Green coconut fiber: a novel carrier for the

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization Raquel O. Cristóvão, Sara C. Silvério, Ana P. M. Tavares, Ana Iraidy S. Brígida, José M. Loureiro, Rui A. R. Boaventura, et al. World Journal of Microbiology and Biotechnology ISSN 0959-3993 World J Microbiol Biotechnol DOI 10.1007/s11274-012-1092-4

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Author's personal copy World J Microbiol Biotechnol DOI 10.1007/s11274-012-1092-4

ORIGINAL PAPER

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization Raquel O. Cristo´vaõ • Sara C. Silve´rio • Ana P. M. Tavares • Ana Iraidy S. Brı´gida • Jose´ M. Loureiro • Rui A. R. Boaventura Eugeńia A. Macedo • Maria Alice Z. Coelho

•

Received: 12 March 2012 / Accepted: 24 May 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2 h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50 °C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff’s bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous

R. O. Cristo´vaõ S. C. Silve´rio A. P. M. Tavares J. M. Loureiro R. A. R. Boaventura E. A. Macedo (&) Laboratory of Separation and Reaction Engineering (LSRE), Associate Laboratory LSRE/LCM, Departamento de Engenharia Quı´mica, Faculdade de Engenharia, Universidade do Porto, Rua do Dr. Roberto Frias, 4200-465 Porto, Portugal e-mail: [email protected] A. I. S. Brı´gida M. A. Z. Coelho Escola de Quı´mica/UFRJ, Av. Horaćio Macedo, 2030, CT, Bloco E, Cidade Universita´ria, Rio de Janeiro 21941-909, Brazil

decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment. Keywords Enzyme immobilization Covalent attachment Commercial laccase Coconut fiber Dye decolourization Abbreviations GPTMS 3-glycidoxypropyltrimethoxysilane RB5 Reactive black 5 RB114 Reactive blue 114 RY15 Reactive yellow 15 RY176 Reactive yellow 176 RR239 Reactive red 239 RR180 Reactive red 180 ABTS 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt E Enzymatic state 0 E1 Enzymatic state 1 k Thermal inactivation parameter A Residual enzyme activity a Ratio of specific activity t Time t1/2 Half-life time F Stabilization factor v Reaction velocity vmax Maximum velocity KM Michaelis–Menten constant [S] ABTS concentration Ai Initial absorbance Af Final absorbance

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Author's personal copy World J Microbiol Biotechnol

Introduction The idea of using enzymes in industrial processes has become very attractive especially because they are highly selective, can operate in a wide range of pH, temperature and salinity, they are biodegradable catalysts and are simple and easy to control (Nicell et al. 1993; Torres et al. 2003). However, they have also a number of drawbacks, such as their high cost of isolation and purification, their instability, their non-reusability and inactivation by inhibitors. It is known that enzymes immobilization favours their industrial application, since their stability and durability are improved, their separation from the reaction medium is facilitated and then it is possible their reuse, reducing the cost of the process (Durań et al. 2002; Cao 2005). Laccase (p-diphenol: oxygen oxidoreductase, EC 1.10.3.2) catalyzes the oxidation of a vast amount of phenolic compounds and aromatic amines (Guo et al. 2011). It is also known that the number of laccase substrates can be extended by its combination with redox mediators (Tavares et al. 2008; Sadhasivam et al. 2009). Because of their broad specificity for the reducing substrates, laccases have been studied in different industrial applications, such as pulp delignification (Gamelas et al. 2005), organic synthesis (Ceylan et al. 2008), textile finishing (Pazarlioglu et al. 2005) and contaminated water or soil remediation (Couto and Herrera 2006). Laccase plays also an important role in the degradation of textile dyes from textile industrial effluents (Cristo´vaõ et al. 2008, 2009). During the industrial process, a significant fraction of the dyes is hydrolysed and released into the environment with the rejected dye baths or wash waters. The presence of colour in water will affect the transmission of light and photosynthesis and reduce aquatic diversity. Therefore, the treatment of these waters is of utmost importance (Saratale et al. 2011). The laccase immobilization for application in dye decolourization has been described on several supports by many methods, such as on poly(4-vinylpyridine) grafted and Cu(II) chelated magnetic beads via adsorption (Bayramoglu et al. 2010), on alumina pellets (Osma et al. 2010) and polyvinyl alcohol carrier (Yingui et al. 2002) by covalent attachment and also by entrapment on hydrogels (Yamak et al. 2009). However, some of these supports are expensive and their replacement by cheaper ones, like green coconut fiber used in this work, will contribute to the reduction of process costs. To our knowledge, the enzyme laccase was only immobilized on green coconut fiber by adsorption (Cristo´vaõ et al. 2011). Regarding laccase immobilization by covalent attachment on modified green coconut fiber no reports were found. Among the various immobilization methods, in this work it was decided to study the covalent binding, since it provides strong and

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stable enzyme attachment, avoiding the enzyme desorption or conformational changes when exposed to some medium variations and making the enzymes more attractive for industrial applications. Two covalent immobilization strategies were compared: one point and multipoint attachment. In the one point attachment, the enzyme has almost the same properties as the free enzyme, since only one covalent bond between an amine residue of the enzyme and an aldehyde group of the support is involved. In the multipoint attachment one molecule of enzyme is bound to several active groups on the support surface, which can significantly increase its stability (Guisań 1988; Cardias et al. 1999). To achieve a multipoint covalent immobilization an enhanced reactivity of some enzyme residues is necessary. Since these surface lysine residues have a pKa of around 10.5, at neutral pH they are not very reactive. Therefore, at neutral pH values, the one point attachment is achieved. On the other hand, at alkaline pH values, the reactivity of surface enzyme residues is enhanced, leading to an increased number of Schiff’s bases formed and making possible the multipoint attachment (Mateo et al. 2000). Sometimes it is necessary to add aldehyde groups to the support, through an activation step, making this method more expensive than the adsorption. The immobilization reaction between the aldehyde groups and the amino groups of the enzyme form Schiff’s bases that can be reduced afterwards with a reducing agent, such as sodium borohydride, converting the linkages in very strong and stable bonds and the remnant aldehydes into inert hydroxyl groups (Blanco and Guisań 1989). In this study, the formation of glyoxyl (aldehyde) groups on green coconut fiber is achieved by silanization with 3-glycidoxypropyltrimethoxysilane (GPTMS). In this work it was studied the immobilization of a commercial laccase formulation (DeniLite II S) by covalent attachment on green coconut fiber, following two different strategies: one point covalent attachment (pH 7.0) and multipoint covalent attachment (pH 10.0) and its ability to decolourize different reactive textile dyes in both situations was evaluated.

Materials and methods Chemicals and enzyme Textile dyes Reactive Black 5 (RB5) (Remazol Black B), Reactive Blue 114 (RB114) (Levafix Brilliant Blue E-BRA), Reactive Yellow 15 (RY15) (Remazol Yellow GR), Reactive Yellow 176 (RY176) (Remazol Yellow 3RS), Reactive Red 239 (RR239) (Remazol Brilliant Red 3BS) and Reactive


Red 180 (RR180) (Remazol Brilliant Red F3B) were kindly provided by DyStar (Porto, Portugal) and were used for degradation experiments without any further purification. Enzyme Commercial laccase formulation (DeniLite IIS; 120 U/g) from genetically modified Aspergillus was kindly provided by Novozymes. This formulation is used for indigo dye decolourization in denim finishing operations and includes a phosphate adipic acid buffer and an enzyme mediator. Support Green coconut fiber was kindly donated by Embrapa Agroindu´stria Tropical, Ceara´ State, Brazil. It was cut and sieved to obtain particles between 32 and 35 mesh and then washed with distilled water and dried at 60 °C before being used as immobilization matrix. Other chemicals 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (98 %) and sodium (meta) periodate (C99 %) were obtained from Sigma. Nitric acid (65 %) and sulphuric acid (95 %) were purchased from Merck. GPTMS (C98 %), and sodium borohydride (99 %) were obtained from Aldrich. Functionalization of support The support was functionalized using a four-step process described by Brı´gida et al. (2007): first the support was protonated with nitric acid (10 %, v/v) under low stirring for 30 min at room temperature; then the support was silanized using GPTMS (1 %, v/v) at pH 8.5 for 5 h at 60 °C; in a third step the epoxy groups were hydrolysed with 0.1 M sulphuric acid at 85 °C during 2 h; finally, the aldehyde groups were formed by oxidation with 0.1 M sodium periodate solution at room temperature for 1 h. Before immobilization, the activated fiber was dried under vacuum. Immobilization procedures To study the enzyme immobilization by covalent attachment, 0.1 g of green coconut fiber were incubated in syringes with 1.0 mL of laccase solution containing 0.067 g/mL of enzyme (optimum concentration established in a previous study of laccase immobilization by adsorption (Cristo´vaõ et al. 2011)) in 50 mM sodium phosphate buffer pH 7.0 and in 50 mM sodium bicarbonate buffer pH 10.0.

The syringes with the solutions were stirred on a rotational shaker TECNAL, model TE-165 at room temperature. At these conditions, the effect of contact time was evaluated in the range 2–6 h. After immobilization, the support was separated by filtration and washed three times with 0.1 M of phosphate buffer (pH 7.0) or 0.1 M of bicarbonate buffer (pH 10.0), respectively. The supernatant was kept for enzyme activity measurements. For each assay duplicate runs were made. Measurements of activity of free and immobilized laccase The free and immobilized laccase activities were measured spectrophotometrically (Thermo Electron, model UV1 spectrophotometer) according to the protocol described in a previous work (Cristo´vaõ et al. 2011), using ABTS as substrate. A control experiment with fiber (without enzyme) and ABTS was also performed and no activity was found. One unit (U) was defined as the amount of enzyme that oxidized 1 lmol of ABTS per minute and the free and immobilized laccase activities were expressed in U/L and U/kg, respectively. Immobilization yield (%) is defined as the difference between enzyme activity in the supernatant before and after immobilization divided by the enzyme activity in the supernatant before immobilization. Thermal stability of free and immobilized laccase The thermal inactivation of the free and immobilized laccase at 50 and 60 °C were also investigated following the same method used in a previous work (Cristo´vaõ et al. 2011). The initial activities were compared with the residual activities. The thermal deactivation curves were described following the simplified deactivation model proposed by Henley and Sadana (1985) referenced by Arroyo et al. (1999). This model involves enzymatic states (E, E1) where k is the thermal inactivation parameter (Eq. 1). k

E ! E1

ð1Þ

The thermal parameters were estimated by fitting the experimental data to Eq. 2 using a nonlinear regression code MicroCal Origin software v.3.01 (SPSS Inc., Northampton, USA). A ¼ ð1 aÞ:ekt þ a

ð2Þ

where A is the residual enzyme activity, a is the ratio of specific activity E1/E to the different states (see Eq. 1) and t the time. As in the previous work (Cristo´vaõ et al. 2011), Eq. 2 was used assuming that the final activity of the enzyme is not zero.

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Biocatalyst half-life (t1/2) was calculated from Eq. 2, using the estimated parameters (k, a) and making A equal to 0.5. In this work, stabilization factor (F) was considered as the ratio of immobilized enzyme’s half-lives to soluble enzyme half-life. Operational stability of immobilized laccase The operational stability of immobilized laccase during 10 reaction cycles was determined following a protocol previously used (Cristo´vaõ et al. 2011). At each cycle, a sample was withdrawn in 1 min intervals, absorbance was measured and then it was returned to reactor (initial reaction rate measurements). The final washing of immobilized enzyme between cycles was carried out with phosphate buffer 100 mM pH 7.0 or with bicarbonate buffer 100 mM pH 10.0, depending on the pH of immobilization. For each assay duplicate runs were made.

Degradation of reactive dyes by immobilized laccase A solution of single reactive dye (50 mg/L) (RB5, RB114, RR180, RR239, RY15 or RY176) was continuously orbital stirred (240 rpm) with 0.4 g of support immobilized with laccase (0.067 g/mL of laccase per g support) in phosphate buffer solution pH 7.0 at 35 °C (reactional volume of 25 mL). Three cycles of dye degradation were carried out. After each cycle, the support was washed three times with phosphate buffer pH 7.0, 100 mM or with bicarbonate buffer pH 10.0, 100 mM. Dye decolourization was determined according to Cristo´vaõ et al. (2011). Decolourization is reported as: % decolourization = (Ai - Af)/ Ai 9 100, where Ai is the initial absorbance and Af is the final absorbance of the dye (Cristo´vaõ et al. 2008). A control with support alone (without immobilized enzyme) was carried out at the same conditions in order to determine the dye adsorption by the support.

Storage stability The storage stability of the immobilized enzyme was evaluated by keeping it at 4 °C and determining its activity every 24 h for 96 h.

Results and discussion Immobilization of commercial laccase on green coconut fiber by covalent attachment

Determination of kinetic parameters The rate of an enzymatic reaction follows the Michaelis– Menten equation: v¼

vmax ½S K M þ ½ S

ð3Þ

where v is the velocity of the reaction (mM/min), vmax is the maximum velocity (mM/min), KM is the Michaelis– Menten constant (mM) and [S] is the ABTS concentration (mM). The Michaelis–Menten kinetic parameters KM and vmax of free and immobilized laccase were determined by measuring the laccase activity using ABTS as substrate over a wide range 0.005–0.1 mM for free laccase and 0.026–23.7 mM for immobilized laccase. The parameter values were obtained by non-linear curve fitting of the plot of reaction rate versus substrate concentration using the MicroCal Origin software v.3.01 (SPSS Inc., Northampton, USA). Effect of sodium borohydride reduction The effect of Schiff’s bases reduction by sodium borohydride 0.5 mg/mL was evaluated. After immobilization, the borohydride was added to the immobilization suspension and the reaction occurred for 30 min at 4 °C. After that, the immobilized enzyme was separated by filtration and thoroughly washed with 0.1 M phosphate buffer (pH 7.0).

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In this work the immobilization of commercial laccase by covalent attachment on green coconut fiber was studied by two strategies: one point covalent attachment (pH 7.0) and multipoint covalent attachment (pH 10.0). Therefore, support was initially activated with GPTMS (1 %, (v/v)) and only after that the enzyme was able to successfully connect to the activated support. The contact time influence between the activated support and the enzyme solution was evaluated at different time intervals (2, 4 and 6 h) for each immobilization procedure (pH 7.0 and pH 10.0, with and without reduction). These results are shown in Fig. 1 for the one point attachment (pH 7.0) and for the multipoint attachment (pH 10.0) with or without borohydride reduction for both cases. It can be observed that for all cases the maximum measured activity of the immobilized enzyme was achieved after 2 h of contact time. For higher contact times, no improvement on activity was observed, on the contrary, the activity decreases with the increase of contact time, probably due to the increase of Schiff’s bases formed between the enzyme and the support, which, in turn, promote the rigidity of the enzyme molecule and, therefore, will cause the decrease of enzyme activity (Rodrigues et al. 2008). In Fig. 1 it is also possible to observe that the enzyme immobilized by one point attachment (pH 7.0) showed higher activity (around 736 U/kg) than the one immobilized by multipoint attachment (pH 10.0) (around 380 U/kg). These two


Immobilized enzyme properties

Activity (U/kg)

1000

800

Thermal stability

600

To be a good biocatalyst for industrial applications, the enzyme must support high temperatures. So, its thermal stability is an important property that should be analyzed. Enzyme immobilization is known to increase enzymes stability against their denaturation, thereby limiting its freedom to undergo drastic conformational changes (AlAdhami et al. 2002). Thus, in this study it was analyzed if the enzyme immobilization by covalent attachment contributed to an increased thermal stability of the enzyme, as well as if there was any difference between one point and multipoint attachment, as it was reported by other authors (Mateo et al. 2000; Rodrigues et al. 2008). Thermal stability experiments were carried out at 50 and 60 °C. First, it was studied the thermal stability at 60 °C of free and immobilized laccase at pH 7.0 and pH 10.0, without borohydride reduction, with an immobilization time of 2 h. However, at this temperature the enzyme deactivation was very fast, preventing to see the behaviour differences between both pH values. Even so, by adjusting the thermal deactivation model (Eq. 2) to experimental data and by the model parameters obtained for free and immobilized enzyme (Table 1), it was possible to observe that the commercial laccase immobilization by covalent attachment led to an increment of the enzyme stability towards heat denaturation at 60 °C, with stabilization factors (F) of 2.5 (pH 7.0) and 3.2 (pH 10.0) in relation to free enzyme. This increased stability is probably due to the enzyme binding to the support that contributes to the stabilization of three-dimensional structure of the immobilized enzyme (Mateo et al. 2007; Rodrigues et al. 2008). The commercial laccase immobilized by the adsorption method showed a stabilizing factor slightly higher than the one immobilized by covalent attachment (Cristo´vaõ et al. 2011), however, to be sure of these results, the thermal deactivation parameters at 50 °C should be compared. In order to observe the behaviour differences between the various immobilization conditions studied, the thermal stability was also evaluated at 50 °C. In this case, the reduction effect by sodium borohydride was analyzed for both cases (pH 7.0 and pH 10.0). Once again, experimental data was fitted to thermal deactivation model (Eq. 2) and the model parameters listed in Table 1 were achieved. As it can be observed from the results in Fig. 2 and Table 1, it is now possible to see the differences between using covalent attachment at pH 7.0 or pH 10.0. The derivatives obtained by immobilization at pH 10.0 showed higher stabilities than those obtained at pH 7.0 (both without reduction). It has to be noted that in the immobilization at pH 10.0 case, it was not even possible to calculate the stabilization factor,

400

200

0 0

1

2

3

4

5

6

7

Contact time (h)

Fig. 1 Effect of contact time on the activity of the immobilized commercial laccase on coconut fiber by covalent attachment at pH 7.0 without (filled diamond) and with (open diamond) sodium borohydride reduction and at pH 10.0 also without (filled circle) and with (open circle) sodium borohydride reduction. Error bars indicate standard deviations

points present an immobilization yield of 74 and 50 %, respectively. The decrease in activity at alkaline pH may be also correlated to a significant increment on the enzyme stability promoted by the several bonds established between the enzyme and the support and to the lower number of enzymes linked. Comparing these activity values with the ones achieved with the adsorption immobilization method (Cristo´vaõ et al. 2011), it is also possible to verify that the chemical methods tend to reduce the enzyme activity, since the covalent bonds formed may perturb the enzyme native structure. The Schiff’s bases reduction by sodium borohydride was also evaluated for both cases. This reduction is important to avoid undesired enzyme-support interactions and, consequently, a decrease on enzyme activity and stability, i.e., after the activation step it should be ensured that the reactions are catalyzed only by the enzyme and not by the support, which should be inert (Mateo et al. 2006). However, as it is possible to verify by Fig. 1, in both cases, the reduction caused a loss in enzymes catalytic activity, being this loss more pronounced for pH 7.0. This behaviour has also been observed by Tardioli et al. (2003). It is also known that sodium borohydride can cause deleterious effects on protein structures, such as disulfide bond splitting and reductive cleavage of peptide bonds (Blanco and Guisań 1989), which could be the cause of the observed effect. Making a comparison with others covalently attached laccases, the higher activity of the biocatalyst (0.74 U/g) was a slightly lower than that observed for laccase of Panus conchatus, 1.2 U/g, immobilized in commercial support with high superficial area (Yingui et al. 2002).

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Author's personal copy World J Microbiol Biotechnol Table 1 Kinetic parameters of thermal deactivation of free commercial laccase and immobilized by covalent attachment on coconut fiber at 50 and 60 °C

k (h-1)

a

t1/2 (h)

Free

34.8

0.03

0.02

1.0

0.998

Immobilized at pH 7.0 without reduction

16.2

0.12

0.05

2.5

0.990


13.3

0.15

0.07

3.2

0.962

Free

13.0

0.04

0.06

1.0

0.998


20.4

0.25

0.05

0.97

0.981

Immobilized at pH 7.0 with reduction

10.6

0.36

0.15

2.6

0.871


3.3

0.56

–

–

0.894


2.7

0.44

0.83

14.8

0.859

Enzyme

F

R2

60 °C

50 °C

since the loss of the activity was less than 50 %. These results suggest that in this case, multipoint covalent attachment has occurred. The additional bonds established between the enzyme and the support during the immobilization process at pH 10.0 promoted the stabilization of the immobilized enzyme (Cardias et al. 1999). Analyzing the results of both immobilized enzymes after a final reduction with sodium borohydride (Fig. 2, Table 1), it was found that the derivative reduction caused a small increase in stability for pH 7.0 and for pH 10.0 the stability decreased. The stabilization effect was more pronounced when the immobilization was performed at alkaline pH values, even without reduction. This result for pH 10.0 suggests that, in some conditions, the sodium borohydride may modify the structure of the enzymes reducing

Fig. 2 Thermal stability of (multiplication symbol) free and immobilized commercial laccase obtained by covalent attachment at pH 7.0 with (open diamond) and without (filled diamond) sodium borohydride reduction and at pH 10.0 with (open circle) and without (filled circle) sodium borohydride reduction at 50 °C. The solid line represents the fit of thermal deactivation model to experimental data

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its stability. Similar result was also observed by other authors (Tardioli et al. 2003; Rodrigues et al. 2008), who suggested the addition of competitive inhibitors to the system in order to circumvent this drawback. Nevertheless, both derivatives (one point and multipoint) were more stable than the free commercial laccase, which was completely inactivated in less than 40 min (Fig. 2). Operational stability In order to be economically interesting, these biocatalysts should be reusable. Thus, the operational stability of commercial laccase immobilized in coconut fiber by covalent attachment was tested by 10 cycles of ABTS

Author's personal copy World J Microbiol Biotechnol Fig. 3 Operational stability of commercial laccase immobilized on coconut fiber by covalent attachment at pH 7.0 and pH 10.0, with and without sodium borohydride reduction. Error bars indicate standard deviations

oxidation. The results are shown in Fig. 3. It can be observed that the enzyme immobilized at pH 7.0 without borohydride reduction loses little activity in the first three cycles (around 20 %) and from the fourth cycle its residual activity stabilizes around 45–50 %. The enzyme immobilized at pH 10.0, also without borohydride reduction, had a similar behaviour, however it loses more activity in the first three cycles (around 50 % at the third cycle), probably due to the verified lower enzyme loading in the support. The effect of Schiff’s bases reduction was evaluated with sodium borohydride as reducing agent. The long term stability of the immobilized commercial laccase by one point and multipoint covalent attachment treated with sodium borohydride was also investigated by assaying after repeated washes. As shown in Fig. 3 the reducing agent effect on the operational stability was very remarkable. Both derivates did not lose activity over the ten oxidation cycles. This probably means that in this case the reduction contributed to the stabilization of the Schiff’s bases formed between the enzyme and the support. Park et al. (2003) also observed a similar result when treated GL-7-ACA acylase immobilized on silica gel with sodium borohydride. Comparing these results with the results obtained with the commercial laccase immobilized on green coconut fiber by adsorption (Cristo´vaõ et al. 2011) it is possible to see the difference between both immobilization methods. In the immobilization by adsorption method there is still a residual activity of around 55 % after 10 oxidation cycles. However, it appears that there is a higher loss of activity from cycle to cycle, probably due to the low binding forces between enzyme and support. The immobilization by covalent attachment promotes stronger bonds that help to

prevent the loss of enzyme during the washings (Durań et al. 2002). Storage stability Another important property of immobilized enzymes is their storage stability. Immobilized enzymes were stored at 4 °C and their activities were tested at different times. The obtained results are shown in Fig. 4 in terms of residual activity. By the results it can be observed that there was a significant loss of activity in the first 24 h in all cases and from there an activity stabilization was verified. The

Fig. 4 Storage stability at 4 °C of commercial laccase immobilized on coconut fiber by covalent attachment a at pH 7.0 without (filled diamond) and with (open diamond) sodium borohydride reduction and b at pH 10.0 also without (filled circle) and with (open circle) sodium borohydride reduction. Error bars indicate standard deviations

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derivatives without sodium borohydride treatment showed a higher loss of activity. In the first 24 h the immobilized enzyme at pH 7.0 lost 70 % of its initial activity and the one immobilized at pH 10.0 lost 90 %. These activity losses can be associated with continued reaction between enzymes and support functional groups, as this can lead to enzyme inactivation depending on the bindings that are established, or associated to reversible Schiff’s base. Despite the strong covalent bonds involved between the enzyme and the support, the reaction between amino groups and aldehydes is reversible, which can lead to loss of bounded enzyme (Isgrove et al. 2001). The derivatives reduction with sodium borohydride led to a milder inactivation (around 45–50 % in the first 24 h) and after 96 h both reduced derivatives lost around 65 %, when the ones without reduction have only around 25 % (one point attachment) and around 8 % (multipoint attachment) of its initial activity. Thus, the reduction step led to the Schiff’s bases stabilization and, consequently, the enzyme losses were reduced. Kinetic properties Measuring the initial reactions rates over a wide range of ABTS concentrations on the reaction of commercial laccase immobilized on green coconut fiber by covalent attachment, it was possible to verify that the immobilized enzymes followed a traditional behaviour of an enzymatic reaction. Thus, the kinetic parameters (KM and vmax) were determined by fitting the experimental data to Eq. 3. Table 2 and Fig. 5 show, respectively, the kinetic constants determined and the initial reaction rates of increasing concentrations of ABTS, for free and immobilized commercial laccase by covalent attachment at pH 7.0 and pH 10.0, with and without sodium borohydride reduction. The kinetic constant KM is a measure of the enzyme affinity for a particular substrate. The lesser the value of KM, the larger is the affinity of the enzyme to the substrate. Comparing with the kinetic parameters of the free enzyme, an increase in the KM values of the immobilized enzymes was observed, which means that the free enzyme has higher Table 2 Kinetic parameters of free and immobilized commercial laccase by covalent attachment on coconut fiber at pH 7.0 and pH 10.0 with and without sodium borohydride reduction Enzyme

KM (mM)

vmax (mM.min-1)

Free

0.0044

0.024


0.0717

0.247


0.7651

0.240


1.553

0.243


1.276

0.185

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Fig. 5 Initial reaction rates for different concentrations of ABTS a with free and b with immobilized commercial laccase on coconut fiber by covalent attachment at pH 7.0 without (filled diamond) and with (open diamond) sodium borohydride reduction and at pH 10.0 also without (filled circle) and with (open circle) sodium borohydride reduction. c Scale amplification of the immobilized enzyme at pH 7.0 without reduction kinetics. The solid lines represent the fit of Michaelis-Menten model to experimental data

affinity for ABTS than the immobilized ones. The increase in KM parameter upon immobilization is commonly reported in literature due to structural changes promoted by the enzyme immobilization process (Jiang et al. 2005; Wang et al. 2010). Another parameter to be considered is the presence of diffusional effects influencing the value of KM for the immobilized enzymes. This was observed in other studies with this support (Brı´gida et al. 2008). From the immobilized enzymes KM values, it was possible to observe by Table 2 that the affinity decreased in the following order of the different immobilization conditions: pH 7.0 without reduction [ pH 7.0 with reduction [ pH 10.0 with reduction [ pH 10.0 without reduction. Once again, the commercial laccase immobilized by one point covalent attachment (pH 7.0) without sodium borohydride reduction showed the closest properties to those of free laccase. The multipoint attachment and the reduction step

Author's personal copy World J Microbiol Biotechnol Fig. 6 Dye degradation (%) by immobilized commercial laccase onto green coconut fiber by covalent attachment at pH 7.0 and pH 10.0, with and without sodium borohydride reduction at the end of the a first cycle, b second cycle and c third cycle of reaction

(a)

(b)

(c)

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probably led to the increase of steric hindrance, limiting the ABTS entrance into the enzyme. As a result of structural changes induced by immobilization and of the new environment, the immobilized enzymes by covalent attachment have also higher Michaelis–Menten constants than the immobilized by adsorption one (Cristo´vaõ et al. 2011). On the other hand, vmax values for immobilized enzymes were found to be all similar between them and higher than that of the free enzyme, which could probably be due to the adsorption by the support: the local concentration increases, increasing the reaction rate. Despite of not being the normal behaviour, several other authors have reported an increase in the vmax value with the enzyme immobilization (Roy et al. 2003; Yamak et al. 2009). Dye decolourization by immobilized laccase In this work, the degradation of several reactive dyes (RY15, RY176, RR180, RR239, RB5, RB114) by commercial laccase immobilized on green coconut fiber by one point and multipoint covalent attachment, with and without sodium borohydride reduction was investigated for three decolourization reaction cycles. The results are shown in Fig. 6, and it is possible to observe that the most degraded dyes in all cycles were RY15, RB5 and RB114, as expected, since they are the ones that have lower redox potential (Tavares et al. 2009). The RR239 and RR180 dyes were also slightly degraded, but the RY176 dye was practically not degraded by any of the immobilized enzymes. Since the enzyme immobilized under the different conditions are in contact with the dyes for 24 h, the decolourizations achieved are similar for most of the cases. Only the laccase immobilized at pH 10 without sodium borohydride reduction provided slightly lesser degradations than the others (Fig. 6). However, the one point or multipoint immobilization, as well as the sodium borohydride reduction, do not seem to significantly affect the dyes oxidation process in 24 h cycles. It has been reported that in the treatment of dyes with immobilized enzymes, the colour removal is the result of both enzymatic catalysis and support adsorption (PeraltaZamora et al. 2003; Lu et al. 2007). In order to verify this situation, a control experiment of dyes degradation by the support without immobilized enzyme was carried out. The dyes adsorption by the functionalized coconut fiber was around 56 % for RY15, 2 % for RY176, 12 % for RR180, 37 % for RB5, 50 % for RB114 and no adsorption of RR239 was detected. Thus, the support adsorption had shown to have a great influence in the total degradation of some of the dyes, probably mainly in the first cycle of reaction. In the following cycles, it is expected a support saturation and an increase in the

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laccase contribution for the degradation of the dyes. However, in the second and third cycles there was a decrease in the dyes removal, even in cases where there was no decrease in activity in the operational stability made with the ABTS (Fig. 3). It is known that laccase needs a mediator to achieve the oxidation of these reactive dyes (Tavares et al. 2009). As already observed in the commercial laccase immobilization by adsorption (Cristo´vaõ et al. 2011), probably the mediator that is included in the commercial formulation is not being reoxidized during the reaction cycles and with the simultaneous support saturation, a decreased in dyes degradation is observed. A possible solution might be to add the mediator at each cycle or every two cycles. Since the first cycle of dyes degradation was very similar to that obtained by the laccase immobilized by adsorption (Cristo´vaõ et al. 2011), the immobilization by covalent attachment is more promising, since in terms of operational stability, the immobilized laccase by covalent attachment shows better results. Commercial laccase immobilized by covalent attachment in coconut fiber proved to be a suitable biocatalyst for continuous treatment of textile industrial effluents.

Conclusions Commercial laccase was successfully immobilized by two different strategies of covalent attachment on green coconut fiber. Compared with the free enzyme, the immobilized enzymes displayed a lower activity and affinity, but had improved thermal and operational stabilities. The final reduction with sodium borohydride contributed to the improvement of operational and storage stabilities of both derivatives. Finally, covalently immobilized commercial laccase on green coconut fiber exhibited a significant activity for the continuous degradation of the majority of the reactive dyes studied in the presence of a mediator. All these interesting properties, along with the cheap support, show the suitability of these biocatalysts for industrial applications. Acknowledgments Financial support for this work was in part provided by the project FCT/CAPES/(CAPES 4.1.3/CAPES/CPLP) and by project PEst-C/EQB/LA0020/2011, financed by FEDER through COMPETE—Programa Operacional Factores de Competitividade and by FCT—Fundaçaõ para a Cieˆncia e a Tecnologia, Portugal, for which the authors are thankful. The authors also wish to thank Novozymes (Denmark) for laccase from Aspergillus and DyStar (Porto, Portugal) for reactive dyes. S. Silve´rio thanks FCT for the Ph.D Scholarship (SFRH/BD/43439/2008), M. A. Z. Coelho thanks CNPq and FAPERJ (Brazil) and A. P. M. Tavares acknowledge the financial support (Programme Cieˆncia 2008).


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