Expression of themelA gene fromRhizobium etli CFN42 inEscherichiacoli and characterization of the encoded tyrosinase
Natividad Cabrera-Valladares, Alfredo Martınez, Silvia Pinero, Victor H. Lagunas-Munoz,Raunel Tinoco, Ramon de Anda, Rafael Vazquez-Duhalt,
Francisco Bolıvar, Guillermo Gosset∗
Departamento de Ingenierıa Celular y Biocatalisis, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico,Apdo. Postal 510-3, Cuernavaca, Mor. 62250, Mexico
Received 23 April 2005; received in revised form 6 July 2005; accepted 2 August 2005
ark pigment when growing in solid or liquid media containingl-tyrosine and copper. This pigment was identified as melanin by compt with analytical grade melanin using a spectrophotometric assay. Melanin was synthesized when recombinantE. coli cells were incubatet 30◦C; however, at 37◦C significantly less polymer was produced. The recombinant tyrosinase expressed intracellularly inE. coli wasurified 40-fold with a 25% yield from a cell extract by ammonium sulfate precipitation and ion exchange chromatography. With theurified tyrosinase, theKm for l-dopa andl-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for mctivity were 50◦C and 6.5–7.5, respectively. Activation energy for thermal inactivation (50.77 kJ/mol; usingl-dopa as substrate at pH 7) aalf-life values indicate a higher thermal stability ofR. etli tyrosinase in comparison with mushroom tyrosinase. Interestingly, for a ba
yrosinase, MelA showed an unusually higher activity forl-tyrosine than forl-dopa.2005 Elsevier Inc. All rights reserved.
Tyrosinase (monophenol monooxygenase EC 1.14.18.1)s a copper-containing enzyme involved in melanin syn-hesis. Using molecular oxygen, this enzyme catalyzes theydroxylation of l-tyrosine to l-dihydroxyphenylalaninel-dopa) (cresolase activity), its subsequent oxidation toopachrome (catecholase activity), which through a seriesf nonenzymatically catalyzed oxidoreduction reactionsolymerizes to form melanin. Tyrosinases contain a pair ofupric ions in the active site, bound to peptide motifs nameduA and CuB that contain conserved histidine residues
1]. Tyrosinases and the capacity to synthesize melaninave been described in many phylogenetic groups[2].
Among them, bacterial speciesPseudomonas maltophila,Rhizobium meliloti, R. leguminosarum, R. phaseoli, She-wanella colwelliana, Marinomonas mediterranea MMB-1,Bacillus thuringiensis subsp. Kurstaki and several specof the genusVibrio are known to be melanin produce[3–10].
The Gram-negative bacteriumR. etli establishes a symbotic relationship with the common beanPhaseolus vulgaris.This microbium induces the formation of nodules inroot, where nitrogen fixation takes place. This associabenefits the plant, especially when it is growing in nitropoor soils. It has been determined that the genes requirsymbiosis usually cluster in plasmids (symbiotic plasmor specific chromosomal regions (symbiotic islands)[11].SeveralRhizobium strains can produce the dark pigmmelanin, and in the case ofR. etli, it produces melanin asresult of nodule injures[12]. Genetic studies have establish
N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779 773
that genes required for melanin production (mel) are usuallypresent in plasmids, both symbiotic and nonsymbiotic[4].Despite the genetic location of somemel genes in symbioticplasmids, melanin production is not required for symbioticnitrogen fixation[13]. At the present time, no function formelanin has been established in the rhizobia.
Tyrosinases and melanins have been studied due to theirpotential industrial applications. Tyrosinases from differentbiological sources have been utilized for the synthesis ofl-3,4-dihydroxyphenylalanine (l-dopa) and the removal ofphenolic compounds from wastewaters[14,15]. On the otherhand, microbial production of melanins has been achieved inliquid cultures of melanogenic microorganisms and recom-binantE. coli [3,4,16]. Due to their chemical composition,melanins have physicochemical properties that allow themto act as UV absorbers, cation exchangers, amorphous semi-conductors, X-ray and�-ray absorbers[17,18].
Most of the reported works on the characterizationof tyrosinases have focused on enzymes from eukaryoticorigins. More recently, tyrosinases from bacterial originshave been reported and biochemically characterized[19,20].These studies provided useful information about the bio-chemical diversity in this family of enzymes and their poten-tial for application in industry.
The complete nucleotide sequence of the symbioticplasmid p42d fromR. etli CFN42 has been recently reported(s -s uctd ses.Ic di zest tialp antE Ther luesa nm nasef umo
2
2
E sedioc toneac n-t aCl.L n of
thetac promoter present in plasmid pTrc99A and derivatives,the gratuitous inducer isopropyl-�-d-thiogalactopyranoside(IPTG) was added to a final concentration of 0.1 mM.Ampicillin was used at a final concentration of 200�g/mLin both solid and liquid media cultures ofE. coli strainsharboring plasmid pTrc99A or pTrcmelA. Casein solidmedium used for melanin production contained, per liter, 1 gglucose, 5 g NaCl, 0.1 g CaCl2, 10 g bactotryptone, 15 g agar,5 g casein, 0.4 mg CuSO4 and 2 gl-tyrosine. Liquid mediumused for melanin production cultures was minimal mediumM9 supplemented with 2 g/L glucose, 0.4 g/Ll-tyrosine,40�g/mL of CuSO4, IPTG and ampicillin. Cultures for thesynthesis of apotyrosine were performed in M9 mediumsupplemented with 2 g/L glucose, 0.4 g/Ll-tyrosine, IPTGand ampicillin. Cultures for comparing melanin productionat 30 and 37◦C were performed in 250 mL baffled shakeflasks.
2.2. Plasmid construction
Total DNA was extracted fromR. etli CFN42 grown inPY medium and used as a template to amplify themelA geneby PCR using primers 5melA (5′CCG AAC GTC CAT GGCGTG GCT GGT CGG C′3) and 3melA (5′CCG GAG CCCGGG TGT TAG GCG GAC AC′3) containingNcoI andXmaI restriction sites, respectively. Primer 5melA generatesa eb thes hangei oteins red att ifiedm bed asp ockt Thep step( ionae n-s tar thep withTt etentE tesw ralc ye serts tionf q FSD asedS emsM ingt
accession number: NC004041) [11]. Analysis of theequence from p42d revealed the presence ofmelA (accesion number: AAM54973), whose predicted protein prodisplays sequence similarity to known polyphenol oxida
n the present work, themelA gene fromR. etli CFN42 wasloned and expressed inE. coli. The MelA protein producen E. coli was found to have tyrosinase activity that catalyhe synthesis of melanin usingl-tyrosine as substrate. Parurification of the MelA tyrosinase produced in recombin. coli enabled biochemical and kinetic analyses.
esults showed that MelA tyrosinase has pH optimum vand Km for l-dopa andl-tyrosine similar to other knowicrobial tyrosinases. On the other hand, the tyrosi
rom R. etli displayed a relatively high-temperature optimf 50◦C.
. Materials and methods
.1. Bacterial strains and growth conditions
Strain CFN42 ofR. etli was a kind gift from G. Davila[11].. coli strain XL1 Blue (Stratagene, La Jolla, CA) was u
n cloning experiments and strain W3110[21] for expressionf genemelA. PY medium used for growingR. etli CFN42ontained per liter, 5 g yeast extract, 10 g casein pepnd 10 mL of 0.7 M CaCl2. Growth media used duringmelAloning was liquid and solid Luria broth (LB) medium, coaining per liter, 10 g tryptone, 5 g yeast extract and 10 g NB plates also contained, per liter, 15 g agar. For inductio
n NcoI restriction site within themelA coding sequency changing the TTG start codon to ATG and changingecond codon CCG to GCG. The nucleotide sequence cn the start codon does not cause a change in the prequence; however, a proline to alanine change occurhe second position in the translated product of the modelA gene. For simplicity, the modified gene will stillesignated asmelA and its protein product MelA. PCR werformed using the Elongase kit (Invitrogen) in a multibl
hermocycler (Robocycler Gradient 96, Stratagene).rotocol used consisted of an initial denaturalization95◦C for 3 min), followed by 30 cycles of denaturalizatt 95◦C for 1 min, primer annealing at 57◦C for 1 min, andlongation at 68◦C for 3 min, plus an additional final exteion step of 10 min at 68◦C. The 1854 bpmelA PCR producnd plasmid pTrc99A were digested withNcoI and XmaIestriction enzymes. DNA was purified from each ofrevious reactions, mixed and an aliquot was incubated4 DNA ligase and incubated 15 h at 16◦C. An aliquot of
he ligation reaction was used to transform electrocomp. coli XL1Blue; transformants were selected in LB plaith ampicillin. Plasmid DNA was extracted from seveolonies, digested withNcoI and XmaI and analyzed blectrophoresis in 1% agarose gels in order to verify inize. DNA from a plasmid containing the expected restricragment pattern was purified and sequenced by Taye Terminator Cycle Sequencing Fluorescence-Bequencing using a Perkin-Elmer/Applied Biosystodel 377–18 sequencer. A plasmid clone hav
774 N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779
used to transformE. coli W3110 to generate strainW3110/pTrcmelA.
2.3. Tyrosinase purification
For purification purposes, strain W3110/pTrcmelAwas grown in 10 L of M9 media and supplemented with10 g/L glucose, 100�g/mL ampicillin, 0.1 mM IPTG,40�g/mL CuSO4 and 0.4 g/L tyrosine in a 14-L fer-mentor (Microferm-Fermentor, New Brunswick Co., NJ)equipped with three Rushton impellers at 30◦C, 0.5 vvm,pH 7.0 and 250 rpm. After 20 h of cultivation time, whenoptical density at 600 nm (OD600) reached 2, cells wereharvested by centrifugation in a continuous Sharpless mini-centrifuge (Sharpless super-centrifuge) at 150 mL/min and13,000× g.
All the following purification steps were carried outat 4◦C. The cell pellet from the 10-L fermentor culturewas suspended in 50 mM potassium phosphate buffer pH7.0 (PB7) to get an OD600 close to 310, and treated threetimes in a Pressure Cell Press (French Press, ThermoSpec-tronic, NY) at 1240× 105 Pa. Cell debris were removed bycentrifugation (27,200× g, 45 min). Powdered ammoniumsulfate was added to the supernatant to 20% saturation,centrifuged at 20,000× g for 15 min. The precipitate wasdissolved in 10 mM potassium phosphate buffer pH 8.5( utionwp bedp ) inP ingt ateda mLu reC ne( reda
2
pect at4 me( te( cen-t ed to0 erea mLc uili-b witfi mef e ofa d oui plot-t ed ac ence
of the enzyme. One unit of enzyme activity was defined as1�mol of dopachrome formed per minute at 30◦C. The pro-tein concentration was measured by the dye binding methodof Bradford using a microassay procedure (Bio-Rad) andbovine serum albumin as standard.Km was determined by theLineweaver–Burk plot method usingl-dopa andl-tyrosinein the ranges of 0.05–15 mM and 0.05–2 mM, respectively,at pH 7.0 and 30◦C. Tyrosinase profiles assays for tem-perature and pH were conducted from 20 to 60◦C (at pH7.0) and from pH 4.0 to 9.0, at 30◦C (using three differ-ent buffers, as indicated in the Section3, at a concentrationof 50 mM). Thermostability assays were conducted at 30,37, 40, 45 and 50◦C, pH 7.0. Incubation times for theseassays ranged from 2 to 24 h. The relative reaction rates withsubstratesl-tyrosine, l-tyrosine ethyl ester,N-acethyl-l-tyrosine, caffeic acid and catechol were measured at 475 nmusing 1 mM solutions of each compound in 50 mM PB7at 30◦C.
2.5. Analytical methods
Culture growth was spectrophotometrically determinedas optical density at 600 nm (DU-70, Beckman Instruments,Inc., Fullerton, CA) and converted to dry cell weight perliter using a calibration curve (1 OD600= 0.37 gDCW/L).
cen-ble
ersa,both,
in,w).sass
ofd
rdeditateded
swithetercon-tionifiedume-
PB8.5), and dialyzed against the same buffer, this solas applied to a HiQ (Bio-Rad) column (1.5 cm× 22.5 cm)reviously equilibrated with PB pH 8.5. The adsorroteins were eluted by a linear gradient of NaCl (0–60%B pH 8.5 at a flow rate of 1.2 mL/min. Fractions contain
yrosinase activity were pooled, dialyzed and concentrgainst 10 mM potassium phosphate buffer pH 7.0 to 5sing a Stirred Ultrafiltration Cell (Model 8050, Millipoorporation, MA) with a 30 kDa ultrafiltration membra
Millipore Corporation, MA). The enzyme extract was stot−20◦C.
.4. Tyrosinase assay and kinetic studies
Catecholase activity of tyrosinase was determined srophotometrically (BioMate 5, ThermoSpectronic, NY)75 nm[22] and 30◦C by the appearance of dopachroε = 3600 M−1 cm−1) using 3 mM l-dopa as substraResearch Organics, Inc., OH), in PB7. The final conration of MelA tyrosinase in these assays correspond.022 U/mL. Twenty microliters of enzyme extracts wdded to a total volume of 1 mL reaction mixture in 1uvettes. Before the addition of enzyme, buffers were eqrated to the assay temperature and sparged for 0.5 minltered air at a flow rate of 100 mL/min. Rate for dopachroormation was defined as the slope of the linear zonbsorbance versus time. All determinations were carrie
n triplicate and the mean and standard deviation wereed. Reaction rates in the absence of enzyme were usontrol and were subtracted from the rate in the pres
-
h
t
s
Optical density at 400 nm was used for melanin contration determination. Bacterial cells interfere with solumelanin spectrophotometric determination and vice vhence, OD at 400 and 600 nm were measured forculture with cells and cell free centrifuged broth. OD400of cell free culture broth, after centrifugation (5 m6000× g) was used for melanin quantification (see beloSubtraction of OD600 from the culture with cells minuOD600 of the cell free culture broth was used for biomdetermination.
2.6. Melanin purification and quantification
Purification of melanin produced in liquid culturesstrain W3110/pTrcmelA was performed following a modifieprocedure from della-Cioppa et al.[16]. Briefly, 500 mL ofculture broth containing melanin was centrifuged (6000× g;10 min; room temperature), the cell pellet was discaand the melanin present in the supernatant was precipby lowering the pH to 2 with HCl 6N. The precipitatmelanin was concentrated by centrifugation (12,000× g;12 min; room temperature), dried at 100◦C for 72 h, andstored as a powder in a sealed flask at 4◦C. Dried melaninwere redissolved in water at pH 10, pH was adjusted to 7HCl 6N, and OD400 was measured in a spectrophotom(Beckman DU70), using a 1 cm path length cuvette, atcentrations ranging from 0.01 to 0.05 g/L. The concentraof melanin was calculated from standard curves of purmelanins and using as reference chemically produced elanin (Sigma Chemical Co.).
N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779 775
3. Results and discussion
3.1. Expression of the R. etli melA gene in E. coli andcharacterization of the pigment produced
The melA gene was amplified by PCR from totalR. etliDNA and placed under transcriptional control of the strongtrc promoter present in theE. coli expression vector pTrc99A.The resulting recombinant plasmid pTrcmelA was introducedintoE. coli strain W3110. In strain W3110/pTrcmelA, expres-sion ofmelA is induced by the addition of IPTG to the culturemedia. E. coli strain W3110/pTrcmelA and control strainW3110/pTrc99A were streaked in casein plates containingIPTG plusl-tyrosine and incubated at 30 and 37◦C. After24 h of incubation at 30◦C, a dark and diffusible pigment wasobserved in the area where W3110/pTrcmelA was growing,whereas no pigment was produced by strain W3110/pTrc99A(Fig. 1). After 48 h incubation at 37◦C, a very small amountof dark pigment was observed for strain W3110/pTrcmelA,whereas no pigment was detected in strain W3110/pTrc99A(data not shown).
To characterize the pigment produced by strainW3110/pTrcmelA, shake flask experiments were per-formed at 30◦C in M9 liquid medium supplemented withIPTG, CuSO4 andl-tyrosine. The absorbance spectrum inthe range of 200–800 nm was determined for the pigmenti of ae iden-t nmt enta g toE thatts in,
FgaW
Fig. 2. Characterization of pigment produced byE. coli W3110 express-ing the R. etli melA gene. Absorbance spectra using a concentration of0.01 g/L of eumelanin standard (Sigma Co.) and pigment produced by strainW3110/pTrcmelA.
and thatl-tyrosine is a substrate of MelA. Therefore, thisenzyme can be considered to be a tyrosinase.
To determine the amount of melanin produced byW3110/pTrcmelA in liquid medium, shake flask cultureswere performed at 30 and 37◦C. Media used was M9 supple-mented with IPTG, CuSO4, 2 g/L of glucose and 0.4 g/L ofl-tyrosine. At 30◦C, melanin started accumulating from thestart of the culture. After 12 h, when the culture entered thestationary phase, a large increase in the rate of melanin pro-duction was observed (Fig. 3). This second melanin produc-tion phase lasted 24 h, resulting in the production of 0.41 g/Lof melanin, corresponding to a g melanin/gl-tyrosine yieldof 102.5%. In cultures grown at 37◦C, melanin started accu-mulating at a low rate from the start of the culture, but nosignificant increase in rate was observed upon entry into thestationary phase. This culture produced a melanin concentra-tion of 0.06 g/L after 36 h of culture time.
It should be pointed out that 0.46 g of melanin are expectedto be produced from 0.4 g ofl-tyrosine, which correspondsto a theoretical yield of 100% due to the incorporation of oneatom of oxygen to thel-tyrosine molecule as a result of thecresolase activity of tyrosinase.
Fg ksc n:( ):(
solated from these cultures. It was compared to thatumelanin standard (Sigma Co.) and found to be nearly
ical (Fig. 2). Furthermore, the ratios of absorbance at 400o concentration were determined for the isolated pigmnd the eumelanin standard with results correspondin1%1 cm of 1.6 and 1.5, respectively. These results indicate
he pigment produced by strain W3110/pTrcmelA is melaninynthesized froml-tyrosine, also known as eumelan
ig. 1. Melanin production byE. coli W3110 expressing theR. etli melAene. Cells were streaked on casein plates supplemented withl-tyrosinend incubated 48 h at 30◦C. (A) E. coli W3110/pTrc99A; (B)E. coli3110/pTrcmelA.
776 N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779
3.2. MelA protein purification
MelA tyrosinase was purified from a culture ofW3110/pTrcmelA grown in a 10-L fermentor at 30◦C. Theenzyme was purified by ammonium sulfate treatment, ionicexchange chromatography and concentrated by ultrafiltra-tion. These methods allowed 40-fold tyrosinase activitypurification with a 25% recovery. The final MelA tyrosinaseactivity corresponded to 1.08 U/mL.
3.3. Temperature and pH effect on MelA l-dopa oxidaseactivity
The effect of temperature and pH on thel-dopa oxidaseactivity of MelA tyrosinase were determined with the par-tially purified enzyme. The effect of pH onl-dopa oxidationat 30◦C is shown inFig. 4A. It can be observed that theactivity optimum lies near pH 7.0 (in the range of 6.5–7.5).Relativel-dopa oxidase activity decreased to 50% or lessof the maximum at pH values below 6 or above 8.5. Thetyrosinase from the Gram-negative thermophilic bacteriumThermomicrobium roseum has been extensively character-ized. The pH optimum for this enzyme is 9.5[22]. This valueis similar to the pH optimum of 9.0–9.5, determined for thetyrosinase from pine needle[23]. Like MelA from R. etli, thet
Ftiab
Table 1Biochemical properties of MelA tyrosinase
pH for maximum activity 6.5–7.5Temperature for maximum activity (◦C) 50
Temperature stability (half-life in hours) at pH 730◦C Stable for at least 24 h37◦C 3.940◦C 3.245◦C 2.050◦C 1.9
glaucescens, hamster, mouse and human have a pH optimumnear neutrality[24–27].
The temperature profile of MelA tyrosinase onl-dopa oxi-dation activity is shown inFig. 4B. The maximum relativeactivity was observed at 50◦C at pH 7.0. This value is iden-tical to that determined for human (50◦C) and similar to thatfor pine needle (55–60◦C) tyrosinases[23,27]. On the otherhand, the optimal temperature for MelA tyrosinase activity issignificantly higher that that determined for mouse and ham-ster (37◦C) or chicken (35◦C) tyrosinases[25,26,28]. Theoptimal temperature for MelA is higher than most character-ized tyrosinases, however, it is lower than that reported forthe tyrosinase from the extremely thermophilic bacteriumT.roseum (70◦C) [22].
3.4. MelA thermal stability
The temperature profile of activity was determined at pH7.0 by incubating the reaction at different temperatures. Itwas determined that the tyrosinase fromR. etli is stable for atleast 24 h at 30◦C, and its half-life was 1.87 h at the tempera-ture of maximum activity (50◦C). Table 1shows a summaryof MelA biochemical properties. From this data, an activationenergy value for thermal inactivation of 50.77 kJ/mol (usingl-dopa as substrate at pH 7) was calculated for MelA tyrosi-nase, which is one order of magnitude higher than the one form 7)[ ess om-p t theR onsw
tureo tureo nt tya vedr em ure,w ed at3
yrosinases from the Gram-positive bacteriumStreptomyces
ig. 4. pH and temperature profiles for catecholase activity ofR. etli MelA
yrosinase. (A) Relative activity at different pH values, using 3 mMl-dopan 50 mM acetate, phosphate and Tris–HCl buffers at 30◦C. (B) Relativectivity at different temperatures, using 3 mMl-dopa in 50 mM phosphateuffer pH 7.0.
3
ypek he
ushroom tyrosinase (usingl-tyrosine as substrate at pH15]. Activation energy for inactivation and half-life valuuggest a higher thermal stability of MelA tyrosinase in carison with mushroom tyrosinase, hence, it is likely tha. etli tyrosinase could be useful for industrial applicatihere higher thermal stability is an advantage.The experiments to determine the effect of tempera
n MelA tyrosinase stability revealed that at a temperaf 37◦C or higher, MelA stability is significantly lower tha
hat observed at 30◦C. The lower MelA tyrosinase stabilit 37◦C provides a partial explanation for the obsereduced capacity of strain W3110/pTrcmelA to synthesizelanin in solid media or liquid cultures at this temperathen compared to cultures of the same strain perform0◦C (Fig. 3).
.5. Substrate affinity
MelA tyrosinase showed typical Michaelis–Menten tinetics with l-dopa andl-tyrosine as substrates. T
N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779 777
Km value of MelA for l-dopa was 2.44 mM (±0.4751).This value is one order of magnitude higher than the onereported for human tyrosinase (0.36 mM)[27]. In the caseof tyrosinases from microorganisms, the reportedKm val-ues forl-dopa are: 0.18 mM forThermomicrobium roseum,0.88–1.09 mM forNeurospora crassa and 5.77 mM forStrep-tomyces glaucescens [22,24,29]. TheKm value of MelA forl-tyrosine was 0.19 mM (±0.0062). This is similar to thereportedKm values with this substrate in the case of humantyrosinase (0.17 mM) andS. glaucescens (0.41 mM), andlower than the value reported for the tyrosinase fromVibriotyrosinaticus (3.1 mM) [19,24,27]. Therefore, these resultsshow that MelA tyrosinase displays aKm for l-dopa andl-tyrosine similar to that of enzymes from other microorgan-isms.
3.6. MelA substrate specificity
MelA tyrosinase extract was tested for oxidase activityagainst several phenolic compounds, including: (a) mono-hydroxyphenols: resorcinol,ortho-hydroxybenzoic acid,vanillic acid, syringic acid, ferulic acid,l-tyrosine,l-tyrosineethyl ester andN-acethyl-l-tyrosine; (b) dihydrophenols:l-dopa, caffeic acid and catechol; and (c) the trihydrophenol:pyrogallol. The assay was performed using 1 mM of sub-s toreda wasdh lica ase(d maticp n2t ing to1 witho a-t e2 vedf
These results provide an insight on the substrate specificityof MelA tyrosinase. It displayed characteristics similar tothose of other known tyrosinases, i.e. a high specificity forthe monohydroxyphenoll-tyrosine and the dihydroxyphenoll-dopa. However, the relative substrate specificity of MelAtyrosinase differs from that of the tyrosinases fromT. roseumandS. glaucescens [22,24]. The tyrosinases from these bac-teria oxidize several different mono and dihydroxyphenols.However, in contrast to the MelA tyrosinase, they display arelatively low activity towardsl-tyrosine, when compared toother monophenol substrates. Furthermore, MelA tyrosinasealso oxidized several 4-hydroxyphenyl derivatives (pyru-vate, acetate, acetamine, lactate and glycine) developing,at 24 h and room temperature, yellow colors with differentintensities and no precipitate. Also, tyrosine hydroxamateand phenol were oxidized, but in this case a pink colorwas developed (data not shown). These results indicate thatMelA tyrosinase can recognize severalpara-hydroxyphenylderivatives.
The MelA enzyme oxidized thel-tyrosine derivativesl-tyrosine ethyl ester andN-acethyl-l-tyrosine. The melaninsproduced from oxidation of these compounds display colorsdifferent from eumelanin obtained froml-tyrosine andl-dopa (Table 2), and the one obtained froml-tyrosine ethylester also precipitates forming a dark yellow color. Thesesynthetic forms of melanin, having different colors, couldh
3i
d byg gIr icol( ellsw wasd dt after6 ty
TR binantR. etli es
ctivity (
081.039 w)
.003
038.001.003
uring thndent of avera
aof inc
r 24 h o
trate, incubated for 24 h at room temperature and monit 475 nm. Under these conditions, no oxidation activityetected with the following substrates: resorcinol,ortho-ydroxybenzoic acid, vanillic acid, syringic acid, ferucid and pyrogallol. Substrates oxidized by MelA tyrosinl-tyrosine,l-tyrosine ethyl ester,N-acethyl-l-tyrosine,l-opa, caffeic acid and catechol), were assayed for enzyolymer formation activity (Table 2) as described in Sectio. For substratesl-tyrosine andN-acethyl-l-tyrosine, lagimes before oxidase activity were detected, correspondand 10 min, respectively. No lag times were observedther substrates. In comparison withl-dopa, the highest rel
ive activity was observed withl-tyrosine, displaying a valu.1-fold higher. Low values of relative activity were obser
or N-acethyl-l-tyrosine, catechol and caffeic acid (Table 2).
able 2elative polymer formation activities and melanins generated by recom
a Lag time before first order change of absorbance measurement db Relative polymer formation activity. Average values of three indepell cases.c Color developed, in cuvettes used for enzymatic assays, after 24 hd Precipitate formation, in cuvettes used for enzymatic assays, afte
ave commercial applications.
.7. Copper-chaperone is not essential for copperncorporation into MelA
MelA tyrosinase apoenzyme lacking Cu was obtainerowing strain W3110/pTrcmelA in M9 medium containin
PTG andl-tyrosine but lacking CuSO4. Cells from theesulting liquid culture were harvested and chloramphen30�g/mL) was added to arrest protein translation. Cere ruptured using sonication. No tyrosinase activityetected in the resulting cell extract. CuSO4 was adde
o the extract and tyrosinase activity was measured0 min of incubation at 30◦C. Tyrosinase specific activi
tyrosinase expressed inE. coli W3110/pTrcmelA against different substrat
�A475/min) cColor dPrecipitate
Dark brown Yes (black)Yellow Yes (dark yello
Peach No
Dark brown Yes (Black)Peach NoPeach No
e enzymatic assay.measurements are presented, standard deviation was lower than 6%ge in
ubation at room temperature.f incubation at room temperature.
778 N. Cabrera-Valladares et al. / Enzyme and Microbial Technology 38 (2006) 772–779
corresponded to approximately 8% of the activity determinedfrom cultures grown in the presence of Cu. These resultsshow that Cu can be incorporated to the MelA protein afterit has been synthesized. However, full activity cannot berestored, indicating that most of the incorporation of Cu inMelA apotyrosinase should occur during its synthesis. Cop-per incorporation to some apotyrosinases has been reportedto be dependent on the activity of copper-chaperones.This process has been well characterized inStreptomycesand in mammalian cells, whereas less in known in fungiand plants [1]. Since MelA tyrosinase is active whenexpressed inE. coli grown in medium containing CuSO4,it can be inferred that a copper-chaperone fromR. etliis not required for proper copper incorporation to MelAapotyrosinase.
4. Conclusions
The biochemical characterization of MelA performed inthis work, having an unusually higher activity and affinity forl-tyrosine than forl-dopa when compared to other bacterialtyrosinases, suggests that this enzyme has the potential forapplications in industry. Further studies with this enzyme,obtained by heterologous expression inE. coli or isolatedfrom its native host, should enable a better understanding ofi fe
A
-230a gM et .T ssis-t nf rt,M ndM ndc
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of a
s J.-ssion
[5] Hawkins FK, Johnston AW. Transcription of aRhizobium legu-minosarum biovar phaseoli gene needed for melanin synthesis isactivated bynifA of Rhizobium and Klebsiella pneumoniae. MolMicrobiol 1988;2:331–7.
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ts biochemical properties and its role in the biology oR.tli.
cknowledgement
This work was supported by research grants NCnd 43243 from Consejo Nacional de Ciencia y Tecnoloıa,exico and DGAPA-PAPIIT, UNAM grant IN205005-2. W
hank Guillermo Davila for the gift ofR. etli strain CFN42he authors would like to acknowledge the technical a
ance of Mercedes Enzaldo, Eugenio Lopez and Paul Gaytaor primer synthesis and Jorge Yanez for sequencing suppoario Caro and Veronica Albiter for Pilot Plant support, aarıa E. Rodrıguez for her advise in the purification a
haracterization steps.
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