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REFEREED MANUSCRIPT]Proc. Fla. State Hart. Soc. 121:311-314. 2008.

Biosorption Properties of Citrus Peel DerivedOligogalacturonides, Enzym e-modified Pectin and PeelHydrolysis ResiduesRANDALL G. CAMERON' * , GARY A. LUZIO L ,WILBIJR W . WIDMER1,

AND MUHAMMAD IQBAL2'U.S. Department of Agriculture, A gricultural Research Service, Citrus and Subtropical ProductsLaboratory, 600 A ve. S. N .W . W inter Haven, FL 33881

2 Biotechnology and Food Research Centre, PCSIR Laboratories Complex, Ferozepur Road,Lahore-54600, Pakistan

AonIrIoN1. INDEX WORDS. ion exchange, orange p eel, pectin niethylesterase. waste water retnediationA citrus processing industry priori ty is obtaining adde d value from fruit peel . Appro ximately one -half of each pro cessedfruit is added to the waste stream . Peel residue is compose d mainly of water ( .80%), the remaining 20% (solid Fraction)consis t s of pec t in , solub le sugars , ce l lu lose , prote ins , phen ol ics , e tc . Viewing these con st i tuents in l ight of e xploi t ingpotent ia l functional ity and creat ing added value a t the same t ime as diver t ing mater ia l away f rom the feed m i ll or landfill, pectin provides enormous op portuni ty . To c rea te a new technology cente red on pec t in s t ruc ture and concomitantfunct ional i ty , we a re inves t igat ing m ethods to precisely eng ineer pect in s t ructure and corre la te i t to funct ion. A valu-able pec t in func t iona l i ty , resul t ing f rom i ts polyanionic charac te r , i s i t s b iosorpt ion capab i l it ies . In the past severa lyears we h ave deve loped ana ly t ica l techniques and biochemica l methods to enzym at ica l ly modify pec t in s t ruc ture ,charac terize these structural al terat ions and determ ine their effect on rheolog y and calcium sen sit ivity. Here w e presentdata on the biosorpt ion proper t ies of mo dif ied pec t ins and pec t in f ragments using lead as a model ca t ion . The great-est Pb sorption capacity (Mean = 373.3 mgg-'; S.E. = 1.595 P> 0.001) was observed in the Medium DP s ize-class ofgalacturonic acid ol igom ers . A com par i son of enzymat ical ly demethylated (b lockw ise) hom ogalacturonans indicatedtha t the 60% and 50% D M pec t ins t rea ted a t pH 4.5 had a s ignif icant ly grea te r sorp t ion capac i ty than h igher DM orpH 7.5 treated samples.

Polysaccharidcs, the most abundantly represented class ofhiomolccules in citrus peel (Grohmann et al., 1995), have wellestablished functionality that is amenable for manipulation andconversion to value-added components. Of the polysaccharidespresent in pee l , pec t in offe rs the grea test poten t ia l for enzym at icor chemical conversion to engineer desired properties, i.e. ionexchange capac i ty , in to pee l , p roces s ing re s idues o r o the r was tes t r e am componen t s .Galac turor i ic ac id (GA ). the major sugar found in c i t rus pec t in(Cam eron e t al . , 2008; Ridley et al . . 2001), is locate d largely in thel inear homoga lacturonan region (HG), pect in 's dominant s t ructuraldoma in . A var iable proport ion of these GAs are m ethyl este r i f ieda t the C6 posi t ion , e ffec t ive ly ma sking the nega t ive charge of thecarboxyl group. Pectin functionality is associated with the totalamou nt of methyla ted GAs (degree of methyla t ion; DM) and thedis t ribu t ion of the m e thy la ted and unmethy la ted GA s in the HGregions (Cam eron e t a l . , 2008; Luzio and Cameron , 2007, 2008;W il la ts e t a l ., 2001; ). The demethyla ted GA s may ha ve e i ther anordered or random dist r ibut ion . With an ordered d is t r ibut ion thesize of a contiguous block of demethylated GAs (demethylatedblock DEMB) will vary and its degree of polymerization (DP)can range to g rea te r than 50 GAs in l eng th .W e w o u l d li k e to t h a n k M r . S t e v e n K a u f f m a n , M s. Elena Stewart and M s. SandraMatlack for expert technical assistance.*Corresponding author: email: randall.cameronarS.U5da.gOV: phone: (1163)293-4133. ext. 124

A func t iona l proper ty re la ted to the nega t ive charge impartby the ionized carboxyl group of GA is its ability to interawith heavy m eta l ca t ions of ten assoc ia ted w i th var ious indust rprocesses that require remediation. Both DM and the DP opolygalacturonic acid (PGA) oligorner (equivalent to a DEMin a pec t in molecule) have been show n to e ffec t the i r in te rac twith cations (Kohn. 1987; Malovikova and Kohn, 1979, 198Previous s tudies tes t ing the ef fect of pect in DM on m etal chela thave used base to saponify the pectin and reduce the DM fri t s in i tia l h igher leve l (Kohi i and Luknar . 19 75; Malovikova aKohn, 1979. 1982). Saponification, or alkaline demethylatioequivalent to a degree of enzyme processivity equal to onesingle GA is demethylated with no contiguous GAs being dmethyla ted) , resul ts in a random dist r ibut ion of methyla ted Gand an abundance o f smal l DEM Bs (Cameron c i a l . . 2008) . Ko(1987) has demonstra ted tha t o l igomer DP affec ts the bindingca t ions . the d egree o f a s soc ia tion inc reas ing wi th D P.Pectins demethylated with plant pectin methylesteraswhich demethylate processively (contiguous methylated Gare demethylated until an interference point or intrinsic limireached) producing much larger DEMBs. have not been tesfor the i r abi l i ty to bind m eta l ions. In order to eva lua te the e fof an ordered distribution of DEMBs. differences in averablock s ize (5S ), and number o f DE MBs pe r molecu le o f pe(BN), treated samples were tested for their effectiveness inche la t ion . Samples consis ted of a se r ies of dcmethyla ted pec tf rom 80% to 50% D M gen erated a t e i ther pH 4.5 or 7.5 (Came

Proc, Fla. State Hart. Soc. 121: 2008.

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ci al.. 200>. a scric, of ilarro\v-rallic SILCclass of PGA olio-mers produced by limited digestion with endo-polygalacturonase(Cameron et al., 2005) as well as individual, purified oligomers.and peel materials in which the pectin had been released and thendemcthylated. Additional samples tested were a den-icthylated,commercially extracted pectin; a non-calcium-sensitive-pectin(NCSP) and a sample of hydrolyzed, fermented citrus peel inwhich the ethanol had not been stripped.

Materials and MethodsMATERIALS. Unless other wise noted all chemicals were pur-

chased from Sigma-Aldrich (St. Louis. MO) and at least ACSreagent grade.Biosorbent preparation

DENIETHYLATION suEs. Pectin samples and methods for thepectin demethylation series at pH 4.5 and 7.5 were describedpreviously (Cameron et al.. 2008). Briefly, a model pectin witha DM of 94% . an HG consisting of -97% GA, was dem ethylatedwith the citrus salt-independent PME until the desired DM wasobtained. Deniethylations were run w ith a radiometer P1-1M290pH-Stat controller at 30 C using I MLiOH as the titrant. Thereaction was stopped by allowing the demethylated pectin torapidly flow into a container holding two volumes of ethanol(37 C). The precipitated pectin was lyophilized. A 0.2% (w/v)solution of the lyophilized pectin was used for a limited endopolygalacturonase (EPG) digest to free DEMBs from the largermolecule.BS and BN were calculated as previously described(Cameron et al., 2008).

OLIGOMJR NARROW-RANGE CLASSES. Narrow-range size-classesof GA oligomers were produced b y limited digestion of PGA withEPG followed by differential precipitation and lyophylization(Cameron et al., 2005).

OLIGOlER PURIFICATION. Individual oligoniers were p urifiedby semi-preparative chromatography of the narrow-range size-classes. A CarboPac PA I (Dionex Corporation, Sunnyvale, CA)semi-preparative column was loaded with oligomers frorneithertheLow-DP or Medium-DP size class. An elution gradient from 20%to 60% 1 Mammonium formate was used to separate oligomers inthe Low-DP size class and from 40% to 80% for the Medium DPsize-class. All samples were lyophilized after collection.

iPrOH. The peel mixture was pre-dried in a hood for 2-then dried in a vacuum oven for 16-20 h under vacuum"C. The dried mixture was milled through a 20-mesh scra Wiley Mill Intermediate Model 4276-N.Preparation of block demethylated commercial pecti

Four liters of a 1% pectin solution (70% DM. BB RaPectin DK-4623, CP Kelco, Inc., Lille Skensved, Denwas prepared by dissolving at 80 C and then cooling totemperature. Forty grams of sodium chloride was addPH was adjusted to 7.0 with dilute aqueous sodium hydwith stirring, and then 2 mL of PME containing papaya e(Collupulin(V RTM, Liquid product code 5045. Gist-BrInternational B.V.) was added. The solution was titrated1.000 Nsodium hydroxide and the pH maintained betweand 7.2 until the pectin reached 55% DE as determined volume of base added and the initial DM. The pH of the swas adjusted to 4 w ith dilute HCI and the solution heated toon a stirring hot plate for 2 to 5 min to inactivate the enzympectin solution w as cooled to room temperature and precipwith 70/30 iPrOH water solution. The pectin was collecfiltering through fine nylon mesh cloth and pressed to reexcess iPrOH/water. The block-deesterified pectin was prein a hood for 2-3 h and then dried in a vacuum oven for h under vacuum at 50 C.Extraction of NCSP pectin from citrus peel

NCSP w as extracted from pre-washed oran ge peel accto methods previously described (Joye and Luzio. 2000)Peel hydrolysis and fermentation

The sample consisted of the residue left after hydrolythe complex carbohydrates in citrus processing waste (CPWconversion of fermentable sugars to ethanol. Fresh CPWobtained from a commercial juice processing plant, pretrby steam explosion to reduce the p eel oil concentration to 0.15 wet weight percent, cooled to 38 C using vacuumhydrolyzed with simultaneous fermentation (SSF) in a 3batch using S a c c h a r o n i y c e s c e r e v i s i a e and a hydrolyzing enmixture of Pectinex Ultra SP, Celluclast 1.5 L and Novo188 (Wilkins e t al., 2007).

Synthesis of block dernethylated citrus peelA mixture was prepared from 1 kg ofcornniinuted, pre-washedorange peel and 3 L of deionized water and the p!-1 adjusted to 1.8with concentrated nitric acid. This mixture was heated for3 h at 70C to extract the pectin component. The temperature was reducedto 29C and 40 g of sodium chloride was added with stirring. ThePH was adjusted to 7.0 with dilute aqueous sodium hyd roxide and2 mL of a papaya extract containing PM E activity (Collupulin

RTM. Liquid product code 5045 (Gist-Brocades InternationalB.V.. Charlotte. NC) w as added. The so lution was titrated using1.000 Nsodium hydroxide and the pH maintained between 6.8and 7.2 until the DM of the pectin reached approximately 55%assuming the extracted pectin component had an initial DM was72%. The pH of the solution was adjusted to p1-I 3 .5 with dilutenitric acid and the solution he ated to 85 C on a stirring hot platefor 2 to 5 min to inactivate the enzyme. The reaction mixture wascooled to room temperature and prec ipitated with 8 Lof7O% (v/v)iPrOH in water. The mixture was collected by filtering throughfine nylon mesh cloth and the material pressed to remove excess

31 2

Biosorption studiesThe hiosorption ofPb + 2 by different forms of pectin from ous solutions was carried out in batch biosorption-equilihstudies. Desired concentrations of Pb * 2 solutions were prepardilutin g 10002 nigL I standard Pfr 2 stock solution [Pb(NVWR. W est Chester, PA l . pH of the solution was adjusted tusing 0. 1 M NaOH . The adsorption capacity of different forbiosorbent w as determined in triplicate (except for individuoligomers) by equilibrating 50 rng of biosorbent with 50 m

metal solution (2.41 meq - L- 1 or 500 mg'L-') in 250 mL Erlenmflasks (pre-washed with 1% HNO) on an orbital shaker arpm and 25 2 C for 6 h. After filtration through Whatman40 filter paper, the Pb2 concentrations in the filtrate was dmined by atomic absorption spectrophotometer (AAnalystPerkin-Elmer, Waltham, MA ). The amount of Pb * 2 adsorbedunit hiosorbent mass (milligrams of m etal per gram dry biosorwas determined using the following expression:

q: V (C , -Ce , , )MProc. Fla. State I-fort. Soc. 121:

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40 0

30 0CLE

20 0

CL 100E

250

20 0

cnC Lc n 50

30 0

0. 200U)a,

10 0

where q is the metal uptake (mg Pb 2 g-' dry weight of biosor-bent). V is the volume of metal solution (mL). C is the initialconcent rat ion of Pb 2 in solution (mg'L). C is the residualcon cen t rat ion of Pb * 2 in solution a t equilibrium, an d M is the dryweight of biosorbent.Statistical analysisStatistical differences between treatment means was performedusing GraphPad Prism version 4.03 for Windows (Graph-PadSoftware. San Diego. CA; www.graphpad. com ).Tukey's multiplecom parison test was used to c ompare t reatment m eans.

Results and DiscussionOf all the biosorbents tested the greatest amount of Pb waschelated by the Medium DP size-class of GA oligomers which

was also greater than the other two DP size-classes (Fig. I; F450.9. P< 0.001). This greater sorption capacity by thesecompletely demethylated substrates, compared to the partiallydemethylated substrates disc ussed later, is in agreem en t with thereport by Kohn (198 7) where the author determined that Pb bind-ing w as stoichiometric in relation to the am ount of demethylatedgalacturonan present. The small reduction in sorption c apac ity by

Size Class

the large DP size-class may be due to an inc rease in cross lin kinwith these larger oligomers as previously observed for very lowDM pec t in s (Kohn an d Luknar. 1975).The greatest sorption c apac ity for the citrus salt-in depen denPME demethylated pectins was found in the 60% DM sampldeinethylated at pH 4.5 (Fig. 2). ANO V A in dicated the means wersignificantly different (F= 730.4. P

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400

300EU)

200

C -CM 100

E

\c 3c

SamplePig. 4 . Hiosorption capacit ies of pur if ied GA oligomers .

2000) but it was equivalent in sorption capacity to the 60% and50% DM - pH 4.5 samples (Figs. 2 and 3) and not significantlydifferent from the 50% DM - pH 4.5sample. The demethylatedpeel samples were close to the values foi-the pH 7.5demethylationseries and the 70% DM - pH 4.5 HG. The sample of hydrolyzedand fermented peel had the lowest sorption value (Fig. 3).

The sorption capacity of individual oligomers was similar tothe narrow-range size-classes of GA oligomers (Fig. 4). Unfortu-nately the lack of sufficient samples prevented the replication ofIi is analysis and statistical comparison to other samples. The data

does suggest that a GA trimer is sufficient to complex with Pb2*hut it has been shown (Kohn. 1987) that the DP of an oligomerhas a major impact on the solubility of the oligomer-metal ioncomplex. dependent on the metal species being complexed.

ConclusionIlic data piccntcd demonstrate the need forcontinued studies

on the utilization of citrus peel and peel derived pectin for theemoval of heavy metal ions from other waste streams requiring

i cmediation. To maximize the sorption capacity of pectic materialsImay be necessary to fragment larger molecules and demethylateIhicill either chemicall y or cnyvmaticahl\

Literature CitedCameron, R.G., G. Luzio, E. Baldwin, J. Narciso, and A. Plo

Production of narrow-range size-classes of polygalacturoligoniers. Proc. Fla. State Ilort. Soc. 118:406-409.Cameron, R.G., G.A. Luzio. K. Goodner, and M.A.K. WilliaD e m e t h y l a ti o n o f a m o d e l h o m o g a l a c tu r o n a n w i th a s a l t - in dpectin inethylesterase from citrus: I. Effect of pH on den-tblock size, block number and enzyme mode of action. CarbPolymers 71(2):287-299.Grohmann, K.. R.G. Cameron. and B.S. Buslig. 1995. Fraca nd pre t r e a tme nt of o ra nge pe e l by d i lu te a c id hydroly s i s . BiTechnology 54(2): 129-141.

Joye, D.D., and G.A. Luzio. 2000. Process for selective extrpe c t ins f rom pla nt ma te r ia l by d i f f e re nt ia l pH . C a rbohydra te 43(4):337-342.Kohn, R. 1987. Binding of divalent cations to oligonicric frapectin. Carbohydrate Res. 160:343-353.Kohn, R. and 0. Luknar. 1975. Calcium and strontium ion asolutions of the corresponding pectinates and its dependencdegree of esterification. Collection Czechoslov. Chem. C40:959-970.Luzio. G.A. and R.G. Cameron. 2007. Rheological investipe c t in de e s te r i f i e d us ing sa l t - inde pe nde nt pe c t in me thy le s tec i trus . Proc . Fla . Sta te I- tor t . Soc. 120:304-309.Luzio. G.A. and R.G. Cam eron. 2008. Deme thylat ion of a model

l ac tu r onan w i th the sa l t - i ndependen t pec t in me thyles t e r ase f r oH. S t ruc ture func t ion a na ly s i s . Ca rboh y date Poly. 71:300-3Malovikova. A. and R. Kohn. 1979. Binding of lead and c(Ill) cations to pectin. Collection Czcchoslov. Chem. Co44:2915-2927.

Ma lovikova . A . a nd R. Kohn. 1982. Binding of c a dm ium ion s Collection Czcchoslov. Chem. Commun. 47:702-708.Ridley, B.L., M.A. O'Neill. and D. Mohnen. 2001. Pectins: Sbiosynthe s is , a n dol igoga la c turonide - re la te d s igna l ing . Phy toc57:929-967.W ilkins , MR. , W .W . W idm e r , K. G r o h m a n n . a n d R . G . C a m e r oHydrolysis of grapefruit peel waste with ccllulase and pectizymes. Bioresource Technol. 98(8):1596-160 1.W il la ts , W .G. , C . Or f i l a , G . Lim bc rg, H .C . Buc hhol t , G . J. va n

A.G. Voragen. S.E. Marcus. T.M. Christensen, J. MikkelsMurra y . a n d J .P . Knox. 2001 . Modu la t ion of the de gre e a nd pmethyl-esterification of pectic honiogalacturonan in plant ceImpl ic a t ions for pe c t in m e thy l e s te ra se a c t ion, ma t r ix prope rcell adhesion. J. Biol. Chem. 276 (22):19404-19413.

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