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Journal of Biotechnology 127 (2007) 508–519

Tyrosinase-catalyzed grafting of sericin peptides onto chitosanand production of protein–polysaccharide bioconjugates

Anna Anghileri a, Raija Lantto b, Kristiina Kruus b,Cristina Arosio a, Giuliano Freddi a,∗

a Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano 20133, Italyb VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland

Received 2 May 2006; received in revised form 7 July 2006; accepted 20 July 2006

bstract

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied underomogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase wasble to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lowerhan with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggestinghat the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration ofyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average moleculareight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensationf o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. Theigh temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed theormation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra ofxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized

ericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of theormation of sericin–chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretchingegion seem to support the formation of bioconjugates via the Michael addition mechanism.

2006 Elsevier B.V. All rights reserved.

eywords: Silk sericin; Chitosan; Tyrosinase; HP-SEC; FT-IR; DSC

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∗ Corresponding author. Tel.: +39 02 2665990;ax: +39 02 2362788.

E-mail address: freddi@ssiseta.it (G. Freddi).

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168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2006.07.021

. Introduction

Sericin is the glue protein that binds the two fibroinlaments as they are spun by the silkworm to form theocoon. The term sericin identifies a family of proteins

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A. Anghileri et al. / Journal o

ncoded by two genes, Ser1 and Ser2, and producedhrough alternative splicing at the transcript level inhe middle silk gland cells (Couble et al., 1987; Garelt al., 1997; Huang et al., 2003). Sericin proteins areharacterized by an unusually high content of serine,hich accounts for about 38 mol% (Takasu et al.,002). The hydrogen bonding ability of hydroxylmino acids is considered responsible for the glue-likeroperties of sericin, while a possible hypothesis forhe heterogeneity of this group of proteins is relatedo the need of modulating viscosity and adhesivenessf the protein mixture as the cocoon is spun. Anothermportant biological function that sericin is thought toerform is to lower the shear stress and to absorb theater squeezed from the stretched fibroin mass during

ocoon spinning.Sericin is usually removed from silk textiles before

yeing and finishing by a process called degumming,hich takes advantage of the solubility of sericin

n boiling aqueous solutions containing variousegumming agents, such as soap, alkali, organic acids,r synthetic detergents, including proteolytic enzymesFreddi et al., 2003). As a by-product of the degum-ing process, solubilized and partially hydrolyzed

ericin peptides accumulate into the wastewater, withsubstantial contribution to the total organic charge.his not only entails higher costs for wastewater treat-ent, but also leads to the loss of a still valuable proteinaterial. Fabiani et al. (1996) developed a method for

ericin recovery from wastewater by ultrafiltration.ecovered sericin peptides find effective applications ingredient of cosmetic formulations, including skinnd hair care products, thanks to their moisturizingffect. However, in recent years the range of possiblend-uses of sericin is considerably increasing (Zhang,002). Sericin was used as finishing agent for naturalKongdee et al., 2005) or man-made textiles (Leet al., 2004) with good results in terms of moisturebsorption, antistatic properties, softness, and comfort.

hen air filters made of polyamide or polyester fibresere coated with sericin higher levels of antioxidant

nd antimicrobial activity were detected, suggestingheir potential use as indoor air filters to reduceree radicals and fungi or bacteria contamination

Sarovart et al., 2003). Sericin can be cross-linked,opolymerized, or blended with other polymers toroduce a new range of biodegradable materials withmproved properties (Zhang, 2002; Cho et al., 2003;

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hnology 127 (2007) 508–519 509

hn et al., 2001; Nagura et al., 2001). As a support fornzyme immobilization, sericin improved temperaturetability of immobilized l-asparaginase (Zhang et al.,004).

Biological properties of sericin have recentlyttracted a great deal of interest. In fact, Zhaorigetut al. (2003a,b) reported data on the protective effectf sericin against both chemical- and UV radiation-nduced tumorogenesis by reduction of oxidativetresses. Takeuchi et al. (2005) showed that higholecular weight sericin films effectively induced

ydroxyapatite nucleation under biomimetic condi-ions. Sericin-coated �-tricalcium phosphate ceramicshowed improved durability and desirable bioresorp-ion rate as novel bone repair devices (Miyazaki et al.,004). Tsubouchi et al. (2005) reported that sericinlms enhanced attachment of cultured human skinbroblasts. These findings seems to contrast with

he hypothesis that sericin may be implicated in themmune response observed against virgin silk suturesSoong and Kenyon, 1984). However, Panilaitist al. (2003) showed that the biological responsef sericin is probably dependent on the physicalssociation with the fibres. Moreover, different sericinractions may display different biological activitiesTsubouchi et al., 2005). Therefore sericin, eitherbtained directly from the cocoons or recoveredrom degumming wastewater, can be considered aaluable natural polymer worth of being used for aide range of applications, including those related toiomaterials.

In an attempt to better exploit the properties ofericin proteins, we studied the possibility of usingiotechnological tools to produce new bio-based, high-erforming, and environmentally friendly polymers.nzymes are expected to offer cleaner and safer alter-atives to current chemical practices, owing to theirpecificity and selectivity which allows a more carefulontrol of reaction conditions and final biopolymertructure by targeting selected reactive sites of theubstrate (Shao et al., 1999). We specifically focusedn the production of protein–polysaccharide biocon-ugates via the tyrosinase-catalyzed oxidation-graftingeaction, according to a reaction scheme recently

eveloped for the silk fibroin–chitosan polymerystem (Sampaio et al., 2005; Freddi et al., 2006).yrosinase is a copper-containing enzyme widelyistributed in nature, which has proved to be useful

5 Biotec

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10 A. Anghileri et al. / Journal of

o modify proteins (or other compounds containinghenol groups). The reaction mechanism involvesrotein-bound tyrosine residues which are first hydrox-lated to 3,4-dihydroxyphenylalanine (DOPA), andhen further oxidized to the corresponding o-quinoneDOPA–quinone). These o-quinones are active specieshat can either condense with each other or react withucleophiles, such as the amine and sulfhydryl groupsf amino acid residues (Matheis and Whitaker, 1987)r the amine groups of chitosan. The feasibility ofhe tyrosinase-catalyzed grafting of chitosan has beenemonstrated not only for silk fibroin, but also for aange of proteins, including cytochrome c, horseradisheroxidases, organophosphorus hydrolase (Chen et al.,001), gelatine (Chen et al., 2002), green fluorescentrotein (Chen et al., 2003), and casein (Aberg et al.,004). In this study, the kinetics of the enzymaticeaction of Agaricus bisporus mushroom tyrosinaseith sericin peptides and other silk-derived model

ubstrates was investigated. Then, sericin peptidesxidized with tyrosinase, either alone or in the pres-nce of chitosan, were purified and characterized bysing spectroscopic techniques (UV/vis, FT-IR), sizexclusion chromatography (HP-SEC), and thermalnalysis using differential scanning calorimetryDSC).

. Materials and methods

.1. Chemicals and silk protein substrates

Chitosan (Ch; catalogue number 44,886-9), l-yrosine (Tyr; catalogue number T3754) and mush-oom tyrosinase (MT; EC 1.14.18.1; catalogue number3824) were purchased from Sigma–Aldrich. Accord-

ng to manufacturer specifications, the chitosan usedn this study is a low molecular weight polysaccharidebtained by enzymatic hydrolysis of chitin (deacety-ation degree: 75–85%; viscosity: 20–200 cps; solu-ility: pH < 6.5 in dilute aqueous acid). Silk sericinSS) was purchased from Pecco & Malinverno (IT).t consists of a mixture of sericin polypeptides recov-red from industrial degumming effluents and puri-

ed by ultrafiltration (Fabiani et al., 1996). The pow-er was Soxhlet extracted with ethyl alcohol beforese. The silk fibroin (SF) aqueous solution was pre-ared as described elsewhere (Sampaio et al., 2005).

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hnology 127 (2007) 508–519

he soluble chymotryptic (Cs) or tryptic (Ts) frac-ions of silk fibroin were prepared as follows: a% (w/v) fibroin aqueous solution was diluted 1:1ith ammonium carbonate 0.1 M. �-chymotrypsin

Sigma–Aldrich, catalogue number C-4129) or trypsinSigma–Aldrich, catalogue number T-1426) were dis-olved in a small volume of HCl 1 mM and addedo the fibroin solution. The enzyme-to-substrate ratioas 1:100. The solution was incubated at 37 ◦C for4 h, during which a precipitate was formed. The chy-otryptic (Cp) or tryptic (Tp) precipitates were sepa-

ated by centrifugation. Supernatants were freeze-driedo recover soluble silk fibroin peptides (Cs and Tsractions).

.2. Preparation of the stock chitosan solution

The stock chitosan solution about 1.6% (w/v) wasrepared by adding 1.6 g of chitosan to 100 ml of waternd slowly adding hydrochloric acid (2 M) to attain anal pH of 2–3. After mixing overnight, non-dissolvedaterial was removed from the viscous solution by fil-

ration. Prior to enzymatic reaction the stock solutionas diluted 10-fold to reach a final concentration of

bout 0.16% (w/v), and the pH was raised to about 6.5y adding 1 M NaOH.

.3. Determination of enzymatic activity

Enzyme activity of tyrosinase was measured againstyr, according to Sigma Quality Control Test Pro-edure. The reaction mixture contained 50–100 U ofyrosinase and 0.3 mM Tyr in phosphate buffer 18 mM,H 6.5. The reaction was monitored spectrophotomet-ically at 280 nm (L-DOPA) with UV 1601 spectropho-ometer (Shimadzu, Japan). After an initial lag phaseLand et al., 2003), the absorbance was observed toncrease linearly with time and the activity was esti-

ated from the slope in the linear region. One activitynit (U) is defined as the amount of enzyme whichauses an increase in A280 nm of 0.001/min in the assayonditions.

ion solution containing tyrosinase and 2 mM Tyr dis-olved phosphate buffer 50 mM pH 7. The reaction wasollowed spectrophometrically at 475 nm, to monitorhe formation of DOPA–quinone.

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Differential scanning calorimetry (DSC) measure-ments were performed with a DSC-30 instrument (Met-

A. Anghileri et al. / Journal o

.4. Following tyrosinase-catalyzed reactions byxygen consumption

The activity of tyrosinase on the different silkubstrates (SS, SF, Cs, and Ts) was measured byetermining the consumption of dissolved O2 dur-ng the enzymatic reaction with a Fibox 3 oxygen

eter (PreSens, Germany) equipped with a fibre-opticinisensor at room temperature. Oxygen consumptionas followed in fully filled vials (4.5 ml) under con-

tant stirring. Silk protein substrates were dissolvedn phosphate buffer 50 mM pH 7 at a concentrationf 0.05–0.5 mg/ml. Reaction was started by addingyrosinase (0.1–0.7 U/test) dissolved in the reactionuffer. Test conditions were optimized by performingepeating measurements changing either the substrateoncentration or the amount of enzyme, in order thathe amounts of oxygen and enzyme were not limitinghe reaction rate. The amount of oxidized Tyr residuesas calculated considering that complete oxidation ofmol of Tyr to DOPA–quinone consumes 1 mol of O2.he Tyr content of the different silk substrates was esti-ated by amino acid analysis.

.5. Enzymatic oxidation of sericin and grafting ofhitosan

Different amounts of tyrosinase, from 3.6 to6.2 U/mg, were added to a sericin solution in phos-hate buffer 50 mM pH 7. Oxidation reactions wereonducted at room temperature for 24 h. To preventxygen limitations, the reactions were performed inarge open vessels under continuous stirring. Blankericin samples were incubated in the same condi-ions, without tyrosinase. At the end of the incubationime, samples were extensively dialyzed against watero remove any trace of buffer salts. Afterwards, oxi-ized sericin peptides were recovered in powder formy freeze-drying.

Oxidation-grafting reactions were carried out bydding fixed volumes of chitosan stock solution tohe reaction batches containing sericin and tyrosinase140 U/mg) in phosphate buffer 50 mM pH 6.5. Thehitosan:sericin weight ratio was 1:1. Blank samples

id not contain tyrosinase or sericin. After 24 h of incu-ation at room temperature, the reaction was stoppedith 1 M NaOH and sericin-grafted chitosan was sep-

rated by centrifugation, washed several times with

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hnology 127 (2007) 508–519 511

aOH and water until neutrality, and finally recoveredy freeze-drying.

.6. Determination of amino acid composition

The amino acid composition of silk protein sub-trates was determined after acid hydrolysis with 6 NCl, at 105 ◦C for 24 h, under vacuum. Free amino

cids were analyzed by HPLC (AccQ-Tag Method,aters), at a flow rate of 1 ml/min. Eluate was detected

t 254 nm. Samples were analyzed in duplicate (error:2%). The quantitative amino acid composition was

etermined by external standard calibration (Aminocid Standard H, Pierce).

.7. High-performance size exclusionhromatography (HP-SEC)

Samples were dissolved in a small volume of 50 mModium phosphate buffer, pH 7.2, containing 0.15 MCl, filtered, and analyzed with a BioSuite 125, HR-EC column (Waters). Injection volumes ranged from0 to 100 �l, flow rate was 0.5 ml/min. Eluate wasetected at 254 nm. All samples were analyzed in dupli-ate. Molecular weight calibration was performed bysing the LMW Gel Filtration Calibration Kits (Phar-acia Biotech).

.8. Spectroscopic analyses

UV/vis spectra of untreated and oxidized sericinolutions were measured with a UV 1601 spectropho-ometer (Shimadzu, Japan).

FT-IR spectra were measured in the ATR mode withNexus spectrometer Thermo Nicolet, equipped withZnSe ATR cell (mod. Smart Performer).

.9. Thermal analysis

ler Toledo), from room temperature to 500 ◦C, at aeating rate of 10 ◦C/min, on 2–3 mg samples. Thepen aluminium cell was swept with N2 during thenalysis.

512 A. Anghileri et al. / Journal of Biotec

Fig. 1. Examples of O2 consumption curves obtained by reactionof different silk substrates with tyrosinase. Substrate concentra-tftS

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ion was 0.1 mg/ml for the chymotryptic digest (Cs) and 0.5 mg/mlor the tryptic digest (Ts), sericin (SS), and fibroin (SF). Enzyme-o-substrate ratio: Cs = 0.20 U/mg; Ts = 0.16 U/mg; SS = 0.16 U/mg;F = 0.19 U/mg.

. Results and discussion

.1. Activity of tyrosinase with sericin and otherilk proteins

The activity of tyrosinase with sericin and other silkroteins used as reference substrates, i.e. silk fibroinSF), soluble tryptic (Ts) and chymotryptic (Cs) digestsf silk fibroin, was measured as consumption of the co-ubstrate O2. Typical O2 consumption curves obtainedith the different silk substrates are shown in Fig. 1.n initial time-lag was often observed when a higholecular weight substrate, such as silk fibroin, was

sed. The reactions proceeded effectively with all theubstrates, at a rate that depended on the substrate used.ollowing oxidation, the amount of O2 in the reac-

ion vial decreased reaching a plateau. From the total

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able 1xygen consumption in the reaction of tyrosinase with different silk protein

amplea Total O2 consumption(�g/ml)

Amoby M

ericin 0.160 ± 0.034 56.ibroin 0.158 ± 0.074 58.ryptic digest 0.307 ± 0.084 72.hymotryptic digest 1.052 ± 0.291 100

a Substrate concentration: 0.5 mg/ml.b Tyr concentration: sericin = 0.434 �mol/mg; fibroin = 0.641 �mol/mg;g. Oxidation of 1 mol of Tyr consumes 1 mol of O2.c Calculated from the initial slope of the O2 consumption curve.

hnology 127 (2007) 508–519

mount of O2 consumed during the reaction, and fromhe slope of the initial linear segment of the O2 con-umption curve the percentage of oxidized Tyr residuesnd the reaction rates were calculated for each sub-trate, respectively. From the results listed in Table 1t is possible to observe that both the amount of oxi-ized Tyr residues and the rate of reaction were in theollowing order: Cs > Ts > SS ∼= SF. This trend can beelated to the intrinsic chemical and structural proper-ies of the substrates. The chymotryptic digest consistsf low molecular weight peptides formed by prote-lytic degradation of silk fibroin chains (Freddi et al.,989). Tyr residues in chymotryptic peptides are prefer-ntially located at the C terminus, owing to the cleavagepecificity of �-chymotrypsin. This may explain whyxidation proceeded faster and all the available Tyresidues were oxidized. The size of tryptic peptides ismall as well (Shaw, 1964), but Tyr residues are moreandomly distributed along the peptide sequence, dueo the different cleavage site specificity of trypsin. Thisntailed lowering of both extent of oxidation and reac-ion rate, suggesting that the position of Tyr residueslong the peptide chain is a factor likely to stronglynfluence the accessibility to the enzyme.

Silk fibroin is a high molecular weight fibrous pro-ein with a block-copolypeptide structure comprisingepetitive crystalline and amorphous sequences (Zhout al., 2000). The largest part of Tyr residues (about0%) is located in the so-called “Gly-X” domains, athe end of repeating -(Gly-Ala)n- sequences, which areydrophobic in nature, while the others are distributed

ithin the more heterogeneous and hydrophilic bound-

ry sequences belonging to the amorphous regions.hen dissolved in aqueous solution it preferentially

akes a random coil conformation, with the hydrophilic

substrates

unt of Tyr oxidizedT (%)b

Reaction rate (mg O2/l min U)c

7 ± 7.6 0.477 ± 0.0821 ± 7.9 0.307 ± 0.0938 ± 8.2 0.737 ± 0.179

1.255 ± 0.348

tryptic digest = 0.529 �mol/mg; chymotryptic digest = 0.882 �mol/

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ortions of the protein chain exposed to the solvent, andhe hydrophobic ones folded to form a polar microenvi-onments from which the solvent is excluded (Canetti etl., 1989). It follows that only the Tyr residues exposedo the solvent can be oxidized by tyrosinase, whilehose buried into the core of the hydrophobic pro-ein coils are hardly accessible. The percentage of Tyresidues oxidized under the present experimental con-itions accounted for about 58%.

Silk sericin is more heterogeneous than fibroin inerms of number of polypeptides, molecular weight,hemical composition and physical structure. Thoughonsensus has not been reached yet about the consti-ution of sericin proteins, it has been shown that theer1 gene encodes for various polypeptides which con-ain several repeats of a 38 amino acid motif with

high content of hydroxyl amino acids (44.7 mol%er, 10.5 mol% Thr, 7.9 mol% Tyr), whose composi-

ion is very close to the average composition of sericinGarel et al., 1997). Hence, a large proportion of the Tyresidues is contained in the repetitive motif. Recom-inant sericin proteins based on the 38 amino acidepetitive motif were cloned and their structure in aque-us solution was investigated (Huang et al., 2003).D spectra showed that the secondary structure was aombination of random coil, �-turn, and �-sheet con-ormations. Though these findings do not allow to drawny conclusion on the amount of Tyr residues trapped

nto the partially folded �-turn and/or �-sheet domains,esults reported in Table 1 clearly indicate that a rel-tively large proportion of sericin-bound Tyr residuesere hardly accessible to the enzyme, probably due to

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Scheme 1

hnology 127 (2007) 508–519 513

teric hindrance effects. On the contrary, the reactionate was slightly higher than that of fibroin, probablyecause of the lower average size of sericin peptides,hich did not slow down too much the rate of diffusionf enzyme molecules towards the accessible reactiveites.

.2. Chemical properties of oxidized sericineptides

Previous studies have shown that, in the pres-nce of O2, tyrosinase catalyzes the oxidation to o-uinones of solvent-exposed Tyr residues of variousroteins, including silk fibroin, under both homoge-eous and heterogeneous reaction conditions (Sampaiot al., 2005; Freddi et al., 2006; see reaction diagramn Scheme 1). Solutions containing oxidized sericinere recovered after an overnight incubation and theirV/vis spectra were measured (Fig. 2A). The blank

ample showed the typical UV/vis profile of proteinsith an intense absorption peak at 276 nm attributed

o aromatic amino acids, including Tyr. The absorp-ion intensity in the 250–400 nm range dramaticallyncreased by oxidation. The higher the enzyme-to-ubstrate ratio, the larger the increase in intensity.

oreover, a new broad absorption peak emerged atbove 300 nm. Difference spectra (Fig. 2B) clearlyhowed that these spectral changes are mainly due

o the contribution of two new absorptions centredt about 290 and 350 nm, attributable to DOPA anduinone chromophore, respectively, i.e. the oxidationroducts of Tyr (Kang et al., 2004).

.

514 A. Anghileri et al. / Journal of Biotechnology 127 (2007) 508–519

Fig. 2. (A) UV/vis spectra of blank (a) and oxidized (b–e) sericins1is

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amples. Enzyme-to-substrate ratio: (b) 3.5 U/mg; (c) 7 U/mg; (d)4 U/mg; (e) 30 U/mg. MT: spectrum of a buffered solution contain-ng 30 U of tyrosinase. (B) Difference UV/vis spectra obtained byubtracting the blank sample (a) to samples (b), (c), and (d).

Chemical properties of oxidized sericin were deter-ined by amino acid analysis (Table 2). The amino

cidic pattern of sericin is dominated by the pres-nce of hydroxyl (Ser, Thr, Tyr), acidic (Asp, Glu),nd basic (Lys, His, Arg) amino acid residues, whichotally account for about 73 mol%. Compared to silkbroin, Gly and Ala are minor components, with a totaloncentration of only 20 mol%. Other amino acids areresent in very small amount. As expected, the mosttriking effect of the enzyme-catalyzed oxidation ofericin was the change of Tyr concentration, whichradually decreased from 2.42 mol% (blank sample)own to 1.06 mol%. The loss of Tyr residues at the high-st enzyme-to-substrate ratio accounted for about 56%.

oteworthy, this value is closely similar to that esti-ated by oxygen consumption measurements (56.7%;

ee Table 1). These features confirm that the aromaticroup of Tyr was the main target of the enzymatic reac-

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ig. 3. HP-SEC chromatograms of blank (a) and oxidized (b–d)ericin samples. Enzyme-to-substrate ratio: (b) 3.6 U/mg; (c)8 U/mg; (d) 36.2 U/mg. Reaction time: 24 h.

ion catalyzed by tyrosinase. It is interesting to notehat among the other amino acids the concentrationf Lys showed an average decrease of about 16% inxidized sericin samples. Cys decreased as well, espe-ially at the highest enzyme-to-substrate ratio (−26%).hese changes might be of some meaning in view of

he participation of the amino group of Lys and of theulphydryl group of Cys to the non-enzymatic couplingeaction with the activated quinone species formed byyr oxidation.

To go deeper into the properties of oxidized sericinamples, they were further analyzed by HP-SEC totudy the effect of tyrosinase oxidation on the averageolecular weight (MW) of sericin peptides. The chro-atographic profiles of Fig. 3 clearly show that oxida-

ion caused drastic changes in the MW distribution ofhe oxidized peptides. In particular, the peaks progres-ively shifted to higher MWs, indicating that the size ofhe peptide species increased with increasing the extentf oxidation. The peak MW values were 5800, 8200,1200, and 11700 Da for blank and oxidized sericinamples with increasing extent of oxidation, respec-ively. Judging from the trend of MW values, an enzymeosage of about 18 U/mg was probably sufficient toring the reaction to completion under the experimentalonditions adopted. The loss of low MW species andhe appearance of new components in the high MW

ange were reported for tyrosinase-oxidized fibroinKang et al., 2004). The role of o-quinone residues inhe formation of inter- and/or intra-molecular cross-inks in oxidized proteins is well known (Matheis and

A. Anghileri et al. / Journal of Biotechnology 127 (2007) 508–519 515

Table 2Amino acid composition of blank-treated and oxidized sericin substrates

Amino acid Sericin-0a (mol% ± S.D.) Sericin-1b (mol% ± S.D.) Sericin-2b (mol% ± S.D.) Sericin-3b (mol% ± S.D.)

Asp 18.52 ± 0.45 18.25 ± 0.28 18.24 ± 0.27 18.57 ± 0.38Ser 31.11 ± 0.20 31.46 ± 0.05 31.32 ± 0.04 29.54 ± 0.23Glu 4.90 ± 0.07 4.46 ± 0.08 4.40 ± 0.03 4.80 ± 0.05Gly 14.20 ± 0.13 15.17 ± 0.12 15.27 ± 0.01 15.02 ± 0.11His 1.25 ± 0.04 1.18 ± 0.01 1.13 ± 0.01 1.16 ± 0.03Arg 3.00 ± 0.06 3.23 ± 0.08 3.33 ± 0.04 3.55 ± 0.03Thr 8.71 ± 0.13 8.90 ± 0.13 8.95 ± 0.07 8.62 ± 0.05Ala 5.25 ± 0.05 5.27 ± 0.02 5.21 ± 0.08 5.64 ± 0.01Pro 1.00 ± 0.01 1.07 ± 0.01 1.14 ± 0.04 1.39 ± 0.01Cys 0.49 ± 0.01 0.43 ± 0.01 0.49 ± 0.01 0.36 ± 0.01Tyr 2.42 ± 0.04 1.74 ± 0.02 1.28 ± 0.01 1.06 ± 0.01Val 3.12 ± 0.02 3.17 ± 0.01 3.44 ± 0.02 3.67 ± 0.01Met 0.10 ± 0.01 n.d. 0.10 ± 0.01 n.d.Lys 3.04 ± 0.06 2.60 ± 0.04 2.46 ± 0.03 2.59 ± 0.01Ile 0.89 ± 0.01 0.90 ± 0.01 0.98 ± 0.01 1.18 ± 0.01Leu 1.60 ± 0.01 1.63 ± 0.01 1.63 ± 0.01 1.99 ± 0.01Phe 0.53 ± 0.04 0.59 ± 0.01 0.65 ± 0.01 0.89 ± 0.01

U/mg;

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a Sericin-0: blank sample, without tyrosinase.b Enzyme-to-substrate ratio: sericin-1 = 3.6 U/mg; sericin-2 = 18.1

hitaker, 1987). The non-enzymatic step of the oxida-ion reaction may proceed through several pathways,.e. condensation of quinone species with each otherr coupling with nucleophiles, such as the free amineroups of Lys. Results of amino acid analysis seem toupport the hypothesis that self-condensation probablylayed a major role in cross-linking, though contri-ution of the Lys- and/or Cys-quinone coupling routeannot be excluded, in consideration of the amino acidnalysis results.

.3. Spectroscopic and thermal properties ofxidized sericin peptides

IR spectra of sericin samples in the 1800–700 cm−1

ange are shown in Fig. 4A. Main spectral features arehe strong amide I, II, and III bands at 1647, 1535, and246 cm−1, respectively, in addition to other intenseands falling at 1398 cm−1 (δOH) and 1076 cm−1

overlapping of νCO, νC�N, νC�C� vibrations) (Guptat al., 1997). Compared to the blank-treated sample,ericin samples at increased extent of oxidation didot display strong spectral changes. As previously

bserved for tyrosinase-oxidized silk fibroin, the lackf strong spectral features attributable to oxidation isue to the weakness of the IR absorption bands assignedo the aromatic ring of Tyr and to its oxidation prod-

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sericin-3 = 36.2 U/mg.

cts (Sampaio et al., 2005). Only second derivativepectra (Fig. 4B) allowed to detect small but signif-cant changes in the skeletal (1050 and 1037 cm−1)nd amide III range (1265 cm−1). These changes areifficult to be assigned because they fall in highlyixed vibrational ranges where contributions from dif-

erent chemical groups extensively overlap. However,e can tentatively assign the new band appearing in

he skeletal range at about 1035 cm−1 to the formationf quinone methide tautomer, in agreement with obser-ations previously made on oxidized fibroin (Sampaiot al., 2005). As regards the weak band at 1265 cm−1,hich is present in the blank sample and tends to dis-

ppear following oxidation, it can be related to the lossf Tyr residues because it has been reported that theyontribute to this range, as well as to others (Takeuchit al., 1989). No changes were detected in the νOH andNH modes at above 3000 cm−1 (spectra not shown).

The thermal behaviour of sericin peptides wastrongly affected by oxidation (Fig. 5). The DSC pat-ern of sericin is closely related to the molecular struc-ure and physical properties of constituent polypep-ides. The curve of blank-treated sericin is in good

greement with the DSC profiles previously reportedTsukada, 1978; Lee et al., 2003), with two prominentndothermic transitions at 212 and 319 ◦C. The formers attributed to thermally induced molecular motion

516 A. Anghileri et al. / Journal of Biotec

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ig. 4. (A) FT-IR spectra of blank (a) and oxidized (b–d) sericinamples. Enzyme-to-substrate ratio: (b) 3.6 U/mg; (c) 18 U/mg; (d)6.2 U/mg. (B) Second derivative spectra in the 1300–1000 cm−1

ange of samples (a–d). Reaction time: 24 h.

nd melting of sericin peptides, while the high temper-ture peak is due to thermal decomposition, becausextensive weight loss and evolution of small moleculareight gases were reported to occur at above 300 ◦C

ig. 5. DSC curves of blank (a) and oxidized (b–d) sericin samples.nzyme-to-substrate ratio: (b) 3.6 U/mg; (c) 18 U/mg; (d) 36.2 U/mg.eaction time: 24 h.

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hnology 127 (2007) 508–519

Tsukada, 1978). Following oxidation with tyrosinase,he two intense endo peaks shifted gradually to higheremperature, indicating that the material attained a sub-tantially higher thermal stability. This effect can berimarily attributed to cross-linking and increase ofW, in good agreement with the chromatographic

esults previously discussed.

.4. Tyrosinase-mediated grafting of sericineptides onto chitosan

Oxidized Tyr species (o-quinones and related tau-omers) obtained by reaction of proteins and pep-ides with tyrosinase may undergo a complex set ofon-enzymatic reactions including condensation oroupling with nucleophiles such as the free amineroups of Ch (see Scheme 1). Feasibility of theyrosinase-mediated coupling of proteins to chitosanas recently been demonstrated by various authors, andaluable protein–polysaccharide bioconjugates werebtained with various proteins (Chen et al., 2001,002, 2003; Aberg et al., 2004), including silk fibroinSampaio et al., 2005; Monti et al., 2005; Freddi et al.,006).

Fig. 6A shows the IR spectra of chitosan, blank-reated chitosan (with sericin, without tyrosinase), andf the chitosan–sericin bioconjugate. The main spec-ral features of chitosan are multiple medium bandsn the 1460–1200 cm−1 range (involving δOH, δCH,nd ωCH modes) and many strong bands in the160–1000 cm−1 region, due to νCO modes of COHnd COC groups (Muzzarelli et al., 1980; Sannan et al.,978; Moore and Roberts, 1980; Miya et al., 1980). Theands at 1653, 1589 and 1321 cm−1 are attributable tohe amide I, II, and III modes of the residual N-acetylroups. The spectrum of chitosan exposed to sericinithout tyrosinase showed a closely similar pattern,ith a slight increase in intensity of the amide I and

I bands probably due to physical entrapment of someericin peptides into the chitosan precipitate. There-ore, without tyrosinase the precipitate consisted oflmost pure chitosan, suggesting that washing was ableo effectively remove non-reacted sericin peptides.

On the contrary, the chitosan–sericin bioconjugate

isplayed a mixed spectral pattern, with chitosan andericin bands overlapping over the entire range. Theseesults are consistent with the hypothesis that sericinas effectively incorporated into chitosan. The inter-

A. Anghileri et al. / Journal of Biotec

Fig. 6. (A) FT-IR spectra in the 1800–700 cm−1 range of: (a) chi-t(ao

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osan; (b) blank-treated chitosan (with sericin, without tyrosinase);c) chitosan–sericin bioconjugate. Asterisks in spectrum (b) indicatemide I and II bands. (B) FT-IR spectra in the 4000–2500 cm−1 rangef samples (a–c) and (d) sericin peptides.

ctions between the polysaccharide and protein com-onents were much stronger than those established byhysically mixing the two polymers, because sericineptides became resistant to the washing treatmenthich proved to be very effective in removing any non-

eacted sericin.Fig. 6B shows the νNH spectral range of the same

amples. The IR spectrum of pure sericin was includedor comparison purposes. As a primary amine, chitosanisplays two νNH bands at 3361 and 3421 cm−1. Asxpected, this pattern remains unchanged in the blank-reated chitosan sample. On the other hand, the biocon-ugate showed an IR profile broadly similar to that ofericin, with the presence of a single broad νNH band at290 cm−1. The presence of a single band in the νNH

ange with extensive broadening at higher wavenum-ers is consistent with the conversion of some primarymino groups of chitosan into secondary amino groupsKumar et al., 2000; Sampaio et al., 2005), which are

otns

hnology 127 (2007) 508–519 517

nown to display a single νNH band (Socrates, 1994).his observation allows to make some comments about

he mechanism of the non-enzymatic step of the reac-ion between chitosan and oxidized sericin. The chem-stry of this reaction is still poorly understood. Freemines can be covalently conjugated to quinones viahe Schiff-base reaction mechanism, with formationf imine groups, or via the Michael-type mechanism,hich is expected to result in the formation of sec-ndary amines (Shao et al., 1999; Chen et al., 2001,003; see Scheme 1). Present spectroscopic investiga-ions seem to support the latter reaction mechanism,n good agreement with the results previously obtainedn silk fibroin–chitosan bioconjugates (Sampaio et al.,005). However, this does not allow to exclude theossible contribution of the Schiff-base reaction mech-nism, because the νC N vibrational mode of iminealls at about 1650 cm−1 (Fabian et al., 1956; Monteirond Airoldi, 1999), a spectral range that in our sampless covered by the intense amide I band of sericin. Athis purpose, it is worth noting that Raman spectra ofilk fibroin–chitosan bioconjugates showed changes inhe spectral region where the νC N vibration mode isxpected to fall (Monti et al., 2005). This feature wasnterpreted as a possible involvement of the Schiff-baseechanism in the formation of bioconjugates. In con-

lusion, both reaction pathways may play a role in theeaction of the sericin-bound quinones with the pri-ary amine groups of chitosan. To go deeper into the

hemistry of the reaction, further characterization ofxidized sericin peptides and chitosan–sericin biocon-ugates with Raman and NMR spectroscopy is now inrogress.

. Conclusions

Sericin peptides recovered from degummingastewater were effectively oxidized by tyrosinase.nly a fraction of the Tyr residues available were oxi-ized, probably due to steric hindrance which limitedhe accessibility of the enzyme to the aromatic sidehain groups buried into folded domains. Amino acidnalysis and FT-IR spectroscopy gave evidence of the

xidation of Tyr residues and of the formation of reac-ion products. Tyrosinase-oxidized sericin underwenton-enzymatic cross-linking with chitosan. The FT-IRpectra of the chitosan–sericin bioconjugates demon-

5 Biotec

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18 A. Anghileri et al. / Journal of

trated the formation of covalent bonds between thewo polymers.

The present work shows the potential of the enzy-atic polymer–polymer grafting. On the basis of the

resent results it is possible to envisage the possi-ility of scaling up the process in view of develop-ng new biopolymers with improved properties andunctionalities applicable in various industrial fieldsi.e. textile, coating, packaging, biomedical, etc.).rafting chitosan with sericin peptides may comple-ent the outstanding properties of the polysaccharide

antimicrobial activity) with the new ones brought byericin (antioxidant, UV-resistant, moisturizing, solu-ility, etc.). This will result in the production of valu-ble bio-based polymers from renewable resources,nder the mild reaction conditions assured by the speci-city and selectivity of enzymes. In the context of theenewed interest for sericin proteins, this innovativend environmentally friendly technique will contributeo the exploitation of a valuable industrial by-productnd to its transformation into high-added value biolog-cal materials.

cknowledgements

The work was carried out in the framework of theTReP-project ‘High Performance Industrial Proteinatrices through Bioprocessing’ (HIPERMAX/NMP-

-CT-2003-505790) funded by the European Commis-ion; the contents of the publication reflects only theuthors view.

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