Secretion of Biologically Active Heterologous Oxalate

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 280432 9 pageshttpdxdoiorg1011552013280432

Research ArticleSecretion of Biologically Active Heterologous OxalateDecarboxylase (OxdC) in Lactobacillus plantarum WCFS1Using Homologous Signal Peptides

Ponnusamy Sasikumar1 Sivasamy Gomathi1 Kolandaswamy Anbazhagan2

and Govindan Sadasivam Selvam1

1 Department of Biochemistry Centre for Advanced Studies in Organismal and Functional Genomics School of Biological SciencesMadurai Kamaraj University Madurai 625 021 India

2 INSERM-U844 Institut des Neuroscience de Montpellier Building Hopital St Eloi 34091 Montpellier France

Correspondence should be addressed to Govindan Sadasivam Selvam drselvamgsbiochemrediffmailcom

Received 22 April 2013 Accepted 28 June 2013

Academic Editor Isabel Sa-Correia

Copyright copy 2013 Ponnusamy Sasikumar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Current treatment options for patients with hyperoxaluria and calcium oxalate stone diseases are limited and do not always lead tosufficient reduction in urinary oxalate excretion Oxalate degrading bacteria have been suggested for degrading intestinal oxalate forthe prevention of calcium oxalate stone Here we reported a recombinant Lactobacillus plantarumWCFS1 (L plantarum) secretingheterologous oxalate decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal oxalate Theresults showed secretion and functional expression ofOxdC protein in L plantarum driven by signal peptides Lp 0373 and Lp 3050Supernatant of the recombinant strain containing pLp 0373sOxdC and pLp 3050sOxdC showed OxdC activity of 005Umg and002Umg protein while the purified OxdC from the supernatant showed specific activity of 183Umg and 175Umg proteinrespectively The concentration of OxdC protein in the supernatant was 8ndash12 120583gmL The recombinant strain showed up to 50oxalate reduction in medium containing 10mM oxalate In conclusion the recombinant L plantarum harboring pLp 0373sOxdCand pLp 3050sOxdC can express and secrete functional OxdC and degrade oxalate up to 50 and 30 respectively

1 Introduction

Oxalate is found in a variety of foodstuffs including pepperparsley spinach rhubarb cocoa tea certain dark greenleafy vegetables and plants and those who consume excessdietary oxalate rich food may develop nephrolithiasis Undernormal conditions the daily load of oxalate derived fromendogenous production and intestinal absorption is fullyexcreted by the kidneys Elevated oxalate level in urine knownas hyperoxaluria is one of the major risk factors for recurrenturolithiasis and progressive nephrocalcinosis [1] In additionseveral pathological conditions including Crohnrsquos diseasesteatorrhea and cystic fibrosis or medical procedures suchas jejuno-ileum bypass surgery are associated with enterichyperoxaluria due to enhanced oxalate absorption in thecolon [2ndash4] The lack of new medications and the continued

poor compliance with drug therapy have led to a growinginterest in dietary manipulation and novel therapies aimedat preventing recurrent stone formation Unfortunately anoxalate-free diet is difficult to achieve and would probablybe deficient in essential nutrients Hence other approachesto reducing urinary oxalate for management of stone dis-ease have been explored Normally the intestinal tract iscolonized with bacteria such as Oxalobacter formigenes (Oformigenes) Lactobacillus species and others [5 6] Intestinaloxalate degrading bacteria are capable of degrading oxalateto CO

2and formate the latter is further metabolized and

excreted via feces Thus treatment with oxalate degradingbacteria could be a new therapeutic option in patients withhyperoxaluria and calcium oxalate stone disease Althoughoxalotropic bacterium O formigenes is efficient in oxalatedegradation [7ndash9] its probiotic efficiency is still uncertain

2 BioMed Research International

and not as well established as in lactic acid bacteria (LAB)In addition colonization of O formigenes may require oraloxalate supplements to the gut since it is a strict oxalotrophicbacterium

Probiotics have been evaluated in animals and humanswith respect to antibiotic-associated diarrhea travelerrsquos diar-rhea pediatric diarrhea inflammatory bowel syndrome andirritable bowel syndrome [10] Over the past decade there hasbeen increasing interest in the use of LAB as a delivery systemfor a range of different applications including anti-infectiveallergic diseases and gastrointestinal diseases [11] Del Rioet al [12] developed an oral vaccine based on Lactobacillusplantarum expressing the outer surface protein A (OspA) alipoprotein from Borrelia burgdorferi Evidence suggests thatLAB play a role in controlling intestinal microbiota restor-ing intestinal barrier function and alleviating inflammatoryresponses It is also used in the therapy aid management ofimmunological disorders such as Crohnrsquos disease and Pou-chitis [13 14] Genetically modified LAB such as Lactococcuslactis have been used for delivery of interleukin-2 -6 and -10to combat murine colitis [15 16] Lactobacillus casei has beenused for the delivery of 120573-lactoglobulin in the digestive tractof mice [17] Similarly modified Lactobacillus was shown toprotect against dental caries in rat [18]

Earlier reports showed that wild type LAB have noinfluence on reduction of urinary oxalate excretion [19] Theuse of oxalate decarboxylase (OxdC) enzyme to decomposeintestinal oxalate was broadly demonstrated Orally givenrecombinant Bacillus subtilis (B subtilis) OxdC expressed inEscherichia coli (E coli) has substantially declined the urinaryoxalate level in an experimental rat model [20] Oral therapywith crystalline cross-linked formulation of the OxdC wasdemonstrated to diminish symptoms of hyperoxaluria andable to prevent nephrocalcinosis and urolithiasis inmice [21]Further orally given formulation of B subtilis OxdC wasshown to be safe in rats and dogs during short-term toxicitytests [22] Thus it can be hypothesized that manipulation oflactic acid bacterial component of intestinal microflora withheterologous expression of oxalate degrading protein (OxdC)may decrease the intestinal oxalate and correspondinglyintestinal oxalate absorption and renal excretion

Recently in our laboratory the LAB that heterologouslyexpress OxdC were developed for the possibility of utilizingas a potential probiotics for depletion of the intestinal dietaryoxalate [23] Since the expression of OxdC was at the intra-cellular level the degradation of external oxalate was limitedTherefore further modification with a desired promoter andsecretary elements for its extracellular expression of protein(OxdC) towards the alleviation of hyperoxaluria is vital Thefunctionality of homologous signal peptides (SPs) sequencesof Lactobacillus plantarum WCFS1 (L plantarum isolatefrom human saliva) was studied by Mathiesen et al [24]The study showed that predicted signal peptides in the Lplantarum genome actually are capable of driving the proteinsecretion In the present work we investigated the secretingefficiency of OxdC protein in human microbiota strainL plantarum by using sakacin P controlled Lactobacillusderivatives of the expression vector pSIP401 combined withthe homologous SPs sequence Lp 0373 and Lp 3050 to drive

the secretion of OxdCThe extracellular level expression bio-logical activity of recombinant OxdC and oxalate degradingefficiency of the recombinant L plantarum were evaluated inthis study

2 Materials and Methods

21 Bacterial Strains Media and Growth Conditions Thebacterial strains and plasmids used in this study are listedin Table 1 E coli DH10B (Invitrogen) was grown in Luria-Bertani (LB) broth at 37∘C with shaking and L plantarumWCFS1 was grown in deMan-Rogosa-Sharpe (MRS)media at30∘C without shaking Solid media were prepared by adding15 (wv) agar to the broth Erythromycin (Em) was addedat a final concentration of 200120583gmL for E coli and 5 120583gmLfor L plantarum

22 Standard DNA Techniques and Transformation Allcloning and transformation steps were carried out usingstandard methods [27] Peptide and primers used in thisstudy were synthesized and procured from Sigma AldrichUSA (Table 2) Taq DNA polymerase and the PCR productpurification kit was purchased from Sigma-Aldrich USARestriction enzymes Sal I EcoR I and T4 DNA Ligasewere obtained from Fermentas (Thermo Scientific USA)Restriction enzyme digested DNA was purified with Genelutes gel extract kit (Sigma Aldrich USA) The PCR ampli-fied products were verified by DNA sequencing (EurofinsGenomics India Pvt Ltd India) E coli DH10B was usedas host strain for maintaining plasmids L plantarum waselectrotransformed as described elsewhere [28]

23 Manipulation of Recombinant Plasmids Expression vec-tors constructed in this study are based on pSIP401 a567-Kb vector developed for inducible gene expressionand expression of the gene of interest regulated by a two-component regulatory system (SppK amp SppR) which isactivated by an externally added peptide pheromone [29 30]In pSIP401 vector the homologous signal peptide sequenceLp 0373 and Lp 3050 of L plantarum WCFS1 was clonedat downstream of the sakacin P promoter (P

119904119901119901119860) and its

functionality was studied and discussed elsewhere [24 31] Inthe present work the oxdC gene was amplified from plasmidpLB36 [26] by using OXDC sense and OXDC antisenseprimer harboring the Sal I and EcoR I sites respectivelyThe PCR program consisted of an initial denaturation at95∘C for 5min and 40 cycles of 95∘C for 30 s and 58∘C for1min and extension for 72∘C for 1min followed by finalextension at 72∘C for 10min The 12 kb PCR product wasdigested with Sal I and EcoR I and ligated into Sal IEcoRI digested pLp 0373sAmyA and pLp 3050sAmyA yieldedpLp 0373sOxdC and pLp 3050sOxdC respectively The finalrecombinant vector size of 69 Kb for pLp 0373sOxdC andpLp 3050sOxdC was confirmed by colony PCR restrictionanalysis and sequencing using primer PsppAFThese expres-sion vectors were constructed in E coli DH10B before elec-troporation into the expression host L plantarum WCFS1

BioMed Research International 3

Table 1 Bacterial strains and plasmids used in this study

Strains and plasmids Characteristicsa SourcereferencesStrains

E coli DH10B Host strain for cloning commercial InvitrogenL plantarumWCFS1lowast Host strain plasmid-free silage isolate Kleerebezem et al (2003) [25]

PlasmidpLp 0373sAmyA p256pUC(pGEM)ori PsppA Lp 0373 amyA Emr Mathiesen et al (2009) [24]pLp 3050sAmyA p256pUC(pGEM)ori PsppA Lp 3050 amyA Emr Mathiesen et al (2009) [24]pLB36 pET 32a oxdC Amr Just et al (2004) [26]pSip-OxdC p256pUC(pGEM)ori PsppA oxdC Emr Kolandaswamy et al (2009) [23]pLp 0373sOxdC p256pUC(pGEM)ori PsppA spLp 0373 fused to the oxdC Emr This workpLp 3050sOxdC p256pUC(pGEM)ori PsppA spLp 3050fused to the oxdC Emr This work

aFor strains genotypic and phenotypic characteristics are given for plasmid plasmid and cloned-cassette characteristics are given Emr Amr resistance toerythromycin and ampicillin respectivelylowastL plantarumWCFS1 is a single colony isolate of strain NCIM8826 (Kleerebezem et al 2003 [25])

Table 2 Primers and induction peptide used in this study

Primerspeptide Sequences (51015840 rarr 31015840)a

OXDC sense 51015840-GAGAGTCGACATGAAAAAACAAAATGACATTCCGC-31015840 (Sal I)OXDC antisense 51015840-GGAATTCGTGGTGGTGGTGGTGGTGTTATTTACTGCATTTCTTTTTCACTA-31015840 (EcoR I)PsppA sense 51015840-GTCACTAACCTGCCCCGTTA-31015840

SppIP MAGNSSNFIHKIKQIFTHRlowastaThe restriction sites are underlinedlowastaminoacid sequences of induction peptide (SppIP)

Our laboratory vector construct pSip-OxdC that producesintracellular OxdC was used as control [23]

24 SDS-PAGE Overexpression Analysis of OxdC in Recom-binant L plantarum WCFS1 Protein profiles in the cell-freesupernatants and purified proteins were visualized by run-ning 12 separating gel and 4 stacking gel of sodium dode-cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)[32] The heterologous OxdC proteins in recombinant LplantarumWCFS1 strains were induced by sakacin P (SppIP)as described by Mathiesen et al [33] Briefly the overnightcultures of the clones containing plasmids pLp 0373sOxdCpLp 3050sOxdC and pSip-OxdC were subcultured in MRSmedium containing 5mMMnCl

2 The cultures were allowed

to grow till the absorbance reaches OD600sim 03 and they

were induced by adding SppIP to a final concentration of25ndash50 ngml Cell pellet and the supernatant were collectedseparately at OD

600of sim17 The protein present in cell free

supernatant was precipitated by adding ammonium sulphateat a final concentration of 75 Further the precipitatedprotein was pelleted by centrifugation at 16000 rpm for30min at 4∘CThe pellet was resuspended in 115th volume ofTES buffer (10mM Tris-HCl + 1mM EDTA + 25 sucrose)and analyzed on an SDS-PAGE along with protein markerThe proteins were visualized by silver staining and the gel wasstored in 1 glacial acetic acid

25 Assay for OxdC Activity from Culture Supernatant Ex-pressed from pLp 0373sOxdC and pLp 3050sOxdC Oxalatedecarboxylase activity was assayed in both cell free super-natants and cell lysate of wild type and recombinants

L plantarum WCFS1 strains independently The heterolo-gous OxdC proteins in recombinant L plantarum strainswere induced as described elsewhere in this paper Whenthe OD

600reaches sim17 the cells were harvested and the

protein in culture supernatant was concentrated by adding75 ammonium sulphate Further the precipitated proteinsamples were dialyzed against 50mm Tris-HCl (pH 85)and 05M NaCl at 25∘C for overnight The OxdC assay wasperformed as described by Tanner and Bornemann [34] Inbrief an appropriate volume of reaction mixture containing10mM sodium citrate (pH 40) 150mM potassium oxalatewas taken in 1mL cuvette and concentrated supernatant wasadded to the mixture at 25∘C and incubated for 2min Themixture was neutralized with phosphate buffer (pH 95)Subsequently NAD was added and was monitored for itsreduction in a spectrophotometer (Hitachi) at 340 nm in thepresence of formate dehydrogenase (Sigma Aldrich) Oneunit of enzyme activity was defined as the amount of enzymerequired to reduce 1120583M of NAD per min Total proteinconcentration was measured by biuret method using proteinassay kit (Reckon Diagnostics Pvt Ltd India) in semiauto-mated photometer 5010V5+ (Robert Riele GmbHGermany)against the bovine serum albumin as a standard Similarly theOxdC activity was alsomeasured for corresponding strains atearly exponential growth (OD

600sim 05) and late exponential

growth (OD600sim 1)

To measure the intracellular OxdC activity cell pelletswere washed once with double distilled water and suspendedin sodium citrate buffer (pH 40) and disrupted by soni-cation The cell free extract was obtained by centrifugationat 16000 rpm for 30min at 4∘C The assay for OxdC was


performed To assess the secretion efficiency intracellularOxdC activity wasmeasured and comparedwith extracellularactivity as described elsewhere [31] OxdC activity for thecontrol strain was also assayed

26 Purification of OxdC from Culture Supernatant of Recom-binant L plantarumWCFS1 Supernatant containing recom-binant His tag-fused OxdC protein was purified by FPLCsystem (BioLogic DuoFlow Bio-Rad) equipped with 5mLprofinity column and enzymatic assaywas carried out Brieflythe ammonium sulphate precipitated proteins were collectedby centrifugation at 16000 rpm for 30min at 4∘CThe proteinpellet was dissolved in buffer containing 50mM imidazole-HCl (pH 70) 1M sodium chloride 10 120583MMnCl

2 and 1mM

of protease inhibitor Phenylmethyl Sulfonyl fluoride (PMSF)It was further loaded into previously washed 5mL profinitycolumn The column was equilibrated with 50mM phos-phate buffers (pH 80) containing 05M NaCl

2and 20mM

imidazole OxdC was eluted using 002ndash05M imidazolegradientThe flow rate was maintained at 1mlmin Fractionsof purified proteins were collected in different collectiontube and analyzed on 12 SDS-PAGE along with the proteinmarker and protein was visualized by Coomassie brilliantblue staining The fractions containing purified OxdC werepooled and dialyzed against 50mM Tris-HCl pH 85 con-taining 05MNaClThe purified enzyme was stored at minus80∘Cfor later use The protein quantification and assay for OxdCwere performed as described elsewhere in this paper Specificactivitywas calculated as themean value of three independentassays

27 In Vitro Analysis of Oxalate Degradation by the Recom-binant L plantarum Expressing Heterologous OxdC Theoxalate degrading ability of recombinant L plantarumWCFS1 and respective controls were examined at varioustime intervals In brief bacterial culture was grown inMRS medium supplemented with 10mM potassium oxalateup to OD

600sim 03 Expression of OxdC was induced by

adding 50 ng of induction peptide (SppIP) and 5mM ofMnCl

2 Cell-free medium was collected at different intervals

and assayed for oxalate concentration using oxalate oxidaseenzymatic methods (Trinity Biotech Ireland) according tothe manufacturerrsquos instructions in semiautomated analyzerphotometer 5010 V5+ (Robert Riele GmbH Germany) at OD590 nm Experiments were repeated three times and valueswere expressed as mean

3 Results

31 Construction of Secretion Vectors for the HeterologousExpression of OxdC in L plantarum WCFS1 For the con-struction of OxdC secretion vector in L plantarum WCFS1we selected two different plasmid pLp 0373AmyA andpLp 3050AmyA which harbor homologous signal peptidesequence (Lp 0373 and Lp 3050) of L plantarum at down-stream of the sakacin P promoter (P

119904119901119901119860) In addition both

the signal peptides functionality were well studied in homol-ogous host L plantarum WCFS1 [24 31] Their efficiency of

PsppA

PsppA

RBS oxdC

oxdC

EcoR I

EcoR I

Nde I Sal I

Nde I Sal I

Lp 0373

Lp 3050RBS

Plasmid name pLp 3050sOxdC


Figure 1 Schematic representation of expression cassette for pro-duction and secretion of oxalate decarboxylase under the controlof promoter (P

119904119901119901119860) in L plantarum for details of plasmid con-

structions see the text and Table 1 P119904119901119901119860

indicates the sakacin-Pinducible promoter RBS indicates ribosomal binding site Lp 0373and Lp 3050 indicate the signal peptides sequence oxdC indicatedthe oxalate decarboxylase gene and restriction sites between thesignal peptide sequences and oxdC gene also are indicated

secretion and higher activity of secreted protein promptedus to select these two signal peptides (Lp 0373 and Lp 3050)to study the secretion analysis of OxdC in L plantarumThe resulting recombinant vectors have the size of 69 Kb(pLp 0373sOxdC and pLp 3050sOxdC) Standard modularexpression cassettes developed are depicted in Figure 1 Thevectors were first established in E coli before electroporationinto L plantarum

32 Heterologous Production and Functional Expression ofOxdC by Recombinant L plantarum WCFS1 The samples ofsupernatants were taken in late logarithmic phase (OD

600sim

17) of recombinant L plantarum WCFS1 containing thevectors pLp 0373sOxdC pLp 3050sOxdC and pSip-OxdCSDS-PAGE analysis of concentrated supernatants showedprotein bands equivalent to the 44 kDa of OxdC by sil-ver staining for the strain harboring secretion plasmidspLp 0373sOxdC and pLp 3050sOxdC However the 44 kDacould not be detected on recombinant strain carrying plasmidpSip-OxdC (Figure 2) This shows the secretion of OxdC insupernatant by signal sequences Lp 0373 and Lp 3050 butnot in the control strain containing the plasmid pSip-OxdCdue to the lack of signal peptide sequences

OxdC activity in the concentrated cell-free super-natant of recombinant strains was measured at differ-ent stages of growth Activity of OxdC in L plantarumpLp 0373sOxdC was 001Umg proteins 002Umg pro-teins and 005Umg proteins for early exponential lateexponential and late logarithmic phases respectively Lplantarum pLp 3050sOxdC showed 0002Umg proteins001Umg proteins and 002Umg proteins during threedifferent stages It also indicates that enzymatic activ-ity of OxdC in supernatant of recombinant L plan-tarum WCFS1pLp 0373OxdC and WCFS1pLp 3050OxdCwas increased directly proportional to the growth ofthe strains (Figure 3) The higher activity of OxdC fromrecombinant L plantarum WCFS1 harboring the plas-mid pLp 0373OxdC indicates that secretion of OxdC pro-tein is higher than the strain harboring the plasmid


100857060504030

25

20

15

M 1 2 3

(kD

a)

Figure 2 Silver stained gels after SDS-PAGE of supernatantof lactobacillus strain harboring various plasmids All cells wereinduced with 50 ng inducing peptide mLminus1 at OD

600sim 03 and

harvested at OD600sim 17 lane M molecular weight marker (kDa)

lane 1 strain harboring pSIP-OxdC lane 2 pLp 3050sOxdC lane3 pLp 0373sOxdC arrow indicate the location of OxdC protein(calculated molecular mass of 44 kDa)

21

18

15

12

09

06

03

00 1 2 3 4 6 8 24

Time after induction (hr)

OD

at 6

00 nm

006

005

004

003

002

001

0

Oxd

C ac

tivity

(Um

g) (c

ultu

re su

pern

aten

t)

WCFS1WCFS1pLp 0373OxdCWCFS1pLp 3050OxdC

0373OxdC3050Oxdc

Figure 3 Growth curve analysis andOxdC activity in culture super-natant of different recombinants L plantarum construct againstnonrecombinant L plantarum after induction (arrow indicates theinduction point at OD

600sim 03) The represented data are mean

value of three independent experiments 0373OxdC (solid square)and 3050OxdC (solid triangles) indicates the enzymatic activity ofOxdC in culture supernatant during corresponding growth timeOD600sim 05 (1st hr) late exponential growth OD

600sim 1 (6th hr)

and late logarithmic phase OD600sim 17 (24th hr) of recombinant

strainsWCFS1pLp 0373OxdCWCFS1pLp 3050OxdC respectivelyand WCFS1 indicates the wild type strain of L plantarum

pLp 3050sOxdC No activity was determined in the super-natant of L plantarum pSip-OxdC

The OxdC activity in cellular fraction of recombi-nant strains (OD

600sim 17) showed 28Umg proteins

018Umg proteins and 021Umg proteins for pSip-OxdCpLp 0373sOxdC and pLp 3050sOxdC respectively How-ever no activity was observed in both supernatant and cel-lular fraction of wild type L plantarumWCFS1 To assess the

7060

50

40

30

25

20

15

M 1 2

(kD

a)

Figure 4 SDS-PAGE showing the purified OxdC protein fromculture supernatant of recombinant strains by FPLC lane Mmolecular weight marker in KDa lane 1 purified OxdC from strainharboring plasmid pLp 0373OxdC lane 2 purified OxdC fromstrain harboring plasmid pLp 3050OxdC

secretion efficiency intracellularOxdC activity wasmeasuredat OD

600sim 17 Secretion efficiencies are expressed as the

percentage of the total OxdC activity present in the cell-freesupernatants at OD

600sim 17 The SP Lp 0373 showed highest

secretion efficiency into the medium with 22 whereas SPLp 3050 showed only 9 which suggest that sim78 and sim91of intracellular OxdC were the combination of processed andunprocessed forms of OxdC in recombinant L plantarumpLp 0373sOxdC and pLp 3050sOxdC respectively (Table 3)

33 Purification and Biological Activity of Recombinant OxdCPurified OxdC by FPLC from the supernatant of recom-binant L plantarum WCFS1 was resolved in SDS-PAGE(Figure 4) As expected the purified protein band showscalculated molecular weight corresponding to 44KDa inSDS-PAGE which indicates the correct cleavage of signalpeptides by signal peptidase-I OxdC secreted into themedium by pLp 0373sOxdC was found to be higher whichhas approximately the concentration of 8ndash12120583gmL and thespecific activity of the purified OxdC from the supernatantof L plantarum pLp 0373sOxdC was 183Umg protein Thespecific activity of secreted protein which obtained from Lplantarum pLp 3050sOxdC was 175Umg protein (Figure 5)and concentration of OxdC protein in supernatant was 5ndash8 120583gmL

34 Oxalate Degradation by Recombinant L plantarumStrains Expressing Secretary OxdC The oxalate degrada-tion ability of L plantarum WCFS1 carrying plasmidspLp 0373sOxdC pLp 3050sOxdC was evaluated in thepresence of 10mM potassium oxalate The results showedreduction in oxalate level in the medium of recombinantL plantarum while the medium of wild type L plantarumshowed no reduction in oxalate level The oxalate level inthe medium of L plantarum WCFS1 harboring the plasmidpLp 0373sOxdC and pLp 3050sOxdC decreased from 10mM


Table 3 Oxalate decarboxylase (OxdC) activity in concentrated culture supernatant and cell lysate of various recombinant L plantarumWCFS1 strainsa

Name of strain (plasmid) Protein localizationOxdC (Umg protein)activity in culture

supernatant at OD600 = 17

OxdC (Umg protein)activity in cell fraction at

OD600 = 17Secretion efficiencylowast ()

WCFS1 (Control) NP NA NA NDWCFS1 (pSIP-OxdC) Intracellular NA 28 plusmn 02 NDWCFS1(pLp 0373sOxdC) Extracellular 005 plusmn 001 018 plusmn 007 22

WCFS1(pLp 3050sOxdC) Extracellular 002 plusmn 001 021 plusmn 007 9aData are mean of three independent experiments NP no production NA no activity and ND not determined and lowast secretion efficiency were determinedat OD600 = 17 between the culture supernatant and cell fraction of respective strains

1

6

11

16

21

0373 OxdC WCFS1 3050 OxdC WCFS1

Spec

ific a

ctiv

ity (U

mg)

Figure 5 Specific activity of purified recombinant oxalate decar-boxylase from supernatant of recombinant L plantarum WCFS1(0373 OxdC WCFS1 and 3050 OxdC WCFS1 indicate the OxdCpurified from strain harboring the plasmid pLp 0373sOxdC andpLp 3050sOxdC respectively) both the results are the mean ofthree independent experiments the error bars indicate the standarddeviation Enzyme activities are expressed in Unitsmg of protein

to around 5mM (50) and 7mM (30) respectively in120 h incubationThe result indicates that the oxalate presentin the medium of recombinant strain was degraded as aconsequence of secreted OxdC Results also indicate that theoxalate degrading ability increased proportionately with thegrowth of the strains (Figures 6(a) and 6(b))

4 Discussion

The present study aims to develop a recombinant LABthat can be used as a possible tool for the degradationof intestinal oxalate and prevention of hyperoxaluria andcalcium oxalate stone disease in human Here we developeda genetically modified L plantarum bacterial system forthe delivery of heterologous OxdC at biologically activecondition for therapeutic use L plantarum is found in manyhabitats including dairy and meat products and plant andvegetable fermentations and in the gastrointestinal tract andoral cavity of humans [35] L plantarum WCFS1 is a singlecolony isolate of L plantarum NCIMB8826 which exhibits

efficient secretion of heterologous protein compared withother L plantarum strains Kleerebezem et al [25] identified93 proteins with putative signal peptidase-I cleavage sitein the genome of L plantarum WCFS1 The signal peptidefunctionality of 76 sec-type signal peptides from L plantarumWCFS1 that were predicted to be cleaved by signal peptides-Iwas studied by Mathiesen et al [24] They also reported that82 of 76 tested SPs led to secretion of NucA In addition theSPs Lp 3050 and Lp 0373 of this bacterium showed highersecretion of heterologous protein [31]

In our study activity of OxdC in culture supernatant of Lplantarum pLp 0373sOxdC was higher than the recombinantstrain of pLp 3050sOxdC which is similar to the results onamylase activity reported by Mathiesen et al [31] Secretionefficiency of OxdC in the strain containing pLp 0373sOxdCwas 70 higher than pLp 3050sOxdC This higher secretionindicates that this signal peptide is most promising for futureuse However the overall picture emerging from previousstudies showed the difficulties on rationalizing the secretionlevels and secretion efficiencies [36ndash38] Thus it is clearthat secretion levels and secretion efficiency are not onlysteered by the SP but also by the secretion target The presentwork enables the simple downstream purification step ofOxdCwhich explores the possibility of large scale productionof the OxdC protein for industrial and biotechnologicalapplications

The most extensively investigated form of OxdC is thatisolated from B subtilis The expression of this enzyme inthe cell wall of B subtilis is found to be profound duringacidic stress conditions This bacterial enzyme has beenthe model for numerous studies aimed at deciphering thecatalytic mechanism of oxalate decarboxylase Recent studieshave focused on the use of OxdC as a potential mechanismto decompose intestinal oxalate in humans After the dis-covery that the YrvK gene of B subtilis encodes the 44 kDaoxalate decarboxylase [34] investigators were able to developrecombinant Escherichia coli (E coli) expressing this genePotent oxalate degrading activity was observed when therecombinant E coli was administered orally to hyperoxaluricrat models [20] Extrapolating from these findings Grujic etal [21] demonstrated that oral administration of recombinantB subtilis OxdC to hyperoxaluric mice resulted in decreasedoxalate urinary excretion and prevention of nephrocalcinosis


0

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OD

at 6

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cent

ratio

n of

oxa

late

(mM

)

Time (h)

Growth curveWCFS1 mM oxalate degradation

(a)

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OD

at 6

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Con

cent

ratio

n of

oxa

late

(mM

)

Time after induction (h)

3050 WCFS1 mM oxalate degradation0373 WCFS1 mM oxalate degradationGrowth curve

(b)

Figure 6 Oxalate degradation capabilities of wild type and recombinant L plantarumWCFS1 (a) Oxalate degradation ability of wild type LplantarumWCFS1 corresponding simples of WCFS1 mM oxalate degradation and growth curve indicate concentration of oxalate reductionin supernatant against the growth of the wild type L plantarumWCFS1 strain respectively (b) oxalate degradation capability of recombinantstrains corresponding simples of 3050 WCFS1 0373 WCFS1 mM oxalate degradation and growth curve indicate concentration of oxalatereduction in supernatant of recombinant L plantarum WCFS1 strains harboring plasmid pLp 3050OxdC and pLp 0373OxdC respectivelyagainst the growth of the strains Arrow indicates the point of induction using peptide (sppIP) at OD

600sim 03

Therefore we undertook this study to degrade the intestinaloxalate by secreting OxdC by probiotic strain L plantarum

The reductions of 30 and 50 of oxalate concentrationwere observed in the supernatant of recombinant strainsduring the 120 h incubation period but there was no changein oxalate concentration in wild type L plantarum The finalpH of the supernatant of recombinant strain of L plantarumhas reached nearly to pH 40 (date not shown) Tanner andBornemann [34] reported the enzyme OxdC was induced inacidic growth media and shows maximum activity at pH 50and also found 70 activity at pH 30 Turroni et al [39]experiment of pH controlled batch fermentations revealedthat acidic conditions were a prerequisite for a significantoxalate degradation rate Although the pSIP vectors havebeen successfully applied for the production of differentproteins in L plantarum [23 30 40 41] the heterologousOxdC quantity in L plantarum may be further increased byusage of synthetic propeptide residues for the high oxalatedegradation The fusion of a short synthetic propeptidebetween the SP and the mature moiety is another innovativebiotechnological tool to enhance protein secretion One suchsynthetic propeptide LEISSTCDA a nine amino acid peptidewhich was shown to enhance the secretion efficiency ofseveral heterologous proteins in Lactococcus lactis NucBNucT [42] Even though secretion efficiency is fairly normalthe another possibility to increase the secretion efficiency isto test other SPs from the library made previously and find

the optimal SPs that lead to higher secretion efficiency It isdifficult to compare the present observation with the resultsof previews studies since the studies published so far onengineered secretion in lactic acid bacteria differ with respectto the genetic constructs and host strains used [42]

The accumulation of intracellular precursors and thehigh total level of OxdC activity may be due to the factthat intracellular protein is better protected from proteolyticdegradation than secreted OxdC protein [24] While thepurified OxdC from culture supernatant of recombinantsL plantarum harboring the plasmid pLp 0373sOxdC andpLp 3050sOxdC shows the specific activity of 183U and175Umg protein respectively it is almost similar to theactivity of OxdC purified from the recombinant E coliBL21 [26] Mufarrij et al [43] reported that commerciallyproduced form of oxalate decarboxylase (Oxazyme) degradesthe oxalate completely in the intestinal buffer (pH 65) andoxalate derived from potassium oxalate in the gastric buffer(pH 36) was profoundly digested by Oxazyme AddingOxazyme also substantially reduced the oxalate content ofboth whole and homogenized spinach preparations in eitherbuffer These in vitro findings demonstrate that Oxazymecan metabolize oxalate in both simulated gastric and smallintestinal environments Cowley et al [22] evaluated thetoxicity studies in rats and dogs by orally administered Bsubtilis OxdC enzyme The results of the study showed no


observed adverse effect in both of the animals administeredOxdC (7208mgkgd) in rats and (1872mgkgd) in dogs

Overexpressing OxdC in L plantarum would enable therecombinant strain to efficiently degrade the oxalate in thegut environment However certain optimization needs to bedone for its application in vivoThe optimizationmay includereplacing the antibiotic resistance marker with a food-gradeselection marker that will allow to study directly on animalmodelsThiswas recently demonstratedwith the pSIP vectorsusing alr gene as plasmid selection marker instead of theerythromycin that has been applied for overexpression of120573-galactosidase [44] Similar kind of studies needs to beperformed in future to harness real benefits of geneticallymodified L plantarum strains for humans

5 Conclusions

In conclusion the results indicate that oxalate present in theextracellular environment could be degraded by the recom-binant strains secreting heterologous oxalate decarboxylaseHence the recombinant Lactobacillus bacteria developed inthis study with further modification like change of con-stitutive promoter usage of synthetic propeptide sequencebiological containment status and improved gastrointestinal(GI) track survival will enable us to degrade the oxalate avail-able in intestine of human for therapeutic use Based on thegenetics tools availability themanipulation of recombinant LplantarumWCFS1 with the biologically contained status willease in future focusing

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

The authors express their gratitude to Dr Geir MathiesenNorwegianUniversity of Life Sciences Norway for providingplasmids pLP 3050AmyA and pLP 0373AmyA and professorMichiel Kleerebezem Wageningen Centre for Food Sci-encesTheNetherlands for providing the strain L plantarumWCFS1 The authors also wish to thank Dr Stephen Borne-mann for providing the plasmid pLB36 consisting oxdC geneThis work was supported by University Grand Commission(UGC) New Delhi India and Department of Biotechnologythrough IPLS program The authors also thank UGC andDST for the central instrumentation facility at SBS MKUthrough CEGS CAS NRCBS DST-FIST and DST-PURSEprogramme

References

[1] E Leumann and B Hoppe ldquoThe primary hyperoxaluriasrdquoJournal of the American Society of Nephrology vol 12 no 9 pp1986ndash1993 2001

[2] E Hylander S Jarnum H J Jensen and M Thale ldquoEnterichyperoxaluria dependence on small intestinal resection colec-tomy and steatorrhoea in chronic inflammatory bowel diseaserdquo

Scandinavian Journal of Gastroenterology vol 13 no 5 pp 577ndash588 1978

[3] J H Clark J F Fitzgerald and J M Bergstein ldquoNephrolithiasisin childhood inflammatory bowel diseaserdquo Journal of PediatricGastroenterology and Nutrition vol 4 no 5 pp 829ndash834 1985

[4] L A Matthews C F Doershuk R C Stern and M I ResnickldquoUrolithiasis and cystic fibrosisrdquoThe Journal of Urology vol 155no 5 pp 1563ndash1564 1996

[5] R KumarMMukherjeeM Bhandari A KumarH Sidhu andR D Mittal ldquoRole ofOxalobacter formigenes in calcium oxalatestone disease a study fromNorth Indiardquo European Urology vol41 no 3 pp 318ndash322 2002

[6] B Hoppe E Leumann G von Unruh N Laube and AHesse ldquoDiagnostic and therapeutic approaches in patients withsecondary hyperoxaluriardquo Frontiers in Bioscience vol 8 pp437ndash443 2003

[7] H Sidhu M J Allison J M Chow A Clark and A B PeckldquoRapid reversal of hyperoxaluria in a rat model after probioticadministration of Oxalobacter formigenesrdquoThe Journal of Urol-ogy vol 166 no 4 pp 1487ndash1491 2001

[8] S Hazebrouck R Oozeer K Adel-Patient et al ldquoConstitutivedelivery of bovine 120573-lactoglobulin to the digestive tracts ofgnotobiotic mice by engineered Lactobacillus caseirdquo Appliedand Environmental Microbiology vol 72 no 12 pp 7460ndash74672006

[9] B Hoppe J W Groothoff S-A Hulton et al ldquoEfficacy andsafety of Oxalobacter formigenes to reduce urinary oxalate inprimary hyperoxaluriardquo Nephrology Dialysis Transplantationvol 26 no 11 pp 3609ndash3615 2011

[10] A Ljungh and T Wadstrom Lactobacillus Molecular BiologyFrom Genomics to Probiotics Caister Academic Press NorfolkUK 2009

[11] J M Wells and A Mercenier ldquoMucosal delivery of therapeuticand prophylactic molecules using lactic acid bacteriardquo NatureReviews Microbiology vol 6 no 5 pp 349ndash362 2008

[12] B Del Rio R J Dattwyler M Aroso et al ldquoOral immunizationwith recombinant Lactobacillus plantarum induces a protectiveimmune response in mice with lyme diseaserdquo Clinical andVaccine Immunology vol 15 no 9 pp 1429ndash1435 2008

[13] P Marteau P Seksik and R Jian ldquoProbiotics and intestinalhealth effects a clinical perspectiverdquoBritish Journal of Nutritionvol 88 no 1 pp S51ndashS57 2002

[14] S Prakash and C Martoni ldquoToward a new generation oftherapeutics artificial cell targeted delivery of live cells fortherapyrdquo Applied Biochemistry and Biotechnology vol 128 no1 pp 1ndash21 2006

[15] L Steidler W Hans L Schotte et al ldquoTreatment of murinecolitis by Lactococcus lactis secreting interleukin-10rdquo Sciencevol 289 no 5483 pp 1352ndash1355 2000

[16] L Steidler S Neirynck N Huyghebaert et al ldquoBiological con-tainment of geneticallymodified Lactococcus lactis for intestinaldelivery of human interleukin 10rdquoNature Biotechnology vol 21no 7 pp 785ndash789 2003


[18] C Kruger Y Hu Q Pan et al ldquoIn situ delivery of passiveimmunity by Lactobacillus producing single-chain antibodiesrdquoNature Biotechnology vol 20 no 7 pp 702ndash706 2002


[19] J C Lieske W J Tremaine C De Simone et al ldquoDiet but notoral probiotics effectively reduces urinary oxalate excretion andcalcium oxalate supersaturationrdquo Kidney International vol 78no 11 pp 1178ndash1185 2010

[20] B C Jeong D H Han S I Seo et al ldquoYvrk gene recombinantE coli reduce the concentration of urine oxalate in transientHyperoxaluria rat modelrdquo The Journal of Urology vol 181 p660 2009

[21] D Grujic E C Salido B C Shenoy et al ldquoHyperoxaluriais reduced and nephrocalcinosis prevented with an oxalate-degrading enzyme in mice with hyperoxaluriardquo AmericanJournal of Nephrology vol 29 no 2 pp 86ndash93 2009

[22] A B Cowley D W Poage R R Dean et al ldquo14-Day repeat-dose oral toxicity evaluation of oxazyme in rats and dogsrdquoInternational Journal of Toxicology vol 29 no 1 pp 20ndash31 2010

[23] A Kolandaswamy L George and S Sadasivam ldquoHeterologousexpression of oxalate decarboxylase in Lactobacillus plantarumNC8rdquo Current Microbiology vol 58 no 2 pp 117ndash121 2009

[24] G Mathiesen A Sveen M B Brurberg L Fredriksen LAxelsson and V G H Eijsink ldquoGenome-wide analysis of signalpeptide functionality in Lactobacillus plantarumWCFS1rdquo BMCGenomics vol 10 article 1471 p 425 2009

[25] M Kleerebezem J Boekhorst R Van Kranenburg et al ldquoCom-plete genome sequence of Lactobacillus plantarum WCFS1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 4 pp 1990ndash1995 2003

[26] V J Just C E M Stevenson L Bowater A Tanner DM Lawson and S Bornemann ldquoA closed conformation ofBacillus subtilis oxalate decarboxylase OxdC provides evidencefor the true identity of the active siterdquo The Journal of BiologicalChemistry vol 279 no 19 pp 19867ndash19874 2004

[27] J Sambrook and D Russel Molecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press New York NYUSA 2001

[28] TW AukrustM B Brurberg and I F Nes ldquoTransformation ofLactobacillus by electroporationrdquoMethods inMolecular Biologyvol 47 pp 201ndash208 1995

[29] M B Brurberg I F Nes and V G H Eijsink ldquoPheromone-induced production of antimicrobial peptides in LactobacillusrdquoMolecular Microbiology vol 26 no 2 pp 347ndash360 1997

[30] E Soslashrvig S Gronqvist K Naterstad G Mathiesen V G HEijsink and L Axelsson ldquoConstruction of vectors for induciblegene expression in Lactobacillus sakei and L plantarumrdquo FEMSMicrobiology Letters vol 229 no 1 pp 119ndash126 2003

[31] G Mathiesen A Sveen J-C Piard L Axelsson and V G HEijsink ldquoHeterologous protein secretion by Lactobacillus plan-tarum using homologous signal peptidesrdquo Journal of AppliedMicrobiology vol 105 no 1 pp 215ndash226 2008

[32] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[33] GMathiesen E Soslashrvig J Blatny K Naterstad L Axelsson andV G H Eijsink ldquoHigh-level gene expression in Lactobacillusplantarum using a pheromone-regulated bacteriocin promoterrdquoLetters in Applied Microbiology vol 39 no 2 pp 137ndash143 2004

[34] A Tanner and S Bornemann ldquoBacillus subtilis yvrK is an acid-induced oxalate decarboxylaserdquo Journal of Bacteriology vol 182no 18 pp 5271ndash5273 2000

[35] L Axelsson and S Ahrne ldquoLactic acid bacteriardquo in AppliedMicrobial Systematics F G Priest and M Goodfellow Edspp 365ndash386 Kluwer Academic Publishers Dordrecht TheNetherlands 2000

[36] G Perez-Martinez J Kok G Venema J M Van Dijl H Smithand S Bron ldquoProtein export elements from Lactococcus lactisrdquoMolecular and General Genetics vol 234 no 3 pp 401ndash4111992

[37] A Bolhuis H Tjalsma H E Smith et al ldquoEvaluation ofbottlenecks in the late stages of protein secretion in Bacillussubtilisrdquo Applied and Environmental Microbiology vol 65 no7 pp 2934ndash2941 1999

[38] U Brockmeier M Caspers R Freudl A Jockwer T Noll andT Eggert ldquoSystematic screening of all signal peptides fromBacillus subtilis a powerful strategy in optimizing heterologousprotein secretion in Gram-positive bacteriardquo Journal of Molec-ular Biology vol 362 no 3 pp 393ndash402 2006

[39] S Turroni C Bendazzoli S C F Dipalo et al ldquoOxalate-degrading activity in Bifidobacterium animalis subsp lactisimpact of acidic conditions on the transcriptional levels ofthe oxalyl coenzyme A (CoA) decarboxylase and formyl-CoAransferase genesrdquoApplied and Environmental Microbiology vol76 no 16 pp 5609ndash5620 2010

[40] E Soslashrvig G Mathiesen K Naterstad V G H Eijsink and LAxelsson ldquoHigh-level inducible gene expression in Lactobacil-lus sakei and Lactobacillus plantarum using versatile expressionvectorsrdquoMicrobiology vol 151 no 7 pp 2439ndash2449 2005

[41] E Halbmayr G Mathiesen T-H Nguyen et al ldquoHigh-levelexpression of recombinant 120573-galactosidases in Lactobacillusplantarum and Lactobacillus sakei using a sakacin p-basedexpression systemrdquo Journal of Agricultural and Food Chemistryvol 56 no 12 pp 4710ndash4719 2008

[42] Y Le Loir A Gruss S D Ehrlich and P Langella ldquoA nine-residue synthetic propeptide enhances secretion efficiency ofheterologous proteins in Lactococcus lactisrdquo Journal of Bacteri-ology vol 180 no 7 pp 1895ndash1903 1998

[43] P W Mufarrij J N Lange J Knight et al ldquoSecond placethe effects of oxazyme on oxalate degradation results andimplications of In Vitro experimentsrdquo Journal of Endourologyvol 27 no 3 pp 284ndash287 2013

[44] T-T Nguyen G Mathiesen L Fredriksen et al ldquoA food-gradesystem for inducible gene expression in Lactobacillus plantarumusing an alanine racemase-encoding selection markerrdquo Journalof Agricultural and Food Chemistry vol 59 no 10 pp 5617ndash5624 2011

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


and not as well established as in lactic acid bacteria (LAB)In addition colonization of O formigenes may require oraloxalate supplements to the gut since it is a strict oxalotrophicbacterium

Probiotics have been evaluated in animals and humanswith respect to antibiotic-associated diarrhea travelerrsquos diar-rhea pediatric diarrhea inflammatory bowel syndrome andirritable bowel syndrome [10] Over the past decade there hasbeen increasing interest in the use of LAB as a delivery systemfor a range of different applications including anti-infectiveallergic diseases and gastrointestinal diseases [11] Del Rioet al [12] developed an oral vaccine based on Lactobacillusplantarum expressing the outer surface protein A (OspA) alipoprotein from Borrelia burgdorferi Evidence suggests thatLAB play a role in controlling intestinal microbiota restor-ing intestinal barrier function and alleviating inflammatoryresponses It is also used in the therapy aid management ofimmunological disorders such as Crohnrsquos disease and Pou-chitis [13 14] Genetically modified LAB such as Lactococcuslactis have been used for delivery of interleukin-2 -6 and -10to combat murine colitis [15 16] Lactobacillus casei has beenused for the delivery of 120573-lactoglobulin in the digestive tractof mice [17] Similarly modified Lactobacillus was shown toprotect against dental caries in rat [18]

Earlier reports showed that wild type LAB have noinfluence on reduction of urinary oxalate excretion [19] Theuse of oxalate decarboxylase (OxdC) enzyme to decomposeintestinal oxalate was broadly demonstrated Orally givenrecombinant Bacillus subtilis (B subtilis) OxdC expressed inEscherichia coli (E coli) has substantially declined the urinaryoxalate level in an experimental rat model [20] Oral therapywith crystalline cross-linked formulation of the OxdC wasdemonstrated to diminish symptoms of hyperoxaluria andable to prevent nephrocalcinosis and urolithiasis inmice [21]Further orally given formulation of B subtilis OxdC wasshown to be safe in rats and dogs during short-term toxicitytests [22] Thus it can be hypothesized that manipulation oflactic acid bacterial component of intestinal microflora withheterologous expression of oxalate degrading protein (OxdC)may decrease the intestinal oxalate and correspondinglyintestinal oxalate absorption and renal excretion

Recently in our laboratory the LAB that heterologouslyexpress OxdC were developed for the possibility of utilizingas a potential probiotics for depletion of the intestinal dietaryoxalate [23] Since the expression of OxdC was at the intra-cellular level the degradation of external oxalate was limitedTherefore further modification with a desired promoter andsecretary elements for its extracellular expression of protein(OxdC) towards the alleviation of hyperoxaluria is vital Thefunctionality of homologous signal peptides (SPs) sequencesof Lactobacillus plantarum WCFS1 (L plantarum isolatefrom human saliva) was studied by Mathiesen et al [24]The study showed that predicted signal peptides in the Lplantarum genome actually are capable of driving the proteinsecretion In the present work we investigated the secretingefficiency of OxdC protein in human microbiota strainL plantarum by using sakacin P controlled Lactobacillusderivatives of the expression vector pSIP401 combined withthe homologous SPs sequence Lp 0373 and Lp 3050 to drive

the secretion of OxdCThe extracellular level expression bio-logical activity of recombinant OxdC and oxalate degradingefficiency of the recombinant L plantarum were evaluated inthis study

2 Materials and Methods

21 Bacterial Strains Media and Growth Conditions Thebacterial strains and plasmids used in this study are listedin Table 1 E coli DH10B (Invitrogen) was grown in Luria-Bertani (LB) broth at 37∘C with shaking and L plantarumWCFS1 was grown in deMan-Rogosa-Sharpe (MRS)media at30∘C without shaking Solid media were prepared by adding15 (wv) agar to the broth Erythromycin (Em) was addedat a final concentration of 200120583gmL for E coli and 5 120583gmLfor L plantarum

22 Standard DNA Techniques and Transformation Allcloning and transformation steps were carried out usingstandard methods [27] Peptide and primers used in thisstudy were synthesized and procured from Sigma AldrichUSA (Table 2) Taq DNA polymerase and the PCR productpurification kit was purchased from Sigma-Aldrich USARestriction enzymes Sal I EcoR I and T4 DNA Ligasewere obtained from Fermentas (Thermo Scientific USA)Restriction enzyme digested DNA was purified with Genelutes gel extract kit (Sigma Aldrich USA) The PCR ampli-fied products were verified by DNA sequencing (EurofinsGenomics India Pvt Ltd India) E coli DH10B was usedas host strain for maintaining plasmids L plantarum waselectrotransformed as described elsewhere [28]

23 Manipulation of Recombinant Plasmids Expression vec-tors constructed in this study are based on pSIP401 a567-Kb vector developed for inducible gene expressionand expression of the gene of interest regulated by a two-component regulatory system (SppK amp SppR) which isactivated by an externally added peptide pheromone [29 30]In pSIP401 vector the homologous signal peptide sequenceLp 0373 and Lp 3050 of L plantarum WCFS1 was clonedat downstream of the sakacin P promoter (P

119904119901119901119860) and its

functionality was studied and discussed elsewhere [24 31] Inthe present work the oxdC gene was amplified from plasmidpLB36 [26] by using OXDC sense and OXDC antisenseprimer harboring the Sal I and EcoR I sites respectivelyThe PCR program consisted of an initial denaturation at95∘C for 5min and 40 cycles of 95∘C for 30 s and 58∘C for1min and extension for 72∘C for 1min followed by finalextension at 72∘C for 10min The 12 kb PCR product wasdigested with Sal I and EcoR I and ligated into Sal IEcoRI digested pLp 0373sAmyA and pLp 3050sAmyA yieldedpLp 0373sOxdC and pLp 3050sOxdC respectively The finalrecombinant vector size of 69 Kb for pLp 0373sOxdC andpLp 3050sOxdC was confirmed by colony PCR restrictionanalysis and sequencing using primer PsppAFThese expres-sion vectors were constructed in E coli DH10B before elec-troporation into the expression host L plantarum WCFS1























growth (OD600sim 1)





2 and 1mM


2and 20mM







3 Results


119904119901119901119860) In addition both


PsppA

PsppA

RBS oxdC

oxdC

EcoR I

EcoR I

Nde I Sal I

Nde I Sal I

Lp 0373

Lp 3050RBS









600sim




100857060504030

25

20

15

M 1 2 3

(kD

a)


600sim 03 and



21

18

15

12

09

06

03

00 1 2 3 4 6 8 24


OD

at 6

00 nm

006

005

004

003

002

001

0

Oxd

C ac

tivity

(Um

g) (c

ultu

re su

pern

aten

t)


0373OxdC3050Oxdc




600sim 1 (6th hr)







7060

50

40

30

25

20

15

M 1 2

(kD

a)

















1

6

11

16

21


Spec

ific a

ctiv

ity (U

mg)



4 Discussion






0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























growth (OD600sim 1)





2 and 1mM


2and 20mM







3 Results


119904119901119901119860) In addition both


PsppA

PsppA

RBS oxdC

oxdC

EcoR I

EcoR I

Nde I Sal I

Nde I Sal I

Lp 0373

Lp 3050RBS









600sim




100857060504030

25

20

15

M 1 2 3

(kD

a)


600sim 03 and



21

18

15

12

09

06

03

00 1 2 3 4 6 8 24


OD

at 6

00 nm

006

005

004

003

002

001

0

Oxd

C ac

tivity

(Um

g) (c

ultu

re su

pern

aten

t)


0373OxdC3050Oxdc




600sim 1 (6th hr)







7060

50

40

30

25

20

15

M 1 2

(kD

a)

















1

6

11

16

21


Spec

ific a

ctiv

ity (U

mg)



4 Discussion






0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2 and 1mM


2and 20mM







3 Results


119904119901119901119860) In addition both


PsppA

PsppA

RBS oxdC

oxdC

EcoR I

EcoR I

Nde I Sal I

Nde I Sal I

Lp 0373

Lp 3050RBS









600sim




100857060504030

25

20

15

M 1 2 3

(kD

a)


600sim 03 and



21

18

15

12

09

06

03

00 1 2 3 4 6 8 24


OD

at 6

00 nm

006

005

004

003

002

001

0

Oxd

C ac

tivity

(Um

g) (c

ultu

re su

pern

aten

t)


0373OxdC3050Oxdc




600sim 1 (6th hr)







7060

50

40

30

25

20

15

M 1 2

(kD

a)

















1

6

11

16

21


Spec

ific a

ctiv

ity (U

mg)



4 Discussion






0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100857060504030

25

20

15

M 1 2 3

(kD

a)


600sim 03 and



21

18

15

12

09

06

03

00 1 2 3 4 6 8 24


OD

at 6

00 nm

006

005

004

003

002

001

0

Oxd

C ac

tivity

(Um

g) (c

ultu

re su

pern

aten

t)


0373OxdC3050Oxdc




600sim 1 (6th hr)







7060

50

40

30

25

20

15

M 1 2

(kD

a)

















1

6

11

16

21


Spec

ific a

ctiv

ity (U

mg)



4 Discussion






0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









1

6

11

16

21


Spec

ific a

ctiv

ity (U

mg)



4 Discussion






0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)

Time (h)


(a)

0

02

04

06

08

1

12

14

16

18

2

0

1

2

3

4

5

6

7

8

9

10

11

0 24 48 72 96 120

OD

at 6

00 nm

Con

cent

ratio

n of

oxa

late

(mM

)



(b)


600sim 03








5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































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

Secretion of Biologically Active Heterologous Oxalate