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)
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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.
Morphology engineering of basidiomycetes for improved laccase biosynthesis
Biotechnology Letters, 2015
Objective This work is the first application of a morphological engineering technique called microparticle-enhanced cultivation (MPEC) aimed at the facilitation of laccase production in the submerged cultures by two basidiomycetes species Cerrena unicolor and Pleurotus sapidus. Results The positive effect of the applied 10 lm Al 2 O 3 microparticles at concentrations from 5 to 30 g Al 2 O 3 l-1 was shown. Laccase activity increased 3.5fold for C. unicolor and 2-fold for P. sapidus at 15 g Al 2 O 3 l-1 on 9 and 14 day of the cultivation, respectively, compared to the control culture without microparticles. The increase of laccase activity in the cultivation broths was caused by the action of Al 2 O 3 microparticles on the agglomeration of hyphae. It led to the decrease of the size of the pellets, (on average by 2 mm for C. unicolor), the change of their shape (starshaped pellets for C. unicolor) and the change of their structure (more compact pellets for P. sapidus). Conclusions Application of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.
Effect of Solid State Fermentation Medium Optimization on Pleurotus ostreatus Laccase Production
Acta chimica Slovenica, 2015
The objective of this work was to increase laccase production by Pleurotus ostreatus PLAB through culture medium optimization using solid state culture conditions. Increased laccase activity was obtained through design of experiments (DOE) using the Taguchi orthogonal array (OA). Seven factors, viz. lignocellulose, glucose, yeast extract, peptone, KH(2)PO(4), MgSO(4) 7H(2)O and MnSO(4) H(2)O at three levels and pH at two levels. OA layout of L18 (2(1) x 3(7)) was selected for the proposed experimental design using Minitab 17 software. Data analysis showed that lignocellulose (20 %) and glucose (10 g L1) had positive effect, whereas KH(2)PO(4), MgSO(4)∙7H(2)O and MnSO(4)∙H(2)O did not have significant effect on laccase production. Taguchi OA analysis showed that pH 6, lignocellulose 20 %, glucose 10 g L(-1), yeast extract 6 g L(-1), peptone 15 g L(-1), KH(2)PO(4) 3 g L1, MgSO(4)∙7H(2)O 0.5 g L(-1) and MnSO(4)∙H(2)O 0.1 g L-1 were the optimal conditions to maximize laccase production....
Production of Fungal Laccase Using Orange Peelings as Substrate by Submerged Static Fermentation
Aims: Laccases are diphenol oxidases that have numerous applications in biotechnological processes. In this work, the production of fungal laccase using organic and inorganic feed substrates in submerged static fermentation was investigated. Study Design: One-factor-at-a-time strategy was adopted to optimize the cultural parameters for enhanced laccase production. Place and Duration of Study: Methodology: A total of nine fungal isolates were obtained from wood decaying sites of University of Port Harcourt forest areas and subjected to laccase screening with 2,2-Azinobis-3-ethyl(benzthiazoline-6-sulphonate) (ABTS). The influence of medium components using the basal medium at pH 5.0 as base was evaluated and these cultural parameters include carbon sources (glycerol, rice bran, glucose and ground orange peelings), nitrogen sources (yeast extract, 2 potassium nitrate, peptone and ammonium chloride), metal ions (copper sulfate and manganese sulfate) and inducer compounds (ABTS, Tween 80 and soya oil). Time course study was conducted with the unoptimized and optimized cultural medium. Results: Out of nine cultures tested, seven were found to be laccase-positive with isolates CF-1 and CF-2 being the best potential cultures. Isolate CF-1 which had the highest laccase activity was identified as Pleurotus ostreatus and was chosen for further studies. Ground orange peelings (0.1% w/v) and NH 4 Cl (0.1% w/v) were the most suitable carbon and nitrogen source for laccase production by the fungus. Maximum laccase production was obtained with Cu 2+ at a concentration of 0.05%w/v among other metal ions. Soya oil at concentration of 0.05% (v/v) was the best inducer of the enzyme. The highest laccase production was achieved at an Initial pH 4.5. Under optimal culture medium, the maximum laccase activity was determined to be 7.21 U ml-1 on the 7th day of cultivation; which was approximately three times higher than that in basal medium (2.5 U ml-1). The results obtained indicate that the extracellular laccase production is dependent on various cultural parameters. Conclusion: One-factor-at-a-time strategy adopted in this study proved that the optimum conditions enhanced laccase production by three folds using orange peelings. The results obtained are very interesting since orange peelings are common agricultural wastes in several countries and imply that their re-utilization in the production of enzymes would help solve pollution problems caused by their improper disposal. In addition, Pleurotus ostreatus having shown promise for laccase production using low-cost lignocellulosic substrates could be suggested as a prospective candidate for higher laccase production for several biotechnological applications.
Electronic Journal of Biotechnology, 2013
Background: Enzymatic activity and laccase isoenzymes number of Pleurotus ostreatus grown in different pH values of the growing medium in submerged fermentation and incubated in buffer solutions of different initial pH values were determined. The expression profiles of five laccase genes (Lacc1, Lacc4, Lacc6, Lacc9 and Lacc10) in these cultures were also studied. Results: The highest laccases activity was obtained in cultures grown at initial pH of 4.5 and the lowest in cultures grown at initial pH of 8.5. Isoenzyme profiles were different in all the cases. Lacc1, Lacc4, Lacc6 and Lacc10 were expressed in all the cultures. Conclusions: The initial pH of the growing medium is an important factor for regulating the expression of laccase genes, having an effect on the activity and on the laccase isoenzymes number produced by P. ostreatus in SmF. This is the first report on the influence of different initial pH values of the growing medium on the laccases activity, laccase isoenzymes number and laccases expression profiles of P. ostreatus grown in submerged fermentation.