ISSN : 0976-7045
EISSN : 0976-7053
MDAGHRI ALAOUI S.1, GHANAM J.2, MERZOUKI M.3, PENNINCKX M.J.4, BENLEMLIH M.5*
1Biotechnology Laboratory, Faculty of Science Dhar El Mahraz, PO Box: 1796, Atlas-Fez, University Sidi Mohammed Ben Abdellah, Fez, Morocco.
2Biotechnology Laboratory, Faculty of Science Dhar El Mahraz, PO Box: 1796, Atlas-Fez, University Sidi Mohammed Ben Abdellah, Fez, Morocco.
3Biotechnology Laboratory, Faculty of Science Dhar El Mahraz, PO Box: 1796, Atlas-Fez, University Sidi Mohammed Ben Abdellah, Fez, Morocco.
4Laboratory of Microbial Physiology and Ecology, Université libre de Bruxelles, C/O ISP, 642 Rue Engeland, B-1180, Brussels, Belgium.
5Biotechnology Laboratory, Faculty of Science Dhar El Mahraz, PO Box: 1796, Atlas-Fez, University Sidi Mohammed Ben Abdellah, Fez, Morocco.
* Corresponding Author : benlemlihmo@yahoo.fr
Received : 05-05-2013 Accepted : 27-05-2013 Published : 19-12-2013
Volume : 4 Issue : 2 Pages : 91 - 94
J Biotechnol Lett 4.2 (2013):91-94
Conflict of Interest : None declared
Treatment of Olive oil mill wastewaters with Pycnoporus coccineus laccase entrapped in Ca alginate beads led to significant abatement in their content in phenolic compounds. The immobilized enzyme was more stable over a wide range of temperatures and storage time as compared to the free enzyme and shows an appreciable catalytic capability (560 units g-1 support). Seventy % of laccase activity was retained by the beads after 18 successive cycles of utilization which open the possibility of reuse in a continuous process.
Pycnoporus coccineus, Laccase, Immobilization, olive oil mill wastewater
The annual worldwide production of Olive oil mill wastewaters (OOMW) is estimated to be over 30 million m3, from which 95% is produced in the Mediterranean countries [1] . The OOMW are extremely toxic because of high content of phenolic compounds and represent a serious environmental pollution problem especially for underground and surface water. Conventional wastewater treatment methods are relatively ineffective for removing these kinds of pollutants. New methods for the treatment of this wastewater were developed, in particular, by using White-rot fungi (WRF). WRF produce highly oxidative enzymes, such as ligninase, phenol-oxidase (laccase) and Mn-peroxidase, which are able to degrade lignin, phenol, various xenobiotics and environmental pollutants [2] .
Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.) belongs to a group of multi-copper-containing enzymes requiring oxygen to oxidize various aromatic substrates by one-electron transfer [2] . In recent years, interest in fungal laccases increased strongly because of their potential use in detoxification of environmental pollutants present in wastewaters generated by different industries [3-5] .
Pycnoporus coccineus is a white rot fungus producing large amount of a laccase particularly active in the degradation of several phenolic compounds present in Olive Oil Mill Wastewaters (OOMW) [3] .
Immobilization is an effective procedure for the application of industrial enzymes, in particular laccases [6-8] . In the present paper we present data showing that P. pycnoporus coccineus laccase entrapped in Ca alginate beads could be a useful form for degrading phenols present in OOMW.
Fungal Strain, Culture Conditions and Laccase Purification
The P. coccineus strain (MUCL38527) used in this study was obtained from the BCCM/MUCL (Belgian Coordinated Collections of Microorganisms/Mycotheque de l’Université Catholique de Louvain) and maintained on 2% malt extract agar.
The strain was cultivated during 12 days in high nitrogen content glucose medium (GLUHN) supplemented with ethanol and CuSO4 as inducers [3] .
The crude enzyme was obtained by ultrafiltration from the above mentioned cultivation medium and purified to electrophoretic homogeneity as previously described [3] .
Enzyme Activity, Protein and Total Phenols Analysis
The activity of the free and immobilized enzyme was assayed at pH 4.5 (100 mM Na acetate buffer) and 30°C with guaiacol as substrate [9] . Oxidation of guaiacol was determined by the increase in A465 ( e = 12,000 M-1cm-1). The enzyme activity units (U) were defined as mmol guaiacol substrate oxidised.min-1. Alternatively, laccase activity was determined at pH 5.0 in 100 mM Na acetate buffer using 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonate) (ABTS, Sigma) [3] . Protein was determined by the Bradford procedure, using Bio-Rad assay kit and bovine serum albumin as standard.
Total phenols compounds in OOMW were measured according to Maestro-Duran et al., [10] . The main characteristics of the OOMW produced by an industrial mill unit situated in the city of Fez (Morocco) and used in this study were pH 5.0 ± 0.2; COD 82 ± 5 g l-1; phenols content 3.2 ± 0.3 g l-1.
Procedure for Laccase Immobilization
Two ml of a purified laccase (400 mg protein) solution were mixed with 10 ml of 5% (w/v) sodium alginate solution. The beads were formed by dripping the solution into 100 ml of stirred 0.2 M CaCl2 solution with a syringe fitted with a needle (Gauge 18, Sigma Aldrich) at room temperature. The beads were further consolidated for 1 hr. at 37°C and washed with 0.9% NaCl until no enzyme activity and protein was detected in the washing.
The beads retained 75% of the enzyme activity engaged in the immobilization procedure and exhibited 560 enzyme activity units. g-1 support [Table-1] . The enzyme activity was assayed using guaiacol as substrate; however similar values were obtained when using ABTS as substrate.
pH and Temperature Profile of Free and Immobilized Laccase
The experiments on the effect of pH on the enzyme activity were performed at 30°C in a 2.5- 7.0 pH’s values range (2.5- 4.5, 0.1 M Na citrate-citric acid; 4.0-7.0, 0.1 M Na acetate- acetic acid).
The temperature profile of enzyme activity was investigated by assaying activity at pH 4.5 (0.1 M Na acetate- acetic acid buffer) and temperatures from 20 to 80°C.2.5. pH and temperature stability of free and immobilized laccase.
The pH stability of laccase was determined by incubating the free enzyme and the beads for 24hrs. in the range 2.5-7.0. Suitable aliquots of the enzyme were sampled and the residual activity was measured at pH 4.5 and 30°C.
Thermal stability was determined by incubating the free and immobilized enzyme for 1hr. at different temperatures (20-80°C). The enzyme residual activity was measured at pH 4.5 and 30°C.
Operational Stability of Immobilized Laccase and OOMW Treatment
The operational stability of the immobilized laccase was tested by using several consecutive oxidative cycles of 24 hrs. The beads in the presence of 1 mM guaiacol, were incubated in a 0.2 M acetate buffer solution (pH = 4.5) at 25°C. After each day of incubation, the immobilized laccase was washed three times with the acetate buffer solution and the residual enzyme activity was determined.
Batch experiments were performed by incubating filter-sterilized OO MW during 24hrs. at 25°C with alginate beads entrapped laccase, using an effluent volume/catalyst weight ratio equal to 500:1 [6] .
pH and Temperature Profiles of the Immobilized Laccase
The dependence of free and immobilized laccase activity on pH was assessed in a pH range of 2.5-7.0. Both the free and immobilized laccase exhibited maximal activity at pH values ranging from 3.5 and 4.5 [Fig-1A] . At neutral pH value, the free laccase had a poor activity; however the immobilized laccase maintained 20% of its activity. The enzyme was apparently stable in the pH range from 4.5 to 6 [Fig-1B] ; out of this range, stability decreased significantly for both the free and immobilized laccase.
The temperature-activity profiles [Fig-2A] indicated that the free laccase had optimum temperature around 60°C whereas this value increased to 70°C for the immobilized catalyst. Laccase stability was unaffected by 1 hr. thermal treatment in the range 20-50°C [Fig-2B] .
Ninety percent of activity was safeguarded for the immobilized laccase at 60°C whereas 60% of activity was lost for the free enzyme incubated at this temperature. Fifty percent of the immobilized laccase survived the heat treatment at 80°C whereas the free enzyme was completely destroyed at this temperature.
Cold storage experiments of the enzyme at 4°C showed that the free laccase activity was completely lost after three weeks storage whereas 80% of the activity of the
Immobilized enzyme was safeguarded at that time (result not shown).
Kinetic Properties
Km and Vmax were determined by using the guaiacol substrate in the concentration range of 0.1 to 4 mM at pH 4.5 - 30°C, and using nonlinear regression of the Michaelis-Menten equation [3] . Respective Km values were 1.84 and 1.13 mM for free and immobilized laccases. Calculated Vmax values were 126.6 and 181.8 U.mg -1 protein for free and immobilized laccases, respectively [Table-1] .
Operational Stability of the Immobilized Laccase
The oxidative procedure was repeated during 18 successive days with fresh guaiacol substrate as a model phenolic compound present in OOMW [11] . Complete enzymatic oxidation of guaiacol was checked at the end of each daily cycle. The immobilized P. coccineus laccase oxidized 18 batches of guaiacol while retaining 70% of initial activity [Fig-3] .
Treatment of OOMW with Immobilized Laccase
Treatment of OOMW with immobilized laccase on five consecutive batches showed each time a reduction of about 45% in the total phenols content as shown in [Fig-4] .
Numerous supports have been used for the immobilisation of fungal laccases [6,8,12,13] . Nevertheless, it appears that there is no ideal method and that the choice of the support is largely determined by the source of the laccase and the desired application. The P. coccineus laccase was previously immobilized by covalent linkage on acrylic epoxy-activated resins (Eupergit C and C250 L), however reaching only 110 U g-1 support [13] as compared to the value of 560 U g-1 for the enzyme immobilized in Ca alginate beads [Table-1] . Other protocols using gelatin [14] chitosan [15] also tested here for immobilization of laccase from P. coccineus.
Yet, the enzyme of P. coccineus immobilized on these two carriers lost more than 80% of its catalytic activity (result not shown), while laccase from Lentinula edodes immobilized on chitosan exhibited a catalytic capability of 520 U g-1 support [6] , a value comparable to the 560 U g-1 support obtained for the P. coccineus enzyme immobilized in Ca alginate beads [Table-1] . The reasons why the chitosan procedure was suitable for the L. edodes laccase, and not for P. coccineus, are unknown. It could be related to minor differences in molecular properties of the proteins, although basic features of the L. edodes and P. coccineus enzymes appeared at first glance quite similar [3,16] . The pH activity and stability data of the immobilized P. coccineus laccase are compatible with most of the values reported for OOMW [17] . This constitutes an obvious advantage for using the P. coccineus enzyme for OOMW treatment. Moreover immobilization of the P. coccineus laccase led to a significant stabilizing effect towards heat denaturation in comparison with the free enzyme. The enhanced thermal stability of laccase arising from immobilization could be also a benefit for treating effluents at high temperatures. Finally the good operational stability and durability of the immobilized enzyme open the possibility of its reuse in a continuous process.
The effective use of enzymes in the bio-industry can be enhanced by immobilization. It leads to efficient results in stability and longitivity of the enzyme, and in some cases, it could lead to the possibility of reuse of the enzyme in a continuous process. At present, the enzymes still have their very specific requirements and fragility. That is why, their future depends on industrial solutions are found to enhance stability and make them less susceptible to environmental factors in which they work. The immobilization procedure has a great influence on the activity of the enzyme. In our case, immobilization of laccase was tested on three matrices (sodium alginate, gelatine and chitin). The best result was obtained with sodium alginate, Grafted onto this matrix, the immobilized enzyme showed appreciable catalytic opportunities with properties remarkably improved stability to various parameters.
The results indicated that the immobilized laccase showed a significant catalytic capacity (360 enzyme units / g of support) with 75-86% binding of the enzyme to the matrix studied and a slight loss of activity (28% less than the initial activity). The immobilization has improved the stability of the laccase to the variation of the temperature and pH compared to the free enzyme. Similarly reuse in multiple consecutive catalytic cycles showed that its activity was reduced by 22% after 20 consecutive catalytic cycles. The operational activity decreased only 25.32% after the first week of storage and 90% after several weeks of storage at 4°C in comparison with the free enzyme which has lost almost all of its activity after the first week of storage. Treatment of vegetable with laccase immobilized on five consecutive batches showed each time a reduction of about 45% of the total content of phenols in the OMW.
The future of enzymes will depend to a large part of the cost of their use. This is why encapsulation techniques and grafting have been a technological opening greater opportunities. Research is still needed to reduce the cost of operation enzyme on an industrial scale. The covalent enzyme and alginate beads; neutral polymer to a number of advantages, such as the formation of covalent bonds between the enzyme and the polymer. Allowing the enzyme-polymer conjugate to resist changes in pH and temperature. Sodium alginate derived from seaweed may serve as immobilization matrix at very low cost and used in wastewater recycling.
The broad substrate specificity of laccase is rather significant in association with the stability of its properties, it can be proposed for potential applications in biotechnology and particularly its effectiveness in the treatment of wastewater. Therefore, improving the stability properties of laccase, and the ability to fulfill its reuse by consecutive cycles are targets of considerable importance. If it is used in the treatment reactors and/or repolymerization, immobilized laccase has advantages such as high expenses enzymes, prolonged activity of the enzyme, the very low energy costs and waste by recycling returned algae.
This work was funded through a Wallonia - Morocco bilateral project.
Conflict of Interest
The authors have declared no conflict of Interest.
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 | Fig. 1- Effect of pH on the activity (A) and stability (B) of free and immobilized laccase from P. coccineus Data are mean of three replicates (standard deviations < 8%); (o) free laccase; (â—) immobilized laccase. |
 | Fig. 2- Effect of temperature on the activity (A) and stability (B) of free and immobilized laccase from P. coccineus Data are mean of three replicates (standard deviations <8%); (o) free laccase; (â—) immobilized laccase. |
 | Fig. 3- Operational stability of laccase immobilized in alginate beads Data are mean of three replicates (standard deviations <7%) |
 | Fig. 4- Removal of total phenols in five consecutive batches of OOMW by immobilized laccase from P. coccineus |
 | Table 1- Properties of free and immobilized laccase from P. coccineus |