cross-linking of collagen with laccases and tyrosinases
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Materials Science and Engineering C 31 (2011) 1068–1077
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Cross-linking of collagen with laccases and tyrosinases
S. Jus a,b, I. Stachel c, W. Schloegl d, M. Pretzler b, W. Friess d, M. Meyer c,R. Birner-Gruenberger a,e, G.M. Guebitz a,b,⁎a Austrian Centre of Industrial Biotechnology ACIB, Petersgasse 14, 8010 Graz, Austriab Institute for Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austriac Research Institute for Leather and Plastic Sheeting, Meißner Ring 1-5, 09599 Freiberg, Germanyd Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet Muenchen, Butenandtstraße 5, D-81377 Munich, Germanye Proteomics Core Facility, Center for Medical Research, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, Austria
⁎ Corresponding author at: Institute for Environmentalof Technology, Petersgasse 12, 8010 Graz, Austria. Tel.: +873 8815.
E-mail address: [email protected] (G.M. Guebitz).
0928-4931/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.msec.2011.03.007
a b s t r a c t
a r t i c l e i n f oArticle history:Received 23 September 2010Received in revised form 28 January 2011Accepted 11 March 2011Available online 21 March 2011
Keywords:CollagenTyrosinaseLaccaseCrosslinking
Oxidation of acid soluble collagen (ASC), collagen suspension and BrCN-peptides (BrCN-P) with tyrosinasesfrom B. obtusa (BoT1, BoT2) and A. bisporus (AbT) and laccases from T. versicolor (TvL) and T. hirsuta (ThL)resulted in UV/VIS peaks at 475 nm and 305 nm indicating formation of reactive o-quinones and cross-linkedcomponents. Concomitant oxygen consumption was higher for the low molecular weight enzymes (TvL andBoT2) indicating limited accessibility. SDS-PAGE and SEC bands at higher MW demonstrated the formation ofcross-linked material. LC-MS/MS analysis suggested the involvement of tyrosine residues in cross-linkingwithout major changes of sequence similarities to untreated collagen. However, an increase of the SECα-peaktogether with a decrease of β-peak and the 1235/1450 cm−1 ratio (FTIR) indicated partial degradation.Crosslinking was enhanced by phenolic molecules such as catechine which lead to increased denaturationtemperature and reduced degradation by microbial collagenase. The tensile strength was increased whereasresistance to compressive forces was not influenced.
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1. Introduction
Conventionally, cross-linking of proteins is performed primarily bythe use of aggressive and/or toxic chemicals. Regarding toxicologicaland environmental aspects as well as the low specificity of mostchemicals, there is a high need for alternative technologies. Collagenas biomaterial shows excellent biocompatibility, low antigenicpotential, low toxicity and controllable degradability [1–3]. However,collagen materials are sensitive towards humidity, elevated temper-ature and enzymatic degradation unless stabilized by cross-linking.
Collagen is the primary structural material of vertebrates and is themost abundant protein found in mammalian tissues [4,5]. It is themain structural protein of skin, bone and tendon. Each molecule has acoiled coil structure of three polypeptide chains, two α1(I) and oneα2(I) chain, wound together to form a right handed triple helix ofabout 3000 length and 15 diameter. These chains are twisted andstabilized almost exclusively by hydrogen bonds. The end sections ofthe α-chains – the telopeptides – are formed by 16 amino acids at theN-terminal end and 25 at the C-terminal end. The rod-like triple-helical collagen molecules are arranged in a parallel staggeredorientation to form fibrils. In tissue, the polypeptide chains are
covalently cross-linked both intra- and inter-molecularly. Therefore,the bulk of collagen tissue of skin and bone is not soluble in waterbelow 50 °C. The cross-linking degree increases with increasing age ofmammalians, with a concomitant decrease of solubility [5].
Collagen is an important biomaterialwith applications asprostheses,artificial tissue, drug carrier, cosmetics and inwoundhealing [3,6,7]. It isa good surface-active agent and able to penetrate a lipid-free interface. Itexhibits biodegradability, weak antigenicity and superior biocompati-bility compared with other natural polymers, such as albumin [8,9]. Inmost drug delivery systems made of collagen, in vivo absorption ofcollagen is controlled by the use of cross-linking agents, such asglutaraldehyde [10], chromium tanning [11], formaldehyde [12],polyepoxy compounds [13], acyl azide [14], carbodiimides [15], andhexamethylenediisocyanate [16]. All of these reagents are more or lesstoxic and the main disadvantage is their uncontrollable release.Enzymes could represent an interesting alternative for cross-linkingcompared to these conventional strategies.
Tyrosinases (monophenol, o-diphenol: oxygen oxidoreductase; EC1.14.18.1) are widely distributed in higher plants, animals, andmicroorganisms and are binuclear copper containing enzymes whichcatalyse two different reactions using molecular oxygen: (1) Hydrox-ylation of monophenols to o-diphenols (monophenolase activity)(2) Oxidation of o-diphenols to the corresponding o-quinones(diphenolase activity) [17].
For the hydroxylation, one atom of O2 is incorporated into thearomatic ring of the monophenol substrate and the other is reduced to
1069S. Jus et al. / Materials Science and Engineering C 31 (2011) 1068–1077
water. As the result an o-diphenol moiety is formed from themonophenol. In a second reaction, o-diphenol can be further oxidizedto the corresponding o-quinone. Quinones are reactive compounds andcan react spontaneously to form high molecular weight compounds ormelanin pigments [18–20]. O-quinones are usually unstable in aqueoussolutions and can undergo non-enzymatic reactions, such as cyclizationto amine-chrome, following an intramolecular Michael intramolecular1,4-addition (i.e. o-dopaquinone).
Laccases are polyphenol oxidases (EC 1.10.3.2) with 4 copperatoms in the active site [21]. They oxidize awide range of phenolic andmethoxyphenolic acids but also decarboxylate them and attack theirmethoxy groups (demethylation) [22]. Laccases are capable ofcatalyzing a four-electron transfer reaction necessary to reducemolecular oxygen to form water. Unlike tyrosinases, they do notexhibit hydroxylase activity but oxidize their substrate via one-electron hydrogen abstraction generating radicals. These reactiveradicals can react further and lead to polymerisation, hydration anddisproportionation [23]. Laccases have a wide substrate specificityand in addition to mono and diphenols laccases have been found to becapable of oxidizing various aromatic compounds such as substitutedphenols, diamines, aromatic amines or thiols. The substrate specificityof laccases can be broadened by the addition of small molecularweight compounds called mediators.
Laccases have been shown to directly oxidize both tyrosine andcysteine residues in proteins leading to cross-linking [24–27]. Similarly,tyrosinases catalyze the formation of o-quinones which may eithercondensewith eachother or reactwith amino andsulphydryl residues inproteins resulting in cross-linking [28] (Fig. 1). Quinones have also beensuggested to form tyrosine–tyrosine linkages by coupling [24,29,30]. Indifferent studies, it was shown that tyrosinases oxidize a range of lowmolecular weight natural phenols while the activated quinones weregrafted onto different substrates [31–34]. These grafting reactions ledto drastic changes in the functional properties of the polymers.
CHNH
O
CH2
OH
R
-H+
CH2
O
R
e -
CH2
O
R
OH
ProtA
O
ProtA
O+ 1/2 O2
Tyrosinase
-
-
+ ProtA.
B.
Fig. 1. Tyrosinase (A) and laccase (B) catalyze
Tyrosinases have been used for grafting proteins when usingcomplex mixtures of peptides [33]. The functionalisation of biopoly-mers by tyrosinases and laccases has also been reported for tailoringantioxidant activity of biopolymers [35,36]. Gelatine–catechine con-jugates, synthesized by laccase-catalysed oxidation of catechine in thepresence of gelatine, lead to increased antioxidative properties ascompared to the unconjugated catechine [37]. Other authors haveshown that laccase based cross-linking involves the formation ofisodityrosine bonds in short peptides while generally the mechanismis rather poorly understood [38]. In contrast to other proteins, there isonly very little information available about laccase and/or tyrosinasecross-linking of collagen such as e.g. of tropocollagen macromolecules[39]. Partly, this may be due to the very low tyrosine content incollagen (~0.5%) providing only a few points of possible attack forthese enzymes and thus making collagen an unattractive substrate[40].
Consequently, in this study we compared for the first time thepotential of laccases and tyrosinases for collagen cross-linking. Inaddition, we investigated the effect of phenolic compounds, which havepreviously been shown to stabilize collagen, during this enzymaticprocess [41,42].
2. Materials and methods
2.1. Enzymes
The native and C-terminal processed tyrosinase BoT1 and BoT2,respectively, from Botryosphaeria obtusa were kindly provided byNovozymes, Denmark. The tyrosinase from Agaricus bisporus (AbT)was from Sigma. The Trametes hirsuta laccase (ThL) was isolated andpurified as previously described [43] while the laccase from Trametesversicolor (TvL) is commercially available (BioChemika). The activityof the tyrosinases was assayed according to Duckworth [44] using
NH
C
C O
CH2
O
CH2
O
RCH2
O
R
CH2
O
Rn
OH
ProtA
OH
NH
ProtB
NHC
CHNH
C
O
O
CH2OH
B-NH2
isodityrosine bond
d cross-linking of proteins [27,32,41,42].
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L-tyrosine (Sigma) and L-dihydroxy phenilalanine (L-DOPA, Sigma)as substrates. The activity of the laccases was determined as describedby Nicu-Paavola et al. [45] using 2,2´-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as substrate.
2.2. Substrates
Acid soluble collagen (ASC), its BrCN peptides (BrCN-P) and aninsoluble collagen suspension (CP) were provided by FILK Freiberg,Germany. The BrCN-peptides with defined molecular mass wereobtained from ACS by specific cleavage with cyanogen bromide(BrCN). ASC was prepared from bovine skin by acetic acid extractionand was used as a stock solution of 5 mg/ml in 1% acetic acid solutionafter shaking for 12 h at room temperature. This solution was storedat −20 °C. For the enzymatic treatment with tyrosinase, the collagenstock solution was diluted to a concentration of 1 mg/ml in 50 mMK2HPO4/KOH; pH 5.5. For the laccase treatment, the collagen stocksolution was diluted using 50 mM succinate buffer at pH 4.5. Theinsoluble collagen suspension was used as provided with the additionof a small amount of K2HPO4/KOH buffer at 0.5 ml g− 1. Chlorogenicacid (CHG), caffeic acid (CA), catechine (CAT), ferulic acid (FA) andmethyle gallate (MG) were obtained from Sigma. All other chemicalsused were of analytical grade.
2.3. Enzymatic treatments
The enzymatic oxidation of ASC and BrCN-P was performed in1.5 ml of 0.5 M potassium phosphate buffer (pH 5.5, tyrosinase) or0.5 M succinate buffer (pH 4.5, laccase) using 1 mg ml−1 substrate.Samples were incubated for 24 h at 25 °C (tyrosinase) or at 30 °C(laccase) under constant stirring at 100 rpm. The enzyme dosage was300 U mg−1 for tyrosinases and 22.5 U mg−1 for laccases. Thereactionwas stopped by freezing the samples at−20 °C [46]. To studythe influence of phenolic molecules, 150 μl from a stock solution ofdifferent polyphenolics was added after 20 h of enzyme incubationand the incubation was continued for further 4 h. All experimentswere performed in duplicates. Control samples were incubated at thesame conditions but without addition of the enzyme.
2.4. UV/VIS spectral analysis
A Tecan Infinite 200 plate reader was used for monitoring theenzymatic oxidation of ASC and BrCN-P by recording spectra in a rangefrom250 to 600 nm. For this, UV/VISmicrotiter plateswith a volumeof300 μl were used and the temperature was adjusted to 24±1 °C.
2.5. SDS-PAGE analysis
During electrophoretic analysis, enzymatically modified sampleswere separated using 12% polyacryl amide separating gels and SDS-PAGE protein standards (Broad Range, Biorad). The gels were stainedwith Comassie Brilliant Blue R (Bio-Rad) according to the method ofFurthmayr [47]. To separate proteins in the mass range of 1–100 kDa,Tris-Tricine SDS-PAGE was used with 10–16% gradient gels [48].
2.6. Oxygen consumption measurements
Oxygen consumptionwasmonitored by using an oxygen electrode(Oxygen Meter S, Dual digital model 2, Rank Brothers LTD, GB).Duplicate measurements were carried out under constant mixing insealed 1 ml sample glass flasks.
2.7. Size exclusion chromatography
Samples were analyzed by SEC using an ÄKTA Purifier liquidchromatography system (Amersham Pharmacia Biotech, GE Health-
care) coupled to a UV/Vis detector operating at 280 nm or 214 nm.Samples were centrifuged at 10 G through a 0.25 μm filter membrane(Syringe Driven Filter unit, 25 or 4 mm,Millex-HA,Millipore) and eitherinjected onto a Superdex 200 10/300 GL column or a Superose 610/300 GL column (both Amersham Pharmacia Biotech, Uppsala,Sweden). Separation were performed at 20 °C by injecting 200 μl andusing 0.1 M potassium phosphate and 0.15 M NaCl (pH 6.5) as theeluent at a flow rate of 0.4 ml min−1.
3. ATR-FTIR analysis
IR spectroscopy was performed using a Perkin–Elmer FourierTransform infrared (FT-IR) spectrophotometer with Golden Gateattenuated total reflection (ATR) attachment with a diamond crystal.Spectra were accumulated from 64 scans at a resolution of 4 cm−1. Anoptical bench alignment was performed before each measurement toensure that the spectrometer was fine-tuned and the detector signalmaximized.
3.1. Manufacturing of porous specimens
Collagen specimens were manufactured by freeze drying of thedifferent collagen materials. For sample preparation the collagen wasdispersed 2% in acetic acid solution (pH 3.7). After swelling for 4 h thedispersion was homogenized with an ESGE immersion blender(Unold AG, Germany) and freeze dried in polystyrene wellplates(for cylindrical specimens) or polyethylene-molds. The mold-deriveddried collagen was cut into strips of 1 cm width with a scalpel. Theexact height of the samples (between 2 and 9 mm) was measuredwith a caliper.
3.2. Mechanical testing
To study the mechanical behavior of the collagen samples (n=2),a Texture Analyser (TA) XTPlus (Stable Microsystems, Godalming,Surrey, UK) was used. The compressive strength of the material wasdetermined after at least 2 h of incubation in PBS-buffer at roomtemperature with a cylindrical piston of 10 mm diameter thatcompressed the samples with a speed of 1.0 mm/s at 22 °C. As acharacteristic value the required force for compression to 70% of theinitial height was used. For tensile strength the force upon rupture ofthe wet strips normalized to their cross-section area was evaluated.
3.3. Denaturation temperature
The denaturation temperature was determined using a MettlerToledo DSC821e differential scanning calorimeter. About 3 mg samplewas incubated with 10 μL PBS-buffer in 40 μL aluminum crucibles(Mettler, ME-26763) for at least 2 h at room temperature and thenheated from 20 to 90 °C with a heating rate of 10 K/min. Thedenaturation temperature td was determined by the maximum of theendothermal peak.
3.4. Collagenolytic degradation
To evaluate the resistance of thematerials to enzymatic degradation6 mg samples were incubated in 1 ml TES-buffer (pH 7.4) containing20 μg collagenase (Clostridium histolyticum type H, Sigma). Afterincubation in a water bath at 37 °C for 24 h samples were centrifuged(4 °C, 45 min)with186,000×g (OptimaTLX-CA, BeckmanCoulter, Brea,CA, USA), vacuum dried and gravimetrically analyzed.
3.5. Acidic dialysis
Samples were placed in dialysis membranes (Spectra/Por 1,Spectrum laboratories, USA) and placed in a beaker containing acetic
Table 1Properties of the tyrosinase and laccase enzymes used in this study.
Enzyme Abbreviation Specific activity[U/mg]
MW (literature)[kDa]
Mw (SEC)[kDa]
Botryosphaeriaobtusa
BoT1 986.1 60 68.3
Botryosphaeriaobtusa
BoT2 3675.3 45 41.1
Agaricus bisporus AbT 5528.3 55 53.9Trametes hirsuta ThL 10.8 64 59.9Trametes versicolor TvL 22.5 40 51.1
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acid solution (pH 3.7) for 8 h. Samples were gently stirred and theacetic acid solution was changed twice. Afterwards dialysis wasperformed three times against Milli-Q®-water (MilliporeWaters, UK).
3.6. Protein sequence analysis
Protein identification and internal sequence information wasreceived from LC-MS/MS. Protein bands stained with CoomassieBrillant Blue R-250 were excised from SDS gels (Fig. 4) and reduced,alkylated and digested with Promega modified trypsin as previouslydescribed [49], or with 0.5 μg chymotrypsin (Roche) in 50 mMammonium bicarbonate, and 10 mM CaCl2, or subtilisin (Sigma) in100 mM Tris–HCl, pH 8.5, and 5 mM CaCl2. Alternatively ASC (20 μg)was diluted to 35 μl with 100 mM ammonium bicarbonate, reducedwith 35 μL 10 mM DTT for 20 min at 56 °C using shaking at 550 rpmand alkylated with 8 μL of 55 mM iodoacetamide for 15 min at roomtemperature, using again shaking at 550 rpm. Protein was thendigested in solution with either 0.5 μg trypsin or 0.5 μg chymotrypsinin 50 mM ammonium bicarbonate and 10 mM CaCl2 at 37 °C andshaking at 550 rpm over night.
Digestswere separated by nano-HPLC (Agilent 1200 system, Vienna,Austria) equippedwith a Zorbax 300SB-C18 enrichment column (5 μm,5×0.3 mm) and a Zorbax 300SB-C18 nanocolumn (3.5 μm,150×0.075 mm). 40 μl of sample was injected and concentrated onthe enrichment column for 6 min using 0.1% formic acid as isocraticsolvent at a flow rate of 20 μl/min. The columnwas then switched in thenanoflow circuit, and the sample was loaded on the nanocolumn at aflow rate of 300 nl/min. Separation was carried out using the followinggradient, where solvent A is 0.3% formic acid in water and solvent B is amixture of acetonitrile and water (4 : 1, by vol.) containing 0.3% formicacid: 0–6 min: 13% B; 6–35 min: 13–28% B; 35–47min: 28–50% B,47–48 min: 50–100% B; 48–58 min: 100% B; 58–59 min: 100–13% B;59–70 min: re-equilibration at 13% B. The sample was ionized in thenanospray source equipped with nanospray tips (PicoTipTM Stock#FS360-75-15-D-20, Coating: 1P-4P, 15+/−1 μm Emitter, New Objec-tive, Woburn, MA, USA). It was analyzed in a Thermo LTQ-FT massspectrometer (ThermoFisher Scientific,Waltham,MA,USA) operated inpositive ion mode, applying alternating full scan MS (m/z 400 to 2000)in the ion cyclotron andMS/MSby collision induced dissociation of the 5most intense peaks in the ion trapwith dynamic exclusion enabled. TheLC-MS/MS data were analyzed by searching the SwissProt publicdatabase downloaded onMay26th, 2010, and the aminoacid sequencesof the identified mature collagen alpha 1 and 2 chains (P02453,162–1215 and P02465, 80–1100) for detailed modification analysis,with Mascot 2.2 (MatrixScience, London, UK). Search criteria werecharge 2+ or 3+, precursor mass error 0.05 Da and product mass error0.7 Da, carbamidomethylation as fixed, hydroxylysine, hydroxyproline,oxidation on methionine, N terminal pyro glutamine and tyrosinequinone as variable modifications. A maximum false discovery rate of0.05 using decoy database search, an ion score cut off of 20 and aminimum of 2 identified peptides was chosen as identification criteria.
For detection of possible tyrosine–tyrosine or lysine–tyrosinecrosslinks the respective databases of crosslinked linearized peptidesof the identified mature collagen alpha 1 and 2 chains (P02453, 162–1215 and P02465, 80–1100) were generated by xComb [50] fortrypsin and for chymotrypsin digests, allowing inter- and intramo-lecular crosslinks and up to three missed cleavage sites, and searchedwith Mascot as described above except for allowing−2 H on tyrosineas additional variable modification for the tyrosine–tyrosinecrosslinks.
4. Results and discussion
In this study, the potential of laccases and tyrosinases for collagencross-linkingwasassessed. Threedifferent tyrosinases and twodifferentlaccaseswereused for the enzymatic treatmentof various collagenolytic
substrates. TheMWof the enzymes asmeasured by SEC (Table 1) variesslightly from published data based on SDS-PAGE [43]. In our investiga-tions, SEC was used for determination of MW to allow identification ofthe enzymes during SEC analysis of collagen cross-linking.
Because of the fast precipitation of collagen at higher pH values, allexperiments were carried out at a maximum pH value of 5.5. Whilethe pH optima of the laccases were in the acidic range, the tyrosinaseswere most active at higher pH (6.5). Nevertheless, at pH 5.5 thetyrosinases BoT1, BoT2 and AbT showed 94%, 98% and 88% of theiroptimum activity, respectively. Expectedly, the C-terminal processedBoT2 showed higher activity than the native enzyme which iscommon among plant, bacterial and fungal tyrosinases [51,52].
In a first stage, BrCN peptides (BrCN-P) from acid soluble collagenwere used as model substrates. BrCN treatment results in cleavage ofcollagen into major fragments with defined MW of CB3–5: 61 kDa,CB3–7: 38 kDa, CB7/8: 25 kDa and CB6: 19 kDa [5]. After incubation ofBrCN-P with BoT2, the UV/VIS spectrum showed an increasingabsorption in the area of 280 nm and a new peak appeared at305 nm. This novel peak could be due to Michael-type addition typecross-linking between quinones generated from Tyr-residues and theamino groups of Lys-residues [53,54].
No significant peak was seen at 282 nm corresponding to L-DOPA.The latter was obviously oxidized to dopachrome inducing a peak inthe UV/VIS spectrum at 475 nm [54]. The same peak at 475 nm wasalso seen for the oxidation of collagen with tyrosinases indicating theformation of reactive o-quinones [54]. In addition and similar to theenzymatic treatment of BrCN-P after incubation of collagen withthe tyrosinase BoT2 there was a slight increase of UV absorption in thearea of 305 nm suggesting the presence of cross-links. In contrast, nosuch peaks were observed in case of the laccases which oxidize theirsubstrates to radicals based on one electron abstraction [26,27].
In addition to direct enzymatic cross-linking, the effect of phenolicmolecules to further enhance the enzymatic reaction was assessed.This approach has previously been reported by Thalman [32] whoused small polyphenols for α-lactoalbumin cross-linking. Tyrosinaseoxidation of CAT lead to evolvement of orange color (peak at 450 nm)and a peak at 390 nm appeared in the UV/VIS spectrum indicatingformation of o-quinone [55]. When collagen was oxidized withtyrosinase in the presence of CAT a similar spectrum was obtained inthis study (Fig. 2, left). Compared to collagen alone, only a slightshoulder was seen at 475 nm indicating a possible reaction of catechinwith oxidized collagen [36] (Fig. 2, right).
Enzymatic oxidation of collagen was also studied based onconsumption of oxygen by tyrosinases and laccases. Interestingly,among the laccases, ThL with a molecular weight of 64 kDa showed alower oxygen consumption rate than the smaller laccase TvL with aMWof 40 kDa (Fig. 3). Steric hindrance and denaturation of the nativehelical structure of collagen could play an important role regardingthe accessibility of those internal amino acids which are laccasesubstrates. Similarly, among the tyrosinases, the C-terminal processedtyrosinase BoT2 with a MW of 45 kDa showed a higher oxygenconsumption rate than the native tyrosinase BoT1 (MW=60 kDa).
0.1
0.12
0.14
0.16
0.18
0.2
0.22
0.24
275 325 375 425 475
Abs
orba
nce
Wavelenght (nm)
8 h 9 h 11 h
14 h 18 h
0
0.2
0.4
0.6
0.8
1
300 350 400 450 500 550
Abs
orba
nce
Wavelenght [nm]
t [min]
50 40 30
20
10
0
Fig. 2. UV/VIS spectra of collagen type I oxidized by tyrosinase BoT2 (left) and in the presence of 2 mM catechin.
1072 S. Jus et al. / Materials Science and Engineering C 31 (2011) 1068–1077
Tyrosinases introduce 0.5 equivalents of dioxygen into theirsubstrates (e.g. tyrosyl residues in collagen) leading to quinoneswhile 0.5 equivalents of dioxygen are reduced to water. Thesequinones can couple to aminogroups (e.g. of lysine) or to tryptophaneresidues via Micheals addition [56]. In case of laccase, two equivalentsof tyrosyl radicals can react and lead to cross-linking throughdityrosine or isodityrosine bonds which have been reported to bethe major principle for cross-linking after laccase treatment (amongstother radical reactions) [27,57]. Formation of two tyrosyl radicalswould require two steps of one-electron abstraction by laccasescausing the consumption of 0.5 equivalents dioxygen. Consequently,in this simplified view, complete conversion of all tyrosyl residues bylaccases would require only half the amount of dioxygen whencompared to tyrosinases. However, maximum consumption of oxygenobserved in this study during the reaction of tyrosinases BoT2 and AbTwith collagen corresponded to only 32% conversion of all availabletyrosyl residues again indicating limited accessibility to the substrate.
In initial experiments, SDS-PAGE was used to determine cross-linking effects after tyrosinase and laccase treatment (Fig. 4). Thecollagen polypeptide chains exhibit a considerably lower electropho-retic mobility when compared to other proteins of similar molecularweight [47]. However, according to SDS-PAGE the incubation ofcollagen with the tyrosinase BoT2 led to a clear shift towards highermolecular weights while in case of BoT2 and the laccase TvL bandsdistinctive bands vanished. As seen from Fig. 4, characteristic bands ataround 209 kDa became weaker or even disappeared. Completeabsence of bands was due to precipation/gel formation of collagenupon enzymatic treatment which therefore could not penetrate intothe separating gel during SDS-PAGE analysis. This shift was enhanced
70
80
90
100
0 500 1000 1500
Oxy
gen
cons
umpt
ion
[%]
Time [min]
BlankControlBoT1BoT2AbTThLTvL
Fig. 3. Oxygen consumption during oxidation of collagen with laccases (ThL, TvL) andtyrosinases (BoT1, BoT2, AbT) compared to a blank (buffer only) and a control(substrate and buffer only).
when phenolic molecules were added (data not shown). Compared tothe blank, a band in the region of 100 kDa completely disappearedwhen collagen was incubated with tyrosinases while in the case of thelaccase TvL this band remained but bands in the higher MW regiondisappeared (Fig. 4). This indicates a different mode of action oflaccases and tyrosinases on collagen.
When using BrCN-P as the substrate, a clear shift of individualpeaks to the highermolecularweights regionswas seen in comparisonto the untreated control. This was especially pronounced when smallpolyphenolic substrateswere used as bridging agents. Besides, there isalso a clear formation of smaller oligomeric compounds.
In other experiments, size exclusion chromatography (SEC) wasused to investigate the cross-linking reactions more in detail. Theelution profile of untreated collagen BrCN-P is shown in Fig. 5 andconsists of five individual peak fractions corresponding to molecularweights of 54 kDa, 38 kDa, 25 kDa, 19 kDa, and 13 kDa, respectively[5]. Upon cross-linking of BrCN-P with tyrosinases and laccases, thebroad peak at 15 ml corresponding to fraction 2 (MW=38 kDa)strongly decreased while novel peaks appeared at lower retentionvolumes 12.3, 10.9 and 8.86 ml. This effect was most pronounced forthe tyrosinase BoT2 (Fig. 5). The most significantly shifted peaks wereseen at 10.9 ml corresponding to an MW of 390.6 kDa and at 12.3 mlcorresponding to an MW of 180 kDa. The formation of these peaks isprobably a result of cross-linking BrCN-P. In SEC, elution of moleculesdepends on their hydrodynamic volume and not only on their MWalone. Thus, compact, spherical, cross-linked polymers may elute laterthan flexible proteins [58]. Consequently, intermolecular cross-linkingcan result in apparent higher MW in case of loose bonds whileintramolecular cross-linking can lead to an apparent decrease in MW.
Fig. 4. SDS-PAGE 10% tris-tricine gel of acid soluble collagen after tyrosinase (BoT1,BoT2, AbT) and laccase (TvL) oxidation compared to untreated collagen (blank).
-5
0
5
10
5 10 15 20 25
Abs
orba
nce
[mA
U]
Elution volume [ml]
blankBoT2
-5
15
35
55
75
95
5 10 15 20 25
Abs
orba
nce
[mA
U]
Elution volume [ml]
blank
CA
CHG
CAT
Fig. 5. Size exclusion chromatograms (Superdex 200 10/300 GL column (GE Healthcare)) of collagen BrCN-peptides treated with tyrosinase BoT2 (left) and with tyrosinase BoT2 inthe presence of phenolic molecules (right). Note the larger absorbance range (Y-axis) in the right figure due to incorporation of phenolic compounds.
1073S. Jus et al. / Materials Science and Engineering C 31 (2011) 1068–1077
The large decrease of the peak at 15 ml cannot be quantitativelybalanced with the novel peaks formed since larger cross-linkingproducts escape this analysis method. Indeed, formation of insolubleparticles was observed.
The effect of cross-linking as seen from SEC measurements wasenhanced upon addition of phenolic molecules. Compared to theuntreated control, peaks at 8.5 ml, 8.8 ml, 11.3 ml and 16.8 mlincreased when BrCN-P were incubated with tyrosinase (BoT2) inthe presence of CA and CHG, respectively (Fig. 5). However,incorporation of phenolic molecules into cross-linked BrCN-P stronglyabsorbing at 280 nm mimics a general increase in the amount ofprotein. Thus, increases of peaks around an elution volume of 16 mlmay indicate binding of phenolic molecules to BrCN-P rather thancross-linking. In case of catechine (CAT), strong gel-formationoccurred explaining the low amount of protein in the liquid partseen in SEC.
In a next step enzymatic cross-linking of acid soluble collagen wasstudied. Again, the enzyme treatment of collagen partially leads to theformation of insoluble gel like material especially in the presence ofphenolic molecules. This effect has previously been described forcollagen and catechin where the resulting material became evenresistant to hydrolysis bymammalian collagenase [59]. The remainingliquid fractions of the enzymatically treated samples were theanalyzed by SEC. As shown in Fig. 6, a shift towards higher MW wasobserved after tyrosinase treatment. A novel peak eluting at a volumeof 8 ml was most significant for the enzymatic incubation in thepresence of CAT. A decrease of the β-peak and an increase of the α-peak of the collagen material indicates partial degradation. This effectwas much more pronounced for the laccases used in this study(Fig. 6). Despite the partial degradation, higher aggregates were
6 8 10 12 14 16
0
30
60
90
120
150
cross-linkedmaterial
γ
blank CA CHG CAT
βα
Elution volume [ml]
Abs
orba
nce
[mA
U]
Fig. 6. Size exclusion chromatograms ( Superose 6 10/300 GL column (GE Healthcare)) of aclaccase TvL (right) in the presence of phenolic molecules.
formed (peak above retention volume 8 ml) especially when thelaccases were used alone and in the presence of CAT.
Interestingly, there seems to be a sensitive balance point regardingthe concentration of phenolic molecules which support cross-linking.For CHG, a concentration of 2 mM lead to the formation of aggregateswith high MW eluting at 12 ml and 19 ml during SEC analysis, re-spectively. Previously, cross-linking of thewhey proteinsα-lactalbuminand β-lactoglobulin by tyrosinase in the presence of CA has beendemonstrated by Thalmann et al. [32]. In agreement with our findings,these authors used concentrations of 2 mM CA. With increasingconcentrations of CHG only an increase of the peak eluting at 19 ml inthe SEC chromatogram was seen together with an increase of thecollagen-α-peak indicating partial degradation of the collagen structure(Fig. 7). A decrease in the extent of cross-linking at higher concentra-tions of CHG may be attributed to excessive binding of CHG to collagenleading to blocking of functional groups necessary for cross-linking [32]or steric hindrance/inhibition of the enzyme [34,60]. In addition,oligomerisation of CHG may become an important side reaction [32]which is also indicated during SEC separation by increasing absorptionbetween 30 and 35 ml at higher concentration of CHG.
As a third collagen based product, water-insoluble collagen pastewas incubated with tyrosinase (AbT) and laccase (TvL) in presenceand absence of CAT. Stabilization of collagen by CAT against thecollagenolytic attack has been previously reported [41,61] while thereis less information about the additional effect of enzymes in cross-linking. In FT-IR analysis of films formed from the collagen suspension,bands at 1680, 1530 and 1240 cm−1 represent the amide I, II and IIIbands of collagen, respectively, (Fig. 8) [41]. The amide I band arisespredominantly from protein amide C–O stretching vibrations and theamide II band is due to amide N–H bending vibrations as well as C–N
6 8 10 12 14 16
0
40
80
120
160 blank CAT FA MGγ
βα
Abs
orba
nce
[mA
U]
Elution volume [ml]
id soluble collagen (blank) after treatment with the enzymes tyrosinase BoT2 (left) and
Fig. 7. Size exclusion chromatogram (Superdex 200 10/300 GL column (GE Health-care)) of tyrosinase BoT2 treated collagen in the presence of 2, 5 and 10 mMchlorogenic acid (CHG) (left) and formation of insoluble collagen gel (right).
1074 S. Jus et al. / Materials Science and Engineering C 31 (2011) 1068–1077
stretching vibrations. The amide III band ismore complex consisting ofcomponents from C–N stretching and N–H in plane bending fromamide linkages, as well as absorptions arising fromwagging vibrationsfrom CH2 groups from the glycine backbone and proline side-chains[41].
Upon enzymatic cross-linking of collagen suspension in thepresence of CAT, for both enzymes, a shift to higher transmittanceand significant changes of characteristic amid I, II and III bands wereseen. The 1235/1450 cm−1 ratios was 1.1 for the native collagen anddecreased after treatment with AbT and TvLto 0.96 and 0.94respectively, whereas a denatured collagen (gelatin) would have aratio around 0.6 [41]. This confirms the results obtained with acidsoluble collagen during SEC experiments where an increase of thecollagen-α-peak was seen especially after laccase treatment. Detailedstudies on the water-insoluble collagen paste before and aftercrosslinking by SEC and SDS-Page were not performed since thestartingmaterial already representsmaterial which is too big in size tobe separated by these techniques.
Collagen crosslinked by tyrosinases Bot1 and Bot2 as well asuntreated collagen were subjected to protein sequence analysis byLC-MS/MSafter in solutiondigest byeither trypsin or chymotrypsin. Thebovine collagen alpha 1 (SwissProt identifier P02453) and alpha 2(SwissProt identifier P02465) were identified in all samples as the onlymajor components with high sequence coverage (In detail: alpha 1:untreated collagen: score 13908, 96% sequence coverage; Bot1 treatedcollagen: score 7963, 85% sequence coverage; Bot2 treated collagen:score 10451, 89% sequence coverage; alpha 2: untreated collagen: score12181, 95% sequence coverage; Bot1 treated collagen: score 6827, 95%sequence coverage; Bot2 treated collagen: score 8041, 95% sequence
Fig. 8. FT-IR spectra of insoluble collagen suspension (blank) and after incubation with the tyr(CAT).
coverage). Thisfinding indicates that onlyminor changes are involved incross linking and that cross linking is not extensive.While the sequencecoverage of collagen alpha 2was similar (95%) in all three samples, only85% of Bot1 treated collagen alpha 1 and 89% of Bot2 treated collagenalpha 1 as compared to 96% of untreated collagen alpha 1 was found.Interestingly, while the only tyrosine Y869 of the mature alpha 2 chainwas found in all three samples, only three (Y4, Y6, Y1034) of the fivetyrosines of the mature alpha 1 chain (Y4, Y6, Y1034, Y1053, Y1054)weredetected in theuntreated andonly one (Y1034) in the Bot1 treatedbut none in the Bot2 treated collagen sample suggesting theirinvolvement in the establishment of crosslinks.
For more detailed analysis of the Bot2 treated sample, the distincthighest molecular weight protein band of the Bot2 treated collagenseparated by SDS-PAGE (Fig. 4), which was not visible in theuntreated collagen (Blank), was excised and analysed by LC-MS/MSafter in gel digest with either trypsin, chymotrypsin or subtilisin.Again, bovine collagen alpha 1 (SwissProt identifier P02453) andalpha 2 (SwissProt identifier P02465) were identified in all samples asthe only major components, confirming that the high molecularweight band most likely comprises crosslinked collagen alpha 1 and 2chains. In detail, collagen alpha 1 was identified with a score of 7333and sequence coverage of 58%, and collagen alpha 2 with a score of3239 and 51% sequence coverage (Fig. 9). While the Y869 of themature alpha 2 chainwas found, none of the five tyrosines of the alpha1 chain was detected suggesting their involvement in crosslinking. Adetailed search for crosslinked peptides, however, did not result insignificant identificationswhichmay be due to too low concentrationsof crosslinked as compared to regular peptides.
Crosslinking of collagen for biomaterial use is typically performed inorder to enhance mechanical properties or to increase sustainability. Inorder to study whether laccase or tyrosinase treatment positivelychange these performance parameters of collagen biomaterials, themechanical strength as compression force and force upon rupture aswell as the denaturation temperature and the susceptibility tocollagenolytic digestion representing biodegradability were measured.Porous sponge-like materials were prepared from the water-insolublecollagen paste. Compressive forces measured in the wet state of thespecimens were neither significantly influenced by the enzymetreatment nor by the CAT addition. A possible explanation for thisfinding is the high porosity of the systemswhichwere fully soakedwithPBS after incubation and the compressive forces were mainlydetermined by squeezing out the buffer. Thus, the performance in thistest is not significantly altered by the enzymatic treatment.
The force upon rupture of porous collagen strips was loweredslightly upon treatment with laccase or tyrosinase alone (Fig. 10).Thechanges were statistically significant but rather marginal. However,enzymatic treatment in presence of CAT led to a substantial increase
osinase AbT (left) and the laccase TvL (right) with andwithout the addition of catechine
Fig. 9. Sequence coverage of mature bovine collagen alpha 1 and 2 chains identified in the highest molecular weight band of Bot2 treated collagen separated by SDS-PAGE (Fig. 4).Matched peptides are shown in bold. Tyrosines Y are underlined.
1076 S. Jus et al. / Materials Science and Engineering C 31 (2011) 1068–1077
in tensile strength, indicating stronger interactions of the fibres in thewet state. In comparison, CAT incubation alone did not show an effecton this parameter.
Similarly, upon laccase or tyrosinase treatment followed by neutraldialysis the denaturation temperature of water-insoluble collagen-paste was not significantly (TvL, AbT) or only marginally (ThL)changed when compared to the starting material (Fig. 11). Anincrease by about 2 °C was detected upon addition of catechin alonewhich was further increased by another 0.5 °C upon treatment withtyrosinase AbT or laccase TvL in combination with CAT. In addition, nohints for transitions occurring at lower temperature, which couldpotentially be a result of collagen degradation, were found in the DSCtraces. Furthermore, dialysis in acetic acid (pH 3.7) was performed,that allows a dissociation of intermolecular aldimine-type crosslinks[62]. The resulting repulsive forces lead to swelling of the fibrillarstructures and unbound compounds with a molecular weight belowthe cut-off of the dialysis membrane can be removed by aconcentration gradient. The denaturation temperatures of the CATtreated collagen materials did not change significantly whichindicates that CAT was integrated in the collagen material in a stableway. The denaturation temperatures of the samples not treated withCAT decreased as acid-labile crosslinks might have been cleaved. Thisfinding was not as pronounced for the AbT-treated material andexcludes the TvL-crosslinked material, where acidic-dialysis did notsignificantly decrease denaturation temperature.
In a next step susceptibility of the modified collegens tocollagenase hydrolysis was investigated. Samples that had beenmodified by incubation with laccase of tyrosinase were disintegratedby collagenase treatment into 90% soluble fragments after 24 h ofincubation. The CAT treated collagen, however, shows higherresistance as only 60% were converted to soluble protein material.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Blank Blank-CAT
ThL ThL-CAT
TvL TvL-CAT AbT AbT-CAT
For
ce u
pon
rupt
ure
[mN
/mm
²]
Fig. 10. Tensile strength of enzymatically cross-linked water-insoluble collagen pastespecimens.
Additional 5–20% could be prevented from enzymatic degradation bythe combination of CAT and laccase or tyrosinase revealing again asynergistic effect. Only 40% of the TvL-treated catechin-enriched weredegraded to soluble collagen fractions under the tested conditions.
Thus, laccase and tyrosinase crosslinking in the presence of CATallows to significantly enhance the resistance of collagen materials toenzymatic cleavage and provides an alternative to crosslinking withbifunctional small molecule crosslinkers. In addition it also increasesthe tensile strength which is beneficial for handling of collagenbiomaterials upon implantation or during cell culture work for tissueengineering.
5. Conclusions
In this study, the potential use of laccases and tyrosinases for cross-linking of collagen was demonstrated. Both collagens and BrCN-peptides as model substrates were crosslinked with tyrosinases andlaccases. The cross-linking effect was enhanced in the presence ofphenolic molecules like caffeic acid (CA), chlorogenic acid (CHG) andcatechine (CAT). The determination of the denaturation temperaturerevealed higher values for the catechin-enriched collagen and theenzymatically modified materials. A combination of both rendered thehighest denaturation temperatures. Catechin addition reduced enzy-matic degradation by microbial collagenase, which could be furtherreduced by laccase-treatment whereas the enzymatic modificationsalone were not able to increase the resistance to collagenolyticdigestion. Thus, an enzyme treatment in the presence of phenolicmolecules could indeed replace conventional chemical crosslinkingwith toxic bifunctionalmolecules. The tensile strength of collagen couldbe significantly increased by combining enzymatic treatment withcatechin addition which is an important advantage both when the
56
58
60
62
64
66
68
70
Blank Blank-CAT
ThL ThL-CAT
TvL TvL-CAT
AbT AbT-CAT
Den
atur
atio
n te
mpe
ratu
re [°
C]
Fig. 11. Denaturation temperature of enzymatically cross-linked water-insolublecollagen paste specimens after neutral dialysis (grey) and dialysis at pH 3.7 (black).
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material is used for implants of cell culture in tissue engineering.Interestingly, the very low number of tyrosine residues in collagen stillseems tobehighenough toallowenzymatic crosslinking. SDS-PAGEandSEC results clearly demonstrate crosslinking leading to high MWmaterials while LC-MS/MS shows high sequences similarities ofuntreated and treated materials except for tyrosines which weredetected to a lower extent in crosslinked materials. In future studies,the demonstrated potential of laccases and tyrosinases to incorporatephenolic molecules could be further exploited to add specific function-alities to collagens.
Acknowledgements
This study was performed within the European Project COMET incooperation with the K-Project MacroFun and the COST868 action.Financial support from the EU-CORNET program, the FFG, the SFG andthe Province of Styria is gratefully acknowledged.
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