IMMOBILIZATION OF LACCASE FROM Pleurotus sajor-caju IN POLYAMIDE MEMBRANES

K. Rasera1,2, J. Ferla1,2, R. Riveiros2, M. Zeni2, A.J.P. Dillon1

Institute of Biotechnology, Caxias do Sul University, Caxias do Sul, RS, Brazil 2 Chemistry Laboratory, Caxias do Sul University, Caxias do Sul, RS, Brazil

Abstract Polyamide-6.6 (PA-6.6) membranes were modified with glutaraldehyde, in a phase inversion process. Immobilization of the enzyme laccase from Pleurotus sajor-caju in the modified PA-6.6 film was quantified in the presence of the reducer 2,2-azino-bis-3ethylbenzoazoline-6-sulfonate (ABTS). Under these conditions, phenolic compounds present in the ABTS were degraded by an oxidative process. In the case of the immobilized enzyme, 110 mol of ABTS was degraded after 6 hours of reaction. The same amount of ABTS was degraded after 1 hour in the case of the free enzyme, since the oxidation process is decreased by the low diffusion of ABTS through the membrane containing the immobilized enzyme. This characteristic is relevant since the reaction kinetics may be controlled in immobilized systems. The kinetics of the thermal inactivation (Kd) of laccase activity and the half-life (t1/2) were determined. It was found that there is the possibility for the enzyme to be reused. The process described may be useful for the treatment of textile effluents.

Keywords: Laccase, Pleurotus sajor-caju, Immobilization, Polyamide membranes, Kinetics.

Corresponding Author: Mara Zeni, Chemistry Laboratory, University of Caxias do Sul, Rua Francisco Getlio Vargas 1130, 95070-560, Caxias do Sul, RS, Brazil. Telephone number: 55-5432182100 E-mail: [email protected]

1. Introduction

its stability, facilitating its reuse and separation from the mixture [6]. Among the

The textile industry uses annually 0.7 million tonnes of 20% synthetic of which dyes, are

laccases which have been intensely studied are those produced by Pleurotus sajor-caju, a basidiomycete in which forms part of the human diet, classified as a white-rot fungus since it is an active decomposer of wood, degrading even lignin [7]. The synthesis of polymeric membranes from different polymers, for example, polyamide, has been studied at the

approximately

discharged in industrial effluents. Many of these dyes are stable under different conditions of light, temperature, specific microbial attacks and redox processes, making them resistant to conventional treatment [1]. Since the 1970s, white-rot fungi have been study for the treatment of bleaching effluents of the cellulose and paper industry, along with the decolorization of several dyes in vivo and in vitro [2]. The extracellular enzyme systems of white-rot fungi can degrade a wide variety of recalcitrant compounds, such

Chemistry Department of the University of Caxias to Sul. Griebenow et al. [8]

immobilized laccase by adsorption onto different supports, including polyamide 6.6 membranes. These authors observed that the laccase activity varied according to the support used and the best results were obtained membranes. with the polyamide 6.6

xenobiotics, lignin, and various types of dyes [3]. Leonowicz et al. [2], concluded that cellulose and lignin polymers are degraded simultaneously by enzymatic systems involving demethylation reactions catalyzed by laccases induced by radicals and mediators of low molecular weight resulting from the initial action of lignin peroxidase peroxidase catalyze (LiP) and manganese Laccases of can

2. Materials and methods

2.1 Microorganism The fungal strain used in this study was Pleurotus sajor-caju PS2001 from the collection of the Biotechnology Institute of the University of Caxias do Sul.

(MnP)[4,6]. the oxidation

various

2.2 Culture Media The maintenance medium contained 2% ground sawdust of Pinus sp., 2% ground wheat bran, 0.2% CaCO3, and 2% (w/v)

polyphenols and methoxyphenols [5]. The immobilization of an enzyme in soluble and insoluble substrates increases

agar. The medium was sterilized by autoclaving and placed into Petri dishes. The strains were grown at 28C for 14 days and then kept at 4C (2). For the production of laccases Pinus sp. sawdust (93% w/w), ground wheat bran (6% w/w), CaCO3 (1% w/w), (NH4)2SO4 (1.3% w/v), MnSO4 (0.0015% w/v), CuSO4 (0.0015% w/v) and water were used. Quantities of 180g of the medium were placed in polypropylene bags, autoclaved for 1 hour and inoculated with a 1.5cm agar disk. The inoculated bags were maintained at 28C (2) at a moisture content of 70-80% for 15 days.

contained 0.45 mM ABTS, 90 mM of 0.1M sodium acetate buffer and 1 mL of diluted supernatant to give a final reaction volume of 2.2 mL. The oxidation of the substrate (ABTS) was monitored by the increase in the absorbance at 420 nm, over 90 s at 30C (2C), using =/3.6X104 cm-1M-1 (Wolfenden & Wilson 1982). Enzymatic activity was expressed in the following units: 1U = 1 mol of ABTS oxidized per min at 25C (1). The amount of protein was determined by the Bradford method, using bovine albumin as the standard (Sigma) [9].

2.5. Thermostability of the laccase extract 2.3. Preparation of enzymatic extract The contents of the cultivation bags (180g) were mixed with 360mL of water in Erlenmeyer flasks, which then shaken at 160rpm for 20 min. The mixture was filtered and centrifuged at 5000g for 15min. The supernatant was then precipitated with ammonium sulfate (80% w/v) for 2 hours at 4C. The precipitate was resuspended with half of the initial volume in 0.1 M McIlvaine buffer solution, pH 6.0, and dialyzed in the same buffer [7,8]. 2.6. Kinetics of thermal inactivation The kinetics of the thermal inactivation (Kd) of the laccase activity and the half-life (t1/2) were studied at different temperatures 2.4. Enzyme assay The laccase activity was determined using 2,2-azino-bis(3-ethylbenzthiazoline6-sulfonic acid) (ABTS) (Sigma) as the substrate. The laccase reaction mixture between 2050C (0.5C), with magnetic stirring. The thermal inactivation constant (Kd) of the free laccase was determined using Equation 1, where Ain is the residual The analysis of the thermostability of the enzyme was carried out maintaining the enzymatic solution at a constant

temperature of 20C, 30C, 40C and 50C (1), for a period from 24 hours, using ABTS as the reducing substrate. The laccase activity was measured as described in section 2.3.

activity after thermal treatment during the incubation period, and Ain0 is the initial enzymatic activity. The half-life time (t1/2) was determined using Equation (2) [10].
ln Ain = Kd . t Ain0 (1)

3. Results and Discussion

3.1. Free laccase activity In general, the optimum pH for laccase activity can vary depending on the &

microorganism
(2)

studied.

Bollag

t1/2 = 0.693 Kd

Leonowicz [12] characterized extracellular laccases obtained from Fomes annosus, Pholiota mutabilis, P. ostreatus, praticola T. and

2.7. Immobilization of laccase Initially the supports used were

versicolor,

Rhizoctonia

immobilized using glutaraldehyde (Merck) in 0.1M acetate buffer at pH 7.0. The enzymes were immobilized by covalent bonds between the NH2 of the protein and the aldehyde group of glutaraldehyde [11]. The excess of glutaraldehyde was washed off with distilled water. The films were then suspended in the enzymatic solution, in 0.2M acetate buffer, pH 5.0, for up to 48h, at 20C, 30C and 40C. The quantity of immobilized protein was measured after 2, 4, 6, 8, 10, 12, 24 and 48h.

Botrytis cinerea fungus. They observed that the optimum pH in the case of P. mutabilis remained in the neutral region, while for the other fungi it varied between 3-5. The optimum pH found for the free enzyme was pH 4-5 in McIlvaine buffer. The results showed that the P. sajorcaju laccases are sensitive to temperature (Fig. 1). After 2 h there was a sharp

decrease in the enzymatic activity of the laccases in the solutions maintained at 30C (30%) and 40C (90%), with a total loss for that maintained at 50C. The values for the

2.8. Statistical analysis of the results For the statistical analysis we used analysis of variance (one-way ANOVA), with the post-hoc Tukey test (P<0.05) using the software GraphPad Prism 3.0 for Windows (GraphPad Software, San Diego, EUA).

thermal inactivation constant (Kd) and the half-life (t1/2) can be observed in Fig. 2. From the data given in Fig. 2, it can be seen that the half-life of the enzyme is reduced significantly temperature, with and the also increase the in

thermal

inactivation constant is increased, possibly due to an increase in the denaturation of the enzyme.

100

and 4 h may be due to the action of non

80
Activity (%)

immobilized

laccases

retained

in

the

polymer which were liberated after 4 hours.

60

40

a a a b ba b a b b c b

20

20C 30C 40C

10

15

20

25

Tim (h) e

Fig. 1. Thermostability of the laccase extract at 20, 30, 40 and 50C for 24 h, using ABTS as the substrate in acetate buffer pH 5.

Time (h)

80 70 60 50

7 6 5 4

Fig. 3- Activity of laccase immobilized in PA membranes with different times and temperatures. Values (averages) with the same letters for the same period do not differ significantly according to the Tukey test (P > 0.05).

Kd (h )

t1/2 (h)

40

3
30 20

-1

2
t 1/2 (h)

10 0 10 20 30 40 50

K d (h )

-1

1 0

4. Conclusions It was found that PA membranes can be used for the immobilization of laccases. However, a decrease in the optimum pH

60

T (C )

Fig. 2: Thermal inactivation constant (Kd) for laccase activity and half-life (t 1/2), using ABTS as the substrate reducer, at temperatures of 20 to 50C.

and in the oxidation rate occurred. These membranes also showed a potential for reuse of at least 4 times, with a retention of

In Fig. 3 the data on the oxidation capacity of ABTS for the PA membranes with different immobilization times (2-24 h) and temperatures (20C, 30C and 40C) are shown. As can be observed, the average values for the ABTS oxidation by membranes using an immobilization process of 6 h, at 30 and 40C, are the highest. The low values obtained for ABTS oxidation at 2 h

50-70% of the ABTS oxidation activity.

Acknowledgements The authors acknowledge the University of Caxias do Sul, CAPES and CNPq.

References [1] G.S., Nyanhongo; J. Gomes; G.M., Gubitz; R. Zvauya; J. Read; W., Steiner. Water Res. 36 (2002)1449-1456.

[2] A. Leonowicz; A. Matuszewska; J. Luterek; D. Ziegenhagen; M. WojtasWaslewska; N. Cho; M. Hofrichter; J. Rogalski; Fungal Genetics and Biology 27 (1999) 175-185. [3] T.K., Kirk; R., Farell, Ann Rev. Microbiol. 41 (1987) 465-505. [4] F.W.H.; Obara; G., Vara-Pereira, D.T., Miyagui; M.L.C., da Silva; Acta Sci. Biol. Sci. 27(2005) 303-310. [5] J. Roy, T. E. Abraham, J. of Molecular Catalysis B: Enzymatic 38 (2005) 31-36.

[7] L. Caramelo, M.J. Martinez, A.T. Martinez, Appl. Environ. Microbiol. 65 (1999) 916922. [8] K. Griebenow, A.I. Ruiz, A.J. Malave, C. Felby, Biotechnology Letters, 22 (2000) 229-233. [9] M.M. Bradford., Analytical Biochemistry. 72 (1976) 246-254. [10] M. Camassola, N.T. Sehnen, A.J.P. Dillon, J. Andreaus., Biocatalysis and Biotransformation. 22 (2004) 391-396. [11] G. Carta, J.L. Gainer, A.H. Benton., Biotechnology and Bioengineering. 37

[6] A.J.H., Al-Adhami; J., Bryjak; B., Grez-Markiewickz; W., Peczynska-Czoch; Process Biochemistry. 37 (2002) 1387-1394.

(1990) 1004-1009. [12] J.M. Bollag, A. Leonowicz, Appl. Environ. Microbiol, 48 (1984) 849-854.