BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Extracellular production of lipoxygenase from Anabaenasp. PCC 7120 in Bacillus subtilis and its effect on wheatprotein

Chong Zhang & Tingting Tao & Qi Ying &

Dongliang Zhang & Fengxia Lu & Xiaomei Bie &

Zhaoxin Lu

Received: 28 November 2011 /Revised: 3 January 2012 /Accepted: 9 January 2012 /Published online: 31 January 2012# Springer-Verlag 2012

Abstract In this study, the lipoxygenase (ana-LOX) genefrom Anabaena sp. PCC 7120 was successful expressed andsecreted in Bacillus subtilis. Under the control of the P43promoter, with a signal peptide from the B. subtilis 168 nprBgene, and facilitated by the molecular chaperone PrsA, theproduction of the recombinant ana-LOX (ana-rLOX)reached 76 U/mL (171.9 μg/ml) in the supernatant. Thepurified ana-rLOX was investigated for its effect on doughprotein. Ana-rLOX treatment decreased free sulfhydrylgroups, increased glutenin macropolymer content, promotedthe formation of covalent bonds between gluten protein, andaffected protein crosslinking. The results indicated that largeaggregates involving gliadin and glutenin were formed. Theglutenin macropolymer played a role in the formation of thedough network structure through the exchange of thiol disul-fide bonds and the formation of hydrogen or hydrophobicbonds with other proteins.

Keywords Anabaena sp. PCC 7120 . Recombinantlipoxygenase (ana-rLOX) . Bacillus subtilis . Dough protein

Introduction

Lipoxygenase (LOX) catalyzes the regio- and stereo-selectivedioxygenation of polyunsaturated fatty acid (PUFA) to

hydroperoxides (Brash 1999). PUFA hydroperoxides are fur-ther converted to various metabolites, such as jasmonic acid,which are often bioactive, or serve as defense response signalsin plants (Matui 2006).

LOXs have food-related applications in bread makingand aroma production (Whitehead et al. 1995). Industrialapplications often use crude plant materials or extracts suchas soybean powder, but those materials usually containmultiple enzymes, which may reduce the availability ofPUFAs as a substrate, or metabolize hydroperoxide prod-ucts, thereby decreasing LOX activity (Hughes et al. 1998).The use of pure LOX may increase the catalysis of PUFA,and the best way to obtain purified LOX is by microbialfermentation, with expression of the enzyme from a clonedgene (Casey et al. 1999).

Previously, LOXs were thought to exist exclusively ineukaryotes, but have recently been found in prokaryotes(Koeduka et al. 2007; Vance et al. 2004). The C-terminusof the AOS (allene oxide synthase)-LOX fusion gene fromAnabaena sp. PCC 7120 was found to belong to the LOXfamily (Lang et al. 2008; Niisuke et al. 2009; Schneider etal. 2007), and an active 49–50 kDa ana-LOX has beenexpressed in Escherichia coli (Andreou et al. 2008; Zhenget al. 2008). However, recombinant proteins expressed in E.coli need complex purification steps to remove pathogenicsubstances before they can be used in food, which greatlyincreases their cost.

Bacillus subtilis can be an efficient and safe host forrecombinant protein secretion (Chang 1987; Doi et al.1986). Secreted proteins usually remain in biologically activeform, and secretion simplifies downstream purification(Nakayama et al. 1988). In this work, the LOX gene fromAnabaena sp. PCC 7120 was placed under the control of theP43 promoter, and fused to a sequence encoding the nprB or

Electronic supplementary material The online version of this article(doi:10.1007/s00253-012-3895-5) contains supplementary material,which is available to authorized users.

C. Zhang : T. Tao :Q. Ying :D. Zhang : F. Lu :X. Bie : Z. Lu (*)Laboratory of Enzyme Engineering, College of Food Scienceand Technology, Nanjing Agriculture University,Nanjing 210095, People’s Republic of Chinae-mail: fmb@njau.edu.cn

Appl Microbiol Biotechnol (2012) 94:949–958DOI 10.1007/s00253-012-3895-5

the amyQ signal peptide, for expression and secretion in B.subtilis (Wang and Doi 1984; Cho et al. 2004). PrsA (Jacobs etal. 1993; Kontinen and Sarvas 1993; WahlstrÖm et al. 2003),a B. subtilis chaperone, was coexpressed under control of thePamyE promoter (Ye et al. 1999) to improve ana-LOXsecretion. The production of the recombinant ana-LOX(ana-rLOX) increased to 76 U/mL (171.9 μg/ml) in thesupernatant. In addition, because the proteins of wheat flourare responsible for the formation of a viscoelastic doughcapable of CO2 retention during proofing and posterior baking(Lindsay and Skerritt 1999; Shewry et al. 2001; Steffolani etal. 2010), the effect of the purified ana-rLOX onwheat proteinwas also studied.

Materials and methods

Bacterial strains, plasmids, and oligonucleotides

B. subtilis WB800 (nprE aprE epr bpr mpr::ble nprB::bsrvpr wprA::hyg) (Wu et al. 2002) were used as expressionhosts. The cloning vector pMD19-T was from TaKaRa(Japan), and pHB201 (Bron et al. 1998) was kindlyprovided by the Bacillus Genetic Stock Center (BGSC). Allvectors for ana-rLOX expression in WB800 are pHB201derivatives. Specific primers used for polymerase chain reac-tion (PCR) amplification were synthesized by Invitrogen.

DNA manipulation techniques

Standard recombinant DNA techniques were used, andenzymes were used as specified by the supplier (TaKaRa).Bacillus chromosomal DNA was prepared with a genomicDNA purification kit (Sangon, Shanghai). Transformationswere as described for B. subtilis (Xue et al. 1999).

Construction of secretion expression vectors in WB800

pHP43 and pHP43R were generated using pHB201, a shut-tle plasmid from the B. subtilis cryptic plasmid pTA1060and the E. coli plasmid pUC19, which is fairly stable duringcell passage and fermentation. The primers P1 and P2 wereannealed, and then the cassette was inserted into pHB201 atthe HindIII/ClaI sites to generate pHB-hc. P43, a strongconstitutive promoter in B. subtilis, was amplified usingthe PCR from purified genomic B. subtilis 168 DNA usingprimers P3/P4 and inserted into pHB-hc at the XhoI/BamHI sites to generate pHP43. As a result, the pHB201plasmid was altered by substituting the P59 promoterand cat86::lacZ with the P43 promoter and a multiplecloning site.

The promoter PamyE was amplified using PCR frompurified genomic B. subtilis 168 DNA using primers P5/

P6. The PrsA gene was amplified by two rounds of PCRfrom genomic B. subtilis 168 DNA using primers P7/P8 andP7/P9; as a result, the strong trpA terminator (Kaltwasser etal. 2002) was inserted at the 3′ end of the PrsA gene. ThePamyE-PrsA expression cassette was generated by splicingby overlapped extension PCR (Ho et al. 1989) using primersP5/P6/P7/P9, and then inserted to pHP43 to generatepHP43R.

The signal peptide sequence of SamyQ or SnprB wasamplified by PCR from genomic B. subtilis 168 DNA usingprimers SamyQ-F/SamyQ-R or SnprB-F/SnprB-R, andinserted into BamHI/EcoRI-double-digested pHP43R andpHP43 to generate pHPSQ, pHPSB, and pHPSBR for secret-ed expression.

Cloning ana-LOX gene from Anabaena sp. PCC 7120

LOX from Anabaena sp. 7120 naturally exists as part ofa fusion protein with AOS, according to the NCBIdatabase (NC_003267). The ana-LOX gene was ampli-fied by PCR from purified Anabaena sp. PCC 7120genomic DNA. Because addition of the 6 His tag hadno detrimental effect on catalytic activity, this tag wasadded at the N-terminal of the ana-rLOX for simplifyingthe purification steps.

LOX enzyme assay

The substrate emulsion was prepared on the day of theassay. The substrate solution was prepared by mixing27 μL of pure linoleic acid, 25 μL of Tween-20, and 8 mLdeionized water. The solution was clarified by adding1.1 mL 0.5 M NaOH and diluted to 50 mL with phosphatebuffer (pH 6.0). LOX activity was determined spectropho-tometrically at 35 °C by measuring the increase in absor-bance at 234 nm over 3 min. The reaction mixture contained2.97 mL phosphate buffer, 20 μL substrate solution, and10 μL enzyme solution. One unit of LOX activity wasdefined as an increase absorbance of 0.001 per minute at234 nm (Kermasha and Metche 1986).

Expression of ana-LOX in WB800

The recombinant plasmids pHPSQ-ana-LOX, pHPSB-ana-LOX, and pHPSQR-ana-LOX were electrotransformed intoB. subtilis WB800 and cultured at 15 °C in super-richmedium (25 g/L tryptose, 20 g/L yeast extract, 30 g/Lglucose, and 3 g/L K2HPO4, pH 7.0) (Zhang et al. 2005)containing chloromycetin (10 μg/mL) and erythromycin(3 μg/mL). The culture was incubated with rotary shakingfor 96 h and samples collected every 6 h. After centrifuga-tion at 10,000 rpm for 5 min at 4 °C, the supernatant wasused for enzyme assays. The ana-rLOX activity in the

950 Appl Microbiol Biotechnol (2012) 94:949–958

supernatant (units per milliliter) was used to indicate thesecretion expression level.

Purification of ana-rLOX from WB800

One liter of culture supernatant of recombinant WB800 withpHPSQR-ana-LOX was precipitated with ammonium sulfate,and the precipitate dissolved in 0.05 M Tris–HCl (pH 7.5).The highest activity fraction was loaded onto a DEAE-Sephacel column equilibrated in 0.05 M Tris–HCl (pH 7.5),and bound protein was eluted by a NaCl gradient from 0 to0.8 M. Fractions with LOX activity were collected and loadedonto a Sephadex G-100 column. Then, the active fraction wasbatch-loaded onto a 2-mL nickel-charged NTA agarosecolumn (Pruibest, Shanghai, China) which was previouslyequilibrated with lysis buffer. Expression and the purityof the ana-rLOX were monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE),with a 5% stacking gel and 10% separating gel. Proteinswere stained with Coomassie Brilliant Blue R-250.

Amino acid analysis and partial N-terminal sequence of ana-rLOX

Purified ana-rLOX was hydrolyzed with 6 M HCl at 110 °Cfor 24 h, and the amino acid composition was determined witha Hitachi L-8800 amino acid analyzer (Japan). The N-terminalamino acid sequence analysis by automated Edman degrada-tion was performed with an Applied Biosystems 491 Proteinsequencer (USA).

Biochemical characterizations of ana-rLOX

The iron content of the purified ana-rLOX was determinedby a colorimetric assay using the chromogenic ligand 1,10-phenanthroline monohydrate. In order to eliminate the inter-ference of protein when determining the iron content, 0.5 g ofpurified enzymewas added into a digest tank containing 10mlnitric acid for microwave digestion. When digested fully,nitric acid was volatilized by heating. The residual substancewas dissolved with ultrapure water to a total volume of500 ml. A total of 5 ml of the sample was added to a 50-mlvolumetric flask. Then, 1 ml of 10% hydroxylamine hydro-chloride, 1 ml of 0.15% 1,10-phenanthroline monohydrate,5 ml of 1 mol/L HAc-NaAc (pH 5.0), and ultrapure waterwere added to make a total volume of 50 ml. After mixing andstanding at room temperature for 5 min, the absorbance of thesolution was determined at 510 nm. Reagent blanks weremeasured and a standard curve constructed using a solutionof primary standard grade ammonium ferrous sulfate in0.01 N HNO3. All vessels were cleaned using aqua regia.The protein content of the sample was determined byCoomassie Brilliant Blue staining.

The specificity of ana-rLOX was examined using differentsubstrates including linoleic acid, linolenic acid, and arachi-donic acid at 1.6 mM. The kinetic constants (Km, Vmax) weremeasured using the enzymatic activity assay conditionsdescribed above. In order to determine the effect of the producton the enzyme reaction, different concentrations of the reactionproduct 9R-hydroperoxide (with linoleic acid as a substrate)were added into the reaction system. Enzyme stability wasdetermined at room temperature for different storage times.

The optimal temperature of recombinant ana-LOX wasdetermined by performing the standard assay as describedabove between 20 °C and 55 °C, in 5 °C increments. Theoptimum pH was determined by measuring the enzyme activ-ity from pH 3.0 to 8.0. The buffers (0.1 M) were sodiumcitrate (pH 3.0–5.0) and sodium phosphate (pH 6.0–8.0).

A variety of metal ions were added at 10 mM to 0.5 mLrecombinant ana-LOX solution, including FeCl2, FeCl3,CaCl2, CuSO4, MgSO4, NaCl, MnSO4, and ZnCl2. Theeffect of various ions on ana-LOX activity was determinedby the enzyme assay described above. Residual activity wasexpressed as the percentage of the activity observed in theabsence of any compound.

Each treatment was replicated three times, and the exper-iment was conducted twice.

Preparation of the ana-rLOX modified wheat flour

Flour and purified ana-rLOX were mixed with 10 mMphosphate buffer (pH 6.8) and incubated at 30 °C for 4 h.After incubation, the slurry was frozen immediately and lyoph-ilized, and sieved by a cyclone mill with a 0.5-mm filter. Thecontrol flour was treated the same way but with no enzyme.

Glutenin macropolymer (GMP) isolation

Glutenin macropolymer (GMP) was extracted according to amodified method of Weegels et al. (1999). After ordinalextraction of albumin, globulin, gliadin, and glutenin fromfreeze-dried dough (100 mg) using the Osborne method anddissolving the proteins in SDS, the precipitate was suspended in3 mL GMP extracting solvent (containing 2% SDS, 5% mer-captoethanol, 10% glycerol, and 0.01 M Tris–HCl buffer, pH08.0) and stirred for 10 min at high speed and 1 h at low speed.After centrifugation for 30 min at 10,000 rpm, the supernatantcontaining GMP was separated and stored at −20 °C for furtheranalysis. Total protein content of the precipitates was determinedby the Bradford method and expressed as percentage of proteinof GMP per gram of freeze-dried dough.

Protein fractionation

Protein fractionation was carried out according to a modifica-tion of the sequential Osborne extraction method. Extraction

Appl Microbiol Biotechnol (2012) 94:949–958 951

was performed for 300±2 mg of freeze-dried dough. Eachextraction step was carried out with constant stirring followedby centrifugation for 15 min at 10,000 rpm. Sequential proteinextraction was performed using 0.6 mL distilled water (F1),2% w/v NaCl (F2), 70% v/v ethanol (F3), and 0.05 M aceticacid (F4) for 30 min, respectively. The protein content of eachfraction was determined according to a modification of theBradford method using bovine serum albumin (SigmaChemical Co.) as a standard, and the results were expressedas percentage of protein of each fraction per gram of freeze-dried dough.

Free sulfhydryl groups (SHf) determination

Free sulfhydryl (SHf) groups were determined according toAnderson (2000) with modifications of Hanft and Koeler(2006). Freeze-dried dough (100 mg) was extracted with1.5% (w/v) SDS twice. The precipitate was mixed with1.5 mL buffer A (8 M urea, 3 mM EDTA, 1% SDS, and0.2 M Tris–HCl, pH 8.0), stirred for 1 h, and then was addedto 0.15 mL buffer B (10 mM DTNB, 0.2 M Tris–HCl,pH 8.0) and stirred for another hour. After centrifugationfor 15 min at 8,000 rpm, the absorbance of the supernatantswas measured at 412 nm. The extinction coefficient used totransform absorbance values into concentration values was13,600 M−1 cm−1. Determinations were made in triplicate.

Scanning electron microscopy

The dough was frozen and lyophilized by the samemethod as previously described (Steffolani et al. 2010).The images were taken using a scanning electron microscopewith a 6-kVacceleration voltage. The micrographs were takenusing ×1,000 magnification.

Results

Expression of ana-rLOX from WB800

To express and secrete ana-rLOX from WB800, the 1,254-bp ana-LOX gene was inserted at the XhoI site to generatethe recombinant expression vectors pHPSQ-ana-LOX,pHPSB-ana-LOX, and pHPSBR-ana-LOX. Time-courseexpression assays for secreted LOX activity are shown inFig. 1. Under the control of the P43 promoter, the ana-LOXgene was continuously secreted when directed by either theSamyQ or of SnprB signal peptide. At 84 h of cultivation,LOX activity in the culture medium accumulated to a maxi-mum level of 48 U/mL (108.6 μg/ml) with pHPSQ-ana-LOX,58 U/mL (131.2 μg/ml) with pHPSB-ana-LOX, and 76 U/mL(171.9 μg/ml) with pHPSBR-ana-LOX. SDS-PAGE of thesupernatant from recombinant WB800 cells is shown inFig. 2a, and intracellular ana-rLOX activity was determined(109 U/mL with pHPSQ-ana-LOX, 101 U/mL with pHPSB-ana-LOX, and 85 U/mL with pHPSBR-ana-LOX). The secre-tion efficiency was approximately 28.7% for pHPSQ-ana-LOX, 36.5% for pHPSB-ana-LOX, and 47.2% forpHPSBR-ana-LOX. These results suggested that the SnprBsignal peptide was more effective than SamyQ at directingana-rLOX secretion. The maximal activity in the culture was76 U/mL (171.9 μg/ml) with the PamyE-PrsA fragment in theexpression vector pHPSBR-ana-LOX, suggesting that PrsApromotes protein secretion by the Sec pathway.

Purification of ana-rLOX from WB800

The expressed ana-rLOX was purified from the concentratedsupernatant prepared from the cell culture using three chro-matographic steps as described in the “Materials and

Fig. 1 The time-course profileof the expression of the ana-rLOX gene with pHPSQ(squares), pHPSB (diamonds),and pHPSBR (triangles) inWB800. The cell number isshown as log (cells per millili-ter) (circles)

952 Appl Microbiol Biotechnol (2012) 94:949–958

methods” section. As shown in Table 1, the ana-rLOX waspurified 7.8-fold with a yield of 5%. The purified enzymeexhibited a specific activity of 442.2 U/mg of protein and waselectrophoretically homogeneous (Fig. 2b).

The first 12 N-terminal amino acids of the ana-rLOX wereidentified by Edman degradation. The N-terminal amino acidsequence of ana-rLOX (EESEFHHHHHHP) begins with E-E-S (residues 1 to 3) of the nprB signal peptide cleaved by signalpeptidases. This was followed by E-F (residues 4 and 5),which was translated from the EcoRI sequence (GAATTC).The last seven residues matched the amino acid sequencededuced from the nucleotide sequence containing a 6 His tagand one P residue. The molecular mass of the ana-rLOX wasestimated to be 47 kDa by SDS-PAGE (Fig. 2b), which was

identical to the size predicted from the gene sequence byDANMAN software.

Enzymatic properties of purified ana-rLOX

The chromogenic iron assay using 1,10-phenanthrolinemonohydrate is a highly selective method, as other elementssuch as Sn, Al, Ca, Mg, Zn, and Si at concentrations of up to40-fold higher than iron have no effects on the determinedresults. Measurement of the iron content after nickel affinitycolumn purification using a colorimetric iron-specific assaygave an iron:protein molar ratio of 0.5.

The activity of LOX on a variety of substrates wasdetermined. Linoleic acid is the best substrate for thisenzyme. Arachidonic acid was 54% and linolenic acid was38% relative activity toward linoleic acid. The Vmax, Km, andKcat for the catalysis of linoleic acid oxidation by ana-rLOXwere calculated as 210.5 U/min, 3.5 mM, and 15 s−1,respectively.

As for most enzymes, the reaction activity was inhibitedby the formation of the product. With linoleic acid as thesubstrate, the product 9R-hydroperoxide contributed toproduct inhibition. Ana-rLOX activity declined to 50%when 1.386×10−6 mM of the 9R-hydroperoxide wasadded.

The ana-rLOX stability was determined at room temper-ature where 93% of the enzyme remained active after stor-age for 1 h.

The effects of temperature on ana-rLOX activity areshown in Fig. 3a. The ana-rLOX had an optimum reactiontemperature of 45 °C. Enzyme activity increased slightlyfrom 20 °C to 45 °C then fell sharply at 50 °C. Activity ofana-LOX appeared to be optimal at pH 6.0 and wasonly 50% active at pH 6.6, with no activity at pH 8.0(Fig. 3b), indicating that ana-rLOX is more active in acidicconditions.

The effect of inhibitors and activators on ana-LOX activ-ity was also studied. As shown in Table 2, enzyme activitywas stimulated by up to 181.91% by Fe2+ and was stronglyinhibited by as much as 100% by Fe3+ and Cu2+. It wasslightly inhibited by 11.34% by Zn2+, and 17.8% by Mn2+.Little effect was seen with Ca2+, Mg2+, or Na+.

47kD

47kD

(a)

(b)

Fig. 2 a SDS-PAGE of the supernatant of recombinant WB800 cells.M, molecular mass markers; lane 1, WB800; lane 2, WB800 withpHPSQ; lane 3, WB800 with pHPSQ-ana-LOX; lane 4, WB800 withpHPSB-ana-LOX; lane 5, WB800 with pHPSBR-ana-LOX. The arrowindicates the band corresponding to approximately 47 kD ana-LOX. bSDS-PAGE of the purified ana-rLOX

Table 1 The purification ofrecombinant His6-ana-LOX Purification step Total protein (mg) Total

activity (U)Specificactivity (U/mg)

Purificationfold

Yield (%)

Crude extract 1,340.8 76,000 56.7 1.0 100

(NH4)2SO4 680.2 46,117.6 67.8 1.2 60.7

DEAE–Sephacel 237.1 23,400.3 98.7 1.7 30.8

Sephadex G-100 64.3 10,175.2 158.2 2.8 13.4

Ni-NTA agarosecolumn

8.6 3,803.4 442.2 7.8 5.0

Appl Microbiol Biotechnol (2012) 94:949–958 953

Effects of ana-rLOX on wheat protein

The glutenin protein content from dough significantlydecreased when 10 U/g of ana-rLOX was added to linoleicacid, although a lower dose increased the glutenin content.Ana-rLOX induces crosslinking of gluten proteins, and these

new bonds cause structural changes in the proteins, modifyingtheir solubility. Ana-rLOX catalyzes the oxidation of disulfidebonds between cysteines, as well as intra- and inter-moleculardisulfide bonds. Free radicals from the oxidation reactioncatalyzed by ana-rLOX are transformed to proteins by increas-ing the negative charge density; the increase of repulsiveinteractions among protein chains prevents a stable aggregatefrom forming, and this promotes an increase of solubility inSDS.

However, when the protein solubility in different solu-tions was studied (Table 3), different results from theexpected ones were observed. Samples incubated with highlevels of ana-rLOX and LA showed a reduction of proteinsolubility in water and NaCl solution as compared to thecontrol. The action of LOX on F1 and F2 promoted theformation of large aggregates insoluble in water and NaClsolution. In addition, molecules of albumin and globulinwhich have a higher content of lysine could be participatingin crosslinking with glutenin, decreasing NaCl solubility.These results indicated that large molecules were formedwhen ana-rLOX was included in the formulation.

The fraction extracted with SDS solution correspondsmainly to gliadins (F3), which decreased with the high-level addition of LOX (10 U/g) as compared to the control.This result was contradictory to the content of the otherthree proteins of the same sample (Table 3). In each stepof the sequential extraction, the centrifugal force increasedinteractions between the protein chains, promoting the pre-cipitation of aggregates. The last fraction (F4) was com-pletely solubilized, and the glutenin protein amount washigher in the high level of LOX sample (Table 3).

Effects of ana-rLOX on SHf

The SHf content of the different samples is shown in Fig. 4.All samples with ana-rLOX had smaller SHf content thanthe controls (with or without linoleic acid). The oxidanteffect of the production of hydroperoxide catalyzed by

0

20

40

60

80

100

120

20 25 30 35 40 45 50 55

Re

lati

ve

ac

tiv

ity

(%)

0

20

40

60

80

100

120

3 4 5 5.2 5.3 5.6 6 6.3 6.6 7 8

pH

Rel

ati

ve

ac

tiv

ity

(%)

Fig. 3 Effect of temperature (a) and pH (b) on the activity of thepurified ana-rLOX

Table 2 Effect of metalions (10 mM) on theactivity of recombinantana-LOX

Data expressed asmean ± SD, n03

Metal ions Relative activity (%)

Control 100

Fe2+ 181±5

Mg2+ 108±4

Ca2+ 105±6

Na1+ 97±3

Zn2+ 88±5

Mn2+ 82±2

Fe3+ 0

Cu2+ 0

Table 3 Effect of ana-rLOX on protein content in different solutions

Sample F1 (%) F2 (%) F3 (%) F4 (%)

Control 2.30a 0.64a 0.90a 0.44ac

LOX (1 U/g) 2.06bc 0.57bc 0.74b 0.47a

LOX (10 U/g) 2.08abc 0.59bc 0.79c 0.44a

LA 2.18abc 0.57ab 0.51def 0.33b

LOX (1 U/g) + LA 2.14abc 0.51c 0.56e 0.46a

LOX (10 U/g) + LA 2.03bc 0.52c 0.51f 0.39c

Letters within a column indicate significantly different values (p<0.05)

F1 albumin dissolved in water, F2 globulin dissolved in 2% w/v NaCl,F3 gliadin dissolved in 70% w/v ethanol, F4 glutenin dissolved in0.05 M acetic acid

954 Appl Microbiol Biotechnol (2012) 94:949–958

ana-rLOX promoted the formation of disulfide bondsamong the proteins. However, the high level of ana-rLOX(10 U/g) had a higher SHf content compared to the doughwith a low level of ana-rLOX (1 U/g). Ana-rLOX catalyzes

the formation of isopeptidic bonds, which modifies thethree-dimensional structure of gluten protein.

SEM results

The microstructure of different dough samples producedwith ana-rLOX (Fig. 5c–f) presented a more continuousand closed gluten network structure, as compared to thecontrols (with or without linoleic acid, Fig. 5a, b).However, dough treated with 10 U/g of ana-rLOX (with orwithout linoleic acid, Fig. 5e, f) showed a more openprotein matrix and a greater number of pores than thesamples with a lower ana-rLOX content (Fig. 5c, d).The gluten fibrils of the dough prepared with ana-rLOXwere coarser, indicating the formation of a stronger andmore resistant network.

Discussion

LOX has food-related applications in bread making (Baysaland Demirdöven 2007) and aroma production (Whitehead etal. 1995), and recombinant LOX is a new resource in food

Fig. 4 The SHf content in SDS-soluble protein when treated withdifferent levels of ana-rLOX. L1, control (with no enzyme treatment);L2, LA (with 0.001 mmol/g dough linoleic acid treatment); L3, with1 U/g ana-rLOX treatment; L4, with 10 U/g ana-rLOX treatment; L5,with 1 U/g ana-rLOX and LA treatment; L6, with 10 U/g ana-rLOXand LA treatment

Fig. 5 Scanning electronmicroscopy of the dough withno enzyme treatment (a), with0.001 mmol/g linoleic acidtreatment (b), with 1 U/g ana-rLOX treatment (c), with 10 U/g ana-rLOX treatment (d), with1 U/g ana-rLOX and LA treat-ment (e), and with 10 U/g ana-rLOX and LA treatment (f)

Appl Microbiol Biotechnol (2012) 94:949–958 955

enzymology (Casey et al. 1999). Nonetheless, until now, nostudies have been reported on recombinant LOX for foodapplications because of the very low activity of the currentproduct (Casey et al. 1999).

We cloned the ana-LOX gene from Anabaena sp. PCC7120 and expressed it in E. coli at a high level, achievingana-LOX activity of 216 U/mL, which was more than 12times higher than the previous report (Kermasha and Metche1986). However, recombinant proteins expressed in E. colirequire complex purification steps before using in food, andthus, B. subtilis is a good alternative as a cell factory forproducing recombinant proteins for food applications(Chang 1987; Doi et al. 1986; Nakayama et al. 1988). It iscapable of secreting recombinant proteins directly into theculture medium, and the secreted foreign proteins usuallyremain in an active form, greatly simplifying downstreampurification. In addition, B. subtilis does not produce endo-toxins, and is considered to be generally regarded as safe, orGRAS microorganism.

To secrete ana-LOX in B. subtilis, we constructed thevectors pHPSQ, pHPSB, and pHPSBR, which were basedon pHB201. The P43 promoter was used, which is a well-characterized overlapping promoter that is functional duringboth the exponential and stationary growth phases. As com-ponents of the secretory apparatus, the signal peptides thatchannel exported proteins into the secretion machinery playa key role in translocation across the membrane (Harwoodand Cranenburgh 2008). Different signal peptides can affectthe secretion efficiency of heterogeneous proteins(Brockmeier et al. 2006). In this study, the signal peptidesfrom amyQ and nprB were used to direct ana-LOX secretionby the Sec pathway. The results suggested that the signalpeptide from nprB was more effective. However, sinceintracellular ana-rLOX was more abundant than secreted

ana-rLOX, the optimal signal peptide for ana-rLOX secre-tion requires further study.

The extracytoplasmic folding factor PrsA is a well-defined lipoprotein required for folding mature proteins intostable and active conformations in the Sec pathway (Jacobset al. 1993; Kontinen and Sarvas 1993; WahlstrÖm et al.2003). It is essential for protein secretion in B. subtilis and isthe limiting factor for high-level secretion (Kontinen andSarvas 1993). In this study, the pHP43R plasmid carryingPrsA under the PamyE promoter was constructed. The pro-duction of secreted ana-LOX increased to 76 U/mL(171.9 μg/ml from pHPSBR-ana-LOX). This result corre-sponded to the expression of SCA (Wu et al. 1998).

Table 4 illustrates the comparison of the biochemical char-acterizations of ana-rLOX with other LOXs. The molecularweight ranged from about 47 to 94 kDa, while LOXs frombacteria are generally smaller than those from plants andanimals. The pH critical value for LOXs varied with the originof LOX; the ana-rLOX had a slightly acid pH optima. Theoptimum temperature for LOXs varied from 25 °C to 60 °C.LOXs isolated from plants are the most effective towardlinoleic acid, while arachidonic acid is the best substrate foranimal LOXs. In this study, ana-rLOX was most effective forthe catalysis of linoleic acid like other bacterial and plantLOXs.

The effect of metal ions on ana-LOXs was also deter-mined. Interestingly, recombinant ana-LOX was stronglystimulated by up to 181.91% by Fe2+ and was completelyinactivated by Fe3+ and Cu2+. We propose that the redoxcharacter of metal ions might affect electron transfer duringthe reaction transition state. Fe2+ binding to the enzymemight make the transition state much more stable, but Fe3+

and Cu2+ may change the enzyme conformation to make thetransition state unstable.

Table 4 Biochemical characterizations of LOXs from different sources

LOX (source) Parameter

Molecularweight (kDa)

Optimum pH Optimumtemperature (°C)

Substratespecificity

Km (mM) Kcat (S−1)

Ana-rLOX (this study) 47 6.0 45 Linoleic acid 3.5 15

Soybean LOX1 (Max et al. 1986;Brash 1999)

94 9 25 Linoleic acid 12.2 300

Pea seed LOX (Szymanowska et al. 2009) 93 5.5 NG Linoleic acid 0.44 NG

Rabbit LOX (Claeysa et al. 1982;Belkner et al. 1998)

75 7 20 Linoleic acid 8.1 40

Human 5-LOX (Soberman et al. 1985;Denis et al. 1991)

72 6 37 Arachidonicacid

63.1 1

Nostoc punctiforme LOX2 (Koedukaet al. 2007)

62.9 7 NG Linoleic acid NG NG

Pseudomonas aeruginosa LOX(Vance et al. 2004)

47 6 60 Linoleic acid 3.57 NG

NG not given

956 Appl Microbiol Biotechnol (2012) 94:949–958

The results indicated that moderate oxidation couldincrease the extraction of protein from wheat flour. However,protein content was decreased because of overoxidationinduced by excess ana-rLOX. While glutenin content was at amaximum, the addition of ana-rLOX lowered it below that ofother proteins. This showed that glutenin had a higher sensi-tivity to oxidants. Since it forms the framework of the glutensystem, glutenin could make dough quality more sensitive tothe amount of ana-rLOX enzyme. The effect of ana-rLOXenzyme on the extraction of glutenin was contrary to gliadin,albumin, and globulin and indicated that oxidation couldcause physical and chemical crosslinking among differentprotein components after the fermentation process.

This study shows that a high level of ana-rLOX (10 U/g)addition resulted in a variation in the four protein compo-nents and was different from that obtained from a low levelof ana-rLOX. The high level of ana-rLOX likely inducedoveroxidation which caused a change in the character ofprotein. The specific mechanism of how ana-rLOX acted ongluten protein remains to be further studied.

Peroxide radicals, which result from the reaction of ana-rLOX oxidizing unsaturated fatty acids (such as LA), affectboth the physical and chemical interactions of a variety ofproteins, and change the composition of gluten. Glutenin isthe main component of gluten, along with gliadin. Glutenincontents reduce while gliadin contents increase after fer-mentation, perhaps due to a portion of gliadin combiningwith glutenin. In addition, albumin and globulin contentsalso decrease, indicating that these two proteins could beincorporated into the gluten network under conditions ofoxidation and fermentation. It can be inferred that oxidationof ana-rLOX enzyme and fermentation could change proteincomposition of gluten.

In conclusion, we successfully overexpressed the ana-LOXgene inB. subtilis, and the ana-rLOXwas active against wheatprotein. The results of this study should provide a basis forfuture improvements and food industrial applications of ana-rLOX.

Acknowledgements This work was funded by the National NaturalScience Foundation of China (31071605), Fundamental ResearchFunds for the Central Universities of China (KWZ200910), and YouthScience and Technology Innovation Fund of Nanjing Agriculture Uni-versity (Y201069).

References

Anderson AK (2000) Changes in disulfide and sulfhydryl contents andelectrophoretic patterns of extruded wheat flour proteins. CerealChem 77:354–359

Andreou AZ, Vanko M, Bezakova L, Feussner I (2008) Properties of amini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutantforms. Phytochemistry 9:1832–1837

Baysal Y, Demirdöven A (2007) Lipoxygenase in fruits and vegeta-bles: A review. Enzyme Microb Technol 40:491-496

Belkner J, Stender H, Kühn H (1998) The rabbit 15-lipoxygenaseperferentially oxygenates LDL cholesterol esters, and this reactiondoes not require vitamin E. J Biol Chem 273:23225–23232

Brash AR (1999) Lipoxygenases: occurrence, functions, catalysis, andacquisition of substrate. J Biol Chem 274:23679–23682

Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Eggert T(2006) Systematic screening of all signal peptides from Bacillussubtilis: a powerful strategy in optimizing heterologous pro-tein secretion in Gram-positive bacteria. J Mol Biol 362:393–402

Bron S, Bolhuis A, Tjalsma H, Holsappel S, Venema G (1998) Proteinsecretion and possible roles for multiple signal peptidases forprecursor processing in Bacilli. J Biotechnol 64:3–13

Casey R, West SI, Hardy D, Robinson DS, Wu ZC, Hughes RK (1999)New frontiers in food enzymology: recombinant lipoxygenases.Trends Food Sci Tech 10:297–302

Chang S (1987) Engineering for protein secretion in gram-positivebacteria. Methods Enzymol 153:507–516

Cho HY, Yukawa H, Inui M, Doi RH, Wong SL (2004) Production ofminicellulosomes from Clostridium cellulovorans in Bacillus sub-tilis WB800. Appl Environ Microbiol 70:5704–5707

Claeysa M, Coenea MC, Hermana AG, Jouvenazb GH, Nugterenb DH(1982) Characterization of monohydroxylated lipoxygenasemetabolites of arachidonic and linoleic acid in rabbit peritonealtissue. Biochim Biophys Acta 1:160–169

Denis D, Falgueyret JP, Riendeau D, Abramovitz M (1991)Characterization of the activity of purified recombinant human5-lipoxygenase in the absence and presence of leukocyte factors. JBiol Chem 266:5072–5079

Doi RH, Wong SL, Kawamura F (1986) Potential use of Bacillussubtilis for secretion and production of foreign proteins. TrendsBiotechnol 4:232–235

Hanft F, Koeler P (2006) Studies on the effect of glucose oxidase inbread making. Journal Sci Food Agr 86:1699–1704

Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: anunfolding story. Trends Microbiol 16:73–79

Hughes RK, Wu ZC, Robinson DS, Hardy D, West SI, Fairhurst SA,Casey R (1998) Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mecha-nisms. J Biol Chem 333:33–43

Ho SN, Hunt DH, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerasechain reaction. Gene 77:51–59

Jacobs M, Andersen JB, Kontinen V, Sarvas M (1993) Bacillus subtilisPrsA is required in vivo as an extracytoplasmic chaperone forsecretion of active enzymes synthesized either with or withoutpro-sequences. Mol Biol 8:957–966

Kaltwasser M, Wiegert T, Schumann W (2002) Construction and appli-cation of epitope- and green fluorescent protein-tagging integrationvectors for Bacillus subtilis. Appl EnvironMicrobiol 68:2624–2628

Kermasha S, Metche M (1986) Characterization of seed lipoxygenaseof Phaseolus vulgaris cv, Haricot. J Food Sci 51:1224–7

Koeduka T, Kajiwara T, Matsui K (2007) Cloning of lipoxygenase genesfrom a Cyanobacterium, Nostoc punctiforme, and its expression inEschelichia coli. Curr Microbiol 54:315–319

Kontinen VP, Sarvas M (1993) The PrsA lipoprotein is essential forprotein secretion in B. subtilis and sets a limit for high-levelsecretion. Mol Biol 8:727–737

Lang I, Gobel C, Porzel A, Heimann I, Feussner I (2008) A lipoxyge-nase with linoleate diol synthase activity from Nostoc sp. PCC7120. Biochem J 2:347–357

Lindsay MP, Skerritt JH (1999) The glutenin macropolymer of wheatflour doughs: structure-function perspectives. Trends Food SciTech 10:247–253

Appl Microbiol Biotechnol (2012) 94:949–958 957

Matui K (2006) Green leaf volatiles: hydroperoxide lyase pathway ofoxylipin metabolism. Curr Opin Plant Biol 9:1–7

Max OF, Richard TCl, John FT, William RD (1986) The lipoxygenasesin developing soybean seeds, their characterization and synthesisin vitro. Plant Physiol 82:1139–1144

NakayamaA, AndoK, Kawamura K,Mita I, Fukazawa K, Hori M, HonjoM, Furutani Y (1988) Efficient secretion of the authentic maturehuman growth hormone by Bacillus subtilis. J Biotechnol 8:123–134

Niisuke K, Boeglin WE, Murray JJ, Schneider C, Brash AR (2009)Biosynthesis of a linoleic acid allylic epoxide: mechanistic com-parison with its chemical synthesis and leukotriene A biosynthe-sis. J Lipid Res 7:1448–1455

Schneider C, Niisuke K, Boeglin WE, Voehler M, Stec DF, Proter NA,Brash AR (2007) Enzymatic synthesis of a bicyclobutane fattyacid by a hemoprotein–lipoxygenase fusion protein from thecyanobacterium Anabaena PCC 7120. Proc Natl Acad Sci USA48:18941–18945

Shewry PR, Popineau Y, Lafiandra D, Belton P (2001) Wheat gluteninsubunits and dough elasticity: findings of the Eurowheat Project.Trends Food Sci Tech 11:433–441

Soberman RJ, Harper TW, Betteridge D, Lewis RA, Austen KF(1985) Characterization and separation of the arachidonicacid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase(arachidonic acid 15-lipoxygenase) of human polymorphonu-clear leukocytes. J Bio Chem 260:4508–4515

Steffolani ME, Ribotta PD, Pérez GT, León AE (2010) Effect ofglucose oxidase, transglutaminase, and pentosanase on wheatproteins: relationship with dough properties and bread-makingquality. J Ceral Sci 51:366–373

Szymanowska U, Jakubczyk A, Baraniak B, Kur A (2009)Characterisation of lipoxygenase from pea seeds (Pisum sativumvar. Telephone L.). Food Chem 116:906–910

Vance RE, Hong S, Gronert K, Serhan CN, Mekalanos JJ (2004) Theopportunistic pathogen Pseudomonas aeruginosa carries a secre-table arachidonate 15-lipoxygenase. Proc Natl Acad Sci USA101:2135–2139

Weegels PL, Hamer RJ, Schofild JD (1999) Relationship betweenglutenin macropolymer content and quality parameter. J ofCereal Science 23:1–18

WahlstrÖm E, Vitikainen M, Kontinen VP, Sarvas M (2003) The extrac-ytoplasmic folding factor PrsA is required for protein secretion onlyin the presence of the cell wall in Bacillus subtilis. Microbiology149:569–577

Wang PZ, Doi RH (1984) Overlapping promoters transcribed by Bacillussubtilis σ55 and σ37 RNA polymerase holoenzymes during growthand stationary phases. J Biol Chem 259:8619–8625

Whitehead IM, Muller BL, Dean C (1995) Industrial use of soybeanlipoxygenase for the production of natural green note flavorcompounds. Cereal Foods World 40:193–197

Wu SC, Ye RQ, Wu XC, Ng SC, Wong SL (1998) Enhanced secretoryproduction of a single-chain antibody fragment from Bacillussubtilis by coproduction of molecular chaperones. J Bacteriol180:2830–2835

Wu SC, Yeung JC, Duan YJ, Ye RQ, Szarka SJ, Habibi HR, Wong SL(2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis:effects of molecular chaperones and a wall-bound protease on anti-body fragment production. Appl Environ Microbiol 68:3261–3269

Xue GP, Johnson JS, Dalrymple BP (1999) High osmolarity improvesthe electro-transformation efficiency of the gram-positive bacteriaBacillus subtilis and Bacillus licheniformis. J Microbiol Methods34:183–191

Ye RQ, Kim JH, Kim BG, Szarka S, Sihota E, Wong SL (1999)High-level secretory production of intact, biologically activestaphylokinase from Bacillus subtilis. Biotechnol Bioeng62:87–96

Zhang XZ, Cui ZL, Hong Q, Li SP (2005) High-Level expression andsecretion of methyl parathion hydrolase in Bacillus subtilisWB800. Appl Environ Microbiol 71:4101–4103

Zheng YX, Boeglin WE, Schneider C, Brash RA (2008) A 49-kDamini-lipoxygenase from Anabaena sp. PCC 7120 retains catalyt-ically complete functionality. J Biol Chem 8:5138–5147

958 Appl Microbiol Biotechnol (2012) 94:949–958