HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 47(2) pp. 17–23 (2019) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2019-16 IMMOBILIZATION OF LACCASE IN ALGINATE BEADS BÉLA VARGA*1, MÓNIKA MEICZINGER2, AND VIOLA SOMOGYI1 1Institute of Environmental Engineering, University of Pannonia, Egyetem u. 10, Veszprém, 8200, HUNGARY 2Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Egyetem u. 10, Veszprém, 8200, HUNGARY The elimination or degradation of micropollutants from wastewater is becoming ever more important nowadays. Using ox- idoreductase enzymes to treat different micropollutants seems a promising solution. However, the viability of the process is highly dependent on the availability and stability of the applied enzymes. In order to improve the stability and provide faster reaction rates, enzymes can be immobilized in various carriers. Properties such as simple production, easy reten- tion and biodegradable carrier material are advantageous, e.g. entrapping laccase in alginate beads. This paper shows the results of the preparation and characterization of immobilized laccase entrapped in calcium alginate beads. The tech- nique of adding a mixture of sodium alginate and laccase dropwise into calcium chloride has been applied, improved and standardized to produce laccase-containing beads of uniform size and activity. For the purpose of characterization, a widely used substrate, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, was used to evaluate the performance of the laccase-containing alginate beads. In addition to the characterization of the laccase-containing algi- nate beads, the enzyme kinetic constants (KM = 26.43 µM, Vmax = 0.23 µM/min) were determined. The reduction in the activity during storage has been described by a decay constant (0.26 d−1) that provides information concerning the de- sign constraints of the process. Results will be used to test the method in terms of the removal of organic micropollutants in continuous systems. Keywords: laccase, immobilization, alginate, ABTS, micropollutants 1. Introduction Awareness concerning the spread of micropollutants in water bodies has been raised in many countries [1] due to their toxic effects on ecological systems [2]. Additionally, these pollutants can infiltrate into reservoirs of drinking water in several ways resulting in public health concerns [3]. Since conventional treatment systems were designed to easily remove biodegradable compounds [4], the effi- ciency of micropollutant degradation is insufficient [5]. The partial elimination of these substances is driven by physical adsorption by the sludge [6] and cometabolism during microbial growth [7]. Therefore, the development of advanced technologies to improve the efficiency of re- moval and mitigate ecological effects is needed [8]. Organic micropollutants could be converted by oxi- doreductase enzymes such as tyrosinase, peroxidase and laccase. Laccase has received attention since it does not require additional cofactors. It promotes the production of an organic radical which combines with different trans- formation products [9]. The toxicity of these compounds is a subject of ongoing research [10], although a reduc- tion in toxicity has been shown in many cases [11]. *Correspondence: vargabela@almos.uni-pannon.hu Immobilization can be advantageous compared to the free form of the enzyme [12]. It can enhance the stability of the enzymes under more extreme conditions, namely higher pH and temperature [13], and makes the separa- tion of the catalyst from the effluent easier. For example, separation by applying an external magnetic force is pos- sible when laccase is immobilized on magnetic particles [14]. By using macro-sized supports, e.g. alginate beads [15] – unlike nanoparticles – the carriers can be retained by an outlet sieve, thus using them in continuous packed- bed reactors is possible [16]. Alginates extracted from brown algae [17] are good candidates for water treatment because of their advanta- geous properties [18], low-cost production and low health risks [19]. Calcium alginate microspheres, successfully used to immobilize polygalacturonase for the purpose of cleaning edible products, have shown improved opera- tional stability as the optimum temperature increases and even modifies the pH dependence of the enzyme [20]. Calcium alginate beads are formed by reversible cross- linking bonds with bivalent calcium ions between chains of α-L-guluronic acid and β-D-mannuronic acid [19]. Their unique properties allow them to be used as an ad- sorbent for dye removal [21]. Transformation of the phe- https://doi.org/10.33927/hjic-2019-16 mailto: vargabela@almos.uni-pannon.hu 18 VARGA, MEICZINGER, AND SOMOGYI nolic compound with laccase entrapped in alginate was successfully tested for bisphenol A [22] and the decolour- ization of textile dyes [23]. The aim of this study was to investigate the immobi- lization of laccase in calcium alginate beads and deter- mine its kinetic behaviour by measuring the conversion of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). Since this substrate is widely used, the results may be easily compared with other lac- case immobilization methods. 2. Materials and methods 2.1 Materials Laccase from Trametes versicolor (catechol oxidase ac- tivity ≥ 0.5 U/mg) and ABTS were purchased from Sigma-Aldrich. Food grade sodium alginate was pur- chased from Dragonspice Naturwaren. Citric acid and sodium phosphate used for making McIlvaine buffer so- lution [24] were supplied by VWR International. Calcium chloride was purchased from REANAL. Reagents were of analytical reagent grade except for sodium alginate. 2.2 Measurement of enzyme activity Laccase activity was determined by measuring the change in concentration of the ABTS radical formed in the catalysed oxidation. Measurements were performed in a Macherey-Nagel Nanocolor UV/VIS spectropho- tometer at 420 nm in a citrate-phosphate buffer solution at pH = 5 and within the range of laboratory temperatures (20± 3 °C). The formation of the product was registered by using the extinction coefficient of the ABTS radical (ε = 36, 000 M−1cm−1 [25]) and calculated using an equation A = EVsample lεVtotal (1) based on the Beer-Lambert law [26], whereA denotes the activity (U/l),E is a measure of the change in absorbance at 420 nm over 1 minute (min−1), Vsample represents the amount of enzyme contained in the sample used (ml), l is the pathlength of the beam of light (cm), ε stands for the extinction coefficient of the ABTS radical (M−1cm−1), and Vtotal expresses the total volume used (ml). In the case of immobilized laccase, beads were placed into the tubes instead of the liquid samples. The specific activity was calculated by measuring the weight and num- ber of beads before the activity in a closed tube was mea- sured. Between measurements, the beads were stored in distilled water in order to minimize the loss of water. 2.3 Immobilization of laccase inside an algi- nate matrix The immobilization protocol was based on entrapment of the enzyme before the formation of insoluble algi- nate beads according to Daâssi et al. [23]. The sodium- alginate crystals were dissolved in 5 ml of 1 mg/ml lac- case stock solution to produce a gel with 2 w/v% alginate content. This was added dropwise from a height of 25 cm into a 2 m/m% solution of calcium chloride where beads formed, then the beads were left for 30 mins to solidify. After this procedure, the beads were extracted from the solution by filtration and washed with distilled water in order to eliminate the unbonded laccase. The beads were stored in distilled water until used for further investiga- tions. The efficiency of the immobilization was determined by measuring the activities of the laccase stock solution (Ainitial), the solution of calcium chloride after immobi- lization (ACaCl2 ) and the washing liquid (Awashing) used. Immobilization efficiency (η) was calculated using the following formula η(%) = 100 Ainitial −ACaCl2 −Awashing Ainitial . (2) 2.4 Characterization of the immobilized lac- case The weight was measured frequently in a closed vessel by using an analytical balance (Kern ABJ 220-4NM). The size of the drops was determined by measuring the di- ameter of the freshly prepared beads after filtration on a square membrane filter. In order to determine the wa- ter content, twenty pieces of freshly prepared beads were put into an oven at a temperature of 105 °C and dried until their weight became constant. 2.5 Effect of temperature on alginate beads In order to gather information regarding the effect of tem- perature on enzyme activity within the optimal temper- ature range of wastewater treatment plants [27], the re- action rate of the ABTS transformation was determined inside a thermostatic cabinet (20− 35 °C). 2.6 Storage stability of the beads The storage stability of the immobilized laccase was de- termined by storing the laccase-containing beads in ultra- pure water at 20 °C without mixing. Beads were removed every 24 hours from the vessel in order to determine the change in their activity. The exponential decay constant was calculated by fitting the following equation At = Ainitiale −kdt (3) to the measured activities, where Ainitial and At indicate the specific activities [U/bead] at the initial and given time, respectively, kd denotes the decay constant [d−1] and t stands for the time of sampling [h]. 2.7 Reuse of the alginate beads The laccase-containing beads were mixed with a 0.05 mg/ml ABTS solution in a phosphate-citrate buffer so- lution (pH = 5) and the change in the ABTS radical con- centration was determined spectrophotometrically. After Hungarian Journal of Industry and Chemistry IMMOBILIZATION OF LACCASE IN ALGINATE BEADS 19 Figure 1: Change of size during drying of the beads (A) beads freshly prepared, (B) beads after drying, grid size: 3 mm measuring the activity of the beads (after 5 mins), they were filtered and washed with 5 ml of distilled water which was considered to be one cycle. During the fil- tration, a picture was taken to document the change in the size and shape of the beads. After washing, the beads were put into a fresh volume of buffer solution that con- tained ABTS and the same cycle repeated six more times. 2.8 Determination of kinetic parameters with ABTS Ten beads were put into a pH = 5 citrate-phosphate buffer solution, then different amounts of ABTS stock solution were pipetted in order to obtain different ini- tial substrate concentrations ([S] = 23, 46, 91, 228, 456, and 911 µM). The change in absorbance was recorded at 420 nm to calculate the concentration of ABTS radi- cals (P). The Michaelis-Menten constant (KM) and max- imum rate of reaction (Vmax) were determined by fitting the Michaelis-Menten equation. d[P] dt = Vmax[S] KM + [S] (4) with non-linear regression. 3. Results and Analysis 3.1 Characterization of the alginate beads Using the technique described, it was possible to produce a uniform bead weight: 0.0152 g (SD = 0.0016 g) of high immobilization efficiency (98.2 %). The diameters of the beads were between 2 and 3 mm. The weight and density of the beads constantly changed during their handling due to the rapid rate of evaporation of water. Therefore, the number of beads was used to calculate the specific activ- ity instead of weight, in a similar way to Daâssi et al. [23]. A specific activity of 0.005 U/bead was achieved, which equates to 0.005 µM production of ABTS radicals in one minute. The water content of the beads was determined to be 98± 1 %. The size of the beads shrank twofold whilst being dried as is shown in Fig. 1. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 200 400 600 800 1000 A B T S ra di ca l co nc en tr at io n (µ M ) Time (s) Figure 2: Change of ABTS radical concentration 3.2 Determination of kinetic parameters The change in the concentration of ABTS radicals as a function of time is presented in Fig. 2. A linear curve could be fitted with the results during the initial 2 min- utes indicating a first-order reaction. The rate of the re- action can be determined from the gradient of the curves. By plotting the initial rates of reaction against the con- centration of the substrate, a lower initial substrate con- centration shows a better fit (R2 = 0.96 − 0.99) when compared to the higher range (R2 = 0.95). As a result, common Michaelis-Menten kinetics were shown (Fig. 3). By fitting an equation to the data gathered from three independent experiments with nonlinear least squares regression, the kinetic constants can be deter- mined, namely KM = 26.43 µM, Vmax = 0.23 µM/min. 3.3 Effect of temperature on alginate beads In the range of 20 − 35 °C, the rate of reaction, thus the enzyme activity, increased rapidly as can be seen in Fig. 4. As expected, the highest activity was measured at 35 °C since, in the case of the free laccase from Trametes versicolor, the optimal temperature for maximum activity was approximately 40 °C [26]. Figure 3: Fitting of Michaelis-Menten curve to the mea- sure. Initial concentrations used for calculation: 5 µM, 9 µM, 23 µM, 46 µM, 91 µM. 47(2) pp. 17–23 (2019) 20 VARGA, MEICZINGER, AND SOMOGYI 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 20 25 30 35 A ct iv ity ( U /b ea d) Temperature (�C) Figure 4: Effect of temperature on the activity of the im- mobilized enzyme. 3.4 Storage stability of the beads During storage, a slow decreasing trend can be observed in the activity of the beads, namely a reduction of 33 % over 5 days. By fitting equation to the measured values, the decay coefficient can be determined as 0.26 d−1. The change in activity and the decay curve produced by the equation are presented in Fig. 5. 3.5 Reuse of the beads The activity of the beads also decreased when repeatedly used. As an example, the results of an experiment were presented in Fig. 6. While the activity of the beads de- creased, their weight increased gradually. The change in the size of the beads and accumulation of the green reac- tion products can be seen in Fig. 7. This figure also shows the deformation of the beads in between the experiments. 4. Discussion Using the method described in this study, uniform beads can be produced with regard to their size and weight by controlling the size of their droplets and the height from which they are dropped. Since the alginate pow- der was dissolved directly in the stock solution of lac- case and the thick layer solidified instantly when it came 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0 1 2 3 4 5 6 7 A ct iv ity ( U /b ea d) Storage time (d) Measured Calculated Figure 5: Change of the activity during storage of the im- mobilized laccase. Figure 6: Change of activity and weight of the beads dur- ing reuse into contact with the CaCl2 solution, the entrapment was instant and effective. As a result, a high immobilization efficiency was achieved (98.2 %). Otherwise, the weight of the beads changed quickly whilst being handled after their formation as the beads dried out easily. However, the change in their water content did not influence their activities. Therefore, in order to achieve reliable results for specific activities, the number of beads was calculated rather than their weight. Evaluation of the reaction kinetics resulted in a Michaelis-Menten curve in the case of immobilized lac- case, in a similar manner to its free form [28]. This in- dicates that the mechanism of the reaction was not influ- enced by the entrapment. Nonetheless, the activity of the beads is different from that of free laccase due to steric effects and limitations with regard to the diffusion of re- actants. In order to achieve higher degrees of activity, the temperature could be increased. This would have ad- vantageous effects on the rate of reaction and diffusion. Additionally, by increasing the number of beads used, the rate of reaction could be fine-tuned to suit the re- quirements of their application in a similar way to the use of free enzymes. In order to determine the product yield or space-time yield, further investigations are rec- ommended. Due to the nature of ABTS, from visual inspection of radicals in itself, it was evident that the reaction products accumulate inside the beads (see Fig. 7). This could result in the deactivation of the enzyme by product inhibition. On the other hand, this phenomenon could be used to re- move the transformation products after enzymatic con- version by means of adsorption. As Tanaka et al. [29] concluded, the diffusion properties depend on the molec- ular size of the substrate. Since laccase-catalysed reac- tions tend to result in coupled products of higher molecu- lar weight [9], its release from the beads could be limited by diffusion. In the case of low-molecular-weight com- pounds, e.g. ABTS, free diffusion can be assumed in the alginate matrix [30]. As the experiments concerning repeated usage have shown, the beads suffer from major structural changes Hungarian Journal of Industry and Chemistry IMMOBILIZATION OF LACCASE IN ALGINATE BEADS 21 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Figure 7: Photos of the laccase containing alginate beads during the repeated use in buffer solutions that contain high concentrations of monovalent cations. This is due to the fact that cross- linking with calcium ions is reversible. These structural changes may result in the enzyme leaking and the specific activity decreasing following repeated usage. The reduc- tion in enzyme activity during storage could also be a re- sult of leakage. However, experiments performed in dis- tilled water fitted well with the decay equation, therefore, this can be taken into consideration during the design of the process. Moreover, in the case of a complex solution, e.g. treated wastewater or even tap water, the presence of bivalent cations may limit the deformation of the beads. 5. Conclusions Uniformly sized alginate beads were produced in the lab- oratory using a widely available biodegradable polymer. The mechanism of immobilized laccase was similar to that of its free form with regard to the conversion of ABTS, although the effects of diffusion and adsorption should be taken into consideration. Due to the structure of the alginate beads, the use of monovalent ions that contain buffers for measurements results in major struc- tural changes. The beads produced were reused success- fully over 7 cycles, while a rapid reduction in activity and structural changes were observed. However, the beads can be stored in distilled water and the reduction in ac- tivity estimated by an exponential decay equation which facilitates the design of the process. Although further in- vestigations regarding the efficiency of enzymatic trans- formation are needed, immobilization of laccase in al- ginate beads is a promising technique for the enzymatic treatment of micropollutants in continuous systems. Symbols A Activity [U/L] ACaCl2 activity of calcium chloride solution after immobilization [U/L] Ainitial activity of the laccase stock solution used [U/L] At specific activity at a given time [U/bead] Awashing activity of the washing liquid [U/L] E change in absorbance at 420 nm over 1 minute [min−1] kd decay constant [d−1] KM Michaelis-Menten constant [µM] l path length of light beam [cm] P reaction product, ABTS radical concentration [µM] S initial substrate concentration [µM] t time [min.; d] Vmax maximum reaction rate [µM/min] Vsample amount of enzyme-containing sample used [ml] Vtotal total volume used [ml] ε extinction coefficient of ABTS radicals[M−1cm−1] η immobilization efficiency [%] Acknowledgement The financial support of Széchenyi 2020 under the projects GINOP-2.3.2-15-2016-00016 and TÉT_15_IN- 1-2016-0094 is greatly appreciated. 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