Effect of Different Culture Conditions and Inducers on Production of Laccase by a Basidiomycete Fungal Isolate Pleurotus Ostreatus HP-1 Under Solid State … (original) (raw)
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 NH 4 NO 3 at 10 mM concentration each) at incubation temperature 28 o C. 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.
Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation
Applied Microbiology and Biotechnology, 2008
Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l −1 , with a biomass production of 5.6 g l −1 and four laccase isoforms. However, cultures grown in solidstate fermentation had a much lower laccase activity of 2,430 U l −1 , biomass production of 4.5 g l −1 , and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.
Aims: Effect of culture medium pH (3.5, 4.5, 6.5, 7.5 and 8.5) on the activity and number of laccase isoenzymes of Pleurotus ostreatus grown in solid-state fermentation using polyurethane foam as a support was evaluated. Methodology: Pleurotus ostreatus was grown in solid-state fermentation using polyurethane foam as inert support at different initial pH of the culture medium. The 2567 enzymatic extracts were obtained by pressing the polyurethane foam every day of culture. The laccase activity was measured in each enzymatic extract using 2,6-dimethoxyphenol as substrate and the isoenzymes number were detected through zymograms. Results: In general, the fungus showed high values of specific growth rate grow that all pH tested, and the higher were at pH 3.5 and 8.5 (0.78 and 0.82h -1 , respectively), whereas at pH6.5 was 0.034h -1 and pHs of 4.5 and 7.5 was 0.047h -1 . Furthermore, the maximum biomass values were low, about 3.7g/L in all cases. The maximum values of laccase activity were observed in fermentations development at pH4.5 and 6.5 (approximately 40000 U/L). The largest number of isoenzymes was observed in fermentations carried out at pH 7.5 and 8.5. Conclusion: In solid-state fermentation on polyurethane foam, the pH of the culture medium did not affect the growth of the fungus, however, there were differences in the production and activity of laccases.
BioResources
Kinetic parameters of growth and laccase activity of five ATCC strains of Pleurotus ostreatus in submerged fermentation were evaluated. The best strain for laccase production and the time of maximum laccase activity were also determined. The greatest laccase activity (37490 U/L), laccase productivity (78 U/L h), specific growth rate (0.026/h), and specific rate of laccase production (119 U/gX h) were observed with the strain of P. ostreatus ATCC 32783. In general, the isoenzyme patterns were different in all the cases; however, all the strains showed two laccase bands in the same position in the gel. Not all strains responded in the same way to the addition of Cu in the culture medium. In general, the sensitivity to Cu could be used to select strains having high laccase activity for commercial exploitation.
Aims: To optimize laccase production by submerged fermentation using an edible mushroom Pleurotus ostreatus ARC280. Study Design: Laccase activity was assayed by monitoring the product formation rate of enzymatic oxidation of syringaldazine spectrophotometrically at 525 nm. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt, between May 2009 and October 2010. Methodology: Pleurotus ostreatus ARC280 was maintained on potato dextrose agar medium. The liquid medium used for the laccase production by the fungal culture during its growth in submerged fermentation was selected from eight liquid media for inducing laccase production. Parameters such as incubation period, temperature, pH of the production medium, carbon and nitrogen sources and other nutritional parameters were studied using syringaldazine as a model substrate for laccase activity determination. Results: 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 of75.48 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. Conclusion: Laccase production by Pl. ostreatus ARC280 has been shown to depend markedly on the composition of the culture medium, carbon, nitrogen content and inducer compounds and governed by parameters such as pH of the production medium and other nutrition parameters.
Comparison of physico-chemical characteristics of four laccases from different basidiomycetes
Biochimie, 2004
New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50°C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K 4 Fe(CN) 6 were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.
Journal of Basic Microbiology, 2002
The production of laccase by a Brazilian strain of Pleurotus pulmonarius was studied in solid state fermentation using wheat bran as substrate. Among oxidative and hydrolytic enzymes tested (laccase, aryl alcohol oxidase, lignin peroxidase, Mn peroxidase, xylanase and cellulase), laccase was the main enzyme produced by P. pulmonarius. The most suitable condition for maximum production of laccase (8,600 U/g substrate) was initial moisture content of 75% and 5 days of cultivation at 30 °C. The optimum pH and temperature for laccase activity were found to be 6.5 and 50 °C, respectively. P. pulmonarius laccase was stable at 50 °C for more than 6 hours, and it retained about 73% and 18% of its activity when heated for 1 h at 55 and 60 °C, respectively. The enzyme was greatly stable at alkaline pH, but not at acidic pH. The laccase activity appear to be correlated with the ability of crude extract to decolourize several industrial dyes.
2016
Laccases are enzymes that have a great potential for use in breaking down toxic compounds. Fungal laccases show high enzymatic activity, especially those produced by basidiomycetes. Depending on the culture conditions and the strain used, a variety of isoenzymes and/or enzymatic activities can be obtained. In this study, extracellular laccase enzymes produced by Pleurotus ostreatus was identified in a submerged culture (SmF), with and without copper sulphate as a chemical inducer, and in a solidstate culture (SSF), using wheat straw as natural inducer. This study was conducted to observe the expression of the enzymes produced under the culture conditions tested and their persistence during the culture, as well as the extracellular activity produced and the correspondence that the isoenzymes presented between the intracellular and extracellular media. A positive effect of the inducers on the specific laccase activity was observed either in SmF with copper sulphate or SSF (41.11 and 4...
Production of Fungal Laccase and Its Immobilization and Stability
Journal of the Faculty of Agriculture, Kyushu University, 2008
The extracellular laccase of white-rot fungus, Cerrena unicolor, was purified from culture by Sephadex G-25 and ion-exchange chromatography on DEAE-Toyopearl column and immobilized on various supports. Purified laccase showed two times higher activity after desalting process and 3.7 to 13.4 times activity after DEAE chromatography. During immobilizing process, 93.8% protein and 100% of laccase activity were coupled to the supports. Following immobilization, the optimal pH (5.5) for immobilized laccase was slightly shifted wide pH values (from 5.0 to 6.0) in the case of some supports, and decreased more gradually in alkaline region. Both free and immobilized laccases showed the highest activity at 60 ˚C, however, the immobilized enzyme was more resistant to the wide range of temperature (50~80 ˚C). Even at the 90 ˚C, treated glass beads and Supp-1 maintained almost 80% activity. After 10 days of storage at 4 ˚C, the immobilized laccases retained 95~100% of its initial activity. It was also more stable during storage at 4 ˚C. While, after the same storage time only 34% the initial activity was retained by the free enzyme. Immobilized laccase on glass supports was retained 85~90% activity over 20 days storage, while free enzyme almost zero activity. The immobilized laccases which are more stable and temperature resistant than free enzyme, seem to be more useful.
Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus
Applied and Environmental Microbiology, 2004
Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml ؊1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter ؊1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml ؊1 . These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter ؊1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml ؊1 (i.e., 360 mg liter ؊1 ) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter ؊1 . In this case, maximal activities were 3,900 and 4,660 nkat ml ؊1 , respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.