Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.     

Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation

Summary The effect of additional nitrogen sources on lignocellulolytic enzyme production by four species of white-rot fungi (Funalia trogii IBB 146, Lentinus edodes IBB 363, Pleurotus dryinus IBB 903, and P. tuberregium IBB 624) in solid-state fermentation (SSF) of wheat straw and beech tree leaves was strain- and substrate-dependent. In general, the yields of hydrolytic enzymes and laccase increased by supplementation of medium with an additional nitrogen source. This stimulating effect of additional nitrogen on enzyme accumulation was due to higher biomass production. Only xylanase specific activity of P. dryinus IBB 903 and laccase specific activity of L. edodes IBB 363 increased significantly (by 66% and 73%, respectively) in SSF of wheat straw by addition of nitrogen source to the control medium. Additional nitrogen (20 mM) repressed manganese peroxidase (MnP) production by all fungi tested. The study of the nitrogen concentration effect revealed that 10 mM peptone concentration was optimal for cellulase and xylanase accumulation by P. dryinus IBB 903. While variation of the peptone concentration did not cause the change in MnP yield, elevated concentrations of this nutrient (20–40 mM) led to a 2–3-fold increase of P. dryinus IBB 903 laccase activity. About 10–20 mM concentration of NH₄NO₃ was optimal for cellulase and xylanase production by F. trogii IBB 146. However, neither the laccase nor the MnP yield was significantly changed by the additional nitrogen source.     

Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens

A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB-HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90% decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 °C under static conditions at pH 7.0. MnP activity was increased by the addition of Mn²⁺ · At 50 M Mn²⁺, high MnP (55.3 U/ml) but low laccase activity (8.3 U/ml) was observed. Influence of oxalic acid on MnP activity was also observed.     

Enzymes produced by <Emphasis Type="Italic">Ganoderma australe</Emphasis> growing on wood and in submerged cultures

Enzymes produced by Ganoderma australe in solid-state fermentation and submerged cultures were evaluated. Strain A464 produced laccase activity in liquid medium and in solid-state cultures containing Drimys winteri or Eucalyptus globulus wood chips, while MnP and LiP activities were not detected. On the other hand, strain A272 cultured for 75 days on E. globulus presented MnP activity of 719 IU/kg of wood. The suitability of D. winteri wood as a substrate enabling MnP production was checked with a well-documented MnP-producing basidiomycete, Ceriporiopsis subvermispora, which produced MnP activity of 327 IU/kg of wood in 9-day-old cultures. Data from two different G. australe strains (A272 and A464) indicated that MnP secretion depended on strain origin as well as on culture conditions.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Ligninolytic enzymes of selected white rot fungi cultivated on wheat straw

Ligninolytic enzymes of the white rot fungiCoriolopsis polyzona, Phanerochaete chrysosporium, andTrametes versicolor growing on wheat straw under nearly natural conditions were investigated. Manganese peroxidase (MnP), secreted as early as on day 3, was dominant over other activities during the initial phase (the first 10 days). Its activity profile was similar in all the three fungi. Lignin peroxidase (LIP) was not detected in the extracellular enzyme extracts ofC. polyzona andP. chrysosporium cultures.T. versicolor secreted LIP after 10 d of growth. Another, recently described, enzyme activity of manganese-independent peroxidase (MIP) was detected in all the three fungi tested and it appeared on about day 5 (later than MnP and earlier than LIP); it was the dominant activity after day 10. Laccase activity appeared at basal levels without any significant changes. Pyranose 2-oxidase was probably the major extracellular H₂O₂-generating activity (with all the three fungi) that appeared contemporarily with MnP, increased with time, peaking on day 17–18. Glyoxal oxidase could not be detected with any of the fungi.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

Use of Phanerochaete chrysosporium Immobilized on Kissiris for Synthetic Dye Decolourization: Involvement of Manganese Peroxidase

The mineral Kissiris, which is formed from the thickened foam of volcanic lava, was tested to approximate its mineral composition using energy-dispersive X-ray (EDX) analysis. The solid mineral contains silicon dioxide at about 16 (w/w). The considerable surface roughness of Kissiris along with its extensive porosity made this natural solid cell support an attractive candidate for manganese peroxidase (MnP) production for synthetic dye decolourization, at low cost. The white rot fungus Phanerochaete chrysosporium immobilized on the mineral Kissiris was grown in both stationary and agitated cultures (rotary shaker, 100 rev/min) using either carbon- or nitrogen-limited growth medium to study the ability of the fungus to degrade the synthetic dye methylene blue (MB). The value of residual dye for MB used at 60 ppm was 6% within 8 days of the incubation of the nitrogen-limited culture under the shaken conditions. Production of (MnP) occurred simultaneously in nitrogen-limited culture medium with the added MnSO₄ at 100 ppm. The MnP activity was at relatively high level (170 U/l).     

Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology

Abstract

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett–Burman experimental design (PBED) and a central composite design (CCD). In addition, H₂O₂ role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H₂O₂ content was higher in the co-culture (0.33–7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO_4, and inoculum amount were significant factors for laccase and MnP activities and H₂O₂ production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO₄ were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366?g L^?1. Quadratic term of FeSO₄ modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H₂O₂ production; however, the CCD did not enable an increase in H₂O₂ production. Pearson correlation indicated an increase in laccase (r²=0.4411, p?=?0.0436) and MnP (r²=0.5186, p?=?0.0198) activities following increases in H₂O₂ in the co-culture system.     

Production of ligninolytic exoenzymes and14C-Pyrene mineralization byPleurotus sp. in lignocellulose substrate

Pleurotus sp. was grown in liquid medium and on a solid straw substrate, and activities of laccase and manganese-dependent peroxidase (MnP) were recorded. The activities were the highest in a rich, glucose corn-steep liquid medium. In straw cultures, laccase activity was about ten times lower. Under solid state conditions, MnP production was the highest during days 20–40, when laccase activity already had declined. In straw cultures, mineralization of¹⁴C-pyrene was measured as release of¹⁴CO₂. The highest rates of pyrene mineralization occurred during days 20–45,i.e. the period of high MnP activities, suggesting a role of this enzyme in PAH degradation. Within 60d, 24% of pyrene was mineralized.     

Production of the ligninolytic enzymes by immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in an air atmosphere

Summary The production of the ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in air was investigated by adopting different sizes and amounts of the carriers, different medium C/N ratios and different glucose-feeding strategies. No lignin peroxidase (LiP) activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NH₄⁺, 56/2.2 mM) with two sizes and three amounts of the carriers, while comparable levels of manganese peroxidase (MnP) activities were detected only in non-immersed cultures with two sizes of the carriers. A non-immersed state also stimulated LiP formation under carbon limitation (C/N ratio 28/44 mM). High peak activities of LiP, 197 and 164 U/l, were obtained in non-immersed cultures under carbon limitation at the C/N ratios of 28/44 and 56/44 mM, respectively, the occurrence of the activities coinciding with the complete consumption of glucose. A very low level of MnP was measured at the C/N ratio of 28/44 mM compared with the similar activities at 56/2.2 and 56/44 mM. An addition of 2 g glucose/l after its complete depletion improved both the production of LiP and MnP markedly in non-immersed culture at the initial C/N ratio of 28/44 mM, whereas a replenishment of 5 g/l, still enhancing the formation of MnP, inhibited the production of LiP first before the later reactivation. It is suggested that non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy is one potential way to realize high production of the ligninolytic enzymes by P. chrysosporium in air.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg⁻¹ protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. K _m values for H₂O₂ and Mn²⁺ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn²⁺-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H₂O₂ and Mn²⁺; in addition, low turnover numbers and K _m values higher than 300 μM characterized the Mn²⁺-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.     

Free-radical polymerization of acrylamide by manganese peroxidase produced by the white-rot basidiomycete Bjerkandera adusta

Acrylamide was polymerized to give polyacrylamide using manganese peroxidase (MnP) produced by the basidiomycete Bjerkandera adusta. The molecular weight of the polymer synthesized by MnP was 155000, higher than those obtained with other reaction systems using horseradish peroxidase and a redox initiator. The ¹³C-NMR spectrum showed that polyacrylamide was atactic. Electron spin resonance analysis revealed that 2,4-pentanedione added as an initiator was first oxidized to generate a carbon-centered radical, which initiated radical additive polymerization of acrylamide.     

Decolorization of the azo dye Acid Red 18 by crude manganese peroxidase: Effect of system parameters and kinetic study

Abstract

To optimize operating conditions for the decolorization of the azo dye Acid Red 18 (AR18) by crude manganese peroxidase (MnP), some important factors affecting enzymatic decolorization were systematically investigated. Under the optimal enzyme reaction conditions, a decolorization efficiency of more than 82.3% was achieved after 60 min treatment. Furthermore, the manganese chelators, malate, tartrate, and lactate were found to be more favorable for the decolorization of AR18 than malonate, acetate, succinate, maleate, oxalate, and citrate. However, the presence of NaCl or Na₂SO₄ had a negative impact on the decolorization of AR18. The K_m and V_max values of MnP for AR18 were 169.66 μmol L^{? 1} and 20.63 μmol L^{? 1} min^{? 1}, respectively. The decolorization of AR18 by MnP followed second-order reaction kinetics with respect to the dye concentration. The decolorization rate constant increased with increasing temperature from 20°C to 35°C, which indicated an activation energy (E_a) of 15.87 kcal mol^{? 1} and frequency factor (k₀) of 1.36 × 10⁸ mg^{? 1} L min^{? 1} according to the Arrhenius equation. The results obtained provide experimental data for the application of crude MnP for the decolorization of AR18, and help to elucidate the biochemical mechanism of dye decolorization by the enzyme.     

Optimized production of lignolytic manganese peroxidase in immobilized cultures of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Manganese peroxidase (MnP) is a key enzyme involved in the lignolysis of white-rot fungi. The purpose of this study is to investigate the effect of immobilization and culture conditions on MnP production in cultures of Phanerochaete chrysosporium grown on polyurethane foam. Higher concentrations of foam and lower levels of spore inoculums resulted in the formation of scattered mycelial pellets, increased autolysis of chlamydospore-like cells (a reservoir of MnP), and a higher activity of MnP. Even though MnP was a secondary metabolite, the addition of 5 times more glucose and diammonium tartrate, as carbon and nitrogen sources, resulted in a 4 fold increase in the dry cell mass. However, MnP activity decreased under these conditions to less than half, due to the formation of increasingly dense pellets and the inhibited lysis of chlamydospore-like cells.