Trametes suaveolens as ligninolytic enzyme producer

Species of the genus Trametes represent one of the most efficient lignin-degraders which can be attributed to a well developed ligninolytic enzyme system. Current trends are screening of ability of new species to produce these enzymes, as well as the optimization of conditions for their overproduction. Therefore, the aim of the study was to evaluate the potential of T. suaveolens to synthesize laccase and Mn-oxidizing peroxidases during fermentation of the selected plant raw materials. Level of enzyme activities was measured on 7, 10 and 14th day of submersion, as well as the solid-state fermentation of wheat straw and oak sawdust in the presence of NH4NO3 in previously determined optimal nitrogen concentration of 25 mM. The enzyme activity was determined spectrophotometrically using ABTS and phenol red as the substrates. The highest level of laccase activity (1087.1 U/L) was noted after 7 days of wheat straw solid-state fermentation, while during the submerged cultivation the production of the enzyme was not noted. Submerged cultivation in oak sawdust-enriched medium was the optimal for activity of Mn-dependent peroxidase (1767.7 U/L on day 14) and Mn-independent peroxidase (1113.7 U/L on day 7). Introduction of T. suaveolens to produce ligninolytic enzyme represented the base for further study, as well as the determination of relation between enzyme activity and rate of lignin degradation. It could lead to greater possibility of fungal species selection with high delignification capacity, which could take participation in sustainable production of food, feed, fibres, and energy, environmentally friendly pollution prevention, and bioremediation

Potential of selected fungal species to degrade wheat straw, the most abundant plant raw material in Europe

Background: Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world. Results: During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L-1 and 1045.5 U L-1, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L-1). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%. Conclusions: According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels. © 2017 The Author(s)

Fungicide sensitivity of selected Verticillium fungicola isolates from Agaricus bisporus farms

Five isolates of Verticillium fungicola, isolated from diseased fruiting bodies of Agaricus bisporus collected from mushroom farms in Serbia during 2002-2003, were studied. By observing their colony morphology under different growth conditions and their pathogenic characteristics, the isolates were identified as V. fungicola var. fungicola. The peat/lime casing was the primary source of infection. Testing of sensitivity to selected fungicides showed that all isolates were highly resistant to benomyl (EC50 values were higher than 200.00 mg/l), moderately sensitive to iprodione (EC50 values were between 11.93 and 22.80 mg/l), and highly sensitive to prochloraz-Mn (EC50 values were less than 3.00 mg/l)

Effect of cultivation conditions on ligninolytic enzyme production by Ganoderma carnosum

Ganoderma carnosum has been found in Europe only, at coniferous trees and it is difficult to distinguish it morphologically from G. lucidum. Since G. carnosum has not been studied biochemically yet, the aim of this study was to analyse its ability to produce Mn-dependent peroxidase (MnP), versatile peroxidase (VP) and laccase (Lac) under conditions of solid-state fermentation of selected plant raw materials as carbon sources, in the presence of different nitrogen concentrations in the medium. Wheat straw, corn stem, oak and grapevine sawdust were the analysed plant raw materials. Nitrogen source in synthetic medium was NH4NO3 and its concentrations were: 10mM N and 20 mM N. Enzyme activity was determined spectrophotometrically, using ABTS and phenol red, as the substrates for Lac and Mn-oxidizing peroxidases, respectively. Maximum level of MnP activity (56.82 U/l) was obtained in the medium with wheat straw and nitrogen concentration of 10 mM. Best carbon source for VP production was grapevine sawdust at nitrogen concentration of 10 mM (80.80 U/l). The obtained Lac activity was very low in the medium with wheat straw (1.80 U/l), while it was not detected in the presence of other three analyzed plant raw materials. Maximum of total protein content (0.06 mgml-1) was noted in the medium where oak sawdust was carbon source and nitrogen concentration was 20 mM.Vrste roda Ganoderma su producenti mnogih biološki aktivnih supstanci pa su objekti proučavanja mnogih medicinskih i farmaceutskih studija. U novije vreme se sve više pažnje poklanja upoznavanju njihovog ligninolitičkog enzimskog sistema, u cilju uspešne primene u različitim biotehnološkim procesima. Za razliku od G. lucidum, čiji je ligninolitički sistem intenzivno proučavan, ostale vrste ovog roda su retko bile objekti istraživanja. G. carnosum se teško morfološki razlikuje od G. lucidum. i još uvek nije biohemijski proučena, pa je cilj našeg istraživanja bio analiza produkcije Mn-oksidujućih peroksidaza i lakaza u uslovima čvrste kultivacije, na različitim biljnim ostacima (pšenična slama, stabljike kukuruza, piljevina hrasta i vinove loze) u prisustvu azota u obliku NH4NO3 i u koncentracijama od 10 mM, odnosno 20 mM. Maksimum produkcije Mn-zavisne peroksidaze dobijen je na pšeničnoj slami pri koncentraciji azota od 10 mM (56.82 U/l). Piljevina vinove loze i koncentracija azota od 10 mM bili su optimalni za sintezu verzatil peroksidaze (80.80 U/l). Dobijena aktivnost lakaza je bila izuzetno niska u medijumu sa pšeničnom slamom (1.80 U/l), dok na ostalim biljnim ostacima nije zabeležena. Maksimalni sadržaj ukupnih proteina je bio najveći u medijumu sa piljevinom hrasta kao izvorom ugljenika i koncentracijom azota od 10 mM.Projekat ministarstva br. 14304

WHEAT STRAW CONVERSION BY ENZYMATIC SYSTEM OF GANODERMA LUCIDUM

The purpose of this study was to resolve the question of whether various nitrogen sources and concentrations affect characteristics of selected G. lucidum ligninolytic enzymes participating in wheat straw fermentation. This is the first study reporting the presence of versatile peroxidase activity in crude extract of G. lucidum culture, as well as isoforms profile of Mn-oxidizing peroxidases. NH4NO3 was the optimum nitrogen source for laccase and Mn-dependent peroxidase activity, while peptone was the optimum one for versatile peroxidase activity. Four bands with laccase activity were obtained by native PAGE and IEF separations from medium enriched with inorganic nitrogen source, and only two bands from medium containing organic source. Medium composition was not shown to affect isoenzyme patterns of Mn-oxidizing peroxidases. Four isoforms of Mn-dependent peroxidase and three of versatile peroxidase were obtained on native PAGE. By IEF separation, five isoforms of Mn-dependent peroxidase and only two of versatile peroxidase were observed. The results demonstrated that G. lucidum has potential for mineralization and transformation of various agricultural residues and should take more significant participation in large-scale biotechnological processes

Effect of cultivation conditions on ligninolytic enzyme production by Ganoderma carnosum

Ganoderma carnosum has been found in Europe only, at coniferous trees and it is difficult to distinguish it morphologically from G. lucidum. Since G. carnosum has not been studied biochemically yet, the aim of this study was to analyse its ability to produce Mn-dependent peroxidase (MnP), versatile peroxidase (VP) and laccase (Lac) under conditions of solid-state fermentation of selected plant raw materials as carbon sources, in the presence of different nitrogen concentrations in the medium. Wheat straw, corn stem, oak and grapevine sawdust were the analysed plant raw materials. Nitrogen source in synthetic medium was NH4NO3 and its concentrations were: 10mM N and 20 mM N. Enzyme activity was determined spectrophotometrically, using ABTS and phenol red, as the substrates for Lac and Mn-oxidizing peroxidases, respectively. Maximum level of MnP activity (56.82 U/l) was obtained in the medium with wheat straw and nitrogen concentration of 10 mM. Best carbon source for VP production was grapevine sawdust at nitrogen concentration of 10 mM (80.80 U/l). The obtained Lac activity was very low in the medium with wheat straw (1.80 U/l), while it was not detected in the presence of other three analyzed plant raw materials. Maximum of total protein content (0.06 mgml-1) was noted in the medium where oak sawdust was carbon source and nitrogen concentration was 20 mM.Vrste roda Ganoderma su producenti mnogih biološki aktivnih supstanci pa su objekti proučavanja mnogih medicinskih i farmaceutskih studija. U novije vreme se sve više pažnje poklanja upoznavanju njihovog ligninolitičkog enzimskog sistema, u cilju uspešne primene u različitim biotehnološkim procesima. Za razliku od G. lucidum, čiji je ligninolitički sistem intenzivno proučavan, ostale vrste ovog roda su retko bile objekti istraživanja. G. carnosum se teško morfološki razlikuje od G. lucidum. i još uvek nije biohemijski proučena, pa je cilj našeg istraživanja bio analiza produkcije Mn-oksidujućih peroksidaza i lakaza u uslovima čvrste kultivacije, na različitim biljnim ostacima (pšenična slama, stabljike kukuruza, piljevina hrasta i vinove loze) u prisustvu azota u obliku NH4NO3 i u koncentracijama od 10 mM, odnosno 20 mM. Maksimum produkcije Mn-zavisne peroksidaze dobijen je na pšeničnoj slami pri koncentraciji azota od 10 mM (56.82 U/l). Piljevina vinove loze i koncentracija azota od 10 mM bili su optimalni za sintezu verzatil peroksidaze (80.80 U/l). Dobijena aktivnost lakaza je bila izuzetno niska u medijumu sa pšeničnom slamom (1.80 U/l), dok na ostalim biljnim ostacima nije zabeležena. Maksimalni sadržaj ukupnih proteina je bio najveći u medijumu sa piljevinom hrasta kao izvorom ugljenika i koncentracijom azota od 10 mM.Projekat ministarstva br. 14304

Activity of Mn-Oxidizing Peroxidases of Ganoderma lucidum Depending on Cultivation Conditions

Trunks and stumps of various deciduous species act as natural habitats for Ganoderma lucidum. The chemical composition of their cell wall affects the development of fungal ligninolytic enzyme system as well as its ability to degrade lignin from the plant cell wall. Additionally, numerous compounds structurally similar to lignin can be degraded by the G. lucidum enzyme system which could take important roles in various biotechnological processes. The laccases, which are the dominant enzymes synthesized by G. lucidum, have been studied more extensively than the Mn-oxidizing peroxidases. Therefore, this study aimed to create the dynamics profile of Mn-oxidizing peroxidases activities in four G. lucidum strains, classifying and determining their properties depending on the cultivation type and plant residue as a carbon source in the medium, as well as to establish whether intraspecific variety exists. The findings suggest that submerged cultivation appeared to be a more appropriate cultivation type for enzyme activities compared with solid-state cultivation, and oak sawdust was a better carbon source than wheat straw. Under the optimum conditions, on day 14, G. lucidum BEOFB 431 was characterized by the highest levels of both Mn-dependent and Mn-independent peroxidase activities (4795.5 and 5170.5 U/L, respectively). Strain, cultivation type, and carbon source were factors that affected the profiles of Mn-oxidizing peroxidases isoenzymes

Intraspecific Diversity in the Production and Characterization of Laccase within Ganoderma lucidum

Ganoderma lucidum has a well-developed ligninolytic enzyme system, where laccase is the dominant and sometimes only synthesizing enzyme, and therefore could find an application in the delignification of abundant plant raw materials and in food, feed, paper, and biofuel production. The questions that provided the goals for the present study were whether the profile of G. lucidum laccase depends on cultivation type and carbon source, as well as whether intraspecific diversity exists. Conditions of submerged cultivation proved more preferable for laccase activity compared with solid-state cultivations in all studied strains, while oak sawdust provided a better carbon source than wheat straw. Maximum laccase activity (7241.0 U/L) was measured on day 14 of oak sawdust submerged fermentation by strain BEOFB 431. Intraspecific diversity in synthesized proteins was more significant in wheat straw than in oak sawdust submerged fermentation. The profile of laccase isoforms was dependent on strain, plant residue, type, and period of cultivation. Four acidic laccase isoforms (pI 3.6) were detected in G. lucidum BEOFB 431 at the same cultivation point where maximal enzyme activity was measured

Degradation of beech wood and wheat straw by Trametes gibbosa

The aim of this study was to optimize cultivation conditions for Mn-oxidizing peroxidases and laccase production and selective degradation of beech wood and wheat straw lignin by Trametes gibbosa. To promote lignin degradation, the effect of different carbon and nitrogen sources, type of cultivation, enzyme dynamics and inducers were studied. Solid-state cultivation was optimal for lignin degradation. Wheat straw was the optimal carbon source for peroxidase activities stimulating the synthesis of numerous isoforms. The dynamics of enzymatic activities showed that 19-day-old fermentation is optimal and that activities were directly associated with enzyme production. Alteration of nitrogen sources and inducers was applied to increase the rate of lignin degradation and decrease cellulose degradation. The presence of nitrogen in the form of (NH4)(2)SO4 and concentration of 10 mM significantly increased the extent and selectivity of delignification of wheat straw compared with cellulose (44.1 vs. 36.1%). The most effective and selective wheat straw degradation (52.0% of lignin vs. 31.3% of cellulose) was achieved by enriching the optimum medium, as defined above, with 1.0 mM p-anisidine. The optimum conditions for wheat straw processing to achieve delignification and production of highly active ligninolytic enzymes by T. gibbosa were (NH4)(2)SO4/wheat bran medium supplemented with p-anisidine. This study indicated the significant potential of optimizing external factors as a promising tool for induction and selectivity in the degradation of lignocelluloses by T. gibbosa as a pretreatment for several biotechnological processes

Antioxidative and antimicrobial potentials of Parmelia saxatilis and Pseudoevernia furfuracea

Even though numerous lichen species possess significant medical potentials they are still unexplored, and particularly species and strains originating from Serbia. Therefore, the aim of this study was to evaluate the antioxidative and antimicrobial potential of ethanol extracts of Parmelia saxatilis and Pseudoevernia furfuracea collected in Serbia. The tested extracts were good scavengers of DPPH radicals, with capacities ranging from 14.76% to 79.76% in P. saxatilis and from 21.39% to 90.04% in P. furfuracea. In P. saxatilis level of DPPH• neutralisation was highly correlated with phenol content (r2 = 0.9981) and in P. furfuracea with amount of total flavonoides (r2 = 0.9641). The extract of P. furfuracea inhibited the growth of all tested microorganisms with exception of Aspergillus flavus, while P. saxatilis extract affected only growth of bacterial species. Among tested microorganisms, Staphylococcus aureus and Klebsiella pneumoniae were the most sensitive, while Enterococcus faecalis, Pseudomonas aeruginosa as well as micromycetes were the least sensitive to tested extracts. Because of these potentials and the fact that their long term usage does not have any negative side effects on organism and development of microbial resistance, the extracts could be included in conventional therapy. [Projekat Ministarstva nauke Republike Srbije, br. 173032