Effects of alkyl chain lengths of gallates upon enzymatic wool functionalisation

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<ul><li><p>Journal of Molecular Catalysis B: Enzymatic 67 (2010) 231235</p><p>Contents lists available at ScienceDirect</p><p>Journal of Molecular Catalysis B: Enzymatic</p><p>journa l homepage: www.e lsev ier .com</p><p>Effects nz</p><p>Kh.M. Ga MGroup of Molec versity</p><p>a r t i c l</p><p>Article history:Received 9 MaReceived in reAccepted 18 AAvailable onlin</p><p>Keywords:Alkyl-gallateLaccaseWoolAntimicrobialWater repellenAntioxidant</p><p>s ona ont prolly grn. Thn verth thon w</p><p>ertiesonali</p><p>1. Introdu</p><p>Wool isdue to its lightness, softness, warmness, and smoothness. How-ever, the complex chemical composition and architecture of thewool bres is responsible for some undesirable properties such ashigh water absorption, felting in mechanical wet operations, andlimited UV protection during use. Under proper temperature andhumidity thtion and prdamage, sktextiles leadof elasticityby washingwool goodsto keep cleagents.</p><p>For thesebe protectebres andantibacteriatextile goodant activitrepellence)properties.from their h</p><p> CorresponE-mail add</p><p>sphth thlic a</p><p>used as food additives due to their antioxidant activity [7,8].The alkyl gallates, having a molecular structure composed of ahydrophilic head (phenolic ring) and a hydrophobic alkyl chain,have also been reported to have antifungal and antibacterialactivity [9,10]. Though different studies investigated the relation</p><p>1381-1177/$ doi:10.1016/j.e wool materials could be a good medium for genera-opagation of microorganisms [1] that could cause brein irritations, and infections [2]. Microbial growth ons to odour development, discolouration, and even lossand tenacity [3]. The infestation can be removed onlyat boiling temperature where structural changes of themight occur. Moreover, the protein bres are difcultan and are easily damaged by conventional cleaning</p><p>reasons, thewoolmaterials of high-grade clothesmustd against microbial attack to prevent damage of thegrowth of pathogenic microorganisms. In addition tol nish, nowadays there is a great demand to produceds with multifunctional properties such as antioxi-y and self-cleaning properties. Hydrophobicity (waterof textiles is frequently associated with self-cleaningThe self-cleaning action of hydrophobic coatings stemsighwater contact angles.Water on these surfaces forms</p><p>ding author. Tel.: +34 937398761; fax: +34 937398225.ress: tzanko.tzanov@upc.edu (T. Tzanov).</p><p>between the hydrophobic chain lengths and alkyl gallates prop-erties, there is no clear consensus regarding the effect of the chainlength upon the antioxidant and antibacterial properties of gallates[1115].</p><p>In addition to the antioxidant and antibacterial activities, thealiphatic chain present in the chemical structure of these com-pounds would provide hydrophobic properties as well. Therefore,it could be expected that the modication of wool with alkyl gal-lates would provide a combination of self-cleaning, antimicrobialand antioxidant properties.</p><p>A proof-of-concept one-step enzymatic process using laccasein aqueousorganic media for covalent grafting of dodecyl gal-late (DG) on wool has been recently described [16]. Laccase isable to catalyse the oxidation of a wide range of aromatic com-pounds using molecular oxygen as electron acceptor and has beenreported to retain activity in some water miscible organic solvents,e.g. ethanol [17,18]. The reaction was carried out in an 80/20 (v/v,%) aqueousethanol mixture to balance the need for dissolving DGwith the need for maintaining the catalytic activity of laccase. Theenzymaticgraftingofdodecyl gallateonwoolprovidedamultifunc-tional textile material with antioxidant, antibacterial and waterrepellent properties.</p><p>see front matter 2010 Elsevier B.V. All rights reserved.molcatb.2010.08.011of alkyl chain lengths of gallates upon e</p><p>ffar Hossain, Mara Daz Gonzlez, Jos Mara Dagular and Industrial Biotechnology, Department of Chemical Engineering, Technical Uni</p><p>e i n f o</p><p>rch 2010vised form 13 August 2010ugust 2010e 24 August 2010</p><p>t</p><p>a b s t r a c t</p><p>The covalent grafting of alkyl gallateaqueousethanol mixture provided inidant, antibacterial and water repellendodecyl gallate have been enzymaticalength on wool functional modicatioan aqueousorganic medium has beegroups in the FTIR spectra, togetherwithe covalent grafting of ester gallatesrepellent as well as antioxidant propplay an important role on wool functi</p><p>ction</p><p>a natural protein material largely used for clothing</p><p>almostdirt wi</p><p>Gal/ locate /molcatb</p><p>ymatic wool functionalisation</p><p>onmany, Tzanko Tzanov </p><p>of Catalonia, C. Colom, 11, 08222 Terrassa, Barcelona, Spain</p><p>wool through a laccase catalysed reaction in 80/20 (v/v, %)e-step process a multifunctional textile material with antiox-perties. Gallic acid and its alkyl esters ethyl, propyl, octyl andafted on wool bres in order to study the effect of alkyl chaine capacity of laccase to oxidise these phenolic compounds inied by electrochemical techniques. The increase of CH2, CH3eXPS analysis of the enzymaticallymodied fabrics conrmedool. The result obtained in this work for antibacterial, watershow that the length of the alkyl chain of gallates molecule</p><p>sation. 2010 Elsevier B.V. All rights reserved.</p><p>erical droplets that readily roll away carrying dust andem [4,5].cid and its alkyl esters are natural compounds [6]</p></li><li><p>232 Kh.M. Gaffar Hossain et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 231235</p><p>Fig. 1</p><p>In the cuent aliphatioctyl gallateication ofwantioxidantcidate theproperties o</p><p>2. Materia</p><p>2.1. Reagen</p><p>Laccaseprotein/g soUK. Enzymdised per msodium tarradical (DP(GA), ethyldodecyl gacompoundssion werePanreac. Allcommercia</p><p>2.2. Electro</p><p>VoltammType III (Eclab GPES soout in a 20tion. The wsurface diamelectrodeswtrode, respeachieved byolish, Buehlwashing inwith laccaswas droppedry for 15m</p><p>2.3. Wool p</p><p>Woven 1washedpresurfactant C20:1 in a labpH 9) at 40</p><p>same bath ratio with 0.1mL/L of 30% H2O2 (0.1M Na2CO3, NaHCO3buffer pH 9) at 55 C for 1h.</p><p>zymatic treatment of wool</p><p>plesEtOHDG</p><p>us w0 rpmpleat tr thein anraturmin.d wa</p><p>urier</p><p>speed uing</p><p>All sond</p><p>ray p</p><p>exp(fro(AluperplineThed resstepstepwer</p><p>n Ar+er atere c</p><p>ater</p><p>conool faGmbd wahe coeposreme. Chemical structures of gallic acid and gallates compounds.</p><p>rrent work gallic acid and its alkyl esters with differ-c chain length, such as ethyl (EG), propyl (PG) and (OG)s were tested as laccase substrates for enzymatic mod-ool targeting increased hydrophobicity, and enhancedand antimicrobial properties. The objective was to elu-inuence of the alkyl chain length on the functionalf wool bres.</p><p>ls and methods</p><p>ts</p><p>(EC 1.10.3.2 Trametes sp. laccase, Laccase L603P; 0.125glid; 0.14U/mg protein) was provided by Biocatalysts,</p><p>e activity (U) was dened as mol of guaiacol oxi-in at pH 4 and 40 C (max 6400M1 cm1). Methanol,trate, hydrochloric acid, 1-diphenyl-2-picrylhydrazylPH)were purchased from SigmaAldrich. Gallic acidgallate (EG), propyl gallate (PG), octyl gallate (OG),</p><p>llate (DG), (chemical structures of all these phenolicin Fig. 1), baird parker agar and egg yolk tellurite emul-</p><p>provided by Fluka. Ethanol 96% was purchased fromchemicals used in this work were of the highest grade</p><p>lly available.</p><p>chemical experiments</p><p>etricmeasurementswere performed using aAutolaboChemie) potentiostat/galvanostat controlled by Auto-ftware version 4.9. All the experiments were carriedmL Metrohm cell with a three-electrode congura-orking electrode was a glassy carbon (GCE) with aeter of 3mm (Metrohm). The counter and referenceere platinum (Metrohm) andAg/AgCl (Metrohm) elec-ctively. The renewal of the glassy carbon surface waspolishing with 1.0 and 0.3m alpha-alumina (Microp-</p><p>2.4. En</p><p>Sambuffer/OG andprevio40 C, 3trol satreated</p><p>Aftesivelytempefor 30distille</p><p>2.5. Fo</p><p>FTIRcollectperformrange.corresp</p><p>2.6. X-</p><p>XPSSystemsourceplaced3d5/20.8 eV.selecte0.8 eV/0.1 eV/mentswith achambysis w</p><p>2.7. W</p><p>Theed wKruss,distille[19]. Tdrop dmeasuer) on amicrocloth polishing pad (Buehler), followedbyan ultrasonic Selecta bath for 2min. For the experimente a 5l sample of a 0.02gprotein/mL enzyme solutiond onto the polished surface of the GCE and allowed toin at room temperature.</p><p>reparation</p><p>00% wool fabric supplied by Lokateks (Slovenia) wasviously to the enzymatic treatmentwith 1g/L non-ionicotemol NI (Colorcenter, Spain) in liquor to good ratiooratory winch machine (0.1M Na2CO3/NaHCO3 buffer</p><p>C for 30min. Thereafter the fabric was bleached at the</p><p>2.8. Antioxi</p><p>The radiwas determdiphenyl-2method, apto test theor hydrogenincubated wroom temped wool wtriplicate anof wool fabric (5 g) were incubated in a 80/20 (v/v, %)solution with 14U/mL laccase and 1mM GA, EG, PG,</p><p>according to the optimised procedure described in ourork [16]. The reaction was allowed to proceed for 2h, atm in a laboratory dyingmachineAhiba (Datacolor). Con-s without enzyme and phenolic compound were alsohe above condition.enzymatic treatment the samples were washed exten-80/20 (v/v, %) buffer/EtOH solution for 1h at room</p><p>e, then with tap water and with distilled water at 80 CAfter the hot wash every sample were washed in coldter and dried at 50 C for 2h.</p><p>transform infrared spectroscopy (FTIR) analysis</p><p>ctra of the wool fabrics before and after treatment weresing a Perkin Elmer Paragon 1000 FT-IR spectrometer,100 scans for each spectrum over the 4004000 cm1</p><p>pectra were normalised against the peak at 1230 cm1</p><p>ing to the amide I group.</p><p>hotoelectron spectroscopy (XPS) analysis</p><p>eriments were performed in a PHI 5500 Multitechniquem Physical Electronics) with a monochromatic X-rayminium Kalfa line of 1486.6 eV energy and 350W),endicular to the analyser axis and calibrated using theof Ag with a full width at half maximum (FWHM) ofanalysed area was a circle of 0.8mm diameter, and theolution for the spectra was 187.5 eV of Pass Energy andfor the general spectra and 23.5 eV of Pass Energy andfor the spectra of the different elements. All measure-e carried out after cleaning by sputtering the surfaceion source (4 keV energy) in a ultra high vacuum (UHV)pressure between 5109 and 2108 Torr. The anal-arried out in triplicate.</p><p>repellence of wool</p><p>tact angles () of water on the unmodied and modi-bric were measured using a 1/2 CCD camera, DSA 100,H, Germany. The measurements were carried out withter as test liquid at 24 C and 65% relative humidityntact angles were followed during up to 600 s after theition on the surface of each sample. Three independentnts were recorded on each sample.</p><p>dant activity</p><p>cal scavenging activity of alkyl gallates modied woolined measuring the decrease in absorbance of 1,1-</p><p>-picrylhydrazyl radical (DPPH) at 515nm [20]. Thisplied for both solid and liquid samples, is widely usedability of compounds to act as free radical scavengersdonors [21]. Samples of modied wool (200mg) wereith 3mL of a 3105 M DPPH solution in MeOH at</p><p>erature in the dark for 1h. Solution containing unmodi-as used as a control. The experiment was carried out ind the results expressed as % inhibition of DPPH accord-</p></li><li><p>Kh.M. Gaffar Hossain et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 231235 233</p><p>ing to the following formulae [22]:</p><p>InhibitionofDPPH (%) =(</p><p>1 ASampleABlank</p><p>) 100 (1)</p><p>2.9. Antimi</p><p>The mosantimicrobiing to ASTMthe reductioquantitativeforming unAlkyl gallatewere incubacus aureuscontact wasnumber of sin terms ofing the numreduction rfollowing fo</p><p>Bacteria red</p><p>where A anunmodied</p><p>3. Results</p><p>3.1. Alkyl gmixture</p><p>Due toan approprdissolving tactivity ofof these co80/20 (v/v,matic modsolvent to wUsing the slates with vof cyclic volfor studyinferences induring theapplied to sexhibit an eistry to studpreviously r</p><p>The elecied can be svoltammogAll gallates378, 374, 34The decreasmost probaelectrode su</p><p>In the prpeak due tolic compouthe peak cuis enzymatcurrent relapresent at e</p><p>Cyclic voltammograms of 0.5mMPG 80/20 (v/v, %) 0.1M tartratetOH mixture pH 4.</p><p>zymatic grafting of GA and alkyl gallates on wool fabrics</p><p>ol fabric was treated with 1.0mM concentration of the dif-gallate compounds and laccase (14U/mL) in 80/20 (v/v, %)EtOHopt</p><p>l galwe</p><p>tOHat rongesool fm in</p><p>GA, Eincrng toisten3]. Fully in. SimincluhenoSchifin wmoded btagel afte</p><p>yclic voltammograms of 0.5mM PG alone () and in the presence of laccase). Scan rate 5mV/s.crobial activity</p><p>t commonly used quantitative method for testing theal activity of textiles is the Shake Flask method accord--E2149-01. This standard method was used to measuren rate in the number of colonies formed and provideddata, which could then be converted to average colony</p><p>its per millilitre (CFU/mL) of buffer solution in the ask.smodied and unmodied fabric swatches (0.5 g each)ted in 50mL suspension of Gram-positive Staphylococ-</p><p>at 37 C for 1h. The suspension both before and afterdiluted and cultured at 37 C for 24h to determine theurviving bacteria. The antimicrobial activity is reportedpercentage of bacteria reduction calculated by compar-ber of surviving bacteria before and after contact. The</p><p>ate in the number of colonies was calculated using thermula:</p><p>uction (%) =(</p><p>A BA</p><p>) 100 (2)</p><p>d B are the average number of bacteria at time 1h forandGA, EG, PG,OGandDGmodiedwool, respectively.</p><p>and discussion</p><p>allates oxidation by laccase in aqueousethanol</p><p>the insolubility of alkyl gallates in aqueous media,iate aqueous/organic mixture to balance the need forhe substrate with the need for maintaining catalyticlaccase is required to develop the enzymatic graftingmpounds on wool. In our previous work a process in%) aqueousethanol mixture was optimised for enzy-ication of wool with dodecyl gallate (DG). At thisater ratio laccase retained 7580% of its activity [16].ame reaction condition the laccase oxidation of gal-arying alkyl chain length was studied here by meanstammetry. UV/vis spectrophotometry was not suitableg the enzymatic oxidation of gallates because no dif-the UV/vis spectra of these compounds were observedreaction. Alternatively, electrochemistry can be easilytudy this reaction since thephenolic compoundsusuallylectrodic process, while the suitability of electrochem-y enzymatic reactions in aqueousorganic media waseported.trochemical behaviour of all gallate compounds stud-ummarized in the example in Fig. 2, where the cyclicrams (1st and 2nd scan) obtained for PG are presented.underwent irreversible oxidationprocesses (Epa) at 374,4 and 383mV for GA, EG, PG, OG and DG, respectively.e in the oxidation current recorded after the rst scan isbly due to phenolic compounds polymerisation on therface.esence of laccase, an important decrease in the anodiccatalytic instead of electrodic oxidation of the pheno-</p><p>nd was recorded for all gallate compounds (Fig. 3). Asrrent is related to the concentration, if one compoundically oxidised by laccase, a decrease in the oxidationted to the minor concentration of non oxidised specielectrode surface is expected.</p><p>Fig. 2.bufferE</p><p>3.2. En</p><p>Woferentbuffer/viouslydodecysurfaceH2O/Ewater</p><p>Chaed wspectru(linesC). Thespondiis consrics [2graduagraftedis alsothese peithergroups</p><p>Theacterizpercenof woo</p><p>Fig. 3. C(. The concentration of phenolic compound was pre-imised for the enzymatic modication of wool withlate (DG) [16]. Unxed...</p></li></ul>

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