Production of lignocellulose-degrading enzymes employing <Emphasis Type="Italic">Fusarium solani</Emphasis> F-552

In this work, capability of Fusarium solani F-552 of producing lignocellulose-degrading enzymes in submerged fermentation was investigated. The enzyme cocktail includes hydrolases (cellulases, xylanases, and proteinases) as well as ligninolytic enzymes: manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). To our knowledge, this is the first report on production of MnP, LiP, and Lac together by one F. solani strain. The enzyme productions were significantly influenced by application of either lignocellulosic material or chemical inducers into the fermentation medium. Among them, corn bran significantly enhanced especially productions of cellulases and xylanases (248 and 170 U/mL, respectively) as compared to control culture (11.7 and 29.2 U/mL, respectively). High MnP activity (9.43 U/mL, control 0.45 U/mL) was observed when (+)-catechin was applied into the medium, the yield of LiP was maximal (33.06 U/mL, control 2.69 U/mL) in gallic acid, and Lac was efficiently induced by, 2,2′-azino-bis-[3-ethyltiazoline-6-sulfonate] (6.74 U/mL, not detected in control). Finally, in order to maximize the ligninolytic enzymes yields, a novel strategy of introduction of mild oxidative stress conditions caused by hydrogen peroxide into the fermentation broth was tested. Hydrogen peroxide significantly increased activities of MnP, LiP, and Lac which may indicate that these enzymes could be partially involved in stress response against H₂O₂. The concentration of H₂O₂ and the time of the stress application were optimized; hence, when 10 mmol/L H₂O₂ was applied at the second and sixth day of cultivation, the MnP, LiP, and Lac yields reached 21.67, 77.42, and 12.04 U/mL, respectively.     

Effect of fungal co-cultures on ligninolytic enzyme activities,H2O2 production,and orange G discoloration

Abstract

In this study, the effects of Aspergillus niger in coculture with the basidiomycetes, Trametes versicolor, T. maxima, and Ganoderma spp., were studied to assess H₂O₂ production and laccase (Lac), Lignin Peroxidase (LiP), and manganese peroxidase (MnP) activities. The results indicated that maximum discoloration was of 97%, in the T. maxima and A. niger coculture, where the concentration of H₂O₂ was 5?mg/L and 6.3?mg/L in cultures without and with dye, respectively. These concentrations of H₂O₂ were 1.6- and 1.8-fold higher than monocultures of T. maxima (3.37?mg/L) and A. niger (3.87?mg/L), respectively. In the same coculture, the LiP and MnP enzyme activities also increased 12-fold, (from 0.08?U/mg to 0.99?U/mg), and 67-fold, (from 0.11?U/mg to 7.4?U/mg), respectively. The Lac activity increased 1.7-fold (from 13.46?U/mg to 24?U/mg). Further, a Box–Behnken experimental design indicated a 1.8-fold increase of MnP activity (from 7.4?U/mg to 13.3?U/mg). In addition, dye discoloration regression model obtained from the Box–Behnken experimental design showed a positively correlation with H₂O₂, (R²?=?0.58) and a negatively correlation with Lac activity (R² = –0.7).     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l⁻¹ and from 55 to 390 U l⁻¹, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.     

Evaluation of bezafibrate,gemfibrozil, indomethacin,sulfamethoxazole, and diclofenac removal by ligninolytic enzymes

Abstract

The laccase (Lac), manganese peroxidases (MnP), and lignin peroxidase enzymes produced by basidiomycete have been studied due to their potential in bioremediation, therefore, in this study, degradation of diclofenac (DCF), sulfamethoxazole (SMX), indomethacin (IND), gemfibrozil (GFB), and bezafibrate (BZF) by enzymes produced by Trametes maxima, Pleurotus sp., and Pycnosporus sanguineus grown in culture was evaluated. The degradation of drugs can mainly be attributed to MnP because a correlation between the activity of this enzyme and the degree of removal was found. The specific activity of Lac did not show correlation with drug removal, while lignin peroxidase was not expressed. Trametes maxima showed the highest specific activity of MnP (387.6?±?67.4?U/mg) and efficiency removal 90.2% of DCF, 72.62% of SMX, 60.76% of IND, 43.39% of GFB, and 32.59% of BZF) followed by Pleurotus sp. with specific activity of MnP of 55.9?±?8.5?U/mg and 89.47% of DCF, 47.61% of GFB and 73% of IND were removed, P. sanguineus had the lowest specific activity of 18?±?1.3?U/mg and was able to remove only 42% of SMX and 10.59% of IND. In order to prove that MnP remove drugs instead of Lac, the pure Lac was tested and only degraded DCF.     

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.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

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.     

PRODUCTION OF LIGNINOLYTIC ENZYMES BY SOLID-STATE FERMENTATION USING Pleurotus eryngii

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ability to produce various ligninolytic enzymes such as laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) by solid-state fermentation (SSF), which was carried out using a support substrate from the fruit juice industry. The chemical content of grape waste from this industry was studied. Also, the production patterns of these extracellular enzymes were researched during the growth of the organism for a period of 20 days and the protein, reducing sugar, and nitrogen levels were monitored during the stationary cultivation. The highest Lac activity was obtained as 2247.62 ± 75 U/L on day 10 in the presence of 750 µM Mn²⁺, while the highest MnP activity was attained as 2198.44 ± 65 U/L on day 15 in the presence of 500 µM Mn²⁺. Decolorization of methyl orange and reactive red 2 azo dyes was also achieved with ligninolytic enzymes, produced in SSF of P. eryngii.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

New and different lignocellulosic materials from Turkey for laccase and manganese peroxidase production by Trametes versicolor

In the present study, the production of laccase (Lac) and manganese‐dependent peroxidase (MnP) by the white‐rot fungus Trametes versicolor grown in submerged cultures with different agricultural residues was investigated. The lignocellulosic materials studied were almond shells, hazelnut husks, sunflower stems, clover straw and hazelnut cobs, because they are common agricultural wastes in Turkey. Among the different lignocellulosic materials studied, hazelnut cobs provided the highest Lac and MnP activities (47.09 and 109.21 U/L, respectively). The optimum conditions were determined for Lac and MnP production in submerged cultures of T. versicolor by using hazelnut cobs as substrate. For Lac production, the optimum incubation time, hazelnut cob concentration, pH, and shaking rate were found as 4 days, 2% w/v, 6.0 and 130 rpm, respectively. For MnP production, the optimum incubation time, hazelnut cob concentration, pH and shaking rate were found as 5 days, 2% w/v, 6.0 and 90 rpm, respectively.     

Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii

Raw mixed-dye wastewater from a textile dye-producing plant was partly decolorized by the agaric white-rot fungus, Clitocybula dusenii. The fungus had higher Mn peroxidase (MnP) and laccase activities when grown with dye effluent than in control cultures. The activity of MnP increased commensurately with the proportion of the raw dye wastewater in the medium (control: 20 U l^–1; 10% v/v effluent: 67 U l^–1; 25% v/v effluent: 130 U l^–1; and 33% v/v effluent: 180 U l^–1). Maximal decolorization rates were achieved over 20 d at 28 °C using four-fold diluted dye-containing effluent on a 5 d pre-grown mycelium.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

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.     

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.     

Cu(II)对一株镰刀菌形态发育及木质素酶系活性的影响

本试验采用室内摇瓶培养,通过添加不同浓度的Cu(II),研究了Cu(II)对降解菌Fusarium sp. ZH-H2形态发育及木质素酶系活性的影响,以期诱导提高木质素酶系的活性。结果表明,当Cu(II)浓度为0.50mmol/L时,菌丝生物量最高达0.38g,菌丝团呈洁白平滑球状且数量最多,此浓度下木质素过氧化物酶（lignin peroxidase,LiP）活性峰值也达到最高,为28 315.41U/L,当Cu(II)浓度为2.00mmol/L时,有利于诱导锰过氧化物酶（manganese peroxidase,MnP）和漆酶（laccase,Lac）的代谢,最高峰值分别为1 612.90U/L、7 829.12U/L,并且明显缩短了峰值的形成时间,浓度过高或过低则对3种木质素酶系活性产生抑制效应,为下一步分子生物学研究奠定了基础。     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.