyzethe stereospecific oxidative deamination of L-amino acidsto the corresponding a-keto acids with release of NH

4

+ andH2O2which is believed to be associated with its biological

activities including inducing apoptosis cytotoxicity edemahemolysis hemorrhage inducing or inhibiting plateletaggregation and parasite-killing and antimicrobial activities[1] When H

2O2is not degraded by catalase it can cause

a decarboxylation of the a-keto acid to the correspondingcarboxylic acid

This enzyme is widely distributed in different sourcesincluding snake venom [2] sea hare [3] insect drugs [4]algae [5] and microorganisms [6 7] So far the LAAOfrom snake venom is the best characterized member of thisenzyme family with respect to not only its toxicology butalso its biochemistry physiology and medicine In contrast

very little is known about LAAO from marine microorgan-ism Although various LAAO-coding sequences have beenpublished to reveal that LAAO family members commonlyhave flavin as coenzyme and possess two conserved andcharacteristic sequence motifs ldquoGGrdquo motif (RxGGRxxST)and dinucleotide-binding (DMB)motif (120573-strand120572-helix120573-strand) [8] only some heterologous expression systems havebeen reported hitherto probably due to the requirement ofposttranslational modification of LAAO In 2003 the firstbacterial heterologous expression system for an LAAO fromRhodococcus opacus was reported The lao-gene from Ropacus was cloned into Escherichia coli and Streptomyceslividans expression vectors It was found that the expressionin E coli resulted in the accumulation of insoluble proteinbut S lividans was a suitable host for the heterologousexpression of LAAO [9] However in 2008 the lao-genefrom Streptococcus oligofermentans was successfully clonedand overexpressed in E coli [10] The LAAO isolated fromsea hare was also functionally expressed in E coli but theexpression level for soluble recombinant LAAOwas relatively

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 979858 8 pageshttpdxdoiorg1011552014979858

2 The Scientific World Journal

low only ca 02mgL culturemedium [11] To our knowledgethe expression of ophidian LAAO has rarely been achievedexcept the successful expression of active recombinant LAAOin the methylotrophic yeast Pichia pastoris [12] To date theheterologous expression of LAAO is still a big challenge

In this study a yellow-pigmented LAAO-producingmarine bacterial strain designated as Pseudoalteromonassp B3 on the basis of the physiological biochemical andmolecular analysis was isolated After retrieval of codingsequence LAAO from Pseudoalteromonas sp B3 (B3-LAAO)was cloned and functionally expressed in E coli strainBL21 (DE3) This communication laid the foundation forfurther studies on enzymatic properties structure biologicalfunction and application of B3-LAAO

2 Materials and Methods

21 Sample Collection and Colony Isolation The intertidalsediment samples were obtained from Dinghai sea areaZhoushan China (3003∘N 12211∘E) Each sample was col-lected at 50 to 100 cm depth below the sea surface Thesampleswere placed in special presterilized plastic bottles andbrought to the lab in aseptic condition After serial dilution(up to 10minus6 dilution) using sterilized sea water 100 120583L of eachdiluted sample was plated on PDA (potato 200 gL sucrose10 gL sea salt 30 gL 20 gL agar) Gausersquos NO 1 (solublestarch 20 gL NaCl 05 gL K

2HPO405 gL FeSO

4001 gL

MgSO4sdot7H2O05 gL KNO

31 gL sea salt 30 gL 20 gL agar

pH 72ndash74) and MM medium (yeast extract 3 gL peptone5 gL sea salt 30 gL 20 gL agar pH 72ndash74) and separatelyincubated at either 28∘C or 25∘C for 2ndash7 days as necessary Tomake broth medium agar was absent The isolated colonieswere purified by streak-plate technique Then each pureisolate was cultured in 50mL of broth medium and culturesupernatant was collected after centrifuge at 8000 rpm for10min at 4∘C for subsequent LAAO activity screening

22 Screening of LAAO-Producing Microorganism LAAOactivity produced by isolate was detected by measuring itsoxidization productions of L-amino acids including H

2O2 a-

keto acid and NH4

+ (1) H2O2measurement 15mL of resul-

tant culture supernatant was mixed with 15mL of 10mM L-Leu for oxidization reaction at 37∘C for 30min Then theproduced H

2O2was measured using Amplex Red Hydrogen

PeroxidePeroxidase Assay kit (Invitrogen USA) accordingto manufacturersquos instruction (2) a-Keto acid measurement15mLof culture supernatantwasmixedwith 15mLof 10mML-Leu for oxidization reaction at 37∘C for 30min Then asreported [13] the produced a-keto leucine was detected using24-dinitrophenylhydrazine (DNP) which can react with car-bonyl group to generate brown-red dinitrophenylhydrazoneBriefly 550 120583L of detection solution was mixed with 450120583Lof 20 trichloroacetic acid and kept at room temperaturefor 30min Next 200120583L of 20mM DNP was added andthe mixture was incubated at room temperature for 15minThe reaction was terminated by adding 4mL of 08MNaOHFinally the mixture was centrifuged at 12000 rpm for 10minand the absorbance of the supernatant was measured at

520 nm (3) NH4

+ detection 25mL of resultant culturesupernatant wasmixedwith 25mLof 10mML-Leu in 250mLglass flask for oxidization reaction at 37∘C for 30min Then1mL of 1MNaOHwas added and paper pH indicator was putabove the glass flask After shaking at 120 rpm for 60min atroom temperature the color change of paper pH indicatordue to the NH

3release was observed

23 Molecular Taxonomy and Phylogenetic Analysis The ge-nomic DNA of LAAO-producing microorganism wasextracted by bacterial DNA isolation method [14] 16S rDNAgene was amplified in 50 120583L containing 37 120583L of ddH

2O

5 120583L of 10timesEasy Taq buffer 4 120583L of 25mM dNTPs 100 nMforward primer 27F (51015840-GAGTTTGATCCTGGCTCAG-31015840)100 nM reverse primer 1527R (51015840-AGAAAGGAGGTGATC-CAGCC-31015840) 1 ng genomic DNA and 1U Taq DNApolymerase with denaturation at 94∘C for 5min followed by30 cycles of 1min at 94∘C 50 s at 55∘C 90 s at 72∘C and a final10min extension at 72∘C At the end of reaction PCR productwas cooled to 4∘C to await further use After size confirmationon 10 agarose gel the PCR product was sent to SangonBiotech (Shanghai) Co Ltd for sequencing of 16S rDNAThesimilarity and homology of 16S rDNA gene sequence wasanalyzed using BLAST search available in genbank of NCBIThe DNA sequences were aligned and phylogenetic tree wasconstructed by neighbor joining method with bootstraptrials 1000 using MegaV402 software

24 Physiological and Biochemical Characterization Theability of the isolate to utilize various carbon and nitrogensources and other physiological and biochemical propertieswas studied according to the recommendation in ldquoTheManual of Systematic Methods of Determinative Bacterialrdquo

25 Retrieval of LAAOGene To retrieve the full length of lao-gene sequence from isolated microbial producer two roundsof PCRwere performed For the first round PCR the 6 degen-erate primers were designed based on the conserved regionsafter alignment of LAAO sequences from general LAAO-producing marine microorganisms (see Table 1) includingComamonas testosteroni KF-1 (ZP 03541004) Dinoroseobac-ter shibae DFL 12 (ABV95616) and Caulobacter sp K31(YP 001683007) and LAAO-producing Pseudoalteromonasmicroorganisms (see Table 2) including Pseudoalteromonashaloplanktis TAC125 (YP 339251) Pseudoalteromonastunicata D2 (ZP 01132853) Pseudoalteromonas atlanticaT6c (YP 662716) and Pseudoalteromonas sp SM9913(YP 004069407) ZZ-1 ZZ-2 and ZZ-3 were forwardprimers and ZF-1 ZF-2 and ZF-3 are reverse ones Asrequired these 6 degenerate primers combined with eachother to form 9 sets of forward and reverse primer pairs PCRamplification was performed with genomic DNA of LAAO-producer as template and PrimeSTAR HS DNA polymerase(TaKaRa Dalian China) as polymerase The Touch-downPCR consisted of denaturation at 94∘C for 5min 15 cycleof 30 s at 94∘C 30 s at 64∘C (minus1∘Ccycle) and 1min at 72∘Cfollowed by another 20 cycles of 30 s at 94∘C 30 s at 48∘C and1min at 72∘C and a final 10min extension at 72∘C After size

The Scientific World Journal 3

Table 1 Conserved regions of LAAOs from common marine microorganisms used for design of degenerate primers

Item Accession no Sequences ofa

Region 1 Region 2Comamonas testosteroni KF-1 ZP 03541004 58VLGAGLAGM66 333ADWCVCTIP341Dinoroseobacter shibae DFL 12 ABV95616 59VLGAGLAGM67 337ADWCVCTIP345Caulobacter sp K31 YP 001683007 57VLGAGLAGM65 331ADWCVCTIP345Degenerate primer ZZ-1 51015840-GTGCTSGGCGCKGGYCT-31015840 ZF-1 51015840-GTGCARACGCASYAGTCG-31015840aBoldface letters indicate the specific amino acids actually used in the design of the degenerate primers (forward primer ZZ-1 reverse primer ZF-1)

Table 2 Conserved regions of LAAOs from Pseudoalteromonasmicroorganisms used for design of degenerate primers


Region 1 Region 2 Region 3Pseudoalteromonashaloplanktis TAC125 YP 339251 13IIGSGAAAGLS22 225AMAWRAGC232 359MTDFNGKTDL368

Pseudoalteromonastunicata D2 ZP 01132853 13IIGSGAAAGLS22 224AMAWRAGC231 357MTDFNGKTDL366

Pseudoalteromonasatlantica T6c YP 662716 13IIGSGAAAGLS22 222AMAWRAGC230 359VTDFNAKTDL368

Pseudoalteromonassp SM9913 YP 004069407 13IIGSGAAAGLS22 225AMAWRAGC232 359MTDFNGKTDL368

Degenerate primerbZZ-2 51015840-

ATTATHGGYAGCGGCGC-31015840

ZZ-351015840-GCHATGGCDTGGCGTGC-31015840

ZF-251015840-CCHGCACGCCAHGCCAT-31015840

ZF-3 51015840-TDSCATTAAAGTCRGTC-31015840

aBoldface letters indicate the specific amino acids actually used in the design of the degenerate primers (forward primers ZZ-2 and ZZ-3 reverse primers ZF-2 and ZF-3)

Table 3 Primers used in cloning of 51015840 and 31015840 flanked sequences of LAAO gene from Pseudoalteromonas sp B3

Primerslowast Sequences (51015840-31015840) Size (bp)Set A

5F-1 TTGCCAAAGACAGAGCCAGGCT 225F-2 AGGTCGCCCTTACTGAGCACTGT 235F-3 TCTTATCAAACACCGCAGCAAT 22

Set B3Z-1 ATTGCCATGGCTTGGCGTG 183Z-2 GTCAAATCCAGACGTTTCCAGT 223Z-3 GCGGTGCCAGTAAAGTGTATCT 22

Set CS-1 ACGATGGACTCCAGAGCGGCCCGCVNVNNNGGAA 34S-2 ACGATGGACTCCAGAGCGGCCCGCBNBNNNGGTT 34S-3 ACGATGGACTCCAGAGCGGCCCGCHNVNNNCCAC 34S-4 ACGATGGACTCCAGAGCGGCCCGCVVNVNNNCCAA 35S-5 ACGATGGACTCCAGAGCGGCCCGCBDNBNNNCGGT 35

B0043 NNNNNN 6B0043-9 NNNNNNNNN 9B0043-10 NNNNNNNNNN 10lowastPrimers in set A are one set of nested specific primers for amplification of 51015840 flanked region of B3-LAAO gene primers in set B are one set of nested primersfor amplification of 31015840 flanked region of B3-LAAO gene primers in set C are arbitrary primers for amplification of 51015840 and 31015840 flanked regions of B3-LAAO gene

confirmation on 1 agarose gel and gel extraction (QiagenCA USA) the PCR product with expected size was clonedinto pMD19-T simple vector (TaKaRa Dalian China) andsequenced by Sangon Biotech (Shanghai) Co Ltd

To obtain the entire lao-gene the second round of inversePCR (using nested-PCR technique) was employed to amplify

the flanked gene sequences (51015840 and 31015840 regions) As shownin Table 3 two nested specific primer sets 5F-1 (1st) 5F-2(2nd) and 5F-3 (3rd) for 51015840 flanked region and 3Z-1 (1st) 3Z-2 (2nd) and 3Z-3 (3rd) for 31015840 flanked region were designedbased on the intermediated lao-gene sequence obtained in thefirst round of PCR Nine arbitrary primers (S-1 S-2 S-3 S-4


S-5 S-6 B0043 B0043-9 and B0043-10) (see Table 3) werepurchased from Invitrogen (USA) and each arbitrary primerwas used to pair with nested specific primer to form forwardand reverse primer set For either 51015840 or 31015840 flanked region oflao-gene a total of 27 nested-PCR amplification reactionswere performed with denaturation at 94∘C for 5min 6 cyclesof 30 s at 94∘C 50 s at 35∘C and 2min at 72∘C followed byanother 30 cycles of 60 s at 94∘C 50 s at 55∘C and 2minat 72∘C and a final 10min extension at 72∘C The desiredPCR product was purified using a gel extraction kit (QiagenCA USA) and cloned into pMD19-T simple vector (TaKaRaDalian China) After sequencing by Sangon Biotech (Shang-hai) Co Ltd the contigs of 51015840 flanked fragment intermediatedfragment and 31015840 flanked fragment were assembled usingldquoCap Conting Assemblyrdquo of BioEdit software V52

26 Cloning and Expression of LAAO in E coli BL21(DE3)The complete lao-gene was amplified using a pair offorward primer (B3laaoF 51015840-CGCGGATCCTATGAAAGA-ACAAGTTC-31015840 with a BamH I restriction site as under-lined) and reverse primer (B3laaoR 51015840-CCCAAGCTTACG-TTTGATTTTACTGG-31015840 with a Hind III restriction site)Pfu DNA polymerase (Promega) was used in a PCR toamplify the 1608 bps lao-geneThe desired PCR product wassubsequently cloned into pMD19-T simple vector and thelao-gene sequence was confirmed by BamH I and Hind IIIdigestion and direct DNA sequencing After BamH I-HindIII digestion the released lao-gene was subsequently clonedinto the BamH I-Hind III restriction sites of expressionvectors pET-28b(+) and pET-20b(+) (Novagen USA) togenerate pET-28b(+)-lao and pET-20b(+)-lao respectivelyThe inserted gene was confirmed by DNA sequencing (San-gon Biotech (Shanghai) Co Ltd)The plasmids pET-28b(+)-lao and pET-20b(+)-lao inserted with a PCR-amplified lao-gene was separately transformed into E coli BL21(DE3)(Novagen) cells and cultured in LB medium supplementedwith 50 120583gmL kanamycin and 100 120583gmL ampicillin Cellswere grown at 37∘C to anOD

600of 04 to 06 Overproduction

of the LAAO protein was induced by addition of 1mMisopropyl-120573-D-thiogalactopyranoside (IPTG) and 01mMFAD together with 01mM Zn2+ at 25∘C for 6 h

After expression the cells from 15mL of culture wereharvested by a centrifugation at 6000 rpm for 5min at 4∘CThen the cell pellets were resuspended in 100120583L of ddH

2O

and mixed with 25 120583L of 4-fold sample loading buffer (10MTris-HCl pH 68 10 SDS 20 120573-mercaptoethanol 50glycerol 1 bromophenol blue) After boiling for 5min30 120583L of resultant sample mixture was subjected to SDS-polyacrylamide gel (SDS-PAGE)with a stacking gel of 5 andseparation gel of 12 as described [15] and the number andmolecular weight of the expressed LAAO were determined

27 Assay of the Expressed LAAO Activity The expressedLAAO activity was determined by measuring the producedH2O2with Prussian blue agar assay [16] In brief 25mL of

the induced cell culture was mixed with 25mL of 10mM L-Leu for oxidation reaction at 37∘C with shaking at 120 rpmAfter incubation for 1 h 50120583L of resultant oxidation mixture

Table 4 Detection of the H2O2 production using Amplex RedHydrogen PeroxidePeroxidase Assay kit

Reactions Amount of H2O2 (mM) Relative activity ()Test 1a 184 100Test 2b 014 761Test 3c 0043 234aCulture supernatant of strain B3 with substrate L-Leu bculture supernatantof strain B3 without substrate L-Leu csame as Test 1 except that the culturesupernatant of strain B3 was denatured with boiling before addition ofsubstrate L-Leu

was subjected to the circular well with diameter of 6mmon Prussian blue agar (10 gL FeCl

3sdot6H2O 10 gL potassium

hexacyanoferrate (III) 2 agar pH 75) After incubation for30min at room temperature the color development resultedfrom Prussian blue formation due to the H

2O2production

was monitored

3 Results and Discussion

31 Isolation of LAAO-Producing Strain Based on morpho-logical identification a total of 157 pure isolates 32 from PDAmedium 51 from Gausersquos NO 1 medium and 74 from MMmedium were obtained Out of those 157 isolates subjected toLAAO-producing screening only one isolate (strain B3) gavethe capability to produce LAAO as determined and verifiedbelow

First LAAOs are able to catalyze L-amino acids to releaseH2O2which can be detected using Amplex Red Hydrogen

PeroxidePeroxidase Assay kit As shown in Table 4 almostno H2O2was detected from either the culture supernatant

of strain B3 without L-Leu (Test 2) or the denatured culturesupernatant by boiling for 10 minutes followed by additionof L-Leu (Test 3) In contrast decent amount of H

2O2

(184mM) was measured from the culture supernatant ofstrain B3 with L-Leu (Test 1) meaning that the culturesupernatant of strain B3 can use L-Leu to release H

2O2 All

these findings indicate that strain B3 may bear the abilityto produce LAAO Second as carbonyl derivative a-ketoacid can react with 24-dinitrophenylhydrazine (DNP) togenerate brown-red dinitrophenylhydrazone with maximumabsorbance at 520 nm [13] As shown in Figure 1 the culturesupernatant of strain B3without L-Leu gave flaxen andOD

520

was low displaying the similar result as the denatured culturesupernatant of strain B3 with L-Leu In contrast both the a-keto leucine (positive control) and the oxidation solution ofL-Leu by the culture supernatant of strain B3 had brown-redcolor with high OD

520value All these findings indicate that

L-Leu can be oxidized by culture supernatant of strain B3to generate the corresponding a-keto acid Therefore strainB3 can produce LAAO Third in the presence of strong baselike NaOH NH

4

+ in solution will become unstable to releaseNH3which can easily be detected by paper pH indicator

The results in Figure 2 showed that after addition of NaOHthe culture supernatant of strain B3 without L-Leu (negativecontrol) did not cause the color change of the paper pHindicatormaintaining the original yellow In contrast like the


1 2 3 4

Figure 1 Detection of a-keto acid production using 24-dinitrophenylhydrazine (DNP) [13] A positive control 120572-ketoleucine in MM medium (OD520 = 0232) B culture supernatantof strain B3 with substrate L-Leu (OD520 = 0198) C culturesupernatant of strain B3 without substrate L-Leu (OD520 = 0032)D boiling-denatured culture supernatant of strain B3 with substrateL-Leu (OD520 = 0045)

controlcontrolPositiveTest Negative

Figure 2 Determination of the presence of NH4

+ after additionof NaOH using paper pH indicator Test culture supernatant ofstrain B3 with substrate L-Leu positive control ammonia solutionnegative control culture supernatant of strain B3 without substrateL-Leu

positive control (ammonia solution) the oxidization solutionof L-Leu by the culture supernatant of strain B3 caused thecolor change of paper pH indicator from original yellow toabsinthe-green All these findings indicate that L-Leu can beused by the culture supernatant of strain B3 to release NH

4

+supporting that strain B3 can produce LAAO

Our quantitative and qualitative measurements togetherclearly showed that the culture supernatant of strain B3 canspecifically oxidize L-Leu to yield H

2O2 a-keto leucine and

NH4

+ thus crossly supporting that the isolate B3 can produceLAAO Among these three detection methods H

2O2assay

kit and DNP assay are quantitative methods with very highsensitivity whereas paper pH indicator can only be used forqualitative measurement with low sensitivity On the otherhand compared with the former two methods the latter onegives advantages including convenience no requirement ofinstrument and cost-effectiveness thus ideal for the first-stepscreening of LAAO-producing microorganisms

32 Brief Characterization of Strain B3 A pair of primers27F and 1527R were successfully used to amplify about 15 kb

16S rDNA gene from strain B3 After sequencing a total of1396 bp were gained The Blast analysis of 16S rDNA at theNCBIwebsite togetherwith the constructed phylogenetic tree(see Figure 3) indicated that strain B3 was very proximal toPseudoalteromonas spp with the highest identity of 985 toPseudoalteromonas viridis On MM medium plate strain B3exhibited yellow smooth round The Gram-staining reactionshowed that it was Gram-negative (photo not shown) Thephysiological and biochemical analysis revealed that it waspositive to starch hydrolysis indole test citrate utilizationand oxidase test and negative to lysine decarboxylationenzyme half a solid agar gelatin hydrolysis H

2S produc-

tion acetamide V-P test methyl red test and melezitosemonohydrate In addition it can consume galactose glucoselactose fructose maltose and rhamnose as carbon sourceand (NH

4)2HPO4 KNO

3 arginine methionine glycine and

tyrosine as nitrogen sources for growth Therefore it wasdesignated as Pseudoalteromonas sp B3

33 Retrieval of lao Gene and Characterization of the DeducedAmino Acid Sequence Among 9 sets of primer pairs onlyone pair from ZZ-2 and ZF-2 gave the desired PCR-amplifiedproduct with around 650 bps in size in the first round of PCRAfter sequencing 654 bps of intermediated LAAO-codingsequence from Pseudoalteromonas sp B3 was obtained toshow 80 identity to L-aspartate oxidase-coding gene fromPseudoalteromonas sp SM9913 In the second round ofinverse PCR only arbitrary primer S-2 was combined withone set of nested specific primers (5F-1 5F-2 and 5F-3)to amplify the desired 51015840 flanked region Similarly onlyarbitrary primer BOO43 was successfully used to pair withthe other set of nested specific primers (3Z-1 3Z-2 and 3Z-3) to amplify the 31015840 flanked region After sequence assemblya full B3-LAAO-coding sequence with 1608 bps in lengthwas obtained This open reading frame (ORF) encodes 535amino acid (AA) residues with a predicted molecular massof 5957146Da (60 kDa) A BLASTN search of the ORFnucleotide sequence at the NCBI website showed that itshared 75 and 73 identity to L-aspartate oxidase fromPseudoalteromonas sp SM9913 and Shewanella denitrificansOS217 respectively According to the deduced amino acidsequence further BLASTP search at the NCBI websiterevealed that B3-LAAO had the highest homology witha number of L-aspartate oxidases giving 80 80 8073 and 70 identity to L-aspartate oxidases from Pseu-doalteromonas sp SM9913 Pseudoalteromonas haloplank-tis ANT505 Pseudoalteromonas tunicata D2 Alishewanellajeotgali KCTC 22429 and Shewanella denitrificans OS217respectively So far most of LAAOs found in Pseudoal-teromonas genus have a strict preference for specific substratefor example L-aspartate oxidase from Pseudoalteromonassp SM9913 Pseudoalteromonas haloplanktis ANT505 andPseudoalteromonas tunicata D2 and L-lysine oxidase fromPseudoalteromonas tunicate D2 [17] In contrast B3-LAAOexhibited a broad substrate spectra with the highest activityto L-Leu followed by L-Lys L-Tys L-Asn L-Gln L-Met L-cystine L-Arg L-Trp and L-Glu but no detectable activityto L-aspartic acid (data not shown) So that B3-LAAO


B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29

Pseudoalteromonas haloplanktis (X67024)Marine bacterium LMG1 (AY082666)Pseudoalteromonas viridis G-364 (AB231329)

QD1-2 (AY626830)Pseudoalteromonas viridis G-1387 (AB231330)Pseudoalteromonas viridis C22c (HQ439520)

1020R (AB373122)Pseudoalteromonas viridis P1 (EU240534)

Pseudoalteromonas rubra G31a (HQ439547)Pseudoalteromonas rubra PC13d (HQ439541)Pseudoalteromonas rubra PC13b (HQ439548)

Pseudoalteromonas sp


Figure 3 Phylogenetic tree of strain B3 based on 16S rDNA The numbers at the nodes indicate the bootstrap level based on the analysis of1000 resampled data sets

10

10

10

10

10

10

Consensus

Pseudoalteromonas haloplanktis ANT505Pseudoalteromonas tunicata D2

Alishewanella jeotgali KCTC 22429

Shewanella denitrificans OS217

Pseudoalteromonas sp B3Pseudoalteromonas sp SM9913

Figure 4 Sequence alignment between B3-LAAO and several homologous LAAOs The highly conserved motifs dinucleotide-bindingdomain motif ohhhhGxGxxGxxxhxxL for FAD binding where o stands for a polar or charged residue and h for a hydrophobic residueand characteristic motif RxGG are indicated

6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)

Figure 5 Maps of the recombinant plasmids containing lao-gene pET28b(+)-lao (a) and pET20b(+)-lao (b)The LAAO-encoding gene wascloned between BamH I and Hind III restriction sites

could represent a novel LAAO which can certainly broadenour view on LAAO from Pseudoalteromonas genus Basedon the alignment of amino acid sequences of 6 homol-ogous LAAOs (see Figure 4) B3-LAAO gave two highlyconserved motifs One is the dinucleotide-binding motifohhhhGxGxxGxxxhxxL which is a typical FAD binding sitewhere o stands for a polar or charged residue and h for ahydrophobic residue the other is the characteristic sequencemotif RxGG These motifs appear in several families of theflavoproteins [1 8]

34 Expression of B3-LAAO in E coli BL2 (DE3) Attemptsto express B3-LAAO in E coli were made and two differ-ent expression vectors pET-28b(+)-lao and pET-20b(+)-lao

were constructed for this purpose (see Figure 5) The lao-gene was cloned into pET28b(+) and pET20b(+) respec-tively and both were separately transformed into E coliBL21(DE3) strain and expressed as described in Section 2PCR amplification restriction endonuclease digestion anddirect sequencing analysis demonstrated that plasmids ofpET28b(+)-lao and pET20b(+)-lao were successfully con-structed (data not shown) SDS-PAGE analysis (see Figure 6)showed that the recombinant B3-LAAOs on the two differentvectors both can be induced and expressed to give the samemolecular mass of about 60 kDa agreeing with the calculatedone based on the deduced amino acid sequence

The activity of the expressed recombinant LAAO wasdetermined with Prussian blue agar assay [16] using L-Leu


RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa

Figure 6 SDS-PAGE analysis of the expressed recombinant B3-LAAO in E coli BL21(DE3) Lane M prestained protein marker(Thermo USA) lane 1 cell culture of E coli BL21(DE3)pET28b(+)-lao with a 6 h-induction by IPTG lane 2 cell culture of E coliBL21(DE3)pET28b(+)-lao without induction lane 3 cell cultureof E coli BL21(DE3)pET20b(+)-lao with a 6 h-induction by IPTGLane 4 cell culture of E coli BL21(DE3)pET20b(+)-lao withoutinduction

as substrate The results in Figure 7 indicated that afterinduction by IPTG the recombinant B3-LAAOs in culturesupernatants from E coli BL21(DE3) transformed with bothpET28b(+)-lao and pET20b(+)-lao can cause the Prussianblue agar to form blue halos which resulted from the H

2O2

production while no LAAO activity could be detected inall tested samples from the sonicated cell pellets (data notshown) In fact accumulation of inclusion bodies was foundin cell pellets These inclusion bodies could be dissolved in8M urea but the enzyme activity could not be restored byslowly diluting the solution All these findings indicate thatB3-LAAO could functionally be expressed in E coli butthe expressed level was relatively low The following reasonsmay account for this phenomenon First H

2O2produced by

LAAOactivity is toxic toE coli [18] and thus this would likelyinhibit the LAAO expression level second the recombinantLAAO fromE colimay bemuch less active than thewild-typeone third E coli may be a relatively unsuitable host for theheterologous production of B3-LAAO Several ways may givesolutions to these commonproblems First other hosts can beused to replace E coli for heterologous expression of LAAO[9] Second coexpression of chaperone protein may preventthe aggregation of the recombinant LAAO in E coli [19]Third the expression conditions can be varied consideringthe inductor concentration time of induction inductiontemperature and duration to reduce the amount of insolublefractions Last but not least inclusion bodies can be dissolvedinto urea for restoration of its activity through dialysis

It has been found that the change in diameter of blue haloon Prussian blue agar is a function of H

2O2concentration

with exponential fit [16] According to the size of halosFigure 7 further showed that E coli BL21(DE3)pET20b(+)-lao with a signal peptide had the higher activity than Ecoli BL21(DE3)pET28b(+)-lao without signal peptide Mostprobably secretion of recombinant LAAO can help to reducethe harm of intracellular active LAAO to cells

4 Conclusion

In the present study an LAAO-producing marine bacteriumB3 was successfully isolated from the intertidal sea area16S rDNA sequence and phylogenetic tree analysis togetherwith physiological and biochemical assays revealed that itcan be designated as Pseudoalteromonas sp B3 Its capabilityof LAAO production was demonstrated by determining therelease of H

2O2 120572-keto leucine and NH

4

+ after oxidizationof L-Leu Through two rounds of PCR entire LAAO-codinggene sequence from Pseudoalteromonas sp B3 was obtainedIts deduced amino acid sequence of 535 residues showed twoconserved motifs GG-motif and 120573120572120573-dinucleotide-bindingdomain motif typical to a number of flavoproteins B3-LAAO had the highest identity of only around 80 toseveral L-aspartate oxidases from different microorganismsIn addition it had high activities to broad L-amino acids butnot to L-aspartic acidTherefore B3-LAAOprobably presentsa novel LAAO which can enrich our knowledge on LAAOfrom Pseudoalteromonas genus

To date heterologous expression of LAAO is still a bigchallenge due to its toxicity to host and the requirement ofposttranslational modification In this study B3-LAAO wassuccessfully and functionally expressed in E coli BL21(DE3)using two different vectors pET28b(+) and pET20b(+) It wasfound that addition of suitable concentration FAD and Zn2+during IPTG induction can increase the activity of recombi-nant B3-LAAO Besides lower induction temperature (25∘C)was better for B3-LAAO expression than higher inductiontemperature (37∘C) Most probably low temperature canreduce the aggregation of B3-LAAO In future further lowerinduction temperature (16∘C) is highly promising No activeintracellular LAAO activity was detected but huge inclusionbodies were formed in cell which is common in heterologousexpression of LAAO [9 11] Overall the expression levelof active B3-LAAO was relatively low We believe that thislevel is only for soluble LAAO while much of the LAAO ispresent in insoluble inclusion bodies Therefore in futurethe expression conditions will be optimized consideringthe inductor concentration time of induction inductiontemperature and duration In addition the inclusion bodieswill be dissolved into urea for its activity restoration Finallycoexpression of chaperone protein with B3-LAAO in E coliis also being performed in our lab

Conflict of Interests

The authors have declared that no conflict of interests exists


12

34

Figure 7 Measurement of the expressed B3-LAAO activity using Prussian blue agar assay [16] Lane 1 E coli BL21(DE3)pET28b(+)-laowithout induction lane 2 E coliBL21(DE3)pET28b(+)-laowith a 6 h-induction lane 3 E coliBL21(DE3)pET20b(+)-laowithout inductionlane 4 E coli BL21(DE3)pET20b(+)-lao with a 6 h-induction

Acknowledgments

This work was supported by the Natural Science Foundationof Zhejiang Province China (Y5100153) the Science andTechnology Planning Project of Zhejiang Province China(Welfare Technology Applied Research Project of ZhejiangProvince Grant no 2011C23007) and the Natural ScienceFoundation of ZJUT (20100213) to Zhiliang Yu

References

[1] Z Yu andHQiao ldquoAdvances in non-snake venom l-amino acidoxidaserdquo Applied Biochemistry and Biotechnology vol 167 no 1pp 1ndash13 2012

[2] Y Sakurai H Takatsuka A Yoshioka et al ldquoInhibition ofhuman platelet aggregation by L-amino acid oxidase purifiedfrom Naja naja kaouthia venomrdquo Toxicon vol 39 no 12 pp1827ndash1833 2001

[3] R Iijima J Kisugi and M Yamazaki ldquoA novel antimicrobialpeptide from the sea hare Dolabella auriculariardquoDevelopmentaland Comparative Immunology vol 27 no 4 pp 305ndash311 2003

[4] M Y Ahn K S Ryu Y W Lee and Y S Kim ldquoCytotoxicityand L-Amino Acid Oxidase Activity of Crude Insect DrugsrdquoArchives of Pharmacal Research vol 23 no 5 pp 477ndash481 2000

[5] O Vallon L Bulte R Kuras J Olive and F-A WollmanldquoExtensive accumulation of an extracellular L-amino-acid oxi-dase during gametogenesis of Chlamydomonas reinhardtiirdquoEuropean Journal of Biochemistry vol 215 no 2 pp 351ndash3601993

[6] G M Brearley C P Price T Atkinson and P M HammondldquoPurification and partial characterisation of a broad-range L-amino acid oxidase from Bacillus carotarum 2Pfa isolated fromsoilrdquo Applied Microbiology and Biotechnology vol 41 no 6 pp670ndash676 1994

[7] Y-L Huang M Li Z Yu and P-Y Qian ldquoCorrelation betweenpigmentation and larval settlement deterrence by Pseudoal-teromonas sp sf57rdquo Biofouling vol 27 no 3 pp 287ndash293 2011

[8] O Vallon ldquoNew sequence motifs in flavoproteins evidence forcommon ancestry and tools to predict structurerdquo Proteins vol38 no 1 pp 95ndash114 2000

[9] B Geueke and W Hummel ldquoHeterologous expression ofRhodococcus opacus L-amino acid oxidase in Streptomyceslividansrdquo Protein Expression and Purification vol 28 no 2 pp303ndash309 2003

[10] H Tong W Chen W Shi F Qi and X Dong ldquoSO-LAAO anovel L-amino acid oxidase that enables Streptococcus oligofer-mentans to outcompete Streptococcus mutans by generatingH2O2from peptonerdquo Journal of Bacteriology vol 190 no 13 pp

4716ndash4721 2008

[11] H Yang P M Johnson K-C Ko et al ldquoCloning charac-terization and expression of escapin a broadly antimicrobialFAD-containing L-amino acid oxidase from ink of the sea hareAplysia californicardquo Journal of Experimental Biology vol 208no 18 pp 3609ndash3622 2005

[12] P R Kommoju P Macheroux and S Ghisla ldquoMolecularcloning expression and purification of l-amino acid oxidasefrom the Malayan pit viper Calloselasma rhodostomardquo ProteinExpression and Purification vol 52 no 1 pp 89ndash95 2007

[13] S Singh B K Gogoi and R L Bezbaruah ldquoOptimization ofmedium and cultivation conditions for L-amino acid oxidaseproduction by Aspergillus fumigatusrdquo Canadian Journal ofMicrobiology vol 55 no 9 pp 1096ndash1102 2009

[14] G L Smith S S Socransky and C M Smith ldquoRapid methodfor the purification of DNA from subgingival microorganismsrdquoOralMicrobiology and Immunology vol 4 no 1 pp 47ndash51 1989

[15] D Kang Y S Gho M Suh and C Kang ldquoHighly sensitive andfast protein detection with Coomassie brilliant blue in sodiumdodecyl sulfate-polyacrylamide gel electrophoresisrdquo Bulletin ofthe Korean Chemical Society vol 23 no 11 pp 1511ndash1512 2002

[16] Z Yu N Zhou C Zhao and J Qiu ldquoIn-gel determinationof L-amino acid oxidase activity based on the visualization ofPrussian blue-forming reactionrdquoPLoSONE vol 8 no 2 ArticleID e55548 2013

[17] S G James C Holmstrom and S Kjelleberg ldquoPurificationand characterization of a novel antibacterial protein from themarine bacterium D2rdquo Applied and Environmental Microbiol-ogy vol 62 no 8 pp 2783ndash2788 1996

[18] T Ehara S Kitajima N Kanzawa T Tamiya and T TsuchiyaldquoAntimicrobial action of achacin is mediated by L-amino acidoxidase activityrdquo FEBS Letters vol 531 no 3 pp 509ndash512 2002

[19] T Nishizawa C C Aldrich and D H Sherman ldquoMolecu-lar analysis of the rebeccamycin L-amino acid oxidase fromLechevalieria aerocolonigenes ATCC 39243rdquo Journal of Bacte-riology vol 187 no 6 pp 2084ndash2092 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


low only ca 02mgL culturemedium [11] To our knowledgethe expression of ophidian LAAO has rarely been achievedexcept the successful expression of active recombinant LAAOin the methylotrophic yeast Pichia pastoris [12] To date theheterologous expression of LAAO is still a big challenge

In this study a yellow-pigmented LAAO-producingmarine bacterial strain designated as Pseudoalteromonassp B3 on the basis of the physiological biochemical andmolecular analysis was isolated After retrieval of codingsequence LAAO from Pseudoalteromonas sp B3 (B3-LAAO)was cloned and functionally expressed in E coli strainBL21 (DE3) This communication laid the foundation forfurther studies on enzymatic properties structure biologicalfunction and application of B3-LAAO

2 Materials and Methods

21 Sample Collection and Colony Isolation The intertidalsediment samples were obtained from Dinghai sea areaZhoushan China (3003∘N 12211∘E) Each sample was col-lected at 50 to 100 cm depth below the sea surface Thesampleswere placed in special presterilized plastic bottles andbrought to the lab in aseptic condition After serial dilution(up to 10minus6 dilution) using sterilized sea water 100 120583L of eachdiluted sample was plated on PDA (potato 200 gL sucrose10 gL sea salt 30 gL 20 gL agar) Gausersquos NO 1 (solublestarch 20 gL NaCl 05 gL K

2HPO405 gL FeSO

4001 gL

MgSO4sdot7H2O05 gL KNO

31 gL sea salt 30 gL 20 gL agar

pH 72ndash74) and MM medium (yeast extract 3 gL peptone5 gL sea salt 30 gL 20 gL agar pH 72ndash74) and separatelyincubated at either 28∘C or 25∘C for 2ndash7 days as necessary Tomake broth medium agar was absent The isolated colonieswere purified by streak-plate technique Then each pureisolate was cultured in 50mL of broth medium and culturesupernatant was collected after centrifuge at 8000 rpm for10min at 4∘C for subsequent LAAO activity screening

22 Screening of LAAO-Producing Microorganism LAAOactivity produced by isolate was detected by measuring itsoxidization productions of L-amino acids including H

2O2 a-

keto acid and NH4

+ (1) H2O2measurement 15mL of resul-

tant culture supernatant was mixed with 15mL of 10mM L-Leu for oxidization reaction at 37∘C for 30min Then theproduced H

2O2was measured using Amplex Red Hydrogen

PeroxidePeroxidase Assay kit (Invitrogen USA) accordingto manufacturersquos instruction (2) a-Keto acid measurement15mLof culture supernatantwasmixedwith 15mLof 10mML-Leu for oxidization reaction at 37∘C for 30min Then asreported [13] the produced a-keto leucine was detected using24-dinitrophenylhydrazine (DNP) which can react with car-bonyl group to generate brown-red dinitrophenylhydrazoneBriefly 550 120583L of detection solution was mixed with 450120583Lof 20 trichloroacetic acid and kept at room temperaturefor 30min Next 200120583L of 20mM DNP was added andthe mixture was incubated at room temperature for 15minThe reaction was terminated by adding 4mL of 08MNaOHFinally the mixture was centrifuged at 12000 rpm for 10minand the absorbance of the supernatant was measured at

520 nm (3) NH4

+ detection 25mL of resultant culturesupernatant wasmixedwith 25mLof 10mML-Leu in 250mLglass flask for oxidization reaction at 37∘C for 30min Then1mL of 1MNaOHwas added and paper pH indicator was putabove the glass flask After shaking at 120 rpm for 60min atroom temperature the color change of paper pH indicatordue to the NH

3release was observed

23 Molecular Taxonomy and Phylogenetic Analysis The ge-nomic DNA of LAAO-producing microorganism wasextracted by bacterial DNA isolation method [14] 16S rDNAgene was amplified in 50 120583L containing 37 120583L of ddH

2O

5 120583L of 10timesEasy Taq buffer 4 120583L of 25mM dNTPs 100 nMforward primer 27F (51015840-GAGTTTGATCCTGGCTCAG-31015840)100 nM reverse primer 1527R (51015840-AGAAAGGAGGTGATC-CAGCC-31015840) 1 ng genomic DNA and 1U Taq DNApolymerase with denaturation at 94∘C for 5min followed by30 cycles of 1min at 94∘C 50 s at 55∘C 90 s at 72∘C and a final10min extension at 72∘C At the end of reaction PCR productwas cooled to 4∘C to await further use After size confirmationon 10 agarose gel the PCR product was sent to SangonBiotech (Shanghai) Co Ltd for sequencing of 16S rDNAThesimilarity and homology of 16S rDNA gene sequence wasanalyzed using BLAST search available in genbank of NCBIThe DNA sequences were aligned and phylogenetic tree wasconstructed by neighbor joining method with bootstraptrials 1000 using MegaV402 software

24 Physiological and Biochemical Characterization Theability of the isolate to utilize various carbon and nitrogensources and other physiological and biochemical propertieswas studied according to the recommendation in ldquoTheManual of Systematic Methods of Determinative Bacterialrdquo

25 Retrieval of LAAOGene To retrieve the full length of lao-gene sequence from isolated microbial producer two roundsof PCRwere performed For the first round PCR the 6 degen-erate primers were designed based on the conserved regionsafter alignment of LAAO sequences from general LAAO-producing marine microorganisms (see Table 1) includingComamonas testosteroni KF-1 (ZP 03541004) Dinoroseobac-ter shibae DFL 12 (ABV95616) and Caulobacter sp K31(YP 001683007) and LAAO-producing Pseudoalteromonasmicroorganisms (see Table 2) including Pseudoalteromonashaloplanktis TAC125 (YP 339251) Pseudoalteromonastunicata D2 (ZP 01132853) Pseudoalteromonas atlanticaT6c (YP 662716) and Pseudoalteromonas sp SM9913(YP 004069407) ZZ-1 ZZ-2 and ZZ-3 were forwardprimers and ZF-1 ZF-2 and ZF-3 are reverse ones Asrequired these 6 degenerate primers combined with eachother to form 9 sets of forward and reverse primer pairs PCRamplification was performed with genomic DNA of LAAO-producer as template and PrimeSTAR HS DNA polymerase(TaKaRa Dalian China) as polymerase The Touch-downPCR consisted of denaturation at 94∘C for 5min 15 cycleof 30 s at 94∘C 30 s at 64∘C (minus1∘Ccycle) and 1min at 72∘Cfollowed by another 20 cycles of 30 s at 94∘C 30 s at 48∘C and1min at 72∘C and a final 10min extension at 72∘C After size
































2O









2O2production

was monitored






2O2


2O2 All


520




4




1 2 3 4






4




NH4


2O2assay




2S produc-


4)2HPO4 KNO





B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29








10

10

10

10

10

10

Consensus






6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)







RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































2O









2O2production

was monitored






2O2


2O2 All


520




4




1 2 3 4






4




NH4


2O2assay




2S produc-


4)2HPO4 KNO





B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29








10

10

10

10

10

10

Consensus






6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)







RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2O









2O2production

was monitored






2O2


2O2 All


520




4




1 2 3 4






4




NH4


2O2assay




2S produc-


4)2HPO4 KNO





B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29








10

10

10

10

10

10

Consensus






6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)







RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4






4




NH4


2O2assay




2S produc-


4)2HPO4 KNO





B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29








10

10

10

10

10

10

Consensus






6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)







RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


B3

90

2032

9821

68

91

82

29B3

90

2032

9821

68

91

82

29








10

10

10

10

10

10

Consensus






6970bp

f1 origin

Kan

ori

Lac I

lao

pET28b(+)-lao

BamH I

-lao

Hind III

(a)

5318bp

ori

Ap

f1 origin

lao

pET20b(+)-lao

BamH I

Hind III

(b)







RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


RecombinantLAAO

M 4321120 kDa

85kDa

50kDa

35kDa

25 kDa

20 kDa



2O2


2O2produced by



2O2concentration


4 Conclusion



4






12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


12

34


Acknowledgments


References











4716ndash4721 2008

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Identification, Cloning, and …downloads.hindawi.com/journals/tswj/2014/979858.pdfResearch Article Identification, Cloning, and Expression of L-Amino Acid Oxidase