Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems

Process Biochemistry 44 (2009) 435–439 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems Karla Mayolo-Deloisa a, Maria del Refugio Trejo-Hernández a, Marco Rito-Palomares b,* a Centro de Investigación en Biotecnologı´a, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico Departamento de Biotecnologı´a e Ingenierı´a de Alimentos, Centro de Biotecnologı´a, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico b A R T I C L E I N F O A B S T R A C T Article history: Received 18 July 2008 Received in revised form 8 December 2008 Accepted 10 December 2008 The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and system pH was carried out to determine under which conditions the laccase concentrates predominantly to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w), phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 95%. The results reported here demonstrated the potential application of ATPS for the valorisation of residual material and the potential establishment of a downstream process to obtain value added products with commercial application. ß 2008 Elsevier Ltd. All rights reserved. Keywords: Laccase Residual compost Agaricus bisporus Aqueous two-phase systems Protein recovery 1. Introduction With the increasing concern to valorise the waste material constantly generated, there is considerable interest in the establishment of efficient processes to obtain valuable products from residues. Currently, the processing of waste material exploiting bioengineering strategies that will rapidly deliver commercial products will definitively draw attention from industry. The establishment of processes that permit the recovery of commercially attractive products from a wide variety of residues is essential. In this context, the potential recovery of enzymes products from microbial residues represents a very interesting case that has been addressed before and continues to raise interest. The design of efficient recovery and purification protocols that allow the recovery of enzymes from residues is one of the key goals in the field. Particularly, in this research laccase produced by Agaricus bisporus was selected as a representative model of this group of value added products of commercial interest that can be recovered from microbial residues. Laccases are multi-copper oxidases that catalyze the one electron oxidation of several aromatic substrates with the simultaneous reduction of dioxygen to two molecules of water [1]. Laccases can be used in * Corresponding author. Tel.: +52 81 8328 4132; fax: +52 81 8328 4136. E-mail address: mrito@itesm.mx (M. Rito-Palomares). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.12.010 bioremediation, beverage (wine, fruit juice and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking and as biosensor [2]. Laccase activity has been extensively demonstrated in more than 60 fungal strains like A. bisporus, Pleurotus ostreatus and Trametes versicolor [3,4]. A. bisporus is considered one most important edible mushroom from the economic perspective. It has been reported that during the growth of the A. bisporus mycelium on composted wheat straw, large amounts of laccases are produced [5–7]. After fruit body harvesting, a considerable amount of residual compost is discarded as by product. This residue is a potential source of ligninolytic enzymes like laccase [6,7]. Several methods for purification of laccase have been reported that consist mainly of ultrafiltration, dialysis, one or more types of chromatography (i.e. exclusion, ion exchange) and electrophoresis [5,8–14]. The use of multi-step process approach results in low yield, high costs of supplies and operation. To overcome some of the disadvantages attributed to the established protocols for the recovery of laccase from residual compost, the use of aqueous twophase systems (ATPS) [15] is proposed as an attractive alternative in this research work. ATPS, assembled from a mixture of polymers (e.g. poly (ethylene glycol); PEG) and salts (e.g. phosphate or sulphate), result in two-phases for the extraction of bio-molecules. ATPS posses several advantages compared to the conventional process, including bio-compatibility, ease of scale-up and low cost [15,16]. In the present research, a practical approach, which 436 K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439 exploits the known effect of system parameters such as PEG and salt (phosphate and sulphate) concentration and the molecular mass of PEG upon product partition, was used. This approach was followed to evaluate the feasibility of using ATPS for the recovery of laccase from residual compost of A. bisporus as a first step in the development of a prototype bio-process. The research was conducted using a crude extract of the residual compost of A. bisporus to obtain different conditions where the target product and the contaminants partitioned preferentially to opposite phases. These conditions were then used to evaluate the effect of increasing crude extract concentration on the system performance. 2. Experimental 2.1. Crude extracts from residual compost of A. bisporus Residual compost of A. bisporus was kindly provided by Dr. Hermilo Leal-Lara from National Autonomous University of Mexico (UNAM). The residual compost (2.0 g) was mixed with distilled water (3.0 mL). The resulting mixture was kept at 4 8C for 24 h. The solid phase was recovered and pressed manually to increase the amount of liquid phase obtained. Subsequently, the liquid phase was passed through a mesh (0.8 mm) and centrifuged (10,000  g) as described by TrejoHernandez et al. [7]. The resulting liquid was referred as crude extract from residual compost of A. bisporus. The laccase enzymatic activity was estimated from the crude extract prior to lyophilization and storage at 20 8C until use. 2.2. Effect of pH on enzyme activity Prior to aqueous two-phase experiments the effect of pH on the enzyme activity of laccase from crude extract of A. bisporus was evaluated. A mixture of lyophilized crude extract with distilled water (10%, w/w) was prepared. pH was changed from 2.5 to 5.5 and 5.5 to 7.0 using sodium acetate–acetic acid buffer (0.1 M) and phosphate buffer (0.1 M) respectively. Samples were taken from each system pH for further biochemical analysis. 2.3. Aqueous two-phase experiments For the aqueous two-phase experiments, systems were selected based upon previous experiences [15,17] to give a volume ratio of 1.0 and a fixed weight of 5.0 g. The strategy behind the selection of the experimental systems is well described elsewhere [15]. The system tie-line length (TLL), which represents the length of the line that connects the compositions of the top and bottom phases in a phase diagram for a defined system, was calculated as described before [18]. Predetermined quantities of stock solutions of potassium phosphate and poly (ethylene glycol) (PEG, Sigma Chemicals, St. Louis, MO, USA) of nominal molecular masses 1000, 1450, 3350 and 8000 g/mol were mixed with lyophilized crude extract from residual compost of A. bisporus (the amount of crude extract added to the ATPS represent the 1.0–7.0% (w/w) of the total system) to give the desired PEG/ salt composition with a final weight of 5.0 g. Additional, 5.0 g experiments were conducted using PEG–sulphate systems to evaluate the effect of system pH upon the behaviour of laccase from crude extract of A. bisporus. Adjustment of the pH (from 3.0 to 7.0) was made by the addition of 1.0 M orthophosphoric acid or potassium hydroxide if needed. Complete phase separation was achieved by lowspeed batch centrifugation at 1500  g for 10 min. Visual estimates of the volumes of top and bottom phases were made in graduated tubes. The volumes of the phases were then used to estimate the experimental volume ratio (Vr, defined as the ratio between the volume of the top phase and the bottom phase). Samples were carefully extracted from the phases (top and bottom phase) and analyzed. The top and bottom phase recovery was estimated as the amount of the target product present in the phase (volume of the phase  volumetric enzymatic activity in the phase) and expressed relative to the initial total activity loaded into the system. Purification factor was estimated as the ratio of the specific activity of laccase after and before the extraction stage. The specific activity of laccase is expressed relative to the total amount of proteins. Results reported are the average of three independent experiments. Fig. 1. Effect of pH on the enzyme activity of laccase recovered from the residual compost of Agaricus bisporus. 3. Results and discussion Initially, laccase rich extracts were subjected to different pH environments to estimate process extraction conditions exploiting ATPS. Fig. 1 shows the impact of pH on the enzyme activity of laccase recovered from the residual compost of A. bisporus. These results can be utilized to define the pH of the ATPS for the potential extraction of laccase. The results of Fig. 1 show that the maximum enzyme activity is obtained at pH range of 4.0–4.5. Decrease of pH below 4.0 and an increase above 4.5 resulted in a reduction of laccase activity. The decrease in laccase activity seen for pH values above and below 4.0–4.5 may be the result of either lower solubility or loss of activity (or both). From these results it was decided that ATPS characterized by system pH below 7.0 will be initially utilized for the potential extraction of laccase from the residual compost of A. bisporus. In order to evaluate the effect of system pH below 7.0 on the recovery of the enzyme from the top PEG-rich phase, PEG–sulphate ATPS were utilized. Based upon previous experience establishing initial process conditions [15], low molecular mass of PEG was selected (i.e. 1000 and 1450 g/mol). PEG–salt systems characterized by molecular mass lower than 1000 g/mol promote the accumulation of cell and cell debris at the top PEG-rich phase [15]. Fig. 2 shows the effect of system pH on the recovery of laccase from the residual 2.4. Analytical techniques The enzymatic activity was estimated following the change in optical density at 436 nm using ABTS as a substrate and the extinction coefficient (e436 = 29,300 M 1 cm 1) as described by Tinoco et al. [19]. Briefly, the assay mixture consisted of 1.775 mL of acetate buffer (0.1 M, pH 4.0), 0.2 mL of substrate (ABTS, 1 mM) and 0.025 mL of enzyme extract-sample. One unit of enzyme activity was defined as the amount of enzyme required to oxide 1.0 mmol of substrate per minute. Protein concentration from the phases was determined using the method of Bradford [20]. Fig. 2. Effect of system pH on the recovery of laccase from the residual compost of A. bisporus in PEG–sulphate ATPS. The selected systems comprising PEG 1000 18% (w/ w), sulphate 15% (w/w) (^) and PEG 1450 18.3% (w/w) and sulphate 11% (w/w) (~), exhibited volume ratio (estimated from blank systems) and tie-line length (TLL) equal to 1.0 and 40% (w/w) respectively. The concentration of crude extract from the compost of A. bisporus in the ATPS was kept constant at 1.0% (w/w). K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439 compost of A. bisporus in PEG 1000 and PEG 1450–sulphate ATPS. In general, increasing the system pH caused an increase in the top phase recovery of laccase from the residual compost. Despite the known laccase stability at acid pH (3–5) [21] (see also Fig. 1), higher enzyme recovery from the top phase was obtained when the pH increased above 4.0. Several authors have discussed the influence of system pH on protein partition behaviour [17]. In general, these reports attributed the increase in the protein concentration in the top phase and a decrease in the bottom phase with increasing pH to free volume effects and potential changes in the structural integrity of the proteins [15,17]. Laccase from A. bisporus is an acidic enzyme, with an isoelectric point (pI) around pH 4.0 [21]. When the pH of the extraction ATPS system is increased above of the pI, the laccase surface charge becomes negative. As a result an increase in the partition coefficient is observed. This behavior was observed with other acidic enzymes [22–25]. Such partition behaviour indicates that laccase is a hydrophobic protein. Andrews et al. [26] have studied the protein behaviour in ATPS and concluded that the hydrophobicity and charge of protein are important factors in the enzyme partition. Laccase exhibited a better partitioning at pH 7.0 under PEG-rich environments. PEG–sulphate systems are better suited for pH values below 6.5, while PEG–phosphate systems are more stable at pH values above 7.0. Therefore, for further evaluation of ATPS performance, at 7.0, PEG–phosphate systems are recommended [15]. Furthermore, in an initial comparison, the enzyme recovery from PEG–phosphate systems at pH 7.0 resulted superior from those obtained from PEG–sulphate systems (see Fig. 2 and Table 1). The enzyme recovery from PEG 1000 and PEG 1450–phosphate systems was 80% and 72%, respectively. In contrast, in PEG 1000 and PEG 1450–sulphate systems the recovery of laccase was 74% and 62%, respectively. Thus it was decided to continue the characterization of the behaviour of laccase obtained from the residual compost of A. bisporus, at pH 7.0, in ATPS formed by PEG and phosphate. PEG–phosphate systems at pH 7.0 can be obtained by the appropriate mixture of the phosphate–salt that form the phases. Further evaluation of ATPS performance at pH above 7.0 was not pursued due to the potential application of the enzyme for bioremediation purpose and to avoid the addition of preparative stage to the process (e.g. adjustment of the system pH). PEG– phosphate systems are the most used and characterized systems in the literature [15]. Initially, the effect of increasing TLL upon the partition behaviour of laccase was evaluated. It has been reported [17] that changes in the TLL affect the free volume available for a defined solute to accommodate in the phase and as a consequence in the partition behaviour of such solute in the ATPS. 437 The effect of increasing TLL upon laccase obtained from the residual compost of A. bisporus when PEG of four different molecular mass (i.e. 1000, 1450, 3350 and 8000 g/mol) was used, is illustrated in Table 1. For all these systems, volume ratio and system pH were kept constant at 1.0 and 7.0, respectively. Overall top phase recovery of laccase decreases with the increase in TLL. An increase in the TLL of an ATPS caused the free volume in the bottom phase to decrease [27] and as a result, the solutes in the lower phase may be promoted to the partition to the top phase. Consequently, the increase of contaminant proteins that concentrate in the top phase with increasing TLL is possible and as a result the amount of the target product that can be accommodate in such phase may be negatively affected [17]. Furthermore, an increase in TLL decreases the volume available in the top phase for solute dissolution. As a result the amount of soluble solute (target and contaminants) in such phase decreases affecting the potential top phase product recovery. It is also important to note that the top phase recovery of laccase decreases when high PEG molar mass were used (see Table 1). The effect of increasing molecular mass of PEG upon protein partition behaviour has been explained based upon the protein hydrophobicity [28] and phase excluded volume [17]. In this case, the decrease in laccase top phase recovery when high PEG molar mass were used may be explained by a migration of the enzyme from the top phase to the bottom phase or interface. ATPS with low molecular mass of PEG (i.e. PEG 1000 and PEG 1450) show the best laccase top phase recovery. ATPS using PEG 1000 (TLL 40 and 45%, w/w) and PEG 1450 (TLL 36 and 39%, w/w) were selected to evaluate the effect of the increase in the concentration of the extract crude loaded to extraction system on the recovery of laccase. An increment in the concentration of crude extract fractionated via ATPS may be of benefit to the potential intensification of the proposed ATPS process. The effect of the concentration of crude extract of the residual compost of A. bisporus upon the laccase recovery is shown in Table 2. It is clear that ATPS was able to fractionate concentrated crude extract. Furthermore, top phase laccase recovery increased when the concentration of the crude extract was raised. PEG of low molecular mass (for example, 1000 and 1450 g/mol) helps to overcome saturation problems at the top phase [17,27,29]. For ATPS selected (i.e. PEG 1000 and PEG 1450), the best performance was obtained at a concentration of crude extract of 5% (w/w). Apparently, when higher concentration of crude extract were used (i.e. 7%, w/w) a negative impact on the laccase recovery was observed. This behaviour can be attributed to the saturation of the top phase system due to the complexity of the crude extract from the residual compost of A. bisporus. It is clear Table 1 Effect of system tie-line lengths and molecular mass of PEG on the recovery of laccase from the residual compost of A. bisporus in PEG–phosphate ATPS. System Molecular mass of PEG (g/mol) % PEG (w/w) % Phosphate (w/w) TLL (% w/w) Top phase recovery (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1000 18.2 18.9 22.2 24.1 15.1 17.5 21.9 23.0 18.0 21.9 22.2 24.1 15.0 16.5 19.0 24.1 15.0 16.0 19.0 20.1 13.0 14.3 18.0 19.8 15.0 18.0 19.0 20.1 11.0 11.5 12.5 20.1 40 45 55 59 36 39 51 56 43 56 58 62 30 33 41 63 80  1.3 78  2.8 67  1.2 49  1.5 72  1.6 72  2.0 53  2.4 40  0.8 59  0.7 44  3.0 40  1.0 39  7.0 40  1.0 39  2.0 35  3.0 32  2.0 1450 3350 8000 The system pH of the selected systems was kept constant at 7.0. The concentration of crude extract from the compost of A. bisporus in the ATPS was kept constant at 1.0% (w/w). K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439 438 Table 2 Effect of crude extract concentration on the recovery of laccase from the residual compost of A. bisporus in ATPS. Molecular mass of PEG (g/mol) % PEG (w/w) % Phosphate (w/w) TLL (% w/w) Crude extract concentration (% w/w) Top phase recovery (%) 1000 18.2 15.0 40 18.9 16.0 45 1.0 2.0 5.0 7.0 1.0 2.0 5.0 7.0 80  1.3 84  4.6 95  6.8 43  0.8 78  2.8 81  4.0 91  5.0 43  1.5 15.1 13.0 36 17.5 14.3 39 1.0 2.0 5.0 7.0 1.0 2.0 5.0 7.0 72  1.6 94  3.0 80  2.2 53  1.5 72  2.0 64  5.0 78  3.8 46  2.2 1450 Table 3 Direct comparison of the developed and the previously reported processes for the recovery of laccase from different sources. Microorganism Main primary recovery stage Process yield (%) Purification factor Type of fermentation Reference Agaricus bisporus Agaricus bisporus Pycnoporus sanguineus Agaricus blazei Panus tigrinus Volvariella volvacea Trametes versicolor Lentinula edodes Perenniporia tephropora ATPS Ultrafiltration Ultrafiltration Ultrafiltration Ultrafiltration Ultrafiltration Ammonium sulphate precipitation Ammonium sulphate precipitation Ammonium sulphate precipitation 95 88 87 83 93 94 56 88 80 2.48 2.7 2.14 1.6 1.18 1.1 2.24 1.98 1.03 Solid (residual compost) Liquid Liquid Liquid Liquid Liquid Liquid Liquid Liquid This research Wood [5] Lu et al. [8] Ullrich et al. [9] Quaratino et al. [10] Chen et al. [11] Han et al. [12] Nagai et al. [13] Ben Younes et al. [14] Selected ATPS (TLL 40% (w/w), PEG 1000 18.2% (w/w), phosphate 15% (w/w), loaded with a concentration of crude extract of 5% (w/w)). from these studies that when a concentration of crude extract of 5% (w/w) was used, ATPS characterized by PEG of 1000 g/mol molecular mass showed a superior performance compared to that from the PEG 1450 systems. Selected extraction conditions (i.e. Vr = 1.0, PEG 1000 18.2% (w/w), phosphate 15% (w/w), TLL of 40% (w/w) and system pH of 7.0) resulted in a laccase top phase recovery of 95%. A further direct comparison based upon the purification factor and enzyme yield achieved between the proposed protocol for the primary recovery of laccase from the residual compost of A. bisporus and previously reported processes (Table 3) highlights the advantages of the ATPS approach. The purification factor obtained from our work is similar to that reported by Wood [5] and higher than those obtained for laccases produced by other fungi. The proposed process involves a onesingle extraction stage to fractionated complex residual compost. The previously reported protocols involve the recovery of laccases when liquid fermentation was used. The use of solid residual compost for the recovery of laccase has been slightly studied due to the complexity of mixture. To date, no previous studies known by the authors have been reported that exploit the potential use of ATPS for the recovery laccase from a complex waste material such residual compost. It is clear that, for certain residual material, this process opens the way to further processing such material as a first step to potentially obtain value added products. 4. Conclusions This study reports the processing of residual compost from A. bisporus in aqueous two-phase systems for the potential primary recovery of laccase. It has been shown that parameters such as tieline length, molecular weight of PEG, type of salt and system pH, influence the recovery of laccase from the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase, since top phase recovery was higher than 90%. The operating conditions selected for the ATPS resulted in a onesingle extraction process for the potential recovery of laccase from residual compost of A. bisporus. Overall, the results reported here demonstrated the potential application of ATPS for the primary recovery of laccase from residual compost material as a first step for the valorisation of such materials and the potential establishment of a downstream process with commercial application. Acknowledgements The authors wish to acknowledge the financial support of Tecnologico de Monterrey, Bioengineering and Nano-bioparticles Research Chair (Grant CAT161). 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