Activity of Lignin-Modifying Enzyme of Selected Medicinal Mushrooms in Submerged Fermentation of Lignocellulosic Materials.

In the present study, wide diversity in the set and activity of lignin-modifying enzymes (LME) was revealed during submerged fermentation of mandarin peel with 15 strains of white rot Basidiomycetes. Among them, Trametes pubescens BCC153 was distinguished by the simultaneous production of laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP). Supplementation of CuSO4 at a concentration of 1 mM in the media for the cultivation of four Trametes species manifold increased the production of laccase. The diverse effects of chemically different lignocellulosic growth substrates and nitrogen sources on the production of individual LME have been established. The maximum laccase activity of T. pubescens was observed when the fungus was cultivated on media containing mandarin peel and wheat bran, whereas the highest MnP and LiP activities were detected in the submerged fermentation of tobacco residue. Peptone and casein hydrolysate appeared to be the best sources of nitrogen to produce laccase and both peroxidases by T. pubescens BCC153 whereas KNO3 was the worst nitrogen-containing compound for the production of all enzymes.

Antifungal Activity of Medicinal Mushrooms and Optimization of Submerged Culture Conditions for Schizophyllum commune (Agaricomycetes).

The main goal of the present study was the exploration of the antifungal properties of Agaricomycetes mushrooms. Among twenty-three tested mushrooms against A. niger, B. cinerea, F. oxysporum, and G. bidwellii, Schizophyllum commune demonstrated highest inhibition rates and showed 35.7%, 6.5%, 50.4%, and 66.0% of growth inhibition, respectively. To reveal culture conditions enhancing the antifungal potential of Sch. commune, several carbon (lignocellulosic substrates among them) and nitrogen sources and their optimal concentrations were investigated. Presence of 6% mandarin juice production waste (MJPW) and 6% of peptone in nutrient medium promoted antifungal activity of selected mushroom. It was determined that, extracts obtained in the presence of MJPW effectively inhibited the grow of pathogenic fungi. Moreover, the content of phenolic compounds in the extracts obtained from Sch. commune grown on MJPW was several times higher (0.87 ± 0.05 GAE/g to 2.38 ± 0.08 GAE/g) than the extracts obtained from the mushroom grown on the synthetic (glycerol contained) nutrient medium (0.21 ± 0.03 GAE/g to 0.88 ± 0.05 GAE/g). Flavonoid contents in the extracts from Sch. commune varied from 0.58 ± 0.03 to 27.2 ± 0.8 mg QE/g. Identification of phenolic compounds composition in water and ethanol extracts were provided by mass spectrometry analysis. Extracts demonstrate considerable free radical scavenging activities and the IC50 values were generally low for the extracts, ranging from 1.9 mg/ml to 6.7 mg/ml. All the samples displayed a positive correlation between their concentration (0.05-15.0 mg/ml) and DPPH radical scavenging activity. This investigation revealed that Sch. commune mushroom has great potential to be used as a source of antifungal and antioxidant substances.

Enhancement of laccase production by Cerrena unicolor through fungal interspecies interaction and optimum conditions determination.

The present study aimed to identify a pair of fungal strains that promote laccase production in the co-cultivation of white-rot basidiomycetes and to determine the optimum conditions to enhance enzyme synthesis under co-fermentation of mandarin peels. Co-cultivation of Cerrena unicolor with Trametes versicolor, Lenzites betulina, and Panus lecomtei led to up-regulation of laccase activity. Moreover, interspecific interaction of Cerrena unicolor and Trametes versicolor induced the production of two new laccase isoenzymes. By contrast, interactions of Cerrena unicolor with Trametes coccineus and Trametes hirsuta resulted in a multiple decreased ability of Cerrena unicolor to produce laccase. Co-cultivation of Cerrena unicolor with other fungi 3- to 12-fold down-regulated manganese peroxidase (MnP) activity. The outcomes of these fungal interactions are closely related to the initial concentration and availability of the nutrients, the partners' inoculum ratio, time, and sequence of their inoculation. Co-cultivation of Cerrena unicolor and Trametes versicolor in fermenter resulted in the accumulation of 476 U/mL laccase and 1.12 U/mL MnP.

Elucidation of the Higher Basidiomycetes Enzyme Activity in Dependence on the Medicinal Mushroom Inoculum Form, Precultivation Medium, Age, and Size.

The impact of five mushroom inoculum form, age, size, and precultivation medium on the lignocellulose-deconstracting enzyme (LCDE) production was evaluated in the submerged fermentation of mandarin marc. The results obtained evidence that an adaptation of individual fungi to lignocellulose during maintenance in culture collection and inoculum cultivation may be useful for the production of individual LCDE. Homogenization of submerged mycelium was beneficial for all LCDE production by Cerrena unicolor 305 and Ganoderna lucidum 447 and for LME secretion by Coriolopsis gallica 142 and Trametes multicolor 511. Finely chopped mycelial agar favored CMCase and xylanase production by T. multicolor 511 and LiP secretion by C. unicolor 305 and G. lucidum 447 while homogenized mycelial agar proved to be the worst form of inoculum for the production of most enzymes. Four-days inoculum was the most appropriate for the laccase and MnP production by G. lucidum 447 and T. multicolor 511 while the 7-days mycelium provided the highest yields of these enzymes in the cultivation of C. unicolor 305. Use of the 12-days homogenized mycelium from the late stationary phase resulted in lowest laccase activity of all fungi but provided the highest cellulase activity. Overall, the study showed that the LCDE activity and their accumulation profiles in the cultures with different inoculum size was species dependent.

Comparison of Mono- and Dikaryotic Medicinal Mushrooms Lignocellulolytic Enzyme Activity.

Mono- and dikaryotic medicinal mushroom strains isolated from four wood-rotting basidiomycete fruiting bodies were comparatively evaluated for laccase, manganese peroxidase, cellulase, and xylanase activities in submerged cultivation in glucose or mandarin peel-containing media. Mandarin peels appeared to be the preferred growth substrate for laccase production by both mono- and dikaryotic Trametes multicolor 511 and T. versicolor 5 while glucose favored laccase activity secretion by Pleurotus ostreatus 2175. Lignocellulose-deconstructing enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains. A distinctive superiority of enzyme activity of the dikaryotic Trametes versicolor 5 and P. ostreatus 2175 over the same species monokaryotic isolates was revealed. By contrast, laccase, cellulase, and xylanase activities of the monokaryotic strain of T. multicolor 511 were rather higher than those in the dikaryotic culture. At the same time, hydrolases activity of Schizophyllum commune 632 was practically independent of the origin of the fungal culture. The results suggest that the monokaryotic isolates derived from the basidiomycetes fruiting bodies inherit parental properties but the capacity of individual monokaryotic cultures to produce lignocellulose-deconstructing enzymes can vary considerably.

Biotransformation of ritalinic acid by laccase in the presence of mediator TEMPO.

Methylphenidate is widely used as a medication for the treatment of attention deficit hyperactivity disorder (ADHD) in children. Less than 1% of methylphenidate is excreted unchanged in urine, while 80% of an oral dose is excreted as ritalinic acid (which is reportedly poorly degradable). This study aims to investigate the biotransformation of ritalinic acid by free and immobilized enzymes. The influence of various laccase mediators on biotransformation efficiency has been tested. Formation of the main transformation products has been monitored and their potential structures suggested. The effective transformation of ritalinic acid was observed only in the presence of 2,2,6,6-tetramethylpiperidine 1-oxyl mediator (TEMPO). The most effective enzyme was the laccase of T. versicolor 159. The main transformation product was an N-methyl derivative of ritalinic acid. Ritalinic acid was also reduced to aldehyde and alcohol, and a broad spectrum of intermediate complexes with oxoammonium ion of TEMPO were detected. This is the first time the biotransformation of ritalinic acid has been investigated in detail.

Evaluation of Lignin-Modifying Enzyme Activity of Trametes spp. (Agaricomycetes) Isolated from Georgian Forests with an Emphasis on T. multicolor Biosynthetic Potential.

In this study, a wide diversity in lignin-modifying enzyme (LME) secretion by 11 Trametes spp. strains isolated from the forests of Georgia was revealed in their submerged cultivation in both synthetic and lignocellulose-based media. Among them, T. multicolor BCC 511 was distinguished by simultaneous production of laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP) in the presence of high carbon and nitrogen concentrations. Mannitol at the concentration of 15 g/L provided an accumulation of 23.7 U/mL laccase and 0.56 U/mL MnP. Significant modulation of LME activity by lignocellulosic substrates, metals, aromatic compounds, and their concentrations was established. Mandarin peels manifold increased the fungus laccase and LiP activities, while the ethanol production residue and banana peels activated manganese-oxidizing and Phenol Red-oxidizing manganese peroxidases, respectively. The addition of 2 mM of copper sulfate to the control medium induced the laccase production 28-fold and did not significantly affect the MnP and LiP activities. Fe2+ at a concentration of 0.1 mM enhanced the fungus volumetric and specific laccase activities almost 8-fold; at a concentration of 0.25-0.5 mM, there was a 2-fold increase in the MnP activity. Mn2+ appeared to be an effective inducer of the Mn-oxidizing MnP, increasing specific activity of the enzyme 14-fold. Supplementation of the copper-containing medium with 1 mM veratryl alcohol or guaiacol favored laccase and MnP production. The high yields of laccase (110 U/mL), MnP (0.62 U/mL), and LiP (0.71 U/mL) obtained in a laboratory fermenter make T. multicolor 511 useful for industrial and environmental applications.

Trinitrotoluene and mandarin peels selectively affect lignin-modifying enzyme production in white-rot basidiomycetes.

Five white-rot basidiomycetes (WRB) species have been evaluated for their potential to tolerate and to degrade 0.2 mM 2, 4, 6-trinitrotoluene (TNT) as well as to produce laccase and manganese peroxidase (MnP) in presence of this xenobiotic. The tested fungal strains produced laccase in both glycerol and mandarin peels-containing media, whereas in the glycerol-containing medium only Cerrena unicolor strains and Trametes versicolor BCC 775 secreted MnP. Replacement of glycerol by milled mandarin peels 3- to 45-fold increased laccase activity, promoted C. unicolor strains and T. versicolor MnP secretion and induced this enzyme production by Fomes fomentarius BCC 38 and Funalia trogii BCC 146. Differential response of the WRB strains to the TNT addition was observed. In particular, laccase activity of C. unicolor increased 2- to 3-fold in both media whereas no stimulation of the laccase production was revealed in cultivation of F. fomentarius. TNT practically did not affect the MnP activity. Two strains of C. unicolor followed by T. versicolor producing laccase and MnP almost completely removed 0.2 mM TNT from the synthetic medium. Increase of TNT concentration from 0 to 0.4 mM in the mandarin peels-based medium and from 0 to 0.3 mM in the glycerol-containing medium stimulated C. unicolor BCC 300 laccase production from 92.4 to 240.7 U/ml and from 17.1 to 48.6 U/ml, respectively. This strain has been resistant to the TNT high concentration and has ability to remove 85 % of initial 0.3 mM TNT content during 6 days of the submerged cultivation.