Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Process Biochemistry 48 (2013) 218–223

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Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization Daniele Spinelli a , Enrico Fatarella a,b , Angelo Di Michele a , Rebecca Pogni a,∗ a b

Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. De Gasperi 2, Siena 53100, Italy Next Technology Tecnotessile, Via del Gelso, Prato, Italy

a r t i c l e

i n f o

Article history: Received 30 August 2012 Received in revised form 20 November 2012 Accepted 7 December 2012 Available online 20 December 2012 Keywords: Fungal laccase Oxidoreductase Amberlite Immobilized enzyme

a b s t r a c t Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 ◦ C. Kinetic parameters, Km and Vmax values, were determined respectively as 0.051 mM and 2.77 × 10−2 mM/s for free and 4.70 mM and 5.27 × 10−3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 ◦ C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Biocatalytic process economics can be enhanced by enzyme reuse and by the improvement in enzyme stability achieved by immobilization. Moreover, the capacity to retain or recover enzymes also promotes easier product separation thereby permitting continuous processes, and preventing carry-through of protein or activity to subsequent process steps [1]. Immobilization can also improve enzyme performance under optimal process reaction conditions, e.g. acidity, alkalinity, organic solvents, and elevated temperatures, aspects that have often restricted enzyme application to industrial chemical synthesis [2–4]. Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are extracellular, multicopper enzymes that use molecular oxygen as a co-substrate to oxidize various aromatic and non-aromatic compounds by a radical-catalyzed reaction mechanism [5]. These enzymes and their applications in several industrial sectors have been studied since the nineteenth century due to their ability to oxidize phenolic compounds [6–9]. A variety of polymeric support materials including natural (chitin, chitosan, agarose, and cellulose derivatives) and synthetic (nylon, polysiloxane/polyaniline, polyvinylalcohol, poly(glycidylmethacrylate) and polyacrylic poly-

∗ Corresponding author at: Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. De Gasperi 2, 53100 Siena, Italy. Tel.: +39 0577 234258; fax: +39 0577 234239. E-mail address: [email protected] (R. Pogni). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.12.005

mers) have been used for the immobilization of laccase from different sources [10–14]. The major advantages of laccase immobilization are the increase in the thermostability of the enzyme and its resistance to extreme conditions and chemical reagents. In addition, immobilized laccases may be easily separated from the reaction products, allowing the enzymes to be employed in continuous bioreactor operations [15–18]. On the other hand, the immobilization processes could result in conformational changes of the enzyme, promoting a loss of activity [17]. As solid support, several types of commercial Amberlite have been successfully used for the immobilization of enzymes like lipase, ␣-amylase and urease for industrial applications [19]. Amberlite represents a good support as it is resistant to biological degradation, compatible with almost all organic solvents and most concentrated acids and above all is inexpensive. Furthermore, Amberlite after a pre-treatment step can be reacted with glutaraldehyde, a commonly used crosslinker, to covalent bind the enzyme to the matrix [19]. Glutaraldehyde can stabilize the enzyme, permitting also a greater enzyme flexibility for conformational changes required for activity [20]. Moreover, charged groups on the surface of a support (i.e. Amberlite) may lead to electrostatic interaction between individual ionic amino acids on the enzyme surface. Both types of charge (positive and negative) as well as charge density on the material surface can alter the enzyme activity upon immobilization. The activity of ␤-galactosidase bound to an anionic support has been shown to be lower than when the enzyme is bound to a cationic support [21],

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while on the contrary, ␣-chymotrypsin has shown to retain better activity when bound to a negatively charged surface in respect to a positively charged one [22]. Moreover, covalent or crosslinking immobilization strategies generally lead to losses in laccase activity [23]. Hence, the maintenance of high enzyme activity after immobilization is a major concern. In this study, T. versicolor laccase has been immobilized on Amberlite IR-120 H beads previously reacted with glutaraldehyde. Amberlite IR-120 H is a gel type strongly acidic cation exchange resin of the sulfonated polystyrene type in the physical form of amber spherical beads. Several works report on laccase immobilization on different matrices [15,24], but it is noteworthy that this is the first report on immobilization of laccase onto this type of support. Other types of Amberlite, like Amberlite MB-150 (a mixture of strongly acidic cationic and strongly basic anionic resins), have been successfully tested for enzymatic immobilization showing improvement of enzyme storage stability and reusability [19]. In this paper, comparisons of various physicochemical properties of immobilized laccase with the soluble one have been carried out. An improvement in storage stability and reusability of the immobilized enzyme have been shown. All the experiments have demonstrated the advantages in using the enzyme in its supported form. 2. Materials and methods ABTS (2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) and glutaraldehyde were purchased from Sigma-Aldrich Srl (Milan, Italy). Milli-Q water (Millipore, Milford, MA, USA) was used throughout the experiments. Laccase from T. versicolor (30.6 U/mg) and Amberlite IR-120 H were purchased from Fluka Chemika (Buchs, Switzerland). 2.1. Immobilization of laccase onto Amberlite The procedure described by Kumari and Kayastha for soybean ␣-amylase has been adopted with few modifications for immobilization of laccase [25]. Amberlite IR-120 H beads (100 mg) of 620–830 ␮m mean diameter were first equilibrated at different pH values ranging from 3 to 6 using 1 mM buffers: sodium acetate (pH = 3 and 4.5) and sodium phosphate (pH = 6). Then the pre-treated Amberlite was incubated using 100 ␮L of 2–5% (v/v) solution of glutaraldehyde prepared in the equilibration buffer and kept for 2 h incubation time at room temperature. Activated Amberlite was washed with the equilibration buffer 3 times to remove the unreacted glutaraldehyde. Later, various amounts of enzyme (2–6 mg) were added to the activated Amberlite, and the coupling was tested at different contact times at room temperature. Finally, the unbound enzyme was washed off with the buffer. The washing buffer was kept for activity measurement. Amberlite–laccase system enzyme was stored in semi-dry condition at 4 ◦ C. To determine the optimum immobilization conditions, the following parameters during the immobilization process were changed: (a) glutaraldehyde concentration (2–5%), (b) protein concentration (2–6 mg/mL), (c) pH (3, 4.5 and 6), (d) time of contact (3, 6, 12, 24 h). One parameter was varied and the others kept constant for each experiment. The efficiency of immobilization (EF) was calculated by using the following relationship:

 

EF(%) =

Ai Af

× 100

(1)

where Ai is the specific activity of immobilized enzyme = specific activity of free enzyme (Af ) − specific activity of the unbound enzyme 2.2. Laccase activity assay Laccase activity, both free and immobilized, was assayed by following the oxidation of 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) [26]. The reaction mixture contained laccase and ABTS 5.1 mM in 1 mM buffer. Oxidation of ABTS was monitored spectrophotometrically (Perkin Elmer Lambda 900 UV/VIS/NIR) at max = 414 nm (ε/dm3 mol−1 cm−1 36,000) for 3 min and laccase activity was expressed as % activity. 2.3. Morphology of amberlite beads The morphological structures of the amberlite beads (before and after immobilization) were investigated by scanning electron microscope (SEM; Phenom G2 pure desktop apparatus) working in the magnification range (20–17,000).

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2.4. Steady-state kinetics The optimum pH for the immobilized laccase activity was determined using sodium acetate buffer for pH range 3–4.5 and phosphate buffer for pH = 6. The enzymatic activity was determined in each buffer by the method described for enzyme assay. The optimum temperature for free and immobilized laccase was studied by assaying the enzyme at temperature ranging from 25 to 50 ± 1 ◦ C. The kinetic parameters were estimated from the Lineweaver–Burk plot by varying the substrate concentration (ABTS) from 1 to 14 mg/mL at the temperature of the maximum activity. Km and Vmax were determined from the intercepts at x and y axes, respectively. All parameters were the mean of triplicate determinations from three independent preparations. The standard deviation for the three replicates was in the range of 1–5%. 2.5. Stability and reusability Storage stability experiment was performed to determine the stabilities of free and immobilized laccase. For storage stability measurements, immobilized laccase was kept at 4 ◦ C without buffer. The activity of immobilized laccase was followed for 7 days and determined by the laccase activity assay procedure reported in Section 2.2 [26]. Then, the immobilized laccase was reused 7 times each day and the variation of activity was measured in respect to the initial one. After each assay, Amberlite–laccase system was washed with buffer and stored at 4 ◦ C for other uses.

3. Results and discussion 3.1. Optimum conditions for laccase immobilization on Amberlite Various conditions for laccase immobilization were tested as summarized in Table 1. A high percentage of protein immobilization (78.7%) has been reached for a glutaraldeyde concentration equal to 3% (v/v). Higher concentration of glutaraldehyde led to aggregation, precipitation, loss of enzyme activity, and distortion of enzyme structure [27]. Furthermore, enzyme aggregation has been recorded not only at higher concentration of glutaraldehyde but also when the protein concentration used was >3 mg/mL. Different glutaraldehyde structures are present in solution depending on pH. Under acidic or neutral conditions, glutaraldehyde exists as a mixture of monomers (free aldehyde form or cyclic hemiacetal) or as a polymer (cyclic hemiacetal oligomer) that would be expected to form Schiff bases upon nucleophilic attack by the ␧-amino groups of lysine residues of the enzyme [28–32]. Schiff bases are unstable under acidic conditions and therefore the reaction of cyclic hemiacetal or its multimeric form is promoted. Under basic conditions, the reaction of ␣,␤-unsatured oligomeric aldehydes with amine can give two products resistant to acid hydrolysis: a Schiff base and a Michael addition product [31,33]. The kinetics of glutaraldehyde crosslinking under acidic condition are known to be slower than the kinetics of crosslinking under neutral or basic condition. Mildly acidic conditions are unfavorable for the above-mentioned reactions as the amino groups of protein are likely to be protonated [33,34]. A number of enzymes have shown that the more suitable pH for immobilization does not correspond with the pH of major activity. In fact, compared with free enzymes, immobilization to polycationic surfaces often results in a more acidic pH for the enzyme while immobilization to polyanionic surfaces results in a more basic optimum pH [25,35]. In Fig. 1, the optimal pH for laccase immobilization is shown. For pH 6 a high percentage of immobilization has been reached (95.5%) as also confirmed in literature [36]. Nevertheless, pH 4.5 has been chosen as the enzyme maintains the higher activity (Fig. 1) [37]. The oxidation of ABTS was examined in the pH range 3.0–6.0 at 25 ◦ C (Fig. 1). Both free and immobilized laccase have the maximum activity at pH 4.5 for the oxidation reaction. The pH profile shows for immobilized laccase improved stability on the whole range of tested pHs in comparison with the free form. This means that the immobilization method preserves the enzyme activity and enhances its stability. This result is in agreement with other studies reporting the effect on activity profile of free and immobilized laccase [38,39]. Therefore, at the pH of

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Table 1 Conditions for laccase immobilization onto Amberlite beads: the volume of enzyme loaded was 100 ␮L (3 mg/mL) and the incubation was performed at room temperature (T = 25 ◦ C) and pH = 4.5. Immobilization (%)

Glutaraldehyde (%) 2

3

Incubation time (h) 4

5

× × × ×

14.0 42.8 71.7 70.2 23.8 56.5 78.7 71.2 22.0 64.2 73.6 69.2 25.2 56.5 70.7 59.6

12

24

× × × × × × × × × × ×

× × × × × × × ×

maximum activity and room temperature, a 78.7% protein immobilization was reached with the following conditions: 100 mg of beads activated with 3% (v/v) glutaraldehyde coupled with 100 ␮L of 3 mg/mL laccase for a contact time of 12 h (see Table 1). Fig. 2 shows microscopic images of Amberlite IR-120 H beads (Fig. 2A control), glutaraldehyde activated (Fig. 2B) and immobilized laccase (Fig. 2C) with different image resolution at the optimum immobilization conditions. Glutaraldeyde activated Amberlite beads clearly showed rough particles sticking on the whole surface (Fig. 2B). The use of glutaraldeyde influences the enzyme immobilization and 3% glutaraldeyde concentration represents the better amount for the maximum enzyme immobilization percentage (Table 1). Nevertheless, a non-homogeneous distribution of enzyme is evident from the image reported in Fig. 2C. 3.2. Steady-state kinetics The activity of free and immobilized laccase was assayed at pH 4.5 in the 25–50 ◦ C temperature range. Maximum activities were observed at T = 40 ◦ C and 45 ◦ C for free and immobilized enzymes respectively, as reported in Fig. 3. The small increase in the optimum temperature for Amberlite–laccase system may derive from the stabilized enzyme structure by covalent bond formation via protein amino groups. The covalent bond formation might also reduce the conformational flexibility of the enzyme molecule and may lead an increase in the activation energy of the laccase to reorganize an optimum catalytic conformation for binding to its substrates [40]. The enhanced thermal stability of laccase arising

100

100

80

80

60

60

40

40

20

20

0

Immobilization (%)

Relative activity (%)

6

×

× × × ×

0 3

3

4.5

6

pH Fig. 1. Effect of pH on laccase immobilization (%) on Amberlite beads () and relative activity for the free form () and immobilized () laccase. Experiments were performed in triplicate. The error bars indicate the standard deviation (3%).

× × × ×

from immobilization would be an advantage for its industrial application due to the high temperatures used in the industrial processes [41,42]. These result is in agreement with the thermostability comparison of free and immobilized Cyclodextrin glucanotransferase on Amberlite IRA-900 [43]. Laccase catalyzes the oxidation of ABTS in presence of molecular oxygen. The initial reaction rates of the ABTS oxidation were measured at different substrate concentrations with the free and immobilized laccases. Optimal condition of pH and temperature were chosen for the kinetic parameters determination. The kinetic data for the oxidation of ABTS were fitted to the Michaelis–Menten equation. The Lineweaver–Burk plot of 1/v vs 1/S, Michaelis constant (Km ) and the maximum reaction velocity (Vmax ) of the free and immobilized enzyme were calculated (Figs. 4 and 5). For the free laccase the Km value was found to be 0.051 mM and the Vmax was calculated to be 2.77 × 10−2 mM/s. For the immobilized laccase the Km value was found to be 4.70 mM and the Vmax was calculated to be 5.27 × 10−3 mM/s. The apparent Km for the immobilized enzyme was increased about 92 times compared to the free enzyme. The data are substantially in agreement with the kinetic parameters of immobilized laccase on the surface of magnetic chitosan beads with a high value of Km and a low value of Vmax respect to the free form [39]. The Km value is known as the affinity of the enzymes to substrates and the lower values of Km emphasize the higher affinity between enzymes and substrates. In our study, the Km value for immobilized laccase is greatly increased compared to other papers [38,39]. The Vmax value of the immobilized enzyme decreased about 5.25 times compared to the free enzyme. As expected, the apparent Km and Vmax values were significantly affected after immobilization. In literature, the Vmax value for immobilized laccase compared to that of the free enzyme is reduced of about 2–5 times compared to the free enzyme [38,39]. The increase of Km can be explained as a result of diffusional limitation of the substrate or to conformational changes of the enzyme resulting in a lower affinity of the substrate for the enzyme. From literature the presence of an unstirred layer of solvent, surrounding suspended water insoluble particles like immobilized enzymes, is known as “Nernst layer”. A concentration gradient of substrate is established across the layer. This will determine the enzyme saturation at a higher substrate concentration than normally required for the saturation of the free enzyme. Therefore, this leads to an increase in the Km value [44–46]. A similar change was observed in several cases of different enzymes immobilized on Amberlite beads [19,25]. Another important aspect to take into consideration to evaluate the immobilization procedure is the efficiency factor . This factor

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Fig. 2. (A) Microscopic image of buffer activated Amberlite beads at resolution 375× (260 ␮m). (B) Glutaraldehyde activated Amberlite beads at resolution 375× (260 ␮m). (C) Glutaraldehyde activated laccase onto Amberlite beads at resolution 765× (130 ␮m).

can be calculated from the higher reaction rates of the immobilized enzyme over that of the free counterpart: =

vimmobilized vfree

(2)

where vimmobilized was the reaction rate of the immobilized enzyme and vfree that of the free enzyme. In our case the Amberlite–laccase system provided an efficiency factor of 0.19. This value is lower than that reported in literature for laccase immobilized onto magnetic chitosan beads and policationic fibrous polymer carrying poly(hydroxyethylmethacrylate) films with an efficiency factor of 0.72 [39,47]. The ratio Vmax /Km is a measurement of the catalytic efficiency of an enzyme–substrate pair. In this study, the catalytic efficiencies of the free and immobilized laccase were found to be 0.54 s−1 and 1.12 × 10−3 s−1 , respectively. The catalytic efficiency of laccase was decreased about 482 times upon immobilization. A comparison between kinetic data for free and immobilized laccase is reported in Table 2.

enhanced upon immobilization. Leonowicz et al. [48] reported an increase in storage stability of laccase from T. versicolor immobilized on glutaraldehyde-activated aminopropyl porous glass. Amberlite–laccase system was stored at 4 ◦ C, without buffer. The loss of activity for free laccase after 7 days of storage at 4 ◦ C was about 18.5% in comparison to Amberlite–laccase system which loss was about 1.4%, during the same period (Fig. 6). Therefore, the storage stability of the immobilized laccase was improved a lot in comparison with the free enzyme. This value is in agreement with other studies reported in literature for immobilized laccase on polyacrilonitrile beads and policationic fibrous polymer carrying poly(hydroxyethylmethacrylate) Table 2 Kinetic data for free and immobilized laccase onto Amberlite beads. Enzyme form

Kinetic parameter

Value

Free laccase

Km (mM) Vmax (mM/s) Vmax /Km (s−1 )

0.051 2.77 × 10−2 0.54

Immobilized laccase

Km (mM) Vmax (mM/s) Vmax /Km (s−1 )

4.70 5.27 × 10−3 1.12 × 10−3

3.3. Storage stability and reusability In general, an enzyme in solution is not stable during storage, and its activity is gradually reduced. The stability of laccase was

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Residual activity (%)

100

Relative Activity (%)

90 80 70 60 50

100 95 90 85 80 75

40

70

30

2

3

4

5

6

7

Time (days) 25

30

35

40

45

50

Temperature (°C) Fig. 3. Effect of temperature on free () and immobilized laccase on Amberlite beads (). The relative activity was determined at various temperatures at pH = 4.5 by ABTS oxidation reaction. Experiments were performed in triplicate. The error bars indicate the standard deviation (3%). 3.30E+02 3.10E+02 2.90E+02 2.70E+02

Fig. 6. Storage stability of free () and covalently immobilized laccase on Amberlite () at T = 4 ◦ C and pH = 4.5 determined by ABTS oxidation reaction. Experiments were performed in triplicate. The error bars indicate the standard deviation (1%). 100 90

Re sidual Activity (%)

20

1/v (mM/s)

1

80 70 60 50 40 30 20

2.50E+02

10

2.30E+02

0

2.10E+02

1

2

1.70E+02 1.50E+02 2.00E-02

3

4

5

6

7

Number of reuses

1.90E+02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

1.20E-01

1.40E-01

1.60E-01

Fig. 7. Reusability (7 reuses) of Amberlite–laccase at the optimal temperature after a storage at 4 ◦ C determined by ABTS oxidation reaction. Experiments were performed in triplicate. The error bars indicate the standard deviation (5%).

1/[s] (mM) Fig. 4. Determination of Km for Amberlite–laccase system by Lineweaver–Burk plot method at T = 45 ◦ C and pH 4.5 by ABTS oxidation reaction. The substrate concentration varied from 1 to 14 mg/mL.

films after 7 days of storage at 4 ◦ C [38,39]. Operational stability of others immobilized enzymes as Cyclodextrin glucanotransferase on Amberlite IRA-900 (strong basic anionic exchanger on polystyrene) was also in agreement with our data [43]. With repeated use, the strength of binding between the matrix and enzyme is weakened, leading to loss in activity. Furthermore, repeated cycles with substrates can reduce the catalytic efficiency as well. The Amberlite–laccase system showed a residual activity of 3.65E+01

1/v (mM/s)

3.64E+01 3.64E+01 3.63E+01 3.63E+01 3.62E+01 3.62E+01 2.00E-02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

1.20E-01

1.40E-01

about 30% after 7 cycles (Fig. 7). Successful reuse of various immobilized laccase systems has been reported in other works showing a residual activity between 40% and 50% after 7 cycles [19,25,48–50]. Other enzymes immobilized onto Amberlite MB-150 showed the same residual activity after 10 cycles [19,25]. 4. Conclusions One of the most important aims of enzyme technology is to enhance the conformational stability of the enzyme through immobization procedures. The extent of stabilization depends on the enzyme structure, immobilization methods and type of support. In this paper, the optimum conditions for laccase immobilization on Amberlite IR-120 H beads were determined. The immobilized biocatalyst displays an improved thermal and storage stability paired with a good performance for the reusability. The loss in enzyme activity of the immobilized enzyme might be due in our case, to the inhomogeneous distribution of the aggregated enzyme. Based on the above results, Amberlite seems a promising matrix for immobilization of laccase due to the satisfactory performance of the immobilized biocatalyst described in this work. Therefore, studies on the use of non-toxic, cheap and renewable matrices like Amberlite could have importance in food industry, bioremediation, biofuel production, cosmetics, biomedical, or pharmaceuticals applications in batch and bioreactors.

1.60E-01

1/[s] (mM) Fig. 5. Determination of Km for free laccase by Lineweaver–Burk plot method at T = 40 ◦ C and pH = 4.5 by ABTS oxidation reaction. The substrate concentration varied from 1 to 14 mg/mL.

Acknowledgement This work has been partially supported by the Eco-Innovation European Project BISCOL (ECO/09/256112).

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