Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum
© Iqbal and Asgher; licensee BioMed Central Ltd. 2013
Received: 16 December 2012
Accepted: 12 July 2013
Published: 13 July 2013
An eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents.
A large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the permissible limits.
In conclusion, G. lucidum MnP was immobilized by sol–gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.
To date phenolic/non-phenolic compounds and some toxic environmental pollutants particularly textile waste water effluents posing serious health hazards to the entire living ecosystem and especially on the animals and humans. Over the past several years, there has been great interest among researchers in the production of ligninolytic- and cellulose-degrading enzymes from various agro-industrial waste materials and their by-products, such as wheat straw, rice husk, banana waste, citrus peel, rice straw, corncobs, corn stover, apple pomace, and sugar cane bagasse [1–8]. These wastes are not properly disposed off in developing countries and have become a major source of ecological pollution. Significant efforts have been made to convert lignocellulosic residues to valuable products such as bio-fuels, chemicals and animal feed with the help of ligninolytic enzymes of WRF, many of which have been successful [2, 4–8]. To degrade all the components of lignocellulosic materials and phenolic organic substrates, white rot fungi (WRF) are so far exclusive in their strong oxidative capabilities due to their extracellular nonspecific enzyme system .
WRF produce two major families of enzymes, generally termed ligninolytic enzymes, i.e., extracellular peroxidases (manganese peroxidase, MnP; manganese independent peroxidase, MIP; lignin peroxidase, LiP; and versatile peroxidase, VP) and phenol oxidases (laccases). MnP and laccases have been applied frequently in various bio-technological processes such as lignocellulosic biomass delignification for ethanol production, oxidation of pollutants, bioremediation processes, textile bio-finishing, beverage processing, bio-bleaching of pulps and detergent manufacturing [1–3, 6–8]. MnPs are heme-containing glycoproteins having capability to catalyze the oxidation of Mn2+ to Mn3+. The Mn3+−organic acid complexes, in turn, oxidize phenolic structures present in lignin and also various lignin related organic compounds . In nature, MnP catalyzes plant lignin de-polymerization as component of ligninolytic enzymes complex. A wide range of substrate oxidizing capability renders it an interesting enzyme for biotechnological applications in several industries.
Textile waste effluents contain several types of chemicals including real dyes itself that are toxic, carcinogenic or mutagenic. Textile industries discharged their waste effluents into water streams with or without some partial treatments which mainly causes water pollution and also dangerous for aquatic life. In literature, various physical/chemical methods have been reported by several authors to effectively process the textile effluents such as adsorption, precipitation, chemical reduction, ionizing radiations and ultra filtration [4, 5, 10]. In spite of the existing physical/chemical technologies for colour removal which are usually expensive and commercially/environmentally unattractive, biological processes provide an alternative to existing physico−chemical technologies because they are cost effective, eco−friendly and can be applied to wide range of dye containing industrial effluents [2, 4, 5].
Enzyme immobilization offers a noteworthy solution to industrial and environmental challenges faced by the modern world predominantly in the applications of enzymes in agro-waste management, textile effluents decolorization, and alternatives of health hazardous chemical based procedures. To date, various techniques have been reported for enhancing activity and operational stability of industrial enzymes. Gel entrapment is preferred over other immobilization techniques as this method is convenient and the structure of the enzyme remains secure. Recently, sol−gels have attracted the attention of biotechnologists due to their ability to produce enzymes in defined thin films that are thermo-stable and non-toxic in nature [2, 4]. Among the various potent lignin-degrading microorganisms, G. lucidum is a potential MnP producer with high catalytic potentials that are suitable for a wide range of environmental/biotechnological applications. Therefore, in the current study MnP from G. lucidum was immobilized using sol–gel matrix entrapment technique. The decolorization of different textile effluents by the sol–gel matrix entrapped MnP was also the main focus of the present study to present its approach for bioremediation applications.
Results and discussion
Production, purification and immobilization of MnP
Purification summary for MnP produced by G . lucidum
Total volume (mL)
Total enzyme activity (U)
Total protein content (mg)
Specific activity (U/mg)
Reusability and shelf life
Decolorization of textile effluents by immobilized MnP
Nitro-amines and formaldehyde
Traces of nitro-amines were found in the maximally decolorized treated sample, indicating that nitro-amines were produced as intermediates during effluent decolorization. The formaldehyde content was 12.2 mg/L for untreated effluent and it reduced to 3.6 mg/L after treating with the enzyme. Significantly lower quantities of nitro-amines and formaldehyde in the treated effluents suggest their formation as intermediates in the mechanism of degradation of dyes and other chemical compounds present in the effluent that were ultimately completely degraded by immobilized MnP. The ligninolytic enzymes enhance the aerobic degradation/ mineralization of dyes and pollutants that does not result in the formation of aromatic ammines . The results of the toxicity studies showed that the effluents were not only decolorized but also detoxified by the action of immobilized MnP.
G. lucidum MnP was immobilized by entrapping in a sol–gel matrix with an objective to enhance its functionalities. The MnP was successfully entrapped into a sol–gel matrix of TMOS and PTMS with maximum of 93.7% immobilization efficiency. The sol–gel entrapped MnP presented potential efficiency as bio-catalyst for decolorization of dye containing textile effluents and it was eco-friendly, chemical free and energy saving approach for bio-remediation on textile industry effluents.
Fungal culture, chemicals, and agro-waste substrate
A pure white rot fungal strain G. lucidum obtained from fungal culture collection of Industrial Biotechnology Laboratory, Department of Chemistry & Biochemistry, University of Agriculture Faisalabad, Pakistan, was used for production of MnP in SSF. Sephadex-G- 100, Polyvinyl alcohol, TMOS, and PTMS were from Sigma-Aldrich (USA), while all other chemicals were of analytical grade and used as such. The lignocellulosic waste wheat straw was collected from the Student Research Farms, University of Agriculture Faisalabad, Pakistan. To avoid moisture, the substrate was oven dried (60°C), ground to fine particle size (40 mm mesh size) using Wiley Mill (electric grinder), and stored in airtight plastic jars. For decolorization studies, four textile effluents were collected from Magna, Crescent, Arzoo, and Chenab textile industries of Faisalabad, Pakistan.
Fungal spore suspension
To develop homogeneous fungal spore suspension (107-108 spores/mL), G. lucidum was cultivated at 30±1°C for 5 days in an Erlenmeyer flask containing a basal salt medium. The constituents of the medium were: (NH4)2SO4, 10 g/L; KH2PO4, 4 g/L; MgSO4·7H2O, 0.5 g/L and CaCl2, 0.5 g/L. Before fungal inoculation, the medium was sterilized at 121°C and 1.035 bar pressure in a laboratory scale autoclave (Sanyo, MLS-3020U, Japan) for 15 min.
MnP production and extraction protocol
MnP production by G. lucidum was carried out in 500 mL capacity Erlenmeyer flasks using wheat straw under optimized growth conditions. The optimized conditions were: moisture, 50%; substrate, 5g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5mL; fermentation time, 4 days. Triplicate flasks were autoclaved (120°C) and inoculated with 5 mL freshly prepared fungal inoculum. The inoculated flasks were kept at 30°C in a still culture incubator (EYLA SLI-600ND, Japan) for 4 days. MnP was extracted by adding 100 mL of distilled water to the 4 day old fermented solid-state cultures, followed by shaking at 120 rpm for 30 min. The contents were filtered through Whatman No.1 filter paper and the filtrates were centrifuged at 3,000 g for 10 min. The supernatants were carefully collected and used as crude MnP extracts for MnP assay, purification, and sol–gel immobilization.
MnP activity and protein contents determination
UV/Visible spectrophotometric method was used to determine the activity of MnP as described earlier [3, 17]. Assay reaction mixture contained 1 mL of 1 mM MnSO4 as a substrate, 1 mL of 0.5 mM sodium malonate buffer of pH 4.5, 0.5 mL of 0.1M H2O2 and 0.1 mL of enzyme solution. Blanks contained the same mixture solution without enzyme. Absorbance of each sample was taken after 10 min interval at 270 nm using UV-Vis spectrophotometer (T60, PG Instruments UK). Unit activity was defined as the amount of enzyme required to produce a unit increase in absorbance at specific wavelength (nm) per mL of reaction mixture. Bradford micro assay was used with bovine serum albumin as standard to determine the protein contents of each sample .
Purification and PAGE
Crude MnP extract obtained from solid state culture of G. lucidum was centrifuged (3,000 g) for 15 min at 4°C to attain maximum clarity, and the supernatant was concentrated by freeze drying. The crude enzyme concentrate was placed in ice bath and crystals of ammonium sulfate were added to attain 60% saturation followed by the centrifugation at 5,000 g for 30 min at 4°C. The pellets were dissolved in 50 mM malonate buffer and dialyzed against the same buffer at room temperature with 4 equal changes of buffer after every 6 h to remove extra salt. Total proteins and MnP activity were determined before and after dialysis as described previously. The dialyzed active fractions were loaded on Sephadex G-100 column (2×25 cm) for further purification. Up to 20 active fractions each of 1mL were collected with flow rate of 0.5 mL/min and monitored for MnP activity as described before. Native and SDS poly acrylamide gel electrophoresis was performed according to the method of Laemmli , using Mini-gel electrophoresis apparatus (V-GES, Wealtec Corp. USA). The molecular mass of the purified MnP was estimated in comparison to standard molecular weight markers (standard protein markers, 21-116kDa; Sigma, USA). The protein bands were visualized by staining with Coomassie Brilliant Blue G-250 (Sigma, USA).
To prepare the sol-gel thin films for enzyme entrapment purposes, TMOS and PTMS were used in molar TMOS: PTMS (T: P) ratios of 1:2 ratio by adopting the methodology as described earlier by Asgher and Iqbal . A purified active MnP fraction (2 mg/mL) was suspended in de-ionized water and centrifuged (4,000 g) for 15 min at 4°C. The separated supernatant fluid was added to an equal ratio mixture of aqueous sodium fluoride, polyvinyl alcohol, and de-ionized water. The solution was shaken and PTMS was added, followed by the addition of TMOS. The reaction mixture was gently mixed for 20 sec in a vortex mixer and placed in an ice bath until gelation occurred. The entrapped enzyme fraction was subjected to the spectrophotometric analysis to determine its activity and selected for further decolorization study. Immobilization efficiency was calculated as the ratio of the enzyme entrapped (difference between the enzyme loaded and the enzyme in the supernatant after washing × 100).
Reusability and shelf life
The reusability of the immobilized MnP was investigated in a batch experiment using MnSO4 as a substrate. The gel entrapped MnP was incubated with 1 mM MnSO4 in 50 mM sodium malonate buffer at room temperature (25°C). At the end of each cycle, sol–gel entrapped biocatalyst washed three times with the same buffer before treating the fresh substrate solution. The remaining activity was calculated relative to the initial MnP activity after each cycle. For the commercial utilization of industrial enzymes shelf life is an important consideration. To investigate the storage stabilities, free and immobilized MnP were incubated at room temperature (25°C) for up to 75 periodic days. After every 15 days the residual activities were measured using standard assay protocol as described earlier.
Decolorization of textile effluents by immobilized MnP
To investigate the decolorization applicability of sol-gel-entrapped MnP, four different textile industry wastewater effluents were collected from Magna, Crescent, Arzoo, and Chenab textile industries of Faisalabad. The working conditions of a single continuous operation were: triplicate flasks containing 5 g of sol-gel-entrapped bio-catalyst (MnP), 100 mL of each textile effluent with 1 mL of 1 mM MnSO4 as MnP mediator, and incubated in a temperature-controlled shaker (120 rpm) for 4 h reaction time. At the end of each h, samples were collected from each flask to determine the percentage enzymatic color removal of textile effluent by considering the initial and final absorbance of treated and untreated effluents. 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 effluents was determined by a reduction in optical density at the wavelength of maximum absorbance of each effluent by UV-Vis spectrophotometer.
Formaldehyde and nitro amines
A spectrophotometric method was used to determine the formaldehyde in treated effluents. The method was based on formaldehyde reaction with chromo-tropic acid in the presence of magnesium sulfate producing a stable complex Mg2+/ cyclotetrachromotropylene. Beer’s Law is obeyed in a concentration range of 3 to 11 mg/L of formaldehyde with a correlation coefficient of 0.999. The color complex formed was analyzed by UV/Vis spectrophotometer at 535 nm (λmax). Treated and untreated wastewater sample was examined through high performance liquid chromatography (HPLC) method, using meta-nitro phenol as internal standard (IS) with a variable-wavelength UV detector.
All the experimental data was statistically analyzed using the statistical software Minitab, version 15. The means and standard errors of means (mean ± S.E.) were computed for each treatment and S.E. values have been displayed as Y-error bars in figures.
The present study was a part of the research project focused on the development of ligninolytic enzymes for industrial applications. The authors are grateful to the Higher Education Commission, Islamabad, Pakistan for providing funds for the project studies.
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