Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.     

Screening of laccase, manganese peroxidase, and versatile peroxidase activities of the genus Pleurotus in media with some raw plant materials as carbon sources

Species of the genus Pleurotus are among the most efficient natural species in lignin degradation belonging to the subclass of ligninolytic organisms that produce laccase (Lac), Mn-dependent peroxidase (MnP), versatile peroxidase (VP), and the H₂O₂-generating enzyme aryl-alcohol oxidase, but not lignin peroxidases. Production of Lac and oxidation of 2,6-dimethoxyphenol (DMP) in the presence and absence of Mn²⁺ were detected both in submerged fermentation (SF) of dry ground mandarine peels and in solid-state fermentation (SSF) of grapevine sawdust in all investigated Pleurotus species and strains. Evidence of cultivation methods having a distinct influence on the level of enzyme activities has been demonstrated. Most of the species and strains had higher Lac activity under SSF conditions than under SF conditions. DMP oxidation in the presence and absence of Mn²⁺ was detected in all investigated species and strains, but was lower under SF conditions than under SSF conditions for most of them. However, relative activities of DMP oxidation in the absence of Mn²⁺, as percentages of activity agasint DMP in the presence of Mn²⁺, were higher under conditions of SF than in SSF cultures in most of the investigated species and strains. The obtained results showed that strains of different origins have different efficiently ligninolytic systems and that conditions of SSF are more favorable for ligninolytic activity than those in SF owing to their similarity to natural conditions on wood substrates.     

Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.     

Advanced oxidation processes for decolorization of aqueous solution containing acid red G azo dye

Some investigations concerning the decolorization of Acid Red G azo dye by photooxidation with hydrogen peroxide were performed. The influences of pH, oxidant concentration, and the presence of Fe²⁺ or other metal ions (Co²⁺, Cu²⁺, Ni²⁺, Mn²⁺) as potential catalysts, were investigated. The best results were obtained in the presence of ferrous ions in acid and neutral media. The other ions are not as effective as Fe²⁺ for dye decolorization. Co²⁺ and Cu²⁺ ions have a catalytic action, at low concentration, within a wide range of pH. Ni²⁺ and Mn²⁺ ions have no catalytic effect in photooxidation with hydrogen peroxide at acid Ni²⁺ and Mn²⁺ ions have no catalytic effect in photooxidation with hydrogen peroxide at acid pH values, but show a weak action in alkaline media.     

Characterization of a Novel Laccase LAC-Yang1 from White-Rot Fungus Pleurotus ostreatus Strain Yang1 with a Strong Ability to Degrade and Detoxify Chlorophenols

In this study, a laccase LAC-Yang1 was successfully purified from a white-rot fungus strain Pleurotus ostreatus strain yang1 with high laccase activity. The enzymatic properties of LAC-Yang1 and its ability to degrade and detoxify chlorophenols such as 2,6-dichlorophenol and 2,3,6-trichlorophenol were systematically studied. LAC-Yang1 showed a strong tolerance to extremely acidic conditions and strong stability under strong alkaline conditions (pH 9–12). LAC-Yang1 also exhibited a strong tolerance to different inhibitors (EDTA, SDS), metal ions (Mn²⁺, Cu²⁺, Mg²⁺, Na⁺, K⁺, Zn²⁺, Al³⁺, Co²⁺, and metal ion mixtures), and organic solvents (glycerol, propylene glycol). LAC-Yang1 showed good stability in the presence of Mg²⁺, Mn²⁺, glycerol, and ethylene glycol. Our results reveal the strong degradation ability of this laccase for high concentrations of chlorophenols (especially 2,6-dichlorophenol) and chlorophenol mixtures (2,6-dichlorophenol + 2,3,6-trichlorophenol). LAC-Yang1 displayed a strong tolerance toward a variety of metal ions (Na²⁺, Zn²⁺, Mn²⁺, Mg²⁺, K⁺ and metal ion mixtures) and organic solvents (glycerol, ethylene glycol) in its degradation of 2,6-dichlorophenol and 2,3,6-trichlorophenol. The phytotoxicity of 2,6-dichlorophenol treated by LAC-Yang1 was significantly reduced or eliminated. LAC-Yang1 demonstrated a good detoxification effect on 2,6-dichlorophenol while degrading this compound. In conclusion, LAC-Yang1 purified from Pleurotus ostreatus has great application value and potential in environmental biotechnology, especially the efficient degradation and detoxification of chlorophenols.     

Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp

The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO₃ completely inhibited the enzyme synthesis while (NH₄)₂SO₄ at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu²⁺ had the most inhibiting effect while K⁺ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation.     

Biodegradation of Fluoranthene by Basidiomycetes Fungal Isolate <Emphasis Type="Italic">Pleurotus Ostreatus</Emphasis> HP-1

The biodegradation of fluoranthene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), was investigated in submerged culture using the wood decaying fungus isolated from forest locality in Gujarat, India. The basidiomycete fungal isolate was found to have an ability to grow on sabaroud dextrose agar containing 50 mgl⁻¹ of each naphthalene, anthracene, acenaphthene, benzo (a) anthracene, pyrene, flouranthene, carbazole, and biphenyl. The involvement of extracellular fungal peroxidases such as manganese peroxidase (MnP) and laccase (Phenol oxidase) in the degradation of fluoranthene was studied. On the eighth day of incubation 54.09% of 70 mg l⁻¹ fluoranthene was removed. There after no PAHs removal was observed till the 20th day of the incubation period. The isolate was identified as Pleurotus ostreatus by 18S rRNA, 5.8S rRNA, and partial 28S rRNA gene sequencing. To the best of our knowledge this is the first time Pleurotus ostreatus have been reported to degrade such a high concentration of fluoranthene within much lower time period of incubation. Depletion in the residual fluoranthene in the culture medium was determined by HPLC. Attempts were made to identify the degradation product in the culture medium with the help of FT-IR, NMR, and HPTLC analysis. In the present study positive correlation between fluoranthene degradation and the ligninolytic enzyme (MnP and laccase) production is observed, thus this isolate can play an effective role for bioremediation of PAHs contaminated sites.     

Purification, characterization, kinetic properties, and thermal behavior of extracellular polygalacturonase produced by filamentous fungus Tetracoccosporium sp

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K _m and V _max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag⁺, Co²⁺, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al³⁺, Ba²⁺, Ca²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Mg²⁺, Mn²⁺, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t _1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min⁻¹, and 35.18 mL/(min·mg), respectively. The activation energy (ΔE ^#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters ΔG ^#, ΔH ^#, and ΔS ^# were also calculated, revealing a potential application for the industry.     

Polyhalite and its analogous triple salts

Polyhalite (K₂SO₄ · MgSO₄ · 2CaSO₄ · 2H₂O) and analogue triple salts, where Mg²⁺ is substituted by Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺, have been synthesized. The salts were characterized by thermal analysis, Raman spectroscopy and X-ray powder diffraction. Diffraction patterns and Raman spectra resemble those of natural polyhalite, except K₂SO₄ · CuSO₄ · 2CaSO₄ · 2H₂O. The latter corresponds to the mineral leightonite, which is structurally different. For polyhalite analogues the cell parameters of the triclinic unit cell have been determined from the powder diffraction patterns. The length of the unit cell vectors varies regularly with the ionic radius of the substituted ion M ²⁺ and is explained by changes in the extension of the coordination octahedron of M ²⁺. Thereby increasing distances of the coordinated water molecules at M ²⁺ parallel with decreasing dehydration temperatures of the corresponding polyhalite. Correspondence: Daniela Freyer, Institut für Anorganische Chemie, TU Bergakademie Freiberg, 09599 Freiberg, Germany.     

Effect of tributylphenyltetraethoxylate on enzyme production of Pleurotus ostreatus

The effect of the addition of the nonionic surfactant tributylphenyl-tetraethoxylate to culture media on pH and extracellular protein content, and on production of β-glucosidase, xylanase, laccase, and manganese-dependent and-independent peroxidases by the edible fungus Pleurotus ostreatus was determined. The relationship between fermentation parameters and concentration of surfactant was assessed by multiple linear regression analysis, and the similarities and differences among the fermentation parameters were elucidated by principal component analysis. Calculations proved that except for xylanase all other cultivation parameters were significantly influenced by the surfactant, with the effect higher at higher surfactant concentrations. Surfactant increased the production of β-glucosidase and inhibited laccase and manganese-dependent and-independent peroxidase activities.     

Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Biochemical and enzymatic properties of a novel marine fibrinolytic enzyme from <Emphasis Type="Italic">Urechis unicinctus</Emphasis>

A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the temperature was <50°C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60#x00B0;C the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0–12.0, especially at slightly alkaline pH values. Mn²⁺, Cu²⁺, and Fe²⁺ ions were activators of UFE, whereas Fe³⁺ and Ag⁺ ions were inhibitors. Fe²⁺ ion along with Fe³⁺ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50°C, respectively. When using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate concentration. Also when using casein as substrate, the determined K _m and V _max of UFE were 0.5298 mg/mL and 3.0845 mol of l-tyrosine equivalent, respectively. Our systematic research results are significant when UFE is applied for medical and industrial purposes.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

Purification and Biochemical Characterization of Polyphenol Oxidases from Embryogenic and Nonembryogenic Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) Cells

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl₂, and FeCl₃ markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg²⁺, Ca²⁺, and Mn²⁺ and was moderately inhibited by Ba²⁺, Cu²⁺, and Zn²⁺. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.     

Isolation and Identification of a Newly Isolated <Emphasis Type="Italic">Alternaria</Emphasis> sp. ND-16 and Characterization of Xylanase

Alternaria sp. ND-16, a bacterium isolated from soil sample, was identified as a strain of Alternaria mali based on the morphology and comparison of internal transcribed spacer rDNA gene sequence studies. Furthermore, it is demonstrated that this strain has xylanase activity, and the activity can be optimized under suitable growing conditions where wheat bran and urea are the primary sources of carbon and nitrogen. Partially purified xylanase from Alternaria sp. ND-16 is shown to have an optimal pH of 6.0 and optimal temperature of 50 °C, making this enzyme potentially suitable for industrial applications. It is also demonstrated that Na⁺ and Mn²⁺ show strong inhibition of the xylanase while K⁺, Li⁺, Fe²⁺, Cu²⁺, and Zn²⁺ have no significant effect on the activity.     

Purification and Characterization of Thermostable α-Galactosidase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis><Subscript>GR</Subscript>

An extracellular thermostable α-galactosidase producing Aspergillus terreus _GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate pNPαGal. α-Galactosidase from A. terreus _GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K _m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg²⁺, Ni²⁺, Ca²⁺, Co²⁺, and Mn²⁺ had no effect on enzyme activity, but Ag⁺, Hg²⁺, and Cu²⁺ have inhibited complete activity.     

Solid-state fermentation of lignocellulosic plant residues from Brassica napus by Pleurotus ostreatus

Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratry alcohol (3,4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-flow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.     

Immobilization of manganese peroxidase fromLentinula edodes on azlactone-functional polymers and generation of Mn3+ by the enzyme-polymer complex

Manganese peroxidase (MnP) purified fromLentinula edodes was covalently immobilized on 3M’s azlactone-functional copolymer, 3M EmphazeTM AB1 Biosupport Medium. Tethered MnP is capable of generating Mn³⁺ from Mn²⁺ and H₂O₂. Mn₃₊, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn³⁺ -generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn²⁺ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations < 2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn²⁺. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40–50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies > 65% with 100% of the coupled enzyme active have been measured.