Purification and characterization of manganese peroxidases from native and mutant Trametes versicolor IBL-04

Extracellular manganese peroxidases （MnPs） produced by native and mutant strains of Trametes versicolor IBL‐04 （EB‐60, EMS‐90） were purified by ammonium sulphate precipitation and dialysis, followed by ion‐exchange and gel‐permeation chromatography. The purified enzymes elucidated a single band in the 43‐kDa region on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The optimum pH and temperature of the purified enzymes were found to be 5.0 and 40 °C, respec‐tively. Mutant strain MnPs exhibited a broader active pH range and higher thermal stability than native MnP. Purified MnPs from selected mutants showed almost identical properties to native MnP in electrophoresis, steady‐state kinetics, and metal ion and endocrine‐disrupting compound （EDC） degradation efficiency. Although the fastest reaction rates occurred with Mn2＋, MnPs displayed the highest affinity for ABTS, methoxyhydroquinone, 4‐aminophenol and reactive dyes. MnP activity was significantly enhanced by Mn2＋and Cu2＋, and inhibited in the presence of Zn2＋, Fe2＋, ethylene‐diaminetetraacetic acid and cysteine to various extents, with Hg2＋ as the most potent inhibitory agent. MnPs from all sources efficiently catalyzed the degradation of the EDCs, nonylphenol and triclosan, removing over 80%after 3 h of treatment, which was further increased up to 90%in the presence of MnP‐mediator system. The properties of T. versicolor MnPs, such as high pH and ther‐mal stability, as well as unique Michaelis‐Menten kinetic parameters and high EDC elimination effi‐ciency, render them promising candidates for industrial exploitation.

Noncovalent immobilization of manganese peroxidases from P.chrysosporium on carbon nanotubes

Manganese peroxidases(MnP)from Phanerochaete chrysosporium were adsorbed onto multi-walled carbon nanotubes(MWNT).Four different loadings of MnP on MWNTs were investigated,and the maximum enzyme loading of 47.5µg/mg of MWNTs was obtained in 12 h.The adsorbed MnP showed a catalytic activity of up to 0.1 U/mg of the weight of the system of MnP/MWNTs,with 23%of its original activity retained.The AFM image of the adsorbed enzymes indicated that a layer of MnP covered the surface of the MWNTs and retained its original three-dimensional shape.Amino-based nonspecific interactions may play the dominant role in the adsorption of MnP on MWNTs.

Studies on Manganese Peroxidase Immobilized in Gelatin-containing Microemulsion-based Gels

The immobilized technique of manganese peroxidase(MnP) in gelatin-containing microemulsion-based gels and the effects of storage time and reuse times on its catalytic activity were studied. The results show that the MnP immobilized together with Mn 2+ and H_2O_2 could effectively oxidize syringaldazine in n-heptane. The immobilized MnP still had a high catalytic activity after one-month storage under a freezing condition. The reuse times have a relation to the amount of the immobilized H_2O_2. When the amount of the immobilized H_2O_2 is sufficient, the microemulsion-based gels containing MnP could be used many times.

Manganese peroxidase production from cassava residue by Phanerochaete chrysosporium in solid state fermentation and its decolorization of indigo carmine

Bioconversion of lignocellulosic wastes to higher value products through fungal fermentation has economic and ecological benefits. In this study, to develop an effective strategy for production of manganese peroxidase(Mn P)from cassava residue by Phanerochaete chrysosporium in solid state fermentation, the stimulators of Mn P production were screened and their concentrations were optimized by one-at-a-time experiment and Box–Behnken design. The maximum Mn P activity of 186.38 nkat·g-1dry mass of the sample was achieved after 6 days of fermentation with the supplement of 79.5 mmol·L-1·kg-1acetic acid, 3.21 ml·kg-1soybean oil, and 28.5 g·kg-1alkaline lignin, indicating that cassava residue is a promising substrate for Mn P production in solid state fermentation. Meanwhile, in vitro decolorization of indigo carmine by the crude Mn P was also carried out, attaining the ratio of 90.18% after 6 h of incubation. An oxidative mechanism of indigo carmine decolorization by Mn P was proposed based on the analysis of intermediate metabolites with ultra-high performance liquid chromatography and gas chromatography tandem mass spectrometry. Using the crude Mn P produced from cassava residue for indigo carmine decolorization gives an effective approach to treat dyeing effluents.

pH-Dependence of Manganese (II) Oxidation Reaction by Novel Wild-Type and Mutants Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3) Enzymes

The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5 – 12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0 - 9.0. The rPr-MnP3 mutants’ maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn2+ binding site is the only productive catalytic site for Mn (II) oxidation.

The Roles of pH in the Modification of Wild-Type Recombinant Phlebia radiata Manganese Peroxidase 3 Activities and Stability of Secondary Structures

This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X−), a rapid alkaline transition occurs. Leaving the X− form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y−, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y− occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min−1, and half-life (T1/2) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.

Polycyclic aromatic hydrocarbon biodegradation and extracellular enzyme secretion in agitated and stationary cultures of Phanerochaete chrysosporium

The extracellular enzyme secretion and biodegradation of polycyclic aromatic hydrocarbons （PAHs） were studied in agitated and shallow stationary liquid cultures of Phanerochaete chrysosporium. Veratryl alcohol and Tween80 were added to cultures as lignin peroxidase （LIP） and manganese peroxidase （MnP） inducer, respectively. Shallow stationary cultures were suitable for the production of enzyme, whereas agitated cultures enhanced overall biodegradation by facilitating interphase mass transfer of PAHs into aqueous phases. The use of a LiP stimulator, veratryl alcohol, did not increase PAH degradation but significantly enhanced LiP activity. In contrast, Tween80 increased both MnP secretion and PAH degradation in shallow stationary cultures. On the other hand, high PAH degradation was observed in agitated cultures in the absence of detectable LiP and MnP activities. The results suggested that extracellular peroxidase activities are not directly related to the PAH degradation, and the increased solubility rather than enzyme activity may be more important in the promotion of PAH degradation.

A Simple Structure Model for Enzyme Production by Phanerochaete chrysosporium

In order to understand the behavior of ligninolytic enzyme production by white rot fungi Phanerochaete chrysosporium, study on time courses and a mathematical model for the production of lignin peroxidase (LiP) and manganese peroxidase (MnP) of the fungi was undertaken. Based on the Monod-Jacob operon model, the ligninolytic enzyme would be synthesized in the absence of a related repressor. The repressor is assumed to be active in the presence of ammonia nitrogen, and as combined as co-repressor, it causes the inhibition of enzyme synthesis. The model can explain the mechanism of extracellular ligninolytic enzyme production by white rot fungi. The results,as predicted by the model, correspond closely to those observed in experimental studies. In addition, some light is also shed on unmeasured variables, such as the concentrations of repressor and mRNA that are related to the enzyme synthesis.

Effects of glucose on the decolorization of Reactive Black 5 by yeast isolates

The cometabolic roles of glucose were investigated in decolorization of an azo dye, Reactive Black 5, by yeast isolates, Debaryomyces polymorphus and Candida tropicalis. The results indicated that the dye degradation by the two yeasts was highly associated with the yeast growth process and glucose presence in the medium. Color removal of 200 mg dye/L was increased from 76.4% to 92.7% within 60 h to 100% within 18-24 h with the increase of glucose from 5 to 10 g/L, although the activity of manganese dependent peroxidase （MnP） decreased by 2-8 times in this case. Hydrogen peroxide of 233.3 μg/L was detected in 6 h in D. polymorphus culture. The cometabolic functions of glucose and hydrogen peroxide could be also confirmed by the further color removals of 95.8% or 78,9% in the second cycle of decolorization tests in which 7 g glucose/L or 250 μg H202/L was superadded respectively together with 200 mg dye/L.

Ligninolytic enzymes production by endophytic fungus Diaporthe phaseolorum(Desm.)Sacc.under the influence of different carbon and nitrogen sources

Endophytic fungi are being investigated for their ability to create industrially relevant secondary metabolites.In recent years,there has been a surge in interest in these fungi as a source of novel enzymes,particularly hydrolytic enzymes.The present study investigated the effect of different carbon,organic and inorganic nitrogens on the growth and ligninolytic enzyme production by the endophytic fungus Diaporthe phaeolorum.The fungus was isolated from the leaves of Dillenia indica and analyzed by morpho-molecular basis.The fungus showed promising results for in vitro production of ligninolytic enzymes.Sucrose was the most favorable carbon compound for growth among all the carbon compounds tested.It displayed maximum lignin peroxidase(Lip)activity in fructose(3.5 U/ml),followed by pectin(2.60 U/ml)and glucose(2.53 U/ml).Glucose gave the highest manganese peroxidase(MnP)activity i.e.,6.88 U/ml followed by starch,sucrose and raffinose.Similarly,the maximum laccase activity was 44.5 U/ml in pectin.Potassium nitrate and L-asparagine were the best inorganic and organic nitrogen for growth.In the case of ligninolytic enzyme production,ammonium acetate and ammonium phosphate were the best media for LiP and MnP,whereas laccase production was highest in ammonium nitrate supplemented medium.In organic nitrogen sources,medium supplemented with DL-tryptophan gave the highest Lip production,whereas MnP and laccase production was observed in the medium containing L-tyrosine and L-asparagine.To the best of our knowledge,this is the first report related to the growth and ligninolytic enzyme production by Diaporthe phaseolorum.The findings from the study will assist researchers in improving the production of ligninolytic enzymes by this fungus under in vitro conditions on an industrial scale.

Microbial enzyme systems for lignin degradation and their transcriptional regulation

Lignocellulosic biomass is the most abundant renewable resource in nature and has received considerable attention as one of the most promising alternatives to oil resources for the provision of energy and certain raw materials. The phenolic polymer lignin is the second most abundant constituent of this biomass resource and has been shown to have the potential to be converted into industrially important aromatic chemicals after degradation. However, due to its chemical and structural nature, it exhibits high resistance toward mechanical, chemical, and biological degradation, and this causes a major obstacle for achieving efficient conversion of lignocellulosic biomass. In nature, lignin-degrading microorganisms have evolved unique extracellular enzyme systems to decompose lignin using radical mediated oxidative reactions. These microorganisms produce a set of different combinations of enzymes with multiple isozymes and isoforms by responding to various environmental stimuli such as nutrient availability, oxygen concentration and temperature, which are thought to enable effective decomposition of the lignin in iignocellulosic biomass. In this review, we present an overview of the microbial ligninolytic enzyme systems including general molecular aspects, structural features, and systematic differences in each microorganism. We also describe the gene expression pattern and the transcriptional regulation mechanisms of each ligninolytic enzyme with current data.

Effect of heavy metals and phenol on bacterial decolourisation and COD reduction of sucrose-aspartic acid Maillard product

Melanodins are amino-carbonyl complex, predominantly present in sugarcane molasses based distillery wastewater as major source of colourant. The microbial decolourisafion of melanoidin is a challenge due to its binding property with other co-pollutants of distillery waste. Results revealed that the presence of Zn2＋ （2.00-20.00 mg/L） in melanoidin solution （1200 mg/L） stimulated the bacterial growth and sucrose-aspartic acid Maillard product （SAA） decolourisation as compared to control, while Fe3＋ and Mn2＋ at the same concentration inhibited the process. However, the presence of phenol （100 mg/L） along with Zn2＋, Fe3＋ and Mn2＋ suppressed the bacterial growth, SAA decolourisation and MnP activity. The shrinkage and reduced number of bacterial cell count at higher concentration of heavy metals in presence of phenol was also observed under scanning electron microscope.

Evaluation of ligninolytic activity in spent mushroom substrate from four cultivated mushrooms

Spent mushroom substrate(SMS)is an abundant residue generated by the mushroom industry,which is mostly discarded.In this study,the SMS of four cultivated mushrooms were evaluated as potential sources of ligninolytic enzymes.Pleurotus ostreatus,P.eryngii,P.djamor and Ganoderma lucidum were cultivated in two different substrates to determine growth,mushroom yields and ligninolytic activity of laccase(Lac)and manganese peroxidase(MnP).Maximum activities in the SMS were 3.731 U/g Lac and 0.191 U/g MnP,both for P.eryngii.Colonized mushroom substrates were also analyzed,and higher activities were found in almost all samples.A correlation test between mushroom yields and ligninolytic activities of each culture was made,and we found that high enzymatic activity in the substrate did not result in high mushroom productivity or vice versa.The SMS was a good source of ligninolytic enzymes.Enzymatic activity depended on the mushroom species and its affinity to the substrate,and it was not influenced by the mushroom yields.Production of mushrooms in diverse lignocellulosic substrates is gaining interest,and ob-taining ligninolytic enzymes from residual SMS is an excellent alternative in the circular economy concept.