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Abstract
Laccase is one such copper protein belonging to the oxidoreductase family that oxidizes a large array of organic substrates i.e phenolic and non-phenolic compounds. An attempt was made to screen Pleurotus ostreatus-P1 by plate test on Kirk medium for laccase production by using guaiacol and syringaldazine as indicators and six different liquid culture media having varied composition were work out for laccase production. Hyper Laccase production (5.5±0.33unit/ml) objective was achieved in medium 09-CBZ6 in combination with promising protein production (3mg/ml) under optimized fermentation conditions (pH: 5.5; Temperature: 30°C; Time: 7th day). Laccase production by a long way intensified by the adding of glucose (1.5%, C/N:15) and inducer (CuSO4) gave promising results (13.72±0.30U/ml) and other inducers followed laccase production as Na2SO4> ZnSO4> FeSO4> CaCl2. Sodium Azide was found a significant inhibitor (100%) for laccase activity than SDS (87%) and EDTA (83%). Study brings to a close potential of Pleurotus ostreatus-P1 for laccase production under optimized conditions for industrial applications.
... Vantamuri and Kaliwal (2015) isolated nine fungal colonies of which two fungal cultures showed higher laccase activity. Pycnoporus cinnobarinum strain SYBC-L14 (Khanam et al., 2012), Ganoderma lucidum (Sasidhara and Thirunalasundari, 2014), Pleurotus ostreatus (Nadeem et al., 2014) and Coprinus Discussion comatus (Nasreen et al., 2015) showed maximum laccase activity with 0.02% guaiacol as a substrate. Sindhu et al. (2014) Many researchers have used different types of fermentation process for enzyme production. ...
... Metal ions affect the laccase activity; they enhanced the activity of laccase enzymes. In the current study ferric chloride enhanced the laccase activity produced (Ding et al., 2014) and Pleurotus ostreatus P1 (Nadeem et al., 2014) and Lentinus squarrosulus MR13 (Mukhopadhyay and Banerjee, 2015). ...
... Pleurotus ostreatus P1 (Nadeem et al., 2014) and dithiothreitol inhibited laccase activity from Lentinus squarrosulus MR13 (Mukhopadhyay and Banerjee, 2015). ...
Enzymes are bio-catalysts and play an important role in biochemical and
metabolic reactions. Microbial enzymes are obtained from a variety of microorganisms.
Microbial enzymes have been deliberated on their isolation, screening, characterization
(enzymatic properties), their production from bench scale to large scale (pilot scale)
and their application in industries (bio-industries). Bio-industries regularly focus on R
& D and update their progress on the efficient and effective productions. Many
industries are already using microbial sources for enzyme production for various
commercial processes. Bacteria, fungi and yeast are favoured microorganisms for the
production of enzymes. Microbial enzymes have special characteristics according to
their capability under abnormal conditions (temperature and pH). On the basis of
temperature, microbial enzymes are divided into thermophilic, acidophilic or
alkalophilic. Microbes perform highest enzyme production in thermostable conditions
compared to normal temperature reactions. Thermostable conditions can probably
decrease the microbial contaminations in the large-scale industrial enzyme production.
Thermostability of the enzyme encourages the digestion and breakdown of raw
materials and higher temperature reaction increases the penetration capability of
microbial enzymes.
... Nearly similar results were found by Kumar et al. (2011) who reported that maximal laccase activity in P. ostreatus was on the 9th day (570 U/L). Nadeem et al. (2014) reported that, there was a sharp increase in laccase production by P. ostreatus from 2 to 6th day and after 10th day its activity decreased slightly. Laccase produced in late phase of P. ostreatus that is on 6th and 8th day, reached its maximum value with activity of 5.53 ± 0.11 and 5.68 ± 0.08 U/ml, respectively and after that decreased subsequently. ...
... The results of the present study indicate that an incubation temperature 25, 28 and 30°C was good for laccase production with 28°C being optimal with activity of 0.142 U/ml (Figure 4). But at 35 and 20°C, the fungus growth decreased and consequently laccase's units dropped, because high temperature causes cell membrane composition alteration, stimulation of protein catabolism, and lower temperature results in suppression of nutrient transportation which was in line with the report given by Nadeem et al. (2014). At 35°C, laccase's activity dropped to 0.062 U/ml which is nearly 47% of its maximum activity at 28°C. ...
... The results obtained in the present study are in agreement with Nadeem et al. (2014) who reported that most of the fungal cultures preferred a slightly acidic pH of medium for growth and enzyme production. Prasad et al. (2005), Adejoye and Fasidi (2009) and Sivakami et al. (2012) also showed that in Schizophyllum commune and P. ostreatus the optimal pH for fungi mycelia biomass yield and laccase activities was 5.5. ...
... Nearly similar results were found by Kumar et al. (2011) who reported that maximal laccase activity in P. ostreatus was on the 9th day (570 U/L). Nadeem et al. (2014) reported that, there was a sharp increase in laccase production by P. ostreatus from 2 to 6th day and after 10th day its activity decreased slightly. Laccase produced in late phase of P. ostreatus that is on 6th and 8th day, reached its maximum value with activity of 5.53 ± 0.11 and 5.68 ± 0.08 U/ml, respectively and after that decreased subsequently. ...
... The results of the present study indicate that an incubation temperature 25, 28 and 30°C was good for laccase production with 28°C being optimal with activity of 0.142 U/ml (Figure 4). But at 35 and 20°C, the fungus growth decreased and consequently laccase's units dropped, because high temperature causes cell membrane composition alteration, stimulation of protein catabolism, and lower temperature results in suppression of nutrient transportation which was in line with the report given by Nadeem et al. (2014). At 35°C, laccase's activity dropped to 0.062 U/ml which is nearly 47% of its maximum activity at 28°C. ...
... The results obtained in the present study are in agreement with Nadeem et al. (2014) who reported that most of the fungal cultures preferred a slightly acidic pH of medium for growth and enzyme production. Prasad et al. (2005), Adejoye and Fasidi (2009) and Sivakami et al. (2012) also showed that in Schizophyllum commune and P. ostreatus the optimal pH for fungi mycelia biomass yield and laccase activities was 5.5. ...
The aim of the current study was to investigate the potential of Pleurotus species for laccase production on different lignocellulosic substrates and determine the optimal levels of physicochemical conditions required for the production. The study was conducted in National Agricultural Biotechnological Research Center, Holetta. The fruiting bodies of fungi were collected based on their morphology and inoculated on potato dextrose agar plate. Six different lignocellulosic substrates were collected and prepared for cultivation of Pleurotus species for laccase production. The highest enzyme production was obtained from bean straw compared to other substrates with an activity of 0.112 U/ml. The best three substrates; bean straw, Eucalyptus sawdust and wheat straw were selected, and a mixture of each of them on equal proportions was tested for laccase production potential. A mixture of Eucalyptus sawdust and bean straw on equal proportion was found to be the best, showing an activity of 0.137 U/ml and hence selected for different parameter optimization. Optimal laccase production (0.292 U/ml) was obtained on the 10th day of incubation period and the optimum temperature and pH were 28°C and 5.5, respectively. Soluble starch and peptone were found to be the most preferred carbon and nitrogen sources for laccase production, respectively. Asparagine and alanine induced more laccase production, with asparagine being the most potent inducer.
Key words: Pleurotus species, lignocellulosic substrates, bean straw, Eucalyptus sawdust, laccase.
... Nearly similar results were found by Kumar et al. (2011) who reported that maximal laccase activity in P. ostreatus was on the 9th day (570 U/L). Nadeem et al. (2014) reported that, there was a sharp increase in laccase production by P. ostreatus from 2 to 6th day and after 10th day its activity decreased slightly. Laccase produced in late phase of P. ostreatus that is on 6th and 8th day, reached its maximum value with activity of 5.53 ± 0.11 and 5.68 ± 0.08 U/ml, respectively and after that decreased subsequently. ...
... The results of the present study indicate that an incubation temperature 25, 28 and 30°C was good for laccase production with 28°C being optimal with activity of 0.142 U/ml (Figure 4). But at 35 and 20°C, the fungus growth decreased and consequently laccase's units dropped, because high temperature causes cell membrane composition alteration, stimulation of protein catabolism, and lower temperature results in suppression of nutrient transportation which was in line with the report given by Nadeem et al. (2014). At 35°C, laccase's activity dropped to 0.062 U/ml which is nearly 47% of its maximum activity at 28°C. ...
... The results obtained in the present study are in agreement with Nadeem et al. (2014) who reported that most of the fungal cultures preferred a slightly acidic pH of medium for growth and enzyme production. Prasad et al. (2005), Adejoye and Fasidi (2009) and Sivakami et al. (2012) also showed that in Schizophyllum commune and P. ostreatus the optimal pH for fungi mycelia biomass yield and laccase activities was 5.5. ...
The aim of the current study was to investigate the potential of Pleurotus species for laccase
production on different lignocellulosic substrates and determine the optimal levels of physicochemical
conditions required for the production. The study was conducted in National Agricultural
Biotechnological Research Center, Holetta. The fruiting bodies of fungi were collected based on their
morphology and inoculated on potato dextrose agar plate. Six different lignocellulosic substrates were
collected and prepared for cultivation of Pleurotus species for laccase production. The highest enzyme
production was obtained from bean straw compared to other substrates with an activity of 0.112 U/ ml.
The best three substrates; bean straw, Eucalyptus sawdust and wheat straw were selected, and a
mixture of each of them on equal proportions was tested for laccase production potential. A mixture of
Eucalyptus sawdust and bean straw on equal proportion was found to be the best, showing an activity
of 0.137 U/ml and hence selected for different parameter optimization. Optimal laccase production
(0.292 U/ml) was obtained on the 10th day of incubation period and the optimum temperature and pH
were 28°C and 5.5, respectively. Soluble starch and peptone were found to be the most preferred
carbon and nitrogen sources for laccase production, respectively. Asparagine and alanine induced
more laccase production, with asparagine being the most potent inducer.
Key words: Pleurotus species, lignocellulosic substrates, bean straw, Eucalyptus sawdust, laccase.
... benzendiol: oxygen oxidoreductase) is an interesting enzyme produced by Pleurotus spp., with several potential applications [1], P. djamor is one of the most commonly found mushroom species in the tropical areas of the world with great interest in several industrial applications [2]. P. ostreatus grows in the pH range 5-7 and temperature 25-30 °C [3], and similar ranges have been reported for other species of this genus [4], however, the effect of growth parameters such as pH, temperature and day of enzyme recovery for effective laccase production have not been reported for P. djamor and other Pleurotus spp. ...
... 3D-response surface plots were generated to show the effects of temperature and day of harvest on laccase yield (Fig. 2). Nadeem et al, [4] reported the positive influence of initial pH, temperature and day of harvest of P. ostreatus. In P. djamor, initial low pH had positive effects but temperature and day of harvest had a major influence on all three responses, i.e. biomass, protein and laccase production. ...
... In contrast Shantaveera and Ramalingappa, [10], showed that laccase production was affected positively by both growth period and temperature, 42 days and 45°C, in solid fermentation. The production of laccase by P. ostreatus was maximum at the seventh day after inoculation, 30°C and a pH of 5.5 in induced and not induced cultures [4]. In this work, P. djamor produced laccase after the third day of cultivation, one maximum peak of laccase activity can be detected between the third and fifth day of cultivation and temperature between 25-28°C. ...
A surface response design methodology was used to optimize laccase production by Pleurotus djamor. Among six strains ECS-0184 was selected to apply the mathematical approach. ECS-0184 showed a specific growth rate () of 0.87 days-1 and a laccase yield (YL/X) of 372 UA g-1 biomass. Predictable optimal conditions obtained were pH 5–6, temperature range 25–28 C and 3–5 days of cultivation to obtain best laccase yield, 1175 UA g-1 biomass. Critical factors for laccase production are temperature and day of harvest according with experimental design. The laccase produced by Pleurotus djamor has a molecular weight of 44 kDa; its activity was detected in PAGE gels with ABTS and pyrogallol as substrates.
... ostreatus, P. florida and Calocybe indica) using sterile forceps and filter papers. The plates were incubated at 37 0 c for 6-7 days [6]. ...
... The flasks were then inoculated with a loopful of mycelium of P. ostreatus and P. florida. The flasks were incubated at 37 0 c for 14 days [6]. ...
Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.) is a type of copper containing polyphenol oxidases. It is present primarily in higher plants and filamentous fungi, but also has been found in several species of bacteria. It is widely used in the food and paper industry, synthetic chemistry, bioremediation and biodegradation of phenolic pollutants or in analytical applications. The aim of the present work is to extract and partially purify the extracellular laccase enzyme from mushroom (Pleurotus ostreatus, Pleurotus florida) and synthesize silver nano particle using the isolated laccase enzyme. For this the fungi that believed to have the laccase producing ability were cultured on a standardized medium (PDA). Isolates were then screened for laccase production by growing them on guaiacol incorporated agar medium. The isolates showing maximum activity were evaluated by submerged fermentation for maximum enzyme production. The extracellular laccase enzyme was then extracted and partially purified by ammonium sulphate precipitation and dialysis. The purity of enzyme preparation was assessed by SDS PAGE analysis. The purified enzyme preparation was then used for the synthesis of silver nano particle using silver nitrate and the presence of silver nanoparticle was confirmed by SEM & FTIR analysis. The SEM analysis revealed the shape of the nanoparticle whereas FTIR results yielded the predominant functional groups present in the molecule.
... This is supported by the opinion of Gianfreda et al. (1999) that laccase production is strongly influenced by the concentration of nitrogen in the culture medium and the carbon source used (Galhaup et al., 2003). Furthermore, Nadeem et al. (2014) that carbon and nitrogen sources at the right ratio are needed for propagation and enzyme production. Basidiomycetes fungi including Pleurotus ostreatus have different responses to carbon sources and their concentration in the medium for growth. ...
... Substrate composition that has a balanced C: N ratio can accelerate the growth of Pleurotus ostreatus, because fungi need carbon and nitrogen for its growth. Nadeem et al., (2014) Fermentation time allows the mycelium to grow more optimally and produce enzymes to degrade crude fiber components. This is supported by Musnandar (2004) where the longer the fermentation, the greater the opportunity for enzyme complex to degrade crude fiber components into simple sugars. ...
... In agreement to that, many workers studied extracellular enzyme explaining the highest specific activity of Xylanase (Getachew et al., 2016), laccase in the Pleurotus species (Nadeem et al., 2014) and laccase activity in cauliflower mushroom (Sparassis latifolia) (Sou et al., 2017). ...
Mushroom cultivation is a prevalent activity worldwide, although the domestication of native wild mushrooms is not fully recognised. For wild mushrooms to be economically feasible, they need to possess the ability to be cultivated. The objective of this study was to cultivate 18 wild mushrooms that were collected from their native environments utilising substrates that are readily available in the local area. Wild mushrooms were gathered and acquired using tissue cultures. All the wild mushrooms studied showed mycelial development on the substrates, except for Podaxis pistallris, Amanita solitaria, and Collybia platyphylla. Pleurotus sapidus and Pleurotus floridanus were able to produce fruit satisfactorily. The study revealed that specific wild mushrooms had the ability to produce fruiting bodies when grown on commercial substrates. While mushroom production was not seen in other natural cultures, these findings offer valuable information for improving growth circumstances in the future. Conducting surveys of natural habitats is crucial to guarantee the ongoing production of wild edible mushrooms, safeguarding endangered species and promoting a hopeful outlook for their sustainable utilisation.
... Most fungal laccase reach their maximum activity when the initial pH of the nutrient medium ranges from 4 to 6 22 . The highest activity of laccase was observed at pH between 4.5 and 6.5, in Pleurotus osteatus 23 . Also Sivakumar et al. 24 , reported highest laccase production at pH 5 in case of Ganoderma sp. ...
Different process parameters for degradation of pine needles
... Besides, we have also observed that at temperature 30 °C, purified ligninolytic enzymes (Lac, LiP and MnP) were more stable [48,49]. The results ( Figure S2-S4) revealed that at temperatures above 60 °C, the enzymes lost their activity [50,51]. ...
The textile industries discharge around 10–15% dyes into effluents, posing threat to human health and environment. White-rot fungi are promising candidates for elimination of variety of dyes, owing to their potential for degradation and mineralization of a broad spectrum of highly toxic, recalcitrant, and organic pollutants. In this study, different species of Pleurotus were evaluated for ligninolytic enzymes production using paddy straw, sugarcane bagasse and wheat straw for 7, 14, 21 and 28 days of incubation, respectively. Among substrates, paddy straw exhibited maximum activity of laccase (Lac) (45968.9 ± 347.2 U/g) and manganese peroxidase (MnP) (16312.1 ± 99.9 U/g) from P. ostreatus D-66 after 28 days, however the lignin peroxidase (LiP) (10823.2 ± 94.5 U/g) activity from P. pulmonarius D-79 following 21 days. Purified Lac, MnP and LiP from P. ostreatus D-66 and P. pulmonarius D-79 showed 68.8, 44.1 and 22.8- fold increase with yield of 57.3%, 36.7% and 30.4%, respectively. These enzymes were stable over wide range of pH (3.5 to 7.5) and temperatures (30 to 50 °C) with optimum pH 4.5–5.0 and temperature 30 °C. Among synthetic dyes, the maximum decolorisation (96.9%) was achieved in Remazol brilliant blue R revealed with Lac from P. ostreatus D-66, while in P. pulmonarius D-79 (88.8%) with malachite green and decolorised dye with optimal pH 4.0–6.0 and temperature 30 °C. The findings demonstrated that paddy straw could be an attractive substrate for Pleurotus spp. to produce ligninolytic enzymes that, after purification, can be exploited effectively for degradation of synthetic dyes from textile effluents.
Graphical abstract
... For nitrogen sources the highest level of enzyme production was obtained with ammonium sulphate followed by L-arginine and urea showed lowest enzyme activity. Nadeem et al., (2014) screened Pleurotus ostreatus P1 by plate test on Kirk medium for laccase production by using guaiacol and syringaldazine as indicators and six different liquid culture media and showed hyper laccase production (5.5±0.33unit/ml) objective was achieved in medium 09-CBZ6 in combination with promising protein production (3mg/ml) under optimized fermentation conditions (pH-5.5, temperature-30°C and Time-7th day). ...
... The results are consistent with the majority of prior research that have been conducted to illustrate the influence of temperature on enzyme production. [20] were found that maximal enzyme production was seen at 28°C in Marasmiellus palmivorus, while 30°C produced more laccase in Pleurotus osteatus [21]. The results obtained are comparable to those obtained by [22] who reported maximal laccase production by Marasmium sp. ...
Laccase is an enzyme that has the ability to oxidize substances. It is one of those enzymes that has innate qualities of reactive radical generation, and its use in many domains has been overlooked due to its commercial unavailability. The ability of Marasimus palmivorus MG717877.1 to produce extracellular enzymes (Laccase enzyme) utilizing media containing substrate, named Guaiacol agar medium, was tested. In submerged culture, the ideal pH, incubation duration, and temperature for laccase synthesis were examined. The maximum enzyme activity was reported when the pH of the media was 5.5; laccase activity was (1.03U/ml). The maximum enzyme activity for laccase enzyme was 1.040 U/ml when the temperature was 25°, and (0.922U/ml) after the third day of incubation.
... The chemicals and reagents used in this investigation include silver nitrate (99.9%), ABTS(100%), potato dextrose agar (100%), KH 2 [21]. The inoculated medium was incubated at 30 °C for 28 days. ...
The resistance of bacteria to conventional antibiotics is a global challenge that requires urgent attention while low-cost treatment of printing press wastewater is still an ongoing research. In this study, the antimicrobial potential and printing press wastewater treatment efficacy of biosynthesized T. polyzona silver nanoparticles (TPAgNPs) and laccase-mediated silver nanoparticles (LAgNPs) using T. polyzona extract and laccase were carefully examined. Visual inspection, UV–Vis spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), dynamic light scattering (DLS), energy dispersive X-ray (EDX), and X-Ray diffraction (XRD) were used to characterize the TPAgNPs and LAgNPs. The development of a brown color indicates the synthesis of nanoparticles, and the presence of a surface plasmon resonance (SPR) band at 400 nm indicated the formation of TPAgNPs and LAgNPs. The functional groups such as amines, carboxyl, hydroxyl and carbonyl were responsible for capping and stabilization of the nanoparticles. The TPAgNPs and LAgNPs were spherical, with an average particle size of 9.3 nm and 8.7 nm, respectively. DLS shows intensity with average diameter of 1329 nm and a poly-dispersity index of 0.507. EDX spectral confirmed the presence of pure silver nanoparticles and XRD confirms face-cubic structured nanoparticles. Overall, TPAgNPs and LAgNPs had the highest antibacterial activity against Bacillus subtilis with a zone of 23 mm and 25 mm, respectively. Laccase, TPAgNPs and LAgNPs also showed effective reduction in physicochemical parameters such as turbidity, COD, BOD, TSS, TDS, DO, salinity as well as the heavy metals composition of the Printing Press wastewater, with the total eradication of cadmium.Graphical Abstract
... In the current study ferric chloride and ferrous chloride enhanced the laccase activity produced from Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 respectively. Most researchers finding showed that copper sulphate was the most effective metal for enhancing the laccase activity from Pleurotus ostreatus IMI 395544 (Periasamy et al., 2011), Pleurotus ferulea (Ding et al., 2014) and Pleurotus ostreatus P1 (Nadeem et al., 2014). Mercury chloride enhances the laccase activity from Trichoderma harzianum WL1 (Sadhasivam et al., 2008), Foames fomentarius (Neifar et al., 2011), Saporothrix carnis CPF-05 (Olajuyigbe and Fatokun, 2017) and Thielavia sp. ...
In the present study, lignocellulolytic fungi were isolated by lignocellulolytic waste (Banana waste) soil from the Bhilai-Durg region of Chhattisgarh, India, which was capable to produce laccase enzyme. The laccase producing potent organisms was identified as Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 using molecular characterization by Internal Transcribed Spacer (ITS). For the better production of laccase enzyme physical (incubation period, temperature and pH) and nutritional (carbon, nitrogen and vitamin source) parameters optimized by one factor-at-a time and response surface methodology approach were used. Laccase enzyme was partially purified by ammonium sulphate precipitation and dialysis. The partially purified laccase was characterized by the effect of substrate concentration, temperature, pH, metal ions and inhibitors. Results showed that out of 5 positive cultures only 2 cultures showed the best laccase production after the quantitative screening. ITS-rDNA gene sequencing and BLAST analysis were used to characterize the isolated fungal species and they were found to be D. funicola NFCCI 4534 and A. padwickii NFCCI 4535. The temperature 24 °C and 32 °C and pH 6.0 and 6.5 were suitable for production of D. funicola and A. padwickii, respectively. It was found that Ferric chloride and ferrous sulphate exhibited maximum laccase activity as compared to other metal ions. However, EDTA and l-arginine inhibited D. funicola and A. padwickii, respectively.
... Optimization studies of extracellular laccase production were also carried out by Sidhu et al. [44] and they demonstrated that maximal laccase production (21.7 U mL −1 ) was attained at pH 6.0 by the fungi Scytalidium lignicola. Nadeem et al. [46] also observed similar trend of laccase production in pH range of 4.5 to 6.5 by Pleurotus ostreatus. These findings revealed that there is high correlation between pH of culture media and enzyme production by microorganisms and optimum value of pH depends on the nature of substrates as reactions for laccases varied with different substrates. ...
The present study investigated the screening of mono and co-culture fungal cultivations for laccase production using extracted lignin as the substrate obtained from cauliflower wastes by two different pretreatment methods. Amongst mono and mixed culture fungal cultivations, monoculture of Aspergillus oryzae exhibited the highest enzymatic activity of 29.7 ± 0.6 U mL−1 under submerged conditions and using alkali extracted lignin as substrate. Under the optimal conditions (pH 4.5, 30 °C, 12 days, 1% (w/v) lignin and 0.5 mM Cu2+ concentration) the maximum laccase activity was estimated to be 41.3 ± 2.8 U mL−1 and production yield of 153.3 ± 2.4 mg L−1. Maximum decolorization of pigment extracted from Aspergillus heteromorphus CBS 117.55 cultivated culture media was achieved by administration of 40 U g−1 of crude enzyme concentration. Thermal and pH stability of crude laccase was observed over wide ranges. The dye decolorization efficiency of crude A. oryzae laccase was studied and Congo Red exhibited maximum decolorization percentage (64 ± 1.3%) at 15 µM, 50 °C and pH 4.5. The kinetic study of different dye (Congo Red) concentrations obtained Vmax and Km values of 0.123 × 10−3 M and 0.724 mol L−1 min−1, respectively.
... Most fungal laccase reach their maximum activity when the initial pH of the nutrient medium ranges from 4 to 6 22 . The highest activity of laccase was observed at pH between 4.5 and 6.5, in Pleurotus osteatus 23 . Also Sivakumar et al. 24 , reported highest laccase production at pH 5 in case of Ganoderma sp. ...
... However, Bollag and Leonowicz [31] have shown that azide, thioglycolic acid, and diethyldithiocarbamic acid inhibit secretion of laccase, whereas EDTA affected laccase secretion to a lesser extent. It is believed that small anions such as halides (excluding iodide), azide, cyanide, and hydroxide are known to bind to type-2 and type-3 copper, resulting in an interruption of internal electron transfer and thus inhibit laccase enzymes [32]. The inducing effect seen in our study may be due to increase in fungal biomass in the culture as azide ion mainly inhibits bacterial contamination in the culture. ...
Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautahat at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1 was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions revealed that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9 U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima were pH 6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.
... Jamur basidiomycetes memiliki respon yang berbeda terhadap sumber karbon dan konsentrasinya dalam medium untuk pertumbuhan. Sekresi lakase secara signifikan terjadi saat konsentrasi sumber karbon dalam medium pertumbuhan mencapai tingkat yang rendah (Nadeem, Baig and Sheikh, 2014). ...
Lakase merupakan salah satu enzim ligninolitik yang memiliki kemampuan mendegradasi lignin. Lakase telah diproduksi menggunakan jamur pelapuk putih Marasmius sp. dalam Fermentasi Kultur Padat (FKP) menggunakan jerami padi sebagai media pertumbuhan. Pengaruh sumber karbon yaitu glukosa, gliserol, dan molase dalam medium produksi lakase digunakan dalam penelitian ini. Konsentrasi 0,5%; 1,0%; dan 2,0% digunakan untuk tiap jenis sumber karbon. Hasil menunjukkan bahwa aktivitas tertinggi lakase diperoleh pada kultivasi hari ke 6-10 dengan masing-masing aktivitas (872,0 U/L (hari ke-6), 1516,67 U/L (hari ke-9) dan 1270,69 U/L (hari ke-10). Aktivitas lakase tertinggi diperoleh pada penggunaan medium gliserol dan molase masing-masing adalah 1422,36 U/L (pada konsentrasi 1%, hari ke-7) dan 113,19 U/L (pada konsentrasi 2%, hari ke-8). Aktivitas tertinggi tersebut sebanding dengan penggunaan medium glukosa. Oleh karena itu, gliserol dan molase dapat digunakan sebagai alternatif sumber karbon untuk produksi lakase dengan fermentasi kultur padat.Kata kunci: glukosa, gliserol, lakase, molase, Marasmius sp., fermentasi kultur padat Influence of Carbon Sources on Laccase Production by White Rot Fungus Marasmius sp. in Solid State FermentationAbstractLaccase is an one of the ligninolytic enzymes that capable to degrade lignin in biomass. Laccase has been produced by white rot fungus Marasmius sp. in Solid State Fermentation (SSF) using rice straw as the solid support media. The influence of carbon sources, i.e. glucose, glycerol and molasses in medium of laccase production were studied in this paper. The concentration of 0.5%, 1.0% and 2.0% were used for each carbon sources. The results showed that the highest lacase activity was obtained within 6-10 days of cultivation. Glucose concentration of 0.5%, 1.0% and 2.0% gave the highest laccase activity were 872.0 U/L (day 6), 1516.67 U/L (day 9) and 1270.69 U/L (day 10) respectively. The highest laccase activity on using glycerol and molasses was 1422.36 U/L (at concentration of 1 % on day 7th) and 1113.19 U/L (at concentration of 2% on day 8th), respectively. This activity was comparable to that of glucose substrate. Therefore, glycerol and molasses gave a potential chance as carbon sources for the strategy on low cost laccase production in solid state fermentation.Keywords: glucose, glycerol, laccase, molasses, Marasmius sp., solid state fermentation.
... Laccase has the ability to act on a number of substrates as a result of its potency. The quick color formation with guaiacol is an easy and reliable source for laccase screening [28]. The strain L5 exhibited fast growth and the biggest colored zone around its colony after 7 days of incubation. ...
The current study includes the screening of indigenous extracellular laccase producing fungus from the soil, identified as Phanerochaete chrysosporium species. The selected strain was applied under the optimized condition for the production of laccase in the solid-state fermentation. The effect of polyethylene glycol and Triton X-100 on the enhancement of enzyme activity and biocatalytic removal of bisphenol A using laccase produced by Phanerochaete chrysosporium was investigated at 40°C, initial laccase activity 3U/mL and initial bisphenol A concentration 2 mM after 3 h of treatment. Bisphenol A concentration was evaluated using a colorimetric assay in the presence of 4-AAP. The effect of PEG molecular weight on BPA removal was determined using PEG with a molecular weight between 1500 and 20000.The results showed that the addition of polyethylene glycol and Triton X-100 to the reaction mixture greatly increased the laccase activity and the residual enzyme activity, but no significant changes were found in the removal of bisphenol A. The maximum removal of BPA was obtained at pH 8.0 and 40° C in 90 mg/L of PEG-1500 and 50, 100 mg/L of PEG-6000, while laccase activity at all concentrations of PEG-1500 and 6000 increased.
... Incubation time is also an important factor which affects the biosynthesis of fungus. Our results are in line with Nadeem et al. (2014) reported that maximum laccase activity in Pleurotus ostreatus after 6 th days of incubation. Asgher et al. (2012) reported that the time taken by microorganism in order to produce laccase enzyme is due to long lag phase and primary metabolism. ...
Laccase is a blue copper containing enzyme which is capable of catalyzing the oxidation of phenolic compounds to phenoxyl radicals. The purpose of recent study was to find optimum conditions for maximum laccase enzyme production by Pleurotus nebrodensis WC 850 by applying an important statistical technique response surface methodology (RSM) through solid state fermentation of lignocellulosic biomass. Firstly, screening experiments were conducted in order to select the best substrate for maximum laccase enzyme production among six different lignocellulosic substrates. Wheat straw was selected as best substrate with enzyme activity 145.10 U/mL after 6th day of fermentation. Different physical parameters including pH, temperature, inoculum size, incubation time and moisture were then optimized by response surface methodology under central composite design (CCD). RSM is an effective strategy which explores the optimum operating conditions for multivariable system as well as it also examines the simultaneous, systematic and effective variation of crucial components and determines the possible interaction among higher order effects which results in enhanced enzyme yield. The increase in laccase enzyme activity (171.63 U/mL) was observed at pH 5, temperature 30°C, inoculum size 4mL, incubation time 144mL and moisture 50% after optimization of different factors at different levels. This study reveal that optimization of different factors have significant effect on laccase enzyme production. Moreover, optimization of different parameters through RSM is time saving and good technique in biotechnology. © 2016, Pakistan Agricultural Scientists Forum. All rights reserved.
Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Charac- terization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its toler- ance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.
A great deal of works has been carried out assessing dye-decolorization capabilities of fungi, but only few species were investigated. In this present study, ten fungal species were screened for laccase activity by indicator plate method, of which five species were found to be laccase-positive. Laccase activity varied during growth and maximal laccase activity was observed during the 9 th day, except for Agaricus bisporus. Pleurotus ostreatus gave the highest laccase activities, showing average value around 570 U/L, which were higher than other strains. Addition of 1 mM copper sulphate induced the laccase production efficiently by 60 to 80%, while veratryl and benzyl alcohol induced laccase production in all the laccase positive species except for A. bisporus. Constitutive expression of laccase was observed in F. solani in the presence of copper sulphate. In addition, the extracellular laccase from the P. ostreatus could decolorize reactive dyes, which suggests the potential application of laccase in textile effluent treatment.
The strain Bacillus sp. WD23 exhibiting laccase activity was screened from forest soil. The M9 medium containing Cu2+ was used for enriching and isolating bacterial strains capable of oxidizing syringaldazine (SGZ). One isolated strain was identified as Bacillus subtilis WD23 based on the results of physiological and biochemical tests and 16S rDNA sequence analysis. The strain WD23 could grow at temperatures ranging from 20 to 55°C and showed optimum growth temperature and pH at 25°C and 7.0, respectively. The sporulation rate of the strain clearly correlated well with the laccase activity. The temperature half-life of the spore laccase was 2.5 h at 80°C and the pH half-life was 15 d at pH 9.0. Its spore laccase could decolorized 50~90% of Remazol brilliant blue R (RBBR), alizarin red, congo red, methyl orange and methyl violet, which suggests the potential application of spore laccase in dyestuff treatment.
The decolorization of the recalcitrant dye Remazol Brilliant Blue R (RBBR) by the culture fil-trate of Polyporus sp. S133 and the effect of various environmental factors were investigated. Both biodeg-radation and biosorption were playing an important role in bioremoval mechanisms. The highest biosorp-tion of RBBR in Polyporus sp. S133 was shown by all carbon sources such as sucrose, glucose, fructose, and starch. No biosorption was shown by the addition of aromatic compounds and metal ions; 97.1 % RBBR decolorization was achieved in 120-rpm culture for 96 h, as compared to 49.5 % decolorization in station-ary culture. Increasing the shaking rotation of the culture to more than 120 rpm was proven to give a negative effect on decolorization. The highest produc-tion of laccase was shown at pH 4 and constantly decreases when the pH level increases. The addition of glucose, ammonium tartrate, Cu 2+ , and protocate-chuic acid was the suitable environmental condition for RBBR decolorization. There was a positive rela-tionship between all environmental conditions and laccase production in the decolorization of RBBR.
In the present work, Eight media with different components were screened. The enzyme formed by Pl. ostreatus ARC280 was localized mainly in the extra-cellular fraction. Laccase formation reaches its maximum value with specific activity of about 140 U/mg protein at the twenty-sixth day of incubation, pH 5.0 and 28ºC. Among the various wastes used, corn stover induces the highest laccase production with specific activity of 75.48 U/mg protein. Soluble starch at 1.5% (w/v) and ammonium sulfate was found to be the best carbon and nitrogen sources for laccase formation, respectively. The optimal concentrations of Tween-80 and CuSO4. 5H2O, were found to be 0.1% (v/v) and 100μM and cause enzyme induction by about 44% and 19% than control, respectively.
The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted.
After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10g, ammonium
tartrate 0.11g, Tween-80 0.5g, MnSO4 7mg, and veratryl alcohol 0.3g in 10mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin
peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture.
Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with
60% (NH4)2SO4 yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1
and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR
gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According
to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes
are 38kDa and 40kDa, respectively.
Laccase production by Penicillium martensii NRC 345 was optimized. Eight media with different components
were screened. The enzyme formed by P. martensii NRC 345 was detected mainly exocellularly under shake culture condition.
Laccase formation reaches its maximum value with specific activities of about 7.18 U/mg protein at the twenty-sixth
day of incubation at pH 5.5 and 30°C. Galactose (5 g/l) and sodium nitrate (0.2 g/l) were found to be the best carbon and
nitrogen sources for laccase formation, respectively. Replacement of galactose instead of glucose at the same concentration
increased laccase production by about more than ten-fold. Among the various wastes used wheat bran induces the highest
laccase production with specific activity of 39.52 U/mg protein. Tween-80 and CuSO4.5H2O, were also tested for laccase induction.
The present work focuses on isolating, screening and optimizing the process parameters to achieve the maximum production of extracellular laccases by Ganoderma sp obtained from natural habitat. Guiacol and ABTS (2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate) were used as substrates for screening the laccase activity. Culture condition such as period of incubation, pH, temperature and various nutrition parameters such as carbon source, nitrogen source, copper sulphate concentration and solvents were optimized. The optimization studiesrevealed that the laccase yield was highest when operated at the following conditions: 10 days of incubation, pH 6.0, 2% starch as carbon source, 0.2% yeast extract as nitrogen source, 4% iso-propanol as solvent and 27μM of copper sulphate in the production medium.
Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289 U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67 kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40 °C, respectively. From the LB plot, K
m value of AAO for oxidizing veratryl alcohol was determined to be 0.6 mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.
A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.
Laccases have received much attention from researchers during the past decades due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases. This makes laccases highly interesting for a wide variety of processes, such as textile dye decolouration, pulp bleaching, effluent detoxification, biosensors and bioremediation. The successful application of laccases to the above-mentioned processes requires the production of large quantities of enzyme at low cost. Filamentous fungi are able to produce laccases in high amounts, however, an efficient production system at bioreactor scale is still lacking. This is mainly due to the fact that laccase production by wild-type strains of filamentous fungi is linked to secondary metabolism, which implies that the following drawbacks must be overcome: uncontrolled fungal growth, the formation of polysaccharides around mycelia and the secretion of certain compounds (i.e. proteases) that inactivate laccases. This review summarizes the current status of laccase production by wild-type strains of filamentous fungi at the bioreactor scale.
Schizophyllum commune (Fries), a Nigerian edible mushroom was assessed for fungus biomass and production of laccase in submerged medium. Cultural conditions such as temperatures, pH, carbon sources and nitrogen sources were optimized. Fungus biomass requirements were determined by the mycelia dry weight method. Results showed that temperature of 28°C was optimal for both fungus mycelial biomass yield (109.18±5.20mg/30cm3) and production of laccase (51.5±2.12 U/min). The optimal pH for, fungi mycelial biomass yield and laccase activities was 5.5. Of the 5 carbon sources tested, glucose supported the highest fungus mycelial biomass yield of (88.32±1.50 mg/ cm3), while mannose supported the highest laccase enzyme production with 47.5 ±1.85 (U/min) of laccase (p≤0.05). The most utilized nitrogen source for fungus mycelial biomass yield and laccase activity was urea and yeast extract, which stimulated fungus mycelial biomass yield (125.25±3.45 mg/30 cm3) and laccase production of (65.5±2.52 U/min). The implications of these findings were discussed.
The present study deals with production of ligninolytic enzymes from an indigenous white rot fungus Schizophyllum commune IBL-06 by using banana stalk as substrate through the process of solid state fermentation. The production process was further improved by optimizing a number of physical parameters such as incubation time, moisture level, pH, temperature, inoculums size and nutritional factors (carbon and nitrogen ratio, mediators and metal ions). By optimization of different parameters, the maximum activities of enzymes synthesized by S. commune IBL-06 were observed as 3745 IU/ml for manganese peroxidase (MnP), 2700 IU/ml for lignins peroxidase (LiP) and 345 IU/ml for laccase after 3 days incubation at pH 4.5 and 35 degrees C temperature with 3 ml inoculum size, 60% moisture content, 20:1 C:N ratio (glucose and ammonium nitrate as carbon and nitrogen supplements), 1ml of 1mM MnSO4 as mediator, and 1ml of 1mM MgSO4 center dot 7H(2)O(2). This High activities of ligninolytic enzymes produced by the fungus suggest its potential for commercial scale production of these enzymes for diverse industrial applications.
This study revealed a dual effect of copper on D. squalens laccase activity, inducing and suppressing. The addition of copper sulfate led to enhancement of the enzyme activity until it reached a maximum value at peak copper sulfate concentration (PC) followed by suppression and even complete termination of the enzyme activity. Maximum laccase yield is a function of fungi tolerance and media composition. Nutrients had a significant impact on copper-independent and copper-induced laccase activity. Some nutrients also improved tolerance of cells and laccase-producing system to copper load. The effect of specific nutrient depended on the ratio of carbon and nitrogen sources. This research can be used to predict the effective copper concentration and nutrient composition to stimulate the laccase production without compromising the fungi growth. The methylated substrates (3-O-methylglucose, methylcellulose) in combination with casein and copper demonstrated high potential to support fungi tolerance and enhanced laccase activity.
In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as laccase secretion indicator. A novel laccase-producing strain was isolated and identified as Shiraia bambusicola Henn. strain GZ11K2 according to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. In further investigation, the production of laccase by S. bambusicola GZ11K2 was greatly enhanced by the nontoxic inducers of copper sulfate and rhodamine B. Copper and rhodamine B were added into the cultivation medium at 24 and 12 h, respectively, and the maximum laccase production was obtained. Under the induction of 2.0 mM copper sulfate and 35 μM rhodamine B, an increment of about 80 times of laccase activity compared with that in the inducer-free medium and about 20 times compared with that in the single copper-supplemented medium was observed. Compared with other species, S. bambusicola GZ11K2 exhibits better laccase-producing characteristics with an activity of 16,400 U/L after 108 h, suggesting its potential ability for industrial application.
The fungus Cladosporium cladosporioides was isolated from a coal sample as a dye decolorizing microorganism. The maximum production of laccase from this fungus was found to be 4160 U/l with 0.05 mM CuSO4, 4% (w/v) glucose and lignin 0.04% (w/v). The laccase was purified from C. cladosporioides to homogeneity in a very active state using ammonium salt precipitation and gel permeation chromatography. Analysis with SDS–PAGE showed that the purified laccase was a monomeric protein of 71.2 kDa, and the apparent molecular mass of this enzyme was 75.17 kDa (m/z = 75,174), which was accurately determined by MALDI/TOF-MS. The UV–vis absorbance and electron paramagnetic resonance spectra of the purified laccase showed the presence of type 1 and type 3 copper ions. The optimum pH and temperature of the purified laccase were 3.5 and 40–70 °C, respectively, and the N-terminal amino acid sequence was determined to be IGPTGDMYIVNEDVS. The purified laccase was effective in the biotransformation of aromatic compounds as well as in the decolorization of various dyes.
Screening is conducted to select a fungus with desired characteristics intended for various applications, e.g. bioremediation and enzyme production. A qualitative method was used in this study for screening of fungal ligninolytic enzyme activities. The fungal ligninolytic activity was correlated with its growth from the screenings to identify a suitable fungus for solid substrate fermentation. Four strains of fungi, namely, Phanerochaete chrysosporium, Pycnoporus sanguineus, Phlebia radiata and Pleurotus sajor-caju, were screened for their ligninolytic enzyme activities using guaiacol and remazol brilliant blue-R (RBB-R) as screening reagents. The screenings were conducted at both, the optimal growth temperature of each fungus and 35 • C. All the fungi, except P. radiata, showed positive guaiacol oxidation and RBB-R decolourisation activities. On the basis of the results, P. sajor-caju may be a suitable fungus for use in bioconversion of lignocellulose because it maintained guaiacol oxidation and RBB-R decolourisation activities at 35 • C. Its halo-to-colony area ratios of guaiacol oxidation and RBB-R decolourisation after 6 days were 7.0 and 2.7, respectively. In the subsequent growth study, the measured colony area was closely correlated with the dry fungal cell weight with a regression value (R 2) of 0.971. Thus, the colony size could be used as an indicator of fungal growth if the screening media do not significantly slow the growth of fungus.
A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l−1 in an optimized medium containing 20g of malt extract l−1, 2g of yeast extract l−1, 1.5mM CuSO4. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%.
The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60kDa. The optimum temperature and pH value
of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten
constant (K
m
) of the laccase was 18μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial
enzyme activity was retained after incubation of the laccase at 70°C for 2h, indicating that the laccase was intrinsically
highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0mM of Mg2+, Mn2+, Zn2+ and Ca2+, while inhibited by 4.0mM of Co2+, Al3+, Cu2+, and Fe2+, showing different profiles of metal ion effects.
KeywordsThermostable laccase–Production and purification–Enzymatic properties–White-rot fungus
Laccases are oxidative enzymes linked to biological degradation of lignin. The aim of this work was to evaluate the effect of inducers and different concentrations of nitrogen on production level of total laccase activity and pattern of laccase isoforms, produced in solid state fermentation of sugarcane bagasse by a selected strain of Pleurotus ostreatus. The addition of yeast extract 5 g/L, copper sulfate 150 μM and ferulic acid 2 mM provided highest enzymatic activity (167 U/g) and zymograms indicated the presence of six laccase isoforms (POXA1b, POXA3, POXC and three other isoforms). Results of protein identification by mass spectrometry confirmed the presence of POXC and POXA3 as the main isoenzymes, and also identified a glyoxal oxidase and three galactose oxidases. The fact that the isoenzyme POXA1b was not identified in the analyzed samples can be possibly explained by its sensitivity to protease degradation.
Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000 Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 °C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 °C. In terms of kcat/Km values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free –OH group with an adjacent free or derivatised –OH. The o-diphenols were preferred above their p-counterparts. Compounds with three adjacent –OH groups displayed higher binding affinities but phloroglucinol, an m-substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology.
Laccase is a widespread group of multi-copper enzymes which can catalyze the oxidation of a variety of organic compounds, with concomitant reduction of molecular oxygen to water. It has a wide application in industrial processes, particularly in renewable bio-energy industry. In this study, Pleurotus ostreatus strain 10969 with high yield of laccase, previously isolated from edible fungus growing on Juncao, was applied for optimization of fermentation media and growth parameters for the maximal enzyme production through response surface methodology and further characterization of the laccase activity. The results show that glucose and Mg2+ are the key ingredients for laccase production with the optimum concentration of 0.0988 g/mL and 7.3 mmol/L respectively. Compared to the initial medium, the highest laccase yield observed is approximately increased by 2.5 times under the optimized conditions. Extracellular laccase was then purified and its characters were analyzed. The molecular weight of the laccase is about 40 kDa, and the optimum pH and temperature for its activity is 4.0 and 50 °C with the corresponding Km and Vmax of 0.31 mmol/L and 303.25 mmol/min respectively. DTT, β-mercaptoethanol and NaN3 nearly inhibit all activity of the laccase, as well as the metal ions especially Ag+. In summary, our results will facilitate the utilization of plant lignin in biomass energy industry.
An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, Km and Vmax were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (ki) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.
In the present paper, the mechanism of overproduction of laccase by microbe interaction was investigated in the co-culture process of Ganoderma lucidum and Candida sp. HSD07A. The results show that nitrogen source, sulfur source, hydrolytic enzymes and inducers do not have the significant influence on laccase activity, and glucose deprivation in the medium is also not the crucial reason why G. lucidum overproduces laccase although it can improve laccase activity at a certain extent. Furthermore, glucose deprivation is made by strain HSD07A, and NMR and GC data reveal that the yeast can convert glucose into glycerol and ethanol. G. lucidum cannot assimilate ethanol; however, glycerol is an efficient carbon source for G. lucidum. In the co-culture process, the appearance of the second carbon source, glycerol produced by the yeast, is the crucial reason why G. lucidum overproduces laccase because glycerol can make G. lucidum cells secrete more laccase by prolonging the secretion time under the condition of glucose deprivation. Thus, it is the carbon source succession in the co-culture process that leads to the overproduction of G. lucidum laccase.
The production of laccase by an indigenous strain of Pleurotus ostreatus HP-1 was studied on solid state fermentation. Culture parameters such as type and concentration of substrate, inoculum size, moisture content, pH, surfactant presence, temperature, and nitrogen source were optimized by conventional “one factor at a time” methodology. A maximum laccase yield of 3952 U g-1 of dry substrate optimized was obtained with wheat straw as substrate with five agar plugs as the inoculum, 60% moisture content, pH 5.0, surfactant concentration 0.015 gl-1, and nitrogen source (combination of L-asparagine and NH4NO3 at 10 mM concentration each) at incubation temperature 28oC. Enhancement in laccase activity was achieved with the use of various aromatic inducers and copper sulphate. Highest laccase activity of 14189 U g-1 of dry substrate was achieved using 0.28 mM copper sulphate under optimized conditions. Thus, the indigenous isolate seems to be a potential producer of laccase using SSF and can be exploited for further biotechnological applications. The process also promises economic utilization and value addition of agro-residues.