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     

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.     

Isolation of Bacterial Strain from Sediment Core of Pulp and Paper Mill Industries for Production and Purification of Lignin Peroxidase (LiP) Enzyme

Five bacterial strains were isolated and purified (CSA101 to CSA105) from the sediment core of the effluent released from the Century Pulp and Paper Mill Ltd., India. These strains were grown in minimal salt medium (MSM) containing pulp (10% as a carbon source). The production of lignin peroxidase, CMCase, Fpase, and xylanase together with protein and reducing sugar by all bacterial strains was observed. All of the bacterial isolates responded differently with respect to growth and ligninocellulolytic enzyme production. The maximum lignin peroxidase (LiP) was obtained from the cell extract of Bacillus sp. (CSA105) strain, which was used for purification, fractionation and characterization. The culture filtrate from Bacillus sp. (CSA105) was purified with ammonium sulfate precipitation. Crude protein was desalted by dialyzing with Tris buffer. The lignolytic enzyme produced in the liquid medium was fractionated by gel filtration on Sephadex G-100. In the present study, 12.4-fold purification of LiP enzyme was obtained and 35.85% yield of lignin peroxidase was achieved in the cell extract of Bacillus sp. (CSA105). Lignin peroxidase enzyme plays an important role in lignin degradation process. The ligninolytic enzymes were produced by all of the bacterial strains but maximum lignin peroxidase activity was found in cell extract of CSA105. On the basis of the results obtained, the bacterial strain (CSA105) was found most suitable for the purification of the LiP enzyme.     

Enzymatic extract containing lignin peroxidase immobilized on carbon nanotubes: Potential biocatalyst in dye decolourization

The majority of the textile dyes are harmful to the environment and potentially carcinogenic. Among strategies for their exclusion, the treatment of dye contaminated wastewater with fungal extract, containing lignin peroxidase (LiP), may be useful. Two fungi isolates, Pleurotus ostreatus (PLO9) and Ganoderma lucidum (GRM117), produced the enzymatic extract by fermentation in the lignocellulosic residue, Jatropha curcas seed cake. The extracts from PLO9 and GRM117 were immobilized on carbon nanotubes and showed an increase of 18 and 27-fold of LiP specific activity compared to the free enzyme. Also, LiP from both fungi extracts showed higher Vmax and lower Km values. Only the immobilized extracts could be efficiently reused in the dye decolourization, contrary, the carbon nanotubes became saturated and they should be discarded over time. This device may offer a final biocatalyst with higher catalytic efficiency and capability to be reused in the dye decolourization process.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Factors influencing the lignin-peroxidase-producing ability of Phanerochaete chrysosporium

The dependence of lignin peroxidase (LiP) activities on agitation was confirmed in medium A containing low vitamin and low trace element concentrations. Vigorous shaking (100 strokes·min^–1) caused a total loss of LiP activity, and detergents, having a protective effect on the enzyme activity, could not prevent a substantial reduction in enzyme activity. More important, LiP activity also depends on the maintenance media (potato dextrose agar or malt extract agar) and enzyme production media (carbon- or nitrogen-limited). High losses of LiP activity were observed by subsequent subculturing. Even storage of the inoculum on the maintenance media at 4° C, although better than subculturing, was not advisable. After long storage periods (50–75 weeks) at 4° C, LiP could not be reactivated in poor medium A, containing low amounts of vitamins and trace elements. In rich medium B, containing higher amounts of vitamins and trace elements, even after long storage periods, LiP activity was higher than when the activity was measured in poor medium A before storage.     

非离子反胶束中木素过氧化物酶催化性能研究

木素过氧化物酶（LiP）在环己烷/Brij30/水反胶束体系中可体现催化活力,然而在水/醇/TritonX100/环己烷反胶束体系中却没有催化活力。对影响Brij30反胶束中LiP催化活力各主要因素进行了优化并测定了LiP在其中的时间稳定性;结果表明,20℃下,使LiP体现最佳活力的Brij30反胶束介质条件为：ω₀＝8.5,pH＝2.2,［Brij30］＝600mmol/L;在此条件下,LiP的半衰期可达到50h;与水介质相比,酶活力下降了,但稳定性却提高了。直链醇是TritonX100形成反胶束的必要组分,为揭示醇的作用,还考察了戊醇对Brij30 反胶束中LiP催化活力的影响,发现高浓度戊醇对LiP有失活作用。据此推测助表面活性剂醇可能是LiP在环己烷/TritonX100/戊醇/水反胶束中不能体现催化活力的主要原因。     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.     

Immobilization of lignin peroxidase on nanoporous gold: Enzymatic properties and in situ release of H2O2 by co-immobilized glucose oxidase

Immobilization of enzymes on porous inorganic materials is very important for biocatalysis and biotransformation. In this paper, nanoporous gold (NPG) was used as a support for lignin peroxidase (LiP) immobilization. NPG with a pore size of 40–50 nm was prepared by dealloying Au/Ag alloy (50:50 wt%) for 17 h. By incubation with LiP aqueous solution, LiP was successfully immobilized on NPG. The optimal temperature of the immobilized LiP was ca. 40, 10 °C higher than that of free LiP. After 2 h incubation at 45 °C, 55% of the initial activity of the immobilized LiP was still retained while the free LiP was completely deactivated. In addition, a high and sustainable LiP activity was achieved via in situ release of H₂O₂ by a co-immobilized glucose oxidase. The present co-immobilization system was demonstrated to be very effective for LiP-mediated dye decolourization.     

Stimulation of Ligninolytic Peroxidase Activity by Nitrogen Nutrients in the White Rot Fungus Bjerkandera sp. Strain BOS55

Bjerkandera sp. strain BOS55, a newly isolated wild-type white rot fungus, produced lignin peroxidase (LiP) in nitrogen (N)-sufficient glucose-peptone medium, whereas no LiP was detectable in N-limited medium. The production of LiP was induced by the peptide-containing components of this medium and also by soy bean protein. Furthermore, the production of manganese-dependent peroxidase was stimulated by organic N sources, although lower production was also evident in N-limited medium. Further research showed that the induction of LiP depended on the combination of pH and the type of N source. An amino acid mixture and ammonium induced LiP only at either pH 6 or 7.3, respectively. Peptone induced LiP activity at all pH values tested; however, the highest activity was observed at pH 7.3. The results presented here indicate that Bjerkandera spp. are distinct from the model white rot fungus, Phanerochaete chrysosporium, which produces ligninolytic peroxidases in response to N limitation.     

Bioelectrocatalytic properties of lignin peroxidase from Phanerochaete chrysosporium in reactions with phenols, catechols and lignin-model compounds

Bioelectrocatalytic reduction of H(2)O(2) catalysed by lignin peroxidase from Phanerochaete chrysosporium (LiP) was studied with LiP-modified graphite electrodes to elucidate the ability of LiP to electro-enzymatically oxidise phenols, catechols, as well as veratryl alcohol (VA) and some other high-redox-potential lignin model compounds (LMC). Flow-through amperometric experiments performed at +0.1 V vs. Ag|AgCl demonstrated that LiP displayed significant bioelectrocatalytic activity for the reduction of H(2)O(2) both directly (i.e., in direct electron transfer (ET) reaction between LiP and the electrode) and using most of studied compounds acting as redox mediators in the LiP bioelectrocatalytic cycle, with a pH optimum of 3.0. The bioelectrocatalytic reduction of H(2)O(2) mediated by VA and effects of VA on the efficiency of bioelectrocatalytic oxidation of other co-substrates acting as mediators were investigated. The bioelectrocatalytic oxidation of phenol- and catechol derivatives and 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulphonate) by LiP was independent of the presence of VA, whereas the efficiency of the LiP bioelectrocatalysis with the majority of other LMC acting as mediators increased upon addition of VA. Special cases were phenol and 4-methoxymandelic acid (4-MMA). Both phenol and 4-MMA suppressed the bioelectrocatalytic activity of LiP below the direct ET level, which was, however, restored and increased in the presence of VA mediating the ET between LiP and these two compounds. The obtained results suggest different mechanisms for the bioelectrocatalysis of LiP depending on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of LiP in biotechnological processes as solid-phase bio(electro)catalyst for decomposition/detection of recalcitrant aromatic compounds.     

Manganese Regulation of Veratryl Alcohol in White Rot Fungi and Its Indirect Effect on Lignin Peroxidase

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Polyoxyethylene stimulates lignin peroxidase production inPhanerochœte chrysosporium

In shaken cultures ofPhanerochœte chrysosporium, different Tweens gave rise to similar and high lignin peroxidase (LiP) activities. The polyoxyethylene-sorbitan (POE-S) moieties isolated from Tweens gave rise to somewhat lower LiP activities, whereas fatty acids isolated from Tweens gave rise to much lower LiP activities than parent Tweens. LiP activity appeared 3 d after addition of Tween 80 if this was added within the first 4 d after inoculation. Of the three chemical moieties contained in Tweens,i.e., fatty acids, sorbitan, and polyoxyethylene (POE), only the latter one significantly stimulated the LiP activity of the culture. The stimulatory effect of POE on the LiP activity increased till its molar mass of approx. 1 kDa, then it levelled off. The quantity of POE in the culture decreased with time. Tween 80, its POE-S moiety and POEs seem to enhance LiP production and not only their release.     

Determination of a catalytic tyrosine in <Emphasis Type="Italic">Trametes cervina</Emphasis> lignin peroxidase with chemical modification techniques

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine¹⁸¹ at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan¹⁷¹ have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine¹⁸¹. These results strongly suggest that tyrosine¹⁸¹ is at the catalytic site in T. cervina LiP.