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Printing of Polymer Microcapsules for Enzyme Immobilization on Paper Substrate Anne Savolainen,† Yufen Zhang,‡ Dominic Rochefort,‡ Ulla Holopainen,† Tomi Erho,† Jouko Virtanen,† and Maria Smolander*,† † ‡
VTT Technical Research Center of Finland, VTT P.O. Box 1000, 02044 VTT, Finland Departement de Chimie, Universite de Montreal, CP6128 Succ. Centre-Ville, Montreal, Quebec, Canada H3C 3J7 ABSTRACT: Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.
’ INTRODUCTION Microencapsulation is widely exploited in industries for various functions: immobilization or entrapment, protection, controlled release, structuration, and functionalization. In every case an active ingredient is surrounded by a membrane or matrix and therefore isolated from its surroundings. The encapsulation procedure consists of three steps: incorporating the active ingredient in the microcapsule core or matrix, mechanical operation and engineering either in liquid or solid matrix, and stabilization of the capsule by a chemical, physicochemical, or physical process. When operating in liquid matrix, the dispersion used in the encapsulation can be performed by prilling, spray technologies, emulsification, or microemulsification. After dispersion formulation, droplets can be transformed as solid particles, that is, by solidification, evaporation, gelation, or polymerization. Other encapsulation methods are coating, agglomeration, and layer-by-layer techniques.1,2 Enzyme immobilization has been studied in different applications, for instance in the pulp and paper sector. Enzymes as biotechnological instruments are used in creating novel functional solutions and products, that is, bioactive paper,3,4 biosensors,58 and biofuel cells.9 By using different physical or chemical methods, that is, cross-linking, covalent coupling, adsorption, and entrapment, biomolecules can be immobilized on different surfaces. For instance, oxidative enzymes like laccases have been bound to cellulose and lignin by physical adsorption.10,11 Chemical coupling methods are more complex than physical adsorption and entrapment techniques, yet they are r 2011 American Chemical Society
more efficient in maintaining the enzyme activity than the physical methods. The benefit of using physical immobilization on techniques is in their consistence with mass production.12 There are many publications of different printing methods which have been used in biomolecule immobilization. Khan et al.13 have studied ink jet printing of horseradish peroxidase (HRP) and alkaline phosphatase enzymes on cellulose substrate, where the main goal was two-fold: the first goal was to investigate the thermal stability of the adsorbed enzymes, and the second was to develop a pattern printed by enzymes on paper surface. It was found that HRP could resist the physical forces of ink jet printing, that is, shear and thermal stress, and maintain its activity on the cellulosic surface. The composition of bioactive ink has been developed in the study of Di Risio and Ning.14 In their research, the focus was to study the effect of different ink additives on enzyme activity, especially additives affecting the viscosity and surface tension of the bioink containing horseradish peroxidase. It indicated that the selection of conventionally used viscosity modifiers had a remarkable effect on the activity of the biomolecules. Carboxymethyl cellulose (CMC) was shown to be the most suitable viscosity modifier for HRP-bioink to achieve the desired ink viscosity while preserving the enzyme activity.
Received: November 9, 2010 Revised: April 14, 2011 Published: May 13, 2011 2008
dx.doi.org/10.1021/bm2003434 | Biomacromolecules 2011, 12, 2008–2015
Biomacromolecules The entrapment of biomolecules has been widely utilized as a physical immobilization method. Zhang and Rochefort15 studied the entrapment of laccase in poly(ethyleneimine) (PEI) microcapsules. Two encapsulation methods were compared by characterizing microcapsules produced either through the emulsion method or the vibration nozzle method. According to Zhang et al. enzymes were efficiently entrapped in PEI-microcapsules produced by the emulsion method or encapsulation device. Utilization of this commercial device resulted in larger particle diameter with smaller particle size distribution. Kouisni and Rochefort16 prepared bioactive paper by mixing microcapsules with the pulp before preparing paper sheets or deposited microcapsules on paper surface by filtration. Microcapsules were prepared by the same procedure as described by Poncelet et al.17 Printing of microcapsules and encapsulated enzymes has been reported to some extent. In the United States patent 4729792,18 glycol-based ink containing microcapsules were press-applied and used in making carbonless copy paper. Perfume-containing microcapsules have been gravure and screen printed according to U.S. patent 3888689.19 Shitanda et al.20 has prepared screen printed electrodes by printing carbon-based ink containing glucose oxidase microcapsules consisting of polyamide. In this work, encapsulation of two different laccases, that is, Trametes hirsuta (ThL) and Trametes versicolor (TvL), in PEI microcapsules and subsequent printing of the resulting microcapsules on paper surface were studied. The main purpose of enzyme immobilization on paper substrate was not only to maintain the enzymatic activity during the printing process but also to prolong storage stability of the enzyme by the microencapsulation method. Laccase-containing microcapsules were applied on paper substrate by using three different methods: rod coating, screen, and flexo printing. The factors involving microcapsule preparation and therefore affecting the size of the microcapsules were analyzed by determining the particle size distribution. The effects of encapsulation, ink composition, and printing to enzyme activity were determined. Additionally, the comparison between the adhesion of free laccase and encapsulated laccase on paper surface was tested. A microscopic analysis was performed to visually observe the agglomeration of microcapsules and the success of the printing.
’ EXPERIMENTAL SECTION Materials. Polyethyleneimine (PEI, Mn = 1200 g/mol), sebacoyl chloride (SC), cyclohexane, Span 85, Tween 20, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), TvL, and FITC (fluorescein isothiosyanate) were purchased from Sigma-Aldrich. Laboratory grade purified ThL was from VTT; Yes Bronze 80 g/m2 (UPM) (referred as YES) was used as the printing substrate. Flexo inks containing HydroRez 1100D sulpho polyester-based polymer (Hexion Specialty Chemicals, referred as HZ 1100D) and Tween 80 surfactant (Sigma Chemical Co.) were also studied. Methods. Preparation of PEI Microcapsules. PEI microcapsules were prepared by an interfacial polymerization reaction, in which sebacoyl chloride was used as a cross-linker. Microemulsion, which consisted of an aqueous phase (PEI and Milli-Q water) and organic phase (cyclohexane and 1% v/v of Span 85), was produced by agitating the aqueous and organic phase together with a “house made” stirrer by 1100 rpm. To evaluate the influence of the mixing head used in the agitation, two different shapes of mixing heads were used in the microcapsule preparation: a straight blade, which is referred to as a “house made”, and blade, which has round holes in the blade. Sebacoyl
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Table 1. Ink Composition ink component
percentage by weight
Milli-Q water
30%
PEI-microcapsules
40%
HZ 1100D-binder polymer
25%
Tween 80 surfactant
5%
chloride was added to the emulsion as a cross-linker of PEI to be able to form a membrane around the aqueous phase. This described method was developed by Poncelet et al.17 Enzyme-containing microcapsules were prepared by dissolving protein in the aqueous phase, which was added to the organic phase to form an emulsion. Finally the microcapsules were thoroughly rinsed with water. Fluorescence tagged microcapsules were prepared by preparing FITC-PEI before microencapsulation by dissolving 2 mg of FITC into 0.2 mL of dimethyl sulfoxide, and 120 μL of this mixture was added to solution containing PEI and 50 mM sodium succinate buffer (pH 4.5). The method of preparing covalently bonded FITC onto PEI was performed as specified by Kouisni and Rochefort.16 As specified below, microencapsulation was carried out either by using FITC-PEI or regular PEI. Printing of PEI Microcapsules. When applying a PEI microcapsule containing ink on paper substrate by printing or coating, the same ink recipe was used with each application technique (Table 1). Ink components were mixed together at room temperature by a magnetic stirrer for two hours to achieve a homogeneous ink solution. Microcapsule-containing ink was applied on paper surface by three different methods: screen printing, rod coating, and flexo printing. In screen printing, the sieve mesh counts of 30 and 40 were used. In rod coating, the K Control Coater from RK Print-coat Instrument Ltd., U.K., was used with a rod marked 5 (thickness of wet ink layer 50 μm). The coating speed was 4 units of the instrument. In flexo, the printing press was IGT F1. The printing was performed in the following conditions: surface volume of the anilox disk was 28 mL/m2, the speed 0.3 m/s, the anilox force 100 N, and the printing force 100 N. The amount of wet ink on the printed paper varied from 250 g/m2 for screen printing, to 50 g/m2 for rod coating, and to 10 g/m2 for flexo printing, respectively. The amount of wet ink for the flexo printed sample was evaluated by calculating the theoretical amount and analyzed both with screen printed and rod coated samples by determining grammage according to a general standard method. Printing was carried out in ambient room temperature, and the samples were dried at room temperature overnight after printing.
’ CHARACTERIZATION OF MICROCAPSULES Particle Size Distribution. For particle size distribution measurements, a sample containing 0.1% PEI-microcapsules was prepared. To obtain a well-dispersed sample, the solution was sonicated (Sonics Vibra Cell A191, 100% amplitude without pulsation) for 1 min; during sonication the integration of the capsules was confirmed by light microscopy. Particle size distribution of PEI microcapsules was determined by using Coulter LS230, which is based on light scattering detection. Measurements were performed immediately after sonication. Enzymatic Activity Measurements. To study the compatibility of laccases (T. hirsuta and T. versicolor) with microcapsules, enzymatic activity of laccase was measured 1 d after microcapsule preparation. Enzymatic activity of free and microencapsulated laccase was determined at pH 4.5 using ABTS as a substrate.12 Enzyme aliquots were mixed into 1.8 mL of 5 mM ABTS solution (pH 4.5), and the activity of laccase was monitored as consumption of dissolved oxygen in the presence of ABTS. The dissolved 2009
dx.doi.org/10.1021/bm2003434 |Biomacromolecules 2011, 12, 2008–2015
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Figure 1. Consumption of oxygen (Δmg/L per min) in ABTS oxidation by laccase as a function of laccase dosage in the reaction vessel (volume 1.8 mL): (a) T. hirsuta; (b) T. versicolor.
oxygen was monitored based on dynamic luminescence quenching using an Oxy10 mini sensor oxygen meter (PreSens, Germany) in a closed 1.8 mL reaction vessel. The oxygen consumption measured as Δmg/L per time unit was converted into corresponding enzymatic activity (nkat) with the aid of a calibration curve, which was determined separately for T. hirsuta (0.54 nkat per 1.8 mL) (Figure 1a) and T. versicolor (0.25 4 nkat per 1.8 mL) laccases (Figure 1b). For the activity determination of printed, microencapsulated laccase, paper samples (0.5 cm 2 cm) were immersed in 1.8 mL of 5 mM ABTS solution (pH 4.5), and the activity of laccase was monitored as the consumption of dissolved oxygen as described above. As references, an equivalent amount of free enzyme or empty microcapsules were printed on paper. These samples were likewise analyzed by determining their oxygen consumption by the aforementioned method. Since the specific activity may differ from free to encapsulated enzymes due to diffusional restrictions, we compared the normalized activity for all types of printing. Confocal Laser Scanning Microscope Study. FITC-tagged PEI microcapsules and paper samples were analyzed by a confocal laser scanning microscope (CLSM) equipment consisting of a Bio-Rad Radiance Plus confocal scanning system (BioRad, Hemel Hempstead, Hertfordshire, UK) attached to a Nikon Eclipse E600 microscope (Nikon Corp., Tokyo, Japan). A water suspension of FITC-PEI microcapsules containing 1% of Tween 20 surfactant was analyzed using a 488 nm argon laser line for excitation and a band-pass emission filter at 500560 nm. For comparison, microcapsules without FITC-tag were imaged in brightfield using an Olympus BX-50 microscope (Olympus Corp., Tokyo, Japan). All paper samples were examined with CLSM using a 488 nm argon laser line for excitation, a band-pass emission filter at 500560 nm for green fluorescence, and a longpass emission filter above 570 nm for red fluorescence. Images of the surface and cross-sectional profile were assembled from the optical sections taken using 10 objective (Nikon Plan Apo, numerical aperture 0.45) at a depth of 20 μm with 5.0 μm z step, 1.0 μm pixel size, and resolution of 512 512. In the superimpositions of green- and red-filtered emission images, the autofluorescence of paper substrate appeared green, and the autofluorescence of the microcapsules was seen as yellow.
Figure 2. Particle size distribution of PEI microcapsules containing TvL prepared by two different types of agitator heads.
Figure 3. Microscopy images of agglomerated PEI microcapsule particles: (a) brightfield image of PEI-microcapsules and (b) FITC-tagged PEI-microcapsules imaged with CLSM.
’ RESULTS AND DISCUSSION Particle Size Distribution of PEI Microcapsules. The average particle size of laccase microcapsules was 50 μm; however, the particle size varied considerably from 20 to 200 μm (Figure 2). The most likely reason for the wide particle size distribution was the preparation technique, because agitation speed and the emulsion method have an influence on the particle formation. It has been reported that the large particle size distribution is a result of emulsion preparation by mechanical stirring. Zhang and Rochefort15 reported a large particle size distribution of PEI 2010
dx.doi.org/10.1021/bm2003434 |Biomacromolecules 2011, 12, 2008–2015
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Scheme 1. Laccase Catalyzed Reaction with ABTS as a Substrate
microcapsules prepared by the emulsion method. Du et al. observed wide distribution in paclitaxel-loaded chitosan oligosaccharide particles by the emulsion method.1 Due to the agglomerated particles, the particle size distribution analysis tends to result in overestimated particle diameter for microcapsules since the analysis is based on light scattering, and therefore the measurement unit does not distinguish the difference between a single microcapsule and agglomerated microcapsules. The agglomeration of PEI-microcapsules is shown in Figure 3: part a is a brightfield microscopy image of a microcapsule sample, and part b is a confocal micrograph of FITC-PEI microcapsules; samples were dispersed in 1% Tween 20. The conjugation of FITC was performed in pH 4.5, which is not the optimal pH for the conjugation. A greenish color, which can be seen inside the capsules, may have been a result of either the fluorescence reflected from capsule walls or from free FITC inside the capsule core. This free FITC had remained in the PEI solution, because it had not been removed by chromatographic means from the solution containing FITC-PEI. Nevertheless, conjugation had sufficed, which was verified by observing these FITC-marked capsules by CLSM. A previous CLSM study demonstrated that most enzymes are located within the wall of the PEI microcapsules.16 It has also been reported earlier that the encapsulation efficiency by this method was 94 ( 2%, and the encapsulated enzyme cannot diffuse across the membranes, since pore size of membranes is smaller than enzyme molecules.15 In the present study, the labeled microcapsules were used for localization purposes and did not contain any laccase. Although irrelevant here, FITC may influence the laccase activity, but this was not evaluated. The capsule wall thickness was roughly estimated to be approximately 1.25 μm. As an example, the particle size distribution of TvL microcapsules prepared by a “hole blade” and “straight blade” are presented in Figure 2. The configuration of the agitation head type did not have an outstanding influence on particle size, nor had the encapsulated material, which was either different laccases or Milli-Q-water. To observe remarkable differences between different agitation head types, these heads should have differed more from each other. For instance, when using an agitator head, which is made of thin metal wire with a figure eight shape, it might have dispersed the PEI-solution more evenly and produced smaller capsules. Actually, this phenomenon could have been seen with any other agitation head style, which would have dispersed the solution more evenly. Enzymatic Activity and Stability of Encapsulated Laccase. Rochefort et al.21 has reported that anionic ABTS diffuses inside the positively charged PEI capsule core and strongly interacts
Figure 4. Relative enzymatic activity as a function of time in both encapsulated and free T. versicolor laccase. Samples were stored at 4 °C.
with the capsule wall. This phenomenon resists the diffusion of ABTS back to bulk solution; thus, enzyme activity based on an amperometric analysis cannot be evaluated by using ABTS as a substrate for laccase. Furthermore, the ABTS interaction with the PEI capsule core is a crucial drawback when applications involving shuttling of ABTS between the enzyme's active center and an electrode are considered (e.g., biofuel cells).21 However, in this study, the diffusion of ABTS back to bulk solution is not required for activity measurement since the assay is based on oxygen consumption, and as a neutral molecule oxygen is not expected to interact with the capsule wall. The same applies to applications where diffusion of ABTS is not required yet induces either the color change of the substrate or the oxygen consumption due to the enzymatic reaction. This enabled the usage of ABTS as a substrate for microencapsulated laccase in this work according to reaction Scheme 1. Enzymatic activity and storage stability of encapsulated T. versicolor laccase were studied by measuring oxygen consumption in sodium succinate buffer (pH 4.5) as a function of time. The measurement was performed at room temperature, and microcapsules were stored at 4 °C. In Figure 4, initial activity of PEI-microcapsules containing T. versicolor (1 d) was set to 100%, and the other results were converted to relative enzyme activity by comparing results after 1 d. These results were compared to activity and storage stability of free enzyme. It was clearly shown that the encapsulation of laccase prolonged storage stability of enzyme, and laccase preserved nearly 70% of its activity even after 5 months, whereas activity of free enzyme decreased almost 50% (Figure 4). Results revealed that during certain storage times, the immobilized enzyme attained a level, after which enzyme activity had been maintained almost constant as could be expected based on previous results.22 Increased stability can be explained by the isolation of the enzymes from the environment and hence protected from agents (e.g., bacteria, proteases) that may have affected their structural integrity. Moreover, enzyme reticulation with a polymer has been shown to improve its stability by restricting conformation changes that occur with time and that are responsible for activity decrease. The initial activity decrease is most likely due to the encapsulation procedure and the interaction between PEI and the copper atoms in the laccase active site. However, in terms of industrial applications the stability of the enzyme is quite relevant, for instance in applications where an enzyme containing ink is manufactured as an individual product which is then distributed to printing houses. 2011
dx.doi.org/10.1021/bm2003434 |Biomacromolecules 2011, 12, 2008–2015
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Figure 5. Enzymatic activity as a function of storage time and the compatibility of polymeric ink components (Milli-Q water, HZ 1100Dbinder polymer and Tween 80 surfactant) with encapsulated T. versicolor and free T. versicolor. Samples were stored at 4 °C.
Compatibility of Ink Components with Encapsulated Laccase. The compatibility of encapsulated T. versicolor laccase and
the ink components (Milli-Q water, HZ 1100D-binder polymer and Tween 80 surfactant) used in this work were studied by measuring the enzymatic activity in aqueous suspension at pH 4.5. Enzymatic activity was preserved in the presence of ink components. However, differences could be observed in the maintenance of the activity and stability between encapsulated and free laccase. Although the activity of microencapsulated T. versicolor was originally lower than that of the free enzyme, the encapsulated enzyme maintained its activity virtually constant throughout eight weeks of storage. The difference in the original level of the activity was likely the result of the osmotic pressure in the solution or molecular interactions between the binder polymer and PEI microcapsules, which affected the diffusion of ABTS into the microcapsule. Additionally, it was decidedly indicated that, in the polymer-based ink solution, the activity of nonmicroencapsulated (free) laccase was quite well-preserved during storage although the activity decreased faster than with microencapsulated enzyme (Figure 5). The polymer-based ink used in this work was compatible with studied enzymes and is capable of maintaining constant activity of both free and encapsulated laccase over a long period of time. The ink as such is compatible with laccase; thus, the potential mass transfer of the ink components is unlikely to have an effect on the enzymatic activity. The MW cutoff of the membrane can be estimated between 2 and 3 nm,15 and the charge of the ink polymers needs to be taken into account since the PEI membrane will be positively charged at the pH which has been used in this study. Performance of Laccase Microcapsules on Printed Paper Samples. In further studies, the enzymatic activity of T. hirsuta and T. versicolor laccases in paper samples printed by two different techniques, flexo and screen printing, was measured by an analysis method based on the oxygen consumption. Furthermore, paper samples containing laccase microcapsules were prepared by rod coating as a reference for printing methods. Commercially available wood-free uncoated paper for black and white laser printing, copying, and fax has been used as a printing substrate as our aim is to examine printing of microencapsulated enzyme in mass scale manufacturing using widely available commercial materials.
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The application technique had a significant impact on the amount of microcapsules which could be applied on paper surface. In industrial printing processes, printed layer thickness achieved by screen and flexo printing are 100300 and 5 μm, respectively.23 In this study, these layer thicknesses, which were estimated according to microscopy images, were found to be comparable. It was possible to apply the highest amount of microcapsules on paper by screen printing technology, which was verified by oxygen consumption measurement and CLSM. Compared to screen printing, the amount of applied microcapsules thereby decreased enzyme activity when microcapsules were applied by rod coating or flexo printing. Enzyme activity and evaluated grammage of the microcapsule ink was the lowest in flexo printed paper samples. It is possible that larger capsules had been broken by the shear effect of doctor blade on the anilox roll. Most probably the largest capsules had not been able to fit in the anilox cells. In screen printing, on the other hand, most of the capsules were able to fit through the mesh opening of 450 μm or larger. The approximate thickness of applied microcapsule layers was determined by CLSM images (Figure 6). In these images, unlabeled microcapsules had a strong autofluorescence, which can be observed as yellow in these images. The green color originated from the autofluorescence of the base paper. The effect of the printed enzyme distribution on paper's X, Y, and Z dimensions on activity and aging has been studied by Matilainen et al.24 According to their results, the higher the local enzyme concentration, the higher is the local enzyme activity. In the case of the free enzyme, the molecules might penetrate into paper bulk if there is not a specified coating layer hindering the penetration of the enzyme. Therefore, the observed activity is less because the enzyme is distributed on a larger area. When the enzyme is microencapsulated, the capsules will not penetrate inside the paper structure (typical pore size 101 μm25), which in turn gives high local enzyme concentration on the paper surface. It should also be kept in mind that the activity of printed, microencapsulated enzymes can be affected by several diffusional barriers. Regarding only the ink, the printing layer thickness and the penetration depth of the bioink can increase the response time due to longer diffusion paths and irregular biomolecule density. For microcapsules, the additional restriction due to attractive electrostatic interactions between ABTS and PEI will also affect the activity measurement. Figure 7a illustrates the effect of the printing method on the oxygen consumption and enzyme activity. As expected, empty microcapsules without enzymatic activity did not consume any oxygen in the reaction. The paper sample containing ThL microcapsules prepared by screen printing had the highest enzymatic activity and the fastest oxygen consumption rate, whereas the lowest oxygen consumption rate indicating rather low enzymatic activity was detected in flexo printed samples, as expected. Figure 7b indicates the oxygen consumption rates of the screen printed samples, which contain TvL or ThL. Printed microcapsules without laccase were used as reference samples. Oxygen consumption of screen printed ThL microcapsules was faster compared to the screen printed TvL microcapsules. This divergence is due to the difference in the loaded enzyme amount and the specific activity of these two laccases. T. hirsuta was loaded with 10 000 nkat/mL enzyme during microencapsulation, whereas T. versicolor was loaded with 5000 nkat/mL solution. The better specific activity of the T. hirsuta laccase (2300 nkat/ mg protein) compared to that of the T. versicolor laccase 2012
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Figure 6. Confocal laser scanning images of rod coated, screen printed, and flexo printed samples. Upper row: surface profile and cross-sectional profile of reference paper. Middle row: surface profiles of screen printed, rod coated, and flexo printed enzyme microcapsules. Lower row: cross-sectional profiles of screen printed, rod coated, and flexo printed enzyme microcapsules.
(1800 nkat/mg protein) capsules could possibly have had a positive influence on the measured enzymatic activity of the capsules, which more than doubled by two times higher activity loading. Figure 7c presents the storage stability of screen printed ThL microcapsules, and it is clearly visible that 40% of enzymatic activity was preserved after seven days storage. Further tests were carried out using samples of microencapsulated TvL applied by screen printing on paper substrate. Enzyme activity, printability, and aging could be affected by both a narrow size distribution and the performance of the microcapsule dispersion; these factors might also have an influence on the results obtained. Narrow size distribution may result in a more uniform capsule distribution and prevent the occurrence of “patches” or regions of high activity on the paper. Agglomeration may cause more printing problems and consequently lower apparent activity due to the slow diffusion and
hindered access of substrate in the microcapsules located in the center of larger aggregates. Activity of Enzyme Microcapsules on the Paper Surface after Rinsing Treatment. According to the literature, there are indications of the higher affinity of M. albomyces laccase on cellulose surface compared to T. hirsuta laccase.10,11 Studies based on quartz crystal microbalance with dissipation have shown a slight affinity between T. hirsuta laccase and cellulose. During rinsing some of the enzyme is desorbed from the cellulose surface. In previous studies it had been proven that laccases can be divided into two groups based on their affinity for cellulose. Microencapsulation has been considered a sufficient method to immobilize enzymes on cellulose substrate.10,11 Adhesion of PEI microcapsules on paper surface was examined by a simple washing test: PEI microcapsules containing TvL or ThL were screen printed on paper substrate. After drying, some 2013
dx.doi.org/10.1021/bm2003434 |Biomacromolecules 2011, 12, 2008–2015
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Figure 7. Oxygen concentration as a function of time by screen printed PEI microcapsules: (a) the effect of printing method on oxygen consumption; (b) the effect of enzyme loading in PEI microcapsules on oxygen consumption; (c) storage stability of screen printed PEI microcapsules containing ThL. Samples were stored at ambient RT conditions.
Table 2. Activity of PEI Microcapsules Containing TvL and Free TvL on Paper Surface, and the Influence of Water Rinsing on the Adhesion of Either Immobilized or Free Enzyme on Paper Substratea enzymatic activity [nkat/cm2] as a function of time paper specimens
a
1 day
2 days
1 week
3 weeks
4 weeks
screen printed TvL microcapsules
0.8 ( 0.08
0.8 ( 0.002
0.9 ( 0.20
0.6 ( 0.02
0.6 ( 0.05
screen printed free TvL
0.6 ( 0.09
0.6 ( 0.02
0.5 ( 0.06
0.5 ( 0.11
0.5 ( 0.05
screen printed TvL microcapsules rinsed by water screen printed free TvL rinsed by water
0.8 ( 0.13 0.8 ( 0.29
0.7 ( 0.30 0.6 ( 0.01
0.8 ( 0.05 0.7 ( 0.12
0.5 ( 0.03 0.5 ( 0.08
0.5 ( 0.02 0.5 ( 0.18
Samples were stored at RT conditions. The data represent the mean ( the range.
of the paper samples were rinsed with equal amounts of Milli-Q water. The enzyme remaining on paper surface was detected by laccase activity assay based on oxygen consumption measurement. Given these results one may conclude that most of the enzyme remained on paper surface even though the surface was rinsed with water. When the activity of free and encapsulated printed enzyme samples was analyzed as a function of storage time, it was found that the encapsulation of enzyme had a stabilizing effect on preserving 63% of the initial enzyme activity even after four weeks of storage, and the adhesion of encapsulated enzyme was comparable with the adhesion of free enzyme on paper surface (Table 2). The membrane of the microcapsule due to its charge and porosity is likely to prevent unfavorable interactions with ink components. In addition, the microcapsules will retain some moisture and quickly absorb water upon
addition of a solution containing the reagent, resulting in a higher activity. If the rinsing method would have been stronger, the microencapsulated enzyme might have had better adhesion on paper surface and preserved its activity more effectively compared to free enzyme.
’ CONCLUSIONS Enzyme immobilization on paper surface was successfully achieved by printing polymer microcapsules containing laccase on paper substrate. In the first stage of the study, the particle size distribution and the agglomeration of the microcapsules were analyzed. It was stated that emulsion technique based microencapsulation produced particles with a large size distribution. Research then continued by comparing the enzymatic activity of 2014
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Biomacromolecules encapsulated TvL to that of the free enzyme. Our study revealed that TvL retained its activity during the encapsulation process, and the encapsulation prolonged the storage stability of the enzyme. In this work the effect of the capsule size was not studied, but it can be presumed that even large-sized capsules would prevent degradation by external contaminants, whereas the stabilizing effect due to the enzyme reticulation in the membrane would not remarkably contribute in the case of large-sized capsules. Moreover, the effect of cross-linking density was not examined in a systematic manner, nonetheless one may stipulate that a highly cross-linked network would reduce the molecular weight cutoff, thus decreasing permeation of larger interferents. We have earlier performed a few elemental analysis experiments, which elucidated that there are no significant changes in the membrane composition from the 0.33 to 0.83 cross-linker (v/v) % (over PEI) ratio.15 Below 0.33, the capsules were unstable since they were insufficiently cross-linked. Above 0.33, there was no significant further cross-linking. Furthermore, the compatibility of encapsulated TvL with polymeric ink components and the effect of printing the laccase microcapsules on paper surface on the enzymatic activity were explored. PEI microcapsules containing TvL were compatible with ink components, and it was found that the enzyme activity was preserved above 40% of the theoretical activity for at least eight weeks of storage, stored at 4 °C. In addition, studies involving printing of laccase microcapsules indicated that the enzyme activity on printed paper samples was maintained after printing. The printing method had a crucial influence on the activity level as would be expected, given that the amount of ink transferred on paper surface will undoubtedly affect the enzyme activity. As a final remark it was feasible to produce screen printed bioactive paper, where the laccase microcapsules were successfully used in enzyme immobilization on the paper substrate.
’ AUTHOR INFORMATION Corresponding Author
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*Telephone numbers: þ358 40 702 9933. Fax numbers: þ 358 20 722 7071. E-mail addresses: maria.smolander@vtt.fi.
’ ACKNOWLEDGMENT The paper is a part of an international collaboration among VTT and Universite de Montreal. M. P. Guerrero Palacios and M. Hebert (Universite de Montreal) are thanked for their assistance and kindness. Dr. M. Andberg, O. Liehunen, and B. Hillebrandt-Chellaoui (VTT) are acknowledged for providing the purified T. hirsuta laccase. Nina Vihersola and Ritva Heinonen (VTT) are much appreciated for their skillful technical assistance in encapsulation and the microscopy study. The financial support from TEKES, the Finnish Funding Agency for Technology and Innovation, to VTT is gratefully acknowledged. Funding from NSERC through the Sentinel Research Network for Bioactive Papers is likewise gratefully acknowledged. ’ REFERENCES (1) Du, Y. Z.; Wang, L.; Dong, Y.; Yuan, H.; Hu, F. Q. Carbohydr. Polym. 2010, 79, 1034. (2) Poncelet, D. Surf. Chem. Biomed. Environ. Sci. 2006, 228, 23. (3) Di Risio, S.; Yan, N. J. Pulp Paper Sci. 2008, 34, 203. 2015
dx.doi.org/10.1021/bm2003434 |Biomacromolecules 2011, 12, 2008–2015
