Improvement of Catalytic Efficiency, Thermo-stability and Dye Decolorization Capability of Pleurotus ostreatusIBL-02 laccase by Hydrophobic Sol Gel Entrapment
© Asgher et al.; licensee Chemistry Central Ltd. 2012
Received: 7 August 2012
Accepted: 27 September 2012
Published: 29 September 2012
In serious consideration of the worldwide environmental issues associated with the extensive use of the textile dyes and effluents generated thereof, the scientists across the world are in search for potential treatment technologies for their treatment. In such scenario the ligninolytic enzymes provide a potential alternative because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents.
Laccase produced from Pleurotus ostreatus IBL-02 during decolorization of the reactive textile dye Drimarene brilliant red K-4BL (DBR K-4BL) was purified and immobilized by hydrophobic gel entrapment. The crude laccase was 4.2-fold purified with specific activity of 573.52 U/mg after passing through the DEAE-Sepharose ion exchange and Sephadex-G-100 chromatography columns. P. ostreatus IBL-02 laccase was found to be a homogenous monomeric protein as evident by single band corresponding to 67 kDa on native and sodium dodesylsulfate polyacrylamide gel electrophoresis (PAGE). The laccase was immobilized by entrapment in Sol–gel matrix of trimethoxysilane (T) and proplytetramethoxysilane (P) prepared using different T:P molar ratios. The free and immobilized laccases were compared to investigate the effect of immobilization on catalytic efficiency and thermo-stability features. Laccase immobilized in the Sol–gel of 1:5 T:P ratio was optimally active and thermo-stable fraction at pH 5, 60°C with half-life of 3 h and 50 min. Laccases immobilized in 1:2 and 1:5 T:P ratio gels had significantly higher Km (83 and100mM) and Vmax (1000 and 1111 mM/mg) values as compared to free laccase. After 5 h reaction time varying decolorization percentages with a maximum of 100% were achieved for different dyes and effluents.
In summary, P. ostreatus IBL-02 laccase was immobilized by entrapping in a Sol–gel matrix with an objective to enhance its catalytic and stability properties. Sol–gel entrapped laccase presented potential efficiency as a biocatalyst when applied for decolorization of different dyes and effluents. The main benefits of the Sol–gel matrix immobilization processes are the eco-friendly approach, chemical free and energy saving reaction conditions.
Laccase (EC 18.104.22.168) is a blue copper oxidase secreted by white rot fungi (WRF) not only an important component of the ligninolytic enzyme system responsible for lignin degradation, but can even degrade non-aromatic compounds in the presence of low molecular weight redox mediator compounds . As single enzyme and/or in combination with other ligninolytic, cellulolytic and xylanolytic enzymes, laccases have important applications in bio-ethanol production, bio-pulping in the paper and pulp industry, denim stone washing, wastewater treatment, oxidation of organic pollutants, extraction and stabilization of fruit juices, biosensor development, textile bio-finishing, beverage processing, decreasing dough extensibility in flour, animal feed, cosmetics, clinical diagnosis enzyme immunoassays, and detergent manufacturing [2–8]. However, their high production cost, low operational stabilities, availability in small amounts, susceptibility to attack by proteases and activity inhibition limit their commercial applications in industrial and environmental biotechnology .
Over the last few decades, intensive research in the area of enzyme technology has provided many approaches that facilitate their practical applications. The various techniques for enhancing operational stability of laccases are enzyme engineering, chemical modification, mutation and immobilization . Among them, the newer technological developments in the field of immobilized biocatalysts can offer the possibility of a wider and more economical exploitation of biocatalysts in industry, waste treatment, medicine, and in the development of bioprocess monitoring devices like biosensors . The method of immobilization is the most important because equipped steadiness and reusability of enzyme depend on it. Physical entrapment and surface binding are the two most commonly used methods. Entrapment is preferred over surface binding as this method is easier and cheaper, stable derivatives are formed and the structure of the enzyme remains secure . A second important component of immobilization on which performance of the enzyme depends, is enzyme support. Two types of commonly used supports are hydrophobic and hydrophilic biomaterials. Hydrophobic biomaterials are preferred because these have the ability to entrap large amounts of enzyme with a much higher degree of immobilization and enzyme activity retention. The physical characteristics of Sol-gels have been extensively manipulated for enzyme immobilization and these gels have attracted the attention of biotechnologists. Sol-gels have the ability to produce enzymes in stable defined thin films that are more vigorous having ability to catalyze reactions under wide environmental conditions [13, 14].
Dye containing textile waste effluents contain several types of hazardous chemicals including synthetic dyes . Most of the textile industries discharged their routine waste effluents into the main water streams without or after some partial chemical / physical treatments. An eco-friendly treatment of industrial effluents is still a major environmental concern for modern world . In spite of the existing physical/chemical technologies that are usually expensive and commercially or environmentally unattractive, biological processes seem as potential alternatives because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents. WRF have the ability to degrade contaminants by virtue of its extracellular ligninolytic enzymes including lignin peroxidases (LiPs), manganese peroxidases (MnPs) and laccases [6, 13–16]. Therefore, over the past several years, there has been great interest among researchers in the production of ligninolytic enzymes using various agro-based waste materials .
This manuscript describes the results of a study aimed at immobilizing a laccase, produced by the indigenous strain of P. ostreatus IBL-02 during decolorization of Drimarene brilliant red K-4BL  in Sol-gels matrix of varying hydrophobicities. The investigation also involved the comparison of kinetic, catalytic and thermo-stability properties of immobilized and free laccases and, their abilities to decolorize different textile dyes and industrial effluents.
Results and discussion
Source of laccase
The laccase produced by P. ostreatus IBL-02 during decolorization of Drimarene brilliant red K-4BL under optimum conditions  was used for purification, immobilization and characterization studies. Under optimum conditions, P. ostreatus IBL-02 produced 321 U/mL of laccase during complete decolorization (100%) of Drimarene Brilliant Red K- 4BL in 24 h. The optimum conditions were: glucose (as carbon supplement), 2 g/ 100mL; ammonium nitrate (nitrogen additive), 0.06 g/100 mL; Cu2+ (1mM), 1mL as metal activator; ABTS (10mM), 2mL as mediator, pH 5 and temperature, 35°C. The culture supernatant was used as crude enzyme extract for purification and immobilization purposes.
Purification of laccase
Purification summary for laccase produced by P . ostreatus IBL-02 during decolorization of DBR K- 4BL
Total volume (mL)
Total enzyme activity (U)
Total protein content (mg)
Specific activity (U/mg)
A variety of purification techniques including, ammonium sulfate precipitation, gel filtration and ion exchange chromatography are required to purify laccase from other pigments and contaminating proteins. In line with our findings, Chen et al.  also reported 80% as the saturation point of ammonium sulfate for laccase isolation from P. ostreatus. Mansur et al.  reported that ammonium sulfate precipitation provided 57% yield with a 5~folds purification of laccase.
Native and SDS-PAGE
[Lane 1, Molecular weights in kDa of standard marker; (β-Galactosidase, 116 kDa; Phosphorylase B, 97 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa and trypsin inhibitor, 21 kDa); lane 2, Crude enzyme extract; lane 3 and 4, Purified Laccase in SDS-PAGE; lane 2 and 3 in native PAGE, Purified Laccase (67 kDa)].
Immobilization of laccase
Activities of P . ostreatus IBL-02 laccase immobilized in sol-gels of different hydrophobicities
Gel precursors TMOS:PTMS (molar ratio)
Specific activity (U/mg)
Degree of immobilization
Specific activity corrected* (U/mg)
Entrapment of laccase in Sol-gels involves adsorption phenomenon that has been reported as the best method for immobilization of laccase . Previously, we  reported that entrapment of lignin peroxidase (LiP) from P. chrysosporium in Sol-gels caused hyper-activation but an increase in hydrophobic character above certain optimum limits caused a decrease in LiP activity. As the concentration of silane increases, the degree of immobilization also increases but the activity of enzyme decreases . However, the covalent binding strategy is much more expensive because it requires glutareldehyde as coupling agent.
Characterization of free and immobilized laccase
Effect of pH on free and immobilized laccase
Effect of temperature on free and immobilized laccase
To make an enzyme industrially applicable, temperature is one of the most important parameters to determine the thermo-stability of the enzyme. Although it is the effect of temperature that determines which proteins lose their three dimensional structure, its effect became negligible in case of laccase due to hydrophobic interaction of laccase with the gel material that may stabilize the three dimensional structure of the enzyme. Immobilization enhances thermo-stability because maintenance of the three dimensional structures of protein competes with denaturation and loss of catalytic activities of the proteins . Arica et al.  reported that immobilized laccase lost its activity at a mud slower rate than free enzyme by the rise in temperature. In line with our findings, immobilized laccase has been reported to withstand a wider range of temperatures (50~80°C) than free laccase . Immobilization probably prevents unfolding of laccase that results in a longer half-life as compared to free laccase .
Effect of substrate concentration: Determination of Km and Vmax
Although immobilized laccases have higher Km values than soluble counterparts, the immobilized laccase have been proved to have excellent reusability, thermal constancy and equipped permanence due to high Vmax. The Km value indicates the interaction of enzyme with its substrate. Rekuc et al.  immobilized laccase in cellular foams and found that Km for the soluble and entrapped laccase were 39.4, 133.4 and the Kcat for free and immobilized laccase was 86 and 117. Wang et al.  reported that laccase, after immobilizing in silica nano-particles, showed 3.28 mM and 155.4 min-1, Km and Kcat respectively.
Decolorization of Textile dyes and effluents
Laccases have been considered as the major dye degrading enzymes and are efficient decolorizers of dyes present in industrial wastewaters . The ability of laccases to decolorize dye containing effluents is correlated to its ability to degrade different dyes present in the effluent. Immobilization modifies the activity, selectivity, and equipped permanence of enzymes. Immobilized laccases are more robust dye degraders as compared to their free counterparts . The variation in effluent composition is responsible for the difficulty of its decolorization by enzyme extracts from different fungi . The present sol–gel matrix-entrapped laccase seems to have potential capabilities to meet the challenges of modern industrial sector, especially for bioremediation in the textile industry.
Laccase isolated from an indigenous fungal strain P. ostreatus IBL-02 was immobilized by Sol–gel entrapment with an objective to improve its catalytic and thermo-stability and reusability. Hydrophobic gel entrapment resulted in increased half-life, Vmax and Km values for the enzyme that may be desirable characteristics for its industrial applications. Moreover, the Sol–gel entrapped laccase presented potential efficiency as a biocatalyst for the decolorization of different dyes and local textile wastewaters. The main benefits of the Sol–gel matrix immobilization processes are the eco-friendly approach, chemical free and energy saving reaction conditions. In view of the long term trend of striving for more environmental friendly industrial processes, the health concerns regarding harmful chemicals, the versatility, non-toxicity and mild reaction conditions, the Sol–gel immobilization technology is likely to remain the subject of intensive research investigations in different sectors of industrial and environmental biotechnology.
Trimethoxysilane, proplytetramethoxysilane, polyvinyl alcohol, Sephadex G-100, 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Coomassie Brilliant Blue G-250, sodium dodecylsulphate (SDS), trizma base and standard Protein markers were purchased from Sigma-Fluka-Aldrich (USA). All other chemicals were of analytical grade and were mainly purchased from Merck (Germany) and Scharlau (Spain). For decolorization studies, four different dyes and textile industry effluents were collected onsite from local textile industries in Faisalabad, Pakistan.
Source of laccase
In a previous study, an indigenous novel strain P. ostreatus IBL-02 was found to produce substantial amount of laccase as a major enzyme during decolorization of the reactive textile dye Drimarene Brilliant Red K- 4BL. For maximum laccase production and dye decolorization, the physical and nutritional growth conditions have also been optimized . Laccase was therefore produced under pre-optimized conditions for purification and immobilization studied.
Laccase activity assay
Laccase activities in the collected samples were determined at room temperature by the UV/Vis spectrophotometric assay . The activity of laccase was determined by monitoring the ABTS oxidation in a reaction mixture containing 1ml of 1mM ABTS and 1ml of 50mM malonate buffer (pH 4.5) and 100μL of culture supernatant. The reaction mixture was incubated at 25°C and absorbance of each sample was taken at 420nm after 10 min. Blanks contained 100μL of distilled water instead of enzyme solution or culture supernatant. Laccase activity was expressed as international units (IU) and defined as the amount of enzyme forming 1 μmol of ABTS·+ per min under the assay conditions.
Proteins were estimated using the Bradford micro assay  using bovine serum albumin (BSA) as standard. To 1mL of Bradford reagent, 100μL of each solution were added and mixed on a vortex mixer. The reagent blank was run by adding 100μL of distilled water to 1mL of the Bradford reagent. The change in absorbance (ΔA) at 595nm for all samples was determined within 15–30 min.
Purification of laccase
The culture filtrate was first filtered and centrifuged at 3000 × g and supernatant was then subjected to ammonium sulfate precipitation. The precipitate obtained was dialyzed and lyophilized and then loaded onto a DEAE-Sepharose anion-exchange column 1.5 × 18 cm, equilibrated with 10 mM sodium acetate buffer (pH 4.5), with a linearly increasing NaCl concentration gradient (0 to 0.5 μM) in the same buffer. The six fractions containing laccase activity were pooled, concentrated, and dialyzed overnight against same buffer. Gel filtration chromatography was performed using sephadex G-100 column 2.0 × 40 cm. The DEAE-purified sample was loaded on to the column and 3 mL fractions were collected. The eluted active fractions were dialyzed and protein content was determined by Bradford method.
Native and SDS-PAGE
The purified and lyophilized sample was dissolved in a minimum amount of 50mM malonate buffer, and subjected to 12% native PAGE and 10% SDS-PAGE using a Vertical Minigel electrophoresis apparatus (V-GES, Wealtec Corporation, U.S.A) to determine sample purity and approximate mass of laccase . The approximate molecular mass of the laccase was determined after gel staining with Coomassie Brilliant Blue G followed by the calibration against broad-range molecular weight markers (Sigma, USA), which contained proteins ranging from 21–116 kDa.
Preparation of hydrophobic gels and immobilization of laccase
To prepare the Sol–gel thin films for enzyme entrapment purposes, TMOS and PTMS were used in different molar TMOS: PTMS (T: P) ratios by adopting the methodology as described earlier by Asgher et al. . TMOS and PTMS were used in molar T: P ratios of 1:1, 1:5, 1:10, 1:15, 1:20 and 1:25 to prepare gels of different hydrophobicity, in ascending order. Laccase isolated from P. ostreatus IBL-02 was suspended in water (12.5 mg/mL), shaken for 5 min and centrifuged. The supernatant fluid (400 μL) was added to a mixture of polyvinyl alcohol and water. The solution was shaken and PTMS was added, followed by TMOS. The reaction mixture was vigorously shaken for 5 sec on a vortex mixer and then gently shaken by hand. After about 30 sec, when the mixture formed a clear homogenous solution, it was placed in an ice bath until gel formation occurred. Laccase activity and protein contents of entrapped enzymes (in gels of different hydrophobicity) were determined. The entrapped enzymes having highest specific enzyme activity and protein contents were selected for further characterization.
Characterization/Comparison of free and immobilized laccases
Purified native and Sol–gel entrapped laccases were characterized to determine and compare their pH and temperature optima, and kinetic constants such as Vmax (maximum rate), Km (Michaelis constant) and Kcat (catalytic efficiency).
Effect of immobilization on optimum pH
The activities of purified native and entrapped laccases were studied over a pH range of 2.0-10.0 using ABTS as substrate. The buffers (0.1M) used were: pH 2–2.8, tartaric acid/sodium tartrate; pH 3–3.6, glycine/HCl); pH 3.8-4.5, glutamic acid/HCl; pH4.6-6.0 sodium acetate/acetic acid; pH 6–7, sodium phosphate; pH 7.5-8, Tris–HCl; and pH 9–10, glycine-NaOH buffer.
Effect of immobilization on optimum temperature and thermo-stability
The temperature activity profile of free and entrapped laccase was determined at different temperatures ranging from 25-75°C for 30 min. For determination of half-lives the enzymes were incubated at 60°C for varying time periods before carrying out standard laccase assay.
Effect of immobilization on kinetic constants Km, Vmax and Kcat
The Michaelis-Menten kinetic constants including Km, Vmaxand Kcat were determined by using varying concentrations of ABTS ranging from 0.1-1 mM. Laccase activity was determined for each concentration of ABTS keeping enzyme concentration constant. Lineweaver-Burk plots were constructed between reciprocals of the initial reaction rates (1/Vo) and varying substrate concentrations [1/S].
Applications of free and immobilized laccase
Decolorization of dyes
Free and immobilized laccases were used for the decolorization of four reactive textile dyes (Drimarine Blue K2RL; Drimarine Orange KGL; Drimarine Brilliant Red K4BL and Remazol Brilliant Yellow 3GL). The working conditions of a single continuous operation were: two parallel batch of triplicate flasks containing 10 mL of free and 5 g of sol–gel-entrapped biocatalyst (laccase) was transferred to 100 mL of 0.01 % individual dye solutions with 1mL of 1mM ABTS as a laccase mediator followed by the incubation at 25°C for 5 h in rotary shaker (120 rpm). The culture supernatants recovered after filtration and centrifugation of the enzyme treated samples collected after every each hour were subjected to the residual dyestuff analysis. Absorbance measurements were done by a UV-Visible spectrophotometer (T-60, PG instruments, UK). The absorbance values for respective supernatants at each time period were corrected by subtracting the values for respective control fraction (containing only the reaction medium without enzyme). Decolorization of dye solution was determined by a reduction in optical density at the wavelength of maximum absorbance at λmax (590 nm) by UV–vis spectrophotometric spectrum.
Decolorization of real textile industry effluents
Different dye containing practical textile industry effluents of different colors were collected from Sitara textile (SIT), Nishat textile (NIT), K&N textile (KNT) and Crescent textile (CRT) units of Faisalabad. The effluent source industries did not disclose the names and structures of dyes being used due to their business secrets. The working conditions of a single continuous operation were: two parallel batches of triplicate flasks containing 10 mL of free and 5 g of sol–gel-entrapped laccase, respectively, 100 mL of individual dye solutions/textile effluents with 1 mL of 1 mM ABTS as laccase mediator, and incubated at 30°C in shaking incubator (120 rpm) for 5 h reaction time. The samples collected at one h intervals from each flask were used to determine the percentage color removal of textile dyes and effluents by considering the initial and final effluent absorbance. All the collected samples were centrifuged at 5,000×g for 15 min at room temperature (25°C) and clear supernatants were analyzed spectrophotometrically. Decolorization of individual effluents was determined by a reduction in optical density at the wavelength of maximum absorbance (λmax).
The present study was a part of the research project funded by Higher Education Commission (HEC), Islamabad, Pakistan under National Research Program for Universities (NRPU). The timely provision of Funds by HEC is highly acknowledged.
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