Development of printable bioactive paper containing laccase

Process Biochemistry 47 (2012) 1496–1502

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Development of printable bioactive paper containing laccase Heini Virtanen a,∗ , Hannes Orelma b , Tomi Erho a , Maria Smolander a a b

VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland Department of Forest Products Technology, School of Science and Technology, Aalto University, P.O. Box 16400, 00076 Aalto, Espoo, Finland

a r t i c l e

i n f o

Article history: Received 22 December 2011 Received in revised form 24 February 2012 Accepted 1 June 2012 Available online 9 June 2012 Keywords: Laccase stability Flexographic printing Bioactive paper Quartz crystal microbalance

a b s t r a c t Compatibility of Trametes versicolor and Trametes hirsuta laccases was studied with polymers used for flexographic inks. The aim was to produce bioactive paper with ability to change color. Optimum pH for the stability of Trametes versicolor and Trametes hirsuta laccases was determined during storage at room temperature for 60 days. The optimum pH for the stability of both laccases was 8–9. The stabilization effect of flexo printing inks on the enzymes was tested in liquid form and when coated on paper. Sulfo polyester resin HZ1100D stabilized the two laccases both in solution and on paper. For example, Trametes versicolor laccase remained stable for at least 8 weeks when coated with HZ1100D polymer. Furthermore, the adsorption of the flexo inks to cellulose was studied with quartz crystal microbalance with dissipation monitoring (QCM-D). It was observed that HZ1100D also adsorbs well on cellulose over a wide pH range. The results suggested that laccases are well suited to bioactive paper applications. Large scale manufacturing of bioactive paper products by flexo printing would be possible because of the compatibility of laccases with flexo inks. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Bioactive paper is a paper or other fibre based product, which includes functionalities based on selective reactions of bioactive compounds. Bioactive paper systems usually consist of a paperbased substrate and a bioactive compound attached onto this substrate [1,2]. Laccases are examples of bioactive compounds, which can be used as the active component of bioactive paper. Various methods such as, coating, printing and spraying can be used to add the bioactive compounds to the surface of paper [2–4]. Printing would allow large scale manufacturing without the need for specialty paper manufacturing. Laccases (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) are oxidoreductases belonging to an enzyme class called multicopper oxidases [5,6]. Laccases are widely distributed in nature and are found in almost all known fungal organisms and in many plants as well as in some insects and bacteria [5–8]. Most studied laccases are of fungal origin [9] and overview of occurrence and properties of fungal laccases was published by Baldrian [10]. Particular attractive are laccases from the class of white-rot fungi, including the Trametes genus, which was studied in this paper. Laccases catalyze reactions with a broad range of organic substrates, particularly phenols and other aromatic compounds, such as monophenols, diphenols, polyphenols, aminophenols, and diamines [11] and they also have

∗ Corresponding author. Tel.: +358 40 1795383; fax: +358 207227071. E-mail addresses: heini.virtanen@vtt.fi (H. Virtanen), hannes.orelma@aalto.fi (H. Orelma), tomi.erho@vtt.fi (T. Erho), maria.smolander@vtt.fi (M. Smolander). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.06.001

very large range of application possibilities [6,7,12]. An overview of application possibilities of oxidoreductases from Trametes species was published by Nyanhongo et al. [13]. The increasing demand of laccase on various applications also brings a need to produce laccase with low cost in large quantities. Recent improvements concerning the laccase production are presented by Alessandra et al. [14] and Lettera et al. [15]. ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), is an example of a laccase substrate. Laccases catalyze the oxidation of ABTS to a stable dark green cation radical ABTS+ . Simultaneously, oxygen is reduced to water. Bioactive paper containing laccase can hence be used in novel package applications as an oxygen absorber or oxygen or integrity indicator. The color-changing bioactive paper would also be suitable for anti-counterfeiting purposes, e.g. to ensure the authenticity of a document or package. Enzymes may be denatured by changes in the environmental conditions, such as temperature, pH and ionic strength [16]. In most enzyme applications, it is important that the active protein structure of the enzyme is resistant to denaturing factors [17], because enzyme denaturation means unfolding of the tertiary structure of the protein, after which the enzyme residues are not close enough to perform their functionalities [18]. To be able to have a bioactive product with long shelf-life, it is important that the active substance such as enzyme remains active as long as possible. The addition of stabilizing substances appears to be the simplest [16], oldest, most reliable and therefore most popular approach stabilizing the enzymes [18]. Surfactants, polyols and polymers and metal ions/salts are examples of additives, which have been reported to increase the stability of laccases [19–24]. For

H. Virtanen et al. / Process Biochemistry 47 (2012) 1496–1502

example, Basto et al. [19] reported that solutions of polyethylene glycol (PEG) stabilized Trametes villosa laccase and Papinutti et al. [20] reported that copper sulfate, glycerol, and mannitol enhanced the stability of Fomes sclerodermeus laccases. In addition, immobilizing laccase on polymeric supports has enhanced the stability of laccases [18,22]. While environmental factors such as pH have high effect on the enzyme activity, the pH has to be taken into account already in the beginning of the product planning. Several publications report the optimum pH for activity of laccases with various substrates [10]. It is widely recognized that optimal pH for laccase activity is highly dependent on the substrate. For example, the activity optimum pH of Trametes versicolor laccase was pH 2.5 with ABTS substrate and pH 3.5 with DMP (2,6-dimethoxyphenol) [25]. Thermostability of laccases and other enzymes is also a widely studied subject [20,26–28]. These studies mainly examine the stability of laccase only few hours or couple of days [21,26,27]. This is partly explained by rather high temperatures of some studies (40–70 ◦ C), where laccase inactivates during few hours [26]. A few papers also study the laccase stability during longer period. The effect of metal ions and mediators on the stability of laccase from Trametes hirsuta was studied for 7 days by Rodríguez Couto et al. [23]. Mai et al. [29] studied stability of Trametes versicolor laccase on solutions in the presence of phenolic compounds for 50–58 days. Sorensen et al. [24] reported that storage-stable liquid formulation comprising a laccase would have pH which is more alkaline than the pH optimum of the laccase. For example, pH optimum of Polyporus pinsitus laccase is 5.5. After 4 weeks storage in solution containing 60% PEG 6000 this laccase had residual activity on of 29% at pH 7 and 65% at pH 9. Bioactive paper has recently raised interest by various research groups and developments in this field have been recently reviewed by Pelton [30]. For example, Di Risio and Yan have studied ink jet printing of horseradish peroxidase on paper substrates [3,31]. Partly same authors with this paper have studied interactions between printing substrate and laccase on bioactive paper produced by ink jet printing [32]. However, in our knowledge there are no studies reported related to bioactive paper and compatibility of laccases with flexographic inks, which is the main aim of our study. In this work, optimum pH for the stability of Trametes hirsuta and Trametes versicolor laccases was studied by monitoring the activity of laccases stored over a wide pH range for up to 60 days. The compatibility of the Trametes versicolor laccase was studied with three different polymeric binders designed for flexographic printing inks (sulfo polyester resin HZ1100D and acrylic polymers GLE 520 and J1602). In addition, the adsorption of these polymers onto cellulose was studied with QCM-D. 2. Materials and methods 2.1. Materials Laccases produced by two different fungi were used: laccase from Trametes versicolor (Sigma–Aldrich, Germany, solid powder, activity 21.8 U/mg) and laccase from Trametes hirsuta (produced at VTT according to Poppius-Levlin et al. [33] and purified and characterized according to Rittstieg et al. [34]. ABTS (2,2- Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt (Roche Diagnostics, Germany) was used as the enzyme substrate. Three typical flexographic ink components were tested as major components of bioactive ink. Sulfo polyester resin dispersion, Hydro-Rez 1100D (referred to as HZ1100D, Hexion), had a solids content of 33% and a pH of 6. Other inks were acrylic emulsion Joncryl 1602 (referred to as J1602, BASF) and carboxylated acrylic copolymer, Glascol LE520 (referred to as GLE520, Ciba). Three kinds of papers were used as coating substrates: Yes Bronze, a commercial wood-free uncoated paper for B&W laser printing, copying and faxing. (UPM, 80 g/m2 ), hand-made sheets (VTT, Jyväskylä) made of eucalyptus cellulose (referred to as EUCA), 80 g/m2 , with polyamide–epichlorohydrin (PAE) resin addition (2.5 g/kg), and filter paper (Whatman No.1., England). The details and development of hand-made EUCA paper are described in Lappalainen et al. [35].

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2.2. Spectrophotometric enzyme activity assays Laccase activities of dissolved laccase samples were determined according to Niku-Paavola et al. (1988) using ABTS as substrate and Lambda 20 (UV/VIS) (PerkinElmer, Germany) or Varioskan (Thermo Electron Corporation, USA) spectrophotometers. The apparent activities of corresponding blank samples without enzyme were always measured as a reference. 2.3. Enzyme activity evaluation form the coated paper samples The laccase activity in the coated samples (0.5 cm × 2 cm) was evaluated by monitoring dissolved oxygen consumption in the presence of ABTS. Dissolved oxygen was measured on the basis of luminescent quenching using an oxygen meter (OXY10 mini sensor, Precision Sensing GmbH, Germany). The sample was placed into a reaction vessel (volume 1.8 ml) narrower side on the bottom so that surface was not covered by other parts of the paper. 1.8 ml of 5 mM ABTS solution (in sodiumsuccinate buffer, pH 4.5) was added to the vessel as a substrate and the reaction vessel was sealed immediately. Each vessel contained magnetic stirrer and stirring speed was 560 rpm. The measurements were performed in room temperature. The oxygen consumption was converted to enzymatic activity with the aid of a calibration curve [36], which was determined separately for T. hirsuta and T. versicolor laccases. The apparent activities of corresponding blank samples without enzyme were always measured as a reference. 2.4. Study of adsorption of binder polymers on cellulose by QCM-D Quartz crystal microbalance with dissipation monitoring (QCM-D) studies were performed using a QCM-D E4 apparatus from Q-Sense (Västra Frölunda, Sweden). The QCM-D technique is based on an oscillating quartz crystal, the resonance frequency and energy dissipation of which are measured in real time [37,38]. The adsorbed mass on the crystal surface decreases the resonance frequency that can be measured. The cellulose model surfaces were deposited using spincoating technique from trimethylsilylcellulose (TMSC) dissolved in toluene as described by Kontturi et al. [39]. Adsorption studies were performed using 1 mg/ml binder polymer dissolved in 10 mM phosphate buffer at pHs 5.0. 6.0, 7.0, and 8.0 using a continuous 100 ␮g/ml flow rate, and a constant temperature of 25 ◦ C. 2.5. Coating of bioactive ink Prior to use the Trametes versicolor laccase was dissolved in 50 mM sodium succinate buffer (pH 6) and only the soluble fraction was used further for ink preparation and the water insoluble fraction was discarded. Ink was applied onto the substrate by coating using the Hand Coater (bar 4, black, providing 40 ␮m wet ink layer, RK Print Coat Instruments Ltd.; United Kingdom). Papers were always dried for one day at room temperature before the first measurements. In between the measurements, papers were stored at room temperature. 2.6. Statistical analyses The statistical analyses were performed using SPSS (SPSS 17.0, SPSS Inc., USA) for the results with a minimum of three replicates. The variation in activity was analyzed using one-way analysis of variance (ANOVA) with the Tukey HSD test.

3. Results and discussion 3.1. Stability of laccases in solution To discover optimal bioactive ink composition the stability of laccases was first studied in solution. First, the optimum pH for stability of Trametes hirsuta laccase was determined. The stability of T. hirsuta laccase was then studied with different polymers (used in flexographic printing) on the optimal pH. The studies were continued with similar Trametes versicolor laccase and optimum pH for stability of Trametes versicolor laccase was studied in solutions containing chosen flexo polymer HZ1100D. The optimum pH for stability of Trametes hirsuta laccase was determined by monitoring the activity of laccase stored in phosphate citrate buffer (pH 3–8) at room temperature. Fig. 1 shows the effect of pH on the activity of T. hirsuta laccase during storage at room temperature. At this pH range, pH 3–8, stability of the laccase improved while pH increased. The samples stored at pH 8 and pH 7 retained most of the initial activity after 30 days of storage. Still after 60 days, 62% of the activity was retained at pH 8 and 33% at pH 7. At pH 4.5–6, stability was reasonable during the first weeks, but laccase was inactivated during prolonged storage. At pH 3–4

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pH 3 pH 4.5 pH 6 pH 8

120 100

120

pH 4 pH 5 pH 7

100

Residual activity (%)

Residual activity (%)

140

80 60 40 20 0 0

20

40

80 60 J2602

40

GL520 HZ1100D

20

60

Time (days)

buffer, pH 8

0 0

Fig. 1. Stability of Trametes hirsuta laccase in room temperature at pH 3–8 in phosphate citrate buffer. The data represent means ± range (n = 2).

10

20

30

Time (days) Fig. 2. Stability of Trametes hirsuta laccase during storage at room temperature in flexo ink solutions and in buffer with pH 8. The data represent means ± range (n = 2).

stability was poor and laccase inactivated rapidly during 14 days of storage. The activities of similar laccase samples stored at +4 ◦ C and −21 ◦ C were also measured, but the effect of pH was less remarkable in these conditions. After 60 days in −21 ◦ C, at pH 3 70% of activity retained and in pH 4.5–8 100%. After 60 days in +4 ◦ C 9% of the activity remained, at pH 4 44%, pH 4.5 82% and at pH 5–8 more than 94%. The best pH for long time storage of Trametes hirsuta laccase was pH 8. Even at room temperature, it was possible to retain T. hirsuta laccase active for at least 60 days at optimal pH. Cold storage (+4 ◦ C and −21 ◦ C) prolonged the stability and the effect of pH was less remarkable. However, the function was similar. At +4 ◦ C laccase stored at pH 3–4 does not retain its activity as well as samples stored at higher pH range. It was discovered, that optimum pH for stability of T. hirsuta laccase was pH 8, thus the stability of the laccase was determined at this pH in solutions containing different polymers used for flexographic printing (Fig. 2). During the first two weeks, no great differences were observed in the stability of laccase with the different polymers. After 30 days of storage, HZ1100D appeared to be the most appropriate polymer. At pH 8 the stability of the laccase in buffer and in buffer containing HZ1100D polymer was similar. T. hirsuta laccase was compatible with all three studied flexo polymers at pH 8. The studies were continued with Trametes versicolor laccase and chosen flexo polymer HZ1100D and the stability optimum pH was determined, by measuring laccase activity in HZ1100D polymer solutions adjusted to pH values 4–9 (Table 1). Similarly, the activities of samples (pH 4.5 and pH 6) without HZ1100D polymer were

also assayed in order to evaluate the stabilizing effect of HZ1100D polymer. After one week some effect of the pH was already noticed. 72% of the laccase activity was retained at pH 7 and only 44% at pH 4 and. After 4 weeks, a remarkable loss of enzyme activity was detected in the samples stored at pH 4–5. Less than 13% of the initial activity was retained in these samples, when samples at pH 6–9 containing HZ1100D retained over 40% of initial activity. As T. hirsuta laccase, also T. versicolor laccase on polymer solution was more stable on the higher pH range (7–9) than on the lower (3–6). T. versicolor laccase was more stable in HZ1100D buffer solution than in buffer solution without HZ1100D. After 4 weeks, laccase samples with HZ1100D at pH 4–5 still retained 8–13% of their initial activity, whereas laccase at pH 4.5 in 50 mM sodium succinate buffer without the polymer was almost completely inactivated (only 1% of the initial activity retained). At pH 6 the stability of T. versicolor laccase was also better with HZ1100D than without. HZ1100D clearly stabilized T. versicolor laccase at pH 4.5–6. To conclude the results above, optimum pH for stability for both of the laccases was over 7, which is clearly higher than pH optimum of Trametes species [10,13]. pH optimum of T. hirsuta and T. versicolor has been determined to be 2.5–5 depending on substrate [10,13]. It has also been reported, that to obtain an improved storage-stable liquid formulation, the pH must be 0.5–5.5 pH units more alkaline than the pH optimum of the laccase in question [24]. In basis of our studies here, we could state that this conclusion seems to be true also in case of T. versicolor and T. hirsuta laccases. T. hirsuta laccase was studied with polymers only at optimal pH 8.

Table 1 Activity of Trametes versicolor laccase in variable pH with and without HZ1100D after 1 h, 1 week and 4 weeks of storage at room temperature. The data represent means ± SD (n = 3–6). HZ1100D contenta

Used buffer

pH

sodium succinate sodium succinate sodium succinate sodium succinate sodium succinate sodium succinate phosphate Tris–HCl Tris–HCl

4 4.5 4.5 5 6 6 7 8 9

Activity (% from the initial) ± standard deviation 1 week

20% 20% – 20% 20% – 20% 20% 20% a

As final solid content (w/w).

44.1 ± 0.4 60.6 ± 1.2 18.9 ± 2.0 65.5 ± 0.8 62.8 ± 2.4 50.4 ± 1.3 72.0 ± 1.4 70.1 ± 7.2 57.3 ± 0.8

4 weeks 12.7 ± 0.5 8.8 ± 0.3 1.1 ± 0.3 9.0 ± 0.3 17.1 ± 0.4 13.7 ± 0.5 42.6 ± 1.7 44.3 ± 1.8 40.8 ± 2.3

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Table 2 Trametes versicolor laccase activity one day after application on different paper substrates. The data represent means ± SD (n = 3). Activitya

Paper

0.4 ± 0.1 2.1 ± 0.1 0.5 ± 0.1 2.5 ± 0.3 3.1 ± 0.1

Filter paper Filter paper + HZ1100D EUCA EUCA + HZ1100D Reference a

Activity (nkat/reaction vessel) ± SD.

It was discovered, that polymers did not have stabilizing effect on this pH. With T. versicolor laccase HZ1100D had a stabilizing effect on pH 6 and highly stabilizing effect on pH 4.5. It appears likely that the stabilizing effect of the polymers is enhanced when the pH decreases to the non-optimal level. 3.2. Laccase activity on papers coated with polymeric binders Sulfo polyester resin HZ1100D, a polymeric binder designed for printing inks, was shown to be compatible with Trametes hirsuta and Trametes versicolor in solution and even having laccase stabilizing effect at least at pH 4.5–6. Trametes versicolor laccase was chosen for used in all tests on paper. Initially, compatibility of Trametes versicolor laccase with paper was shortly tested. T. versicolor laccase was applied onto filter paper and onto EUCA precoated with HZ1100D and onto the same papers without HZ110D. The activities were measured one day after the enzyme additions (Table 2). As a reference, an equal amount of laccase was applied directly to the reaction vessel for the activity assay. T. versicolor laccase clearly lost most of its activity when applied as such directly to the cellulose substrate. However, when HZ1100D ink was first coated on the surface of paper, the laccase remained rather stable for at least one day. The activity was close to the reference value. It has been reported, that proteins are not strongly adsorbed onto pure cellulose and it has been shown that paper strengthening additives, such as PAE, promote the absorption [30]. Furthermore, paper sizing has been demonstrated to improve the color intensity of horseradish peroxidase-catalyzed reactions when the horseradish peroxidase is ink jetted on different papers [3,31]. HZ1100D was found to stabilize laccase both in solution and also as a coating layer applied prior to enzyme application. HZ1100D was shown to be compatible with T. versicolor laccase and appeared to prevent the inactivation of laccase on paper. Therefore, the compatibility of that laccase was studied with HZ1100D polymeric binder and two other polymeric binders designed for printing. Laccase solutions were dissolved in the polymeric binder dispersions (HZ1100D, GLE 520 and J1602) and the bioactive dispersions were coated on copy paper. The enzymatic activity was measured as consumption of dissolved oxygen after 1–30 days of

pH 5 pH 7 pH 8 pH 6

ΔF (Hz)

-10 -20 -30 -40 -50 -60 0

20

40

60

Time (min)

80

3.3. QCM-D study The cellulose model surfaces were used to model the adsorption of binder polymers on a wood fibre surface. The goal of the QCM-D studies was to analyze which binder polymer adsorbed most efficiently on cellulose. The change in frequency and dissipation values for absorbed binder polymers in all tested conditions after buffer rinsing are presented in Table 3. Fig. 4(a) presents the change in

b)

Curve order

0

storage (Fig. 3). Samples with J1602 as the polymeric binder were no longer enzymatically active after one day of storage, and thus J1602 is not compatible with these laccases. Laccase coated on copy paper in both HZ1100D and GLE520 solutions appeared to remain active for at least 30 days. After 30 days of storage, Trametes versicolor laccase on paper retained 64% or 81% of its activity compared to the value after one day when coated with HZ110D or GLE520 solution, respectively. Both of the inks HZ1100D and GLE 520 appeared to be compatible with Trametes versicolor laccase. The effect of coating surface pH on the stability of laccases was also studied (Fig. 3). Laccases were earlier determined to be more stable in mildly alkaline pH than at the acidic pH. The natural pH of GLE 520 is 2.5. Therefore, GLE520 was adjusted to pH 4–5 and to pH 6. Laccase was also dispersed to these adjusted GLE520 inks and coated. It was observed, that the increase in pH did not give the expected stability increase.

100

ΔD (10-6)

a)

Fig. 3. Activity of Trametes versicolor laccase polymeric binder mixture coated on copy paper (activity of the laccase in ink 600 nkat/g ink). The data represent means ± range (HZ1100D and GLE 520 n = 3, GLE520, pH 4.5 and GLE 520, pH 6 n = 2).

18 16 14 12 10 8 6 4 2 0

pH 7 pH 8 pH 5 pH 6 0

-10

-20

-30

-40

-50

-60

ΔF (Hz)

Fig. 4. (a) Change in frequency for adsorption of 1 g/l binder polymer HZ1100D on cellulose as measured by QCM-D. (b) Change in dissipation as a function of change in frequency for adsorption of 1 g/l binder polymer HZ1100D on cellulose.

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Table 3 Change in frequency and dissipation values for adsorbed binder polymers: J1602, GLE520 and HZ1100D and slope values of dissipation as a function of change of frequency. J1602

GLE520

HZ1100D

pH

F (Hz)

D (10−6 )

Slope (D–F)

F (Hz)

D (10−6 )

Slope (D–F)

F (Hz)

D (10−6 )

Slope (D–F)

5 6 7 8

−0.5 −14.5 −4.8 −48.5

0.4 12.7 7.1 14.2

1 0.9 0.4 0.2

−0.4 −8.3 3 1.4

1.8 8.1 5 4.7

0.5 0.7 1 0.4

−43.3 −49.5 −50.5 −48.9

6.4 5.6 14.8 6

0.2 0.1 0.3 0.1

frequency curves of the QCM-D studies for binder polymer HZ100D. The binder polymer HZ1100D adsorbed remarkably at all pH values that can be seen as high negative frequency values, but the adsorption profiles were similar. Moreover, the adsorbed HZ1100D formed a highly viscoelastic adsorption layer which was seen as high dissipation values. J1602 binder polymer adsorbed remarkably only at pH 6 and 8 creating highly viscoelastic adsorption layers. The binder polymer GLE520 adsorbed significantly only at pH 6. The QCM-D instrument is widely used in adsorption analyses of polymers on cellulosic substrates. The slope of the D–F–curve is used to illustrate the affinity of the adsorbed polyelectrolyte to cellulose. There the small slope is stated to demonstrate the high affinity of polymer to adsorb on the substrate, whereas high slope corresponds to opposite [40]. The slope values of D–F curves are also shown in Table 3 and in addition Fig. 4(a) shows the change in dissipation as a function of change in frequency for adsorption of HZ1100D. The slopes of the binder polymer HZ1100D at all studied pH values were significantly lower compared to other analyzed polymers except J1602 at pH 7. Moreover the slope was almost constant at pH 5, 6, and 8, whereas at pH 7 the slope was slightly higher indicating slightly lower affinity. These results demonstrate that HZ1100D has high affinity to cellulose leading to stiff conformation of the adsorbed layer. Whereas, other analyzed polymers created looser adsorption layers on the cellulose surface as can be seen in the high slope of the adsorption curves. Moreover, the constant slope of the adsorption curves of polymer HZ1100D demonstrated also the non-electrostatic interactions droving the adsorption process since the changes in pH did not influence to the slope. The slope of the binder polymer J1602 decreased as a function of pH, which is typical for polyelectrolytes [41] indicating that the properties of the polymer are more compatible with cellulose at neutral and slightly basic pH range. The binder polymer GLE520 created a layer, which has remarkable affinity with cellulose only at pH 6. These results indicate that the binder polymer HZ1100D is most suitable for use in flexographic inks, and that it has high attraction to cellulose over a wide pH range. 3.4. Suitability of EUCA paper as a coating substrate of bioactive paper As HZ1100D was found to be a compatible polymer with laccases, the work was continued by studying different base papers. EUCA paper was chosen for the test to be compared with copy paper. Surface roughness of the two sides of handsheets differs, and the coatings and printings were performed on the smoother side of EUCA paper. EUCA paper is very porous compared to copy paper and therefore oxygen was assumed to penetrate the coating layer better when HZ1100D with laccase was coated on the EUCA paper. A better oxygen penetration level was assumed to help the interaction of laccase with oxygen and ABTS. It was tested, whether coating on EUCA paper would increase the laccase activity compared to coating on copy paper. The EUCA paper used in this work contained only PAE and cellulose. By contrast, copy paper contains many additives used in the papermaking process. These could include retention chemicals, calcium carbonate, clay, dyes, titanium dioxide and other whitening agents. Fig. 5

Fig. 5. Activity of Trametes versicolor laccase HZ1100D solution on copy paper and on EUCA paper 1 day to 8 weeks after coating. Activity of laccase in ink 600 nkat/g. The data represent means ± SD (n = 3). a and b are Tukey groups with P value <0.05 for comparison of laccase activity on different substrates. No significant differences were detected in the comparison of activities at different time points with P value <0.05.

presents the effect of the coating surface on the activity of Trametes versicolor laccase. Laccase activity did not significantly decrease during the storage of 8 weeks. Activity of laccase seems to be higher on EUCA paper than on copy paper (although statistically significant difference only after 1 week of storage). The slight difference in activities between EUCA paper and copy paper may be explained by the porosity. The porosity of paper affects the maximum quantity of biomolecules attached onto cellulose [30]. For non-porous papers, only the macroscopic external surface is available. The porosity of EUCA may therefore afford better accessibility to proteins and thus explain the greater activity on EUCA paper. Furthermore, due to the porosity, the weight of the coating was probably to be higher on the EUCA paper than on the copy paper, although the same coating bar was used. However, the differences are mostly insignificant. According to the Tukey test, the EUCA paper differed significantly from the copy paper only after one week of storage. 3.5. Color change of ABTS on EUCA paper with laccase Laccase-based bioactive paper could function as a colorchanging oxygen indicator or integrity indicator. The aim of the color change tests was to estimate the suitability of the papers for the indicator applications. Laccase can use many substrates. Many of the laccase substrates also change color upon oxidation. One example of these substrates is ABTS, which was used in this work. Laccase catalyses the conversion of ABTS to the dark green ABTS cation radical. For several applications of bioactive paper, it would be beneficial to have a rapid color change. EUCA paper coated with HZ1100D is much more porous and more hydrophilic than copy paper coated

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Fig. 6. Images of EUCA paper coated with HZ1100D with laccase (above, activity of laccase in ink 300 nkat/g ink) and without laccase (below). ABTS solution (5 mM, 50 ␮l) was added to the reverse side of the paper. Images were taken 0, 15, 30, 60, 90, 180 and 300 s after ABTS application, respectively, from left to right.

with HZ1100D. Therefore ABTS solution adsorbs rapidly to the surface layer of EUCA paper, which makes it more beneficial for the application. The test for color change was therefore performed only on EUCA paper. ABTS solution was applied onto EUCA paper on the reverse side, where there was no coating. The solution adsorbed already in 15 s, and the color change reaction was also excellent on the reverse side of the paper (Fig. 6). The color change of ABTS on EUCA paper coated with HZ1100D laccase dispersion was rapid. During 5 min, no clear change in the reference samples was detected, and thus this kind of product would be suitable as an authenticity indicator or other color changing indicator.

4. Conclusions The stability optimum pH for the two studied laccases from Trametes versicolor and Trametes hirsuta was found to be 7–9. Thus it is considerably higher than the optimum pH for activity of Trametes versicolor laccase, which has been determined to be 2.5 with ABTS substrate [25]. When developing a product with these laccases, the conflict between the stability and activity optimum pH should be taken into account. It was also discovered that flexographic ink polymers could stabilise T. versicolor laccase and that the effect of pH on the stability became less important in the presence of the polymers. In addition, it appears that when laccase is coated on the paper, pH is less important than in aqueous solution. However, the stability of enzyme solution is relevant in case of storing the aqueous ink. For industrial process it would be advantageous to also have ink with long shelf-life. Laccases proved to be well compatible with flexo ink binders HZ1100D and GLE520, but one studied flexo ink binder J1602 proved to be incompatible with laccases, although in liquid form it did not cause problems. T. versicolor laccases coated on copy paper with flexo inks (HZ1100D and GLE520) remained active at least during 8 weeks of storage. T. versicolor laccase coated as HZ1100D dispersion onto both EUCA and copy paper remained active for 8 weeks and activity did not significantly decrease during this time. In addition, the color formation of ABTS appeared to be rapid and clear on EUCA paper samples coated with T. versicolor laccase. Consequently, the bioactive paper product developed here clearly appeared to be suitable as a rapidly color changing indicator. The information gained in this work can be further used to develop an indicator to be manufactured by printing. Laccase is compatible with flexographic inks, and thus it would be easy to move on to tests printed with a flexographic printer. Bioactive paper has broad range of application possibilities for example on the field of medical diagnostics, safety, security, public

health and smart packaging. Laccase based bioactive paper could be used for example indicating for package or document authenticity or could be further developed for oxygen detecting or oxygen removing applications. Acknowledgments Nina Vihersola is thanked for skilful technical assistance. Dr. M. Andberg, O. Liehunen and B. Hillebrandt-Chellaoui (VTT) are acknowledged for providing the purified T. hirsuta laccase. Thanks to Jouko Virtanen for helping with the polymers. This work was carried out in the BioAct2 project which was made possible by the financial support of Tekes–the Finnish Funding Agency for Technology and Innovation. The authors also acknowledge the financial support of the following companies and the invaluable guidance of the representatives of these companies in the steering group of the project: UPM-Kymmene Oyj, BASF Oy, Tervakoski Oy, Orion Diagnostica Oy, Hansaprint Oy and Oy Medix Biochemica Ab. References [1] Costa SA, Tzanov T, Filipa Carniero A, Paar A, Gübitz GM, Cavaco-Paulo A. Studies of stabilization of native catalase using additives. Enzyme Microb Technol 2002;30:387–91. [2] Aikio S, Gronqvist S, Hakola L, Hurme E, Jussila S. Bioactive paper and fibre products: patent and literary survey. VTT Working Paper 51; 2006. [3] Di Risio S, Yan N. Piezoelectric ink jet printing of horseradish peroxidase on fibrous supports. J Pulp Pap Sci 2008;34:203–11. [4] Gustafsson P, Grönqvist S, Toivakka M, Smolander M, Erho T, Peltonen J. Incorporation of laccase in pigment coating for bioactive paper applications. Nord Pulp Pap Res J 2011;26:118–27. [5] Solomon EI, Sundaram UM, Machonkin TE. Multicopper oxidases and oxygenases. Chem Rev 1996;96:2563–606. [6] Mayer AM, Staples RC. Laccase: new functions for an old enzyme. Phytochemistry 2002;60:551–65. [7] Riva S. Laccases: blue enzymes for green chemistry. Trends Biotechnol 2006;24:219–26. [8] Singh G, Bhalla A, Kaur P, Capalash N, Sharma P. Laccase from prokaryotes: a new source for an old enzyme. Rev Environ Sci Biotechnol 2011;10:309–26. [9] Kiiskinen LL, Viikari L, Kruus K. Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 2002;59:198–204. [10] Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev 2006;30:215–42. [11] Lahtinen M, Kruus K, Boer H, Kemell M, Andberg M, Viikari L, et al. The effect of lignin model compound structure on the rate of oxidation catalyzed by two different fungal laccases. J Mol Catal B 2009;57:204–10. [12] Widsten P, Kandelbauer A. Laccase applications in the forest products industry: a review. Enzyme Microb Technol 2008;42:293–307. [13] Nyanhongo GS, Gübitz G, Sukyai P, Leitner C, Haltrich D, Ludwig R. Oxidoreductases from Trametes spp. in biotechnology: a wealth of catalytic activity. Food Technol Biotechnol 2007;45:250–68. [14] Alessandra P, Cinzia P, Paola G, Vincenza F, Sannia G. Heterologous laccase production and its role in industrial applications. Bioeng Bugs 2010;1:252–62. [15] Lettera V, Del Vecchio C, Piscitelli A, Sannia G. Low impact strategies to improve ligninolytic enzyme production in filamentous fungi: the case of laccase in Pleurotus ostreatus. C R Biol 2011;334:781–8.

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