Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Total Enzyme Syntheses of Napyradiomycins A1 and B1.

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon-carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.

Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata.

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K d = 2960 ± 940 µM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCPinterior, suggesting that the other site, TCPexterior, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCPinterior binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.

Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold nanoparticles by using purified fungal phenol oxidases.

The work shows the ability of cultured Basidiomycetes of different taxonomic groups-Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa-to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl4 to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

[Peroxidase activity of octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus].

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s­1; k cat/K m = 855 mM­1 s­1) has been 100­300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Differential production of lignocellulolytic enzymes by a white rot fungus Termitomyces sp. OE147 on cellulose and lactose.

White-rot fungi are the only organisms known to degrade all basic wood polymers using different strategies of employing a variety of hydrolytic and oxidative enzymes. A comparative secretome analysis of Termitomyces sp. OE147 cultivated on cellulose and lactose was carried out by two-dimensional gel electrophoresis followed by MALDI-TOF/TOF-MS analysis to identify the enzymes coordinately expressed on cellulose. A total of 29 proteins, belonging to CAZy hydrolases (11), CAZy oxidoreductases (13) and some 'other' (5) proteins were identified. Among the CAZy hydrolases, a distinct repertoire of cellulolytic and hemicellulolytic enzymes were produced while among the CAZy oxidoreductases, cellobiose dehydrogenase and laccase were the predominant enzymes along with H2O2 dependent peroxidases. This coordinated expression indicated a unique and integrated system for degradation of not only crystalline cellulose but also other components of lignocellulolytic substrates, namely lignin and xylan. Activities of the identified proteins were confirmed by plate assays and activity measurements. Many of the enzyme activities were also correlated with reduction in the crystallinity index of cellulose. Based on the enhanced production of CDH, ß-glucosidases and several oxidoreductases, a more prominent role of these enzymes is indicated in this fungus in cellulose breakdown.

Purification and characterization of a melanin biodegradation enzyme from Geotrichum sp.

OBJECTIVE: Melanin is a black or brown phenolic polymer present mainly in skin and hair. Although melanin can be degraded by some microbial species, the melanin degradation capacity of Geotrichum sp. is unknown. The aim of this study was to characterize a melanin biodegradation enzyme from Geotrichum sp. METHODS: In this study, we assessed the melanin degradation activity of Geotrichum sp. in comparison with the major melanin-degrading enzymes, manganese-dependent peroxidase (MnP), manganese-independent peroxidase, lignin peroxidase and laccase. Furthermore, the effect of several carbohydrates on melanin degradation by Geotrichum sp. was determined. The MnP enzyme was purified using ammonium sulphate precipitation and Sephadex G-200 column chromatography, and then the conditions for optimal enzymatic activity were determined by adjusting the pH, temperature and Tween-80 concentration. RESULTS: Compared with extracellular ligninolytic enzymes of Geotrichum sp., MnP had the highest ligninolytic enzyme activity; and the highest enzymatic activity was observed in the presence of glucose. The final purified MnP enzyme exhibited 6 U mL-1 activity and had a molecular weight of 54.2 kDa. The enzymatic activity was highest at pH 4.5 and 25-35°C in the absence of Tween-80. CONCLUSION: These results indicate the potential of MnP purified from Geotrichum sp. as a skin-lightening agent in the cosmetic industry.

A novel alkyl hydroperoxidase (AhpD) of Anabaena PCC7120 confers abiotic stress tolerance in Escherichia coli.

In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

Dye decolorization and detoxification potential of Ca-alginate beads immobilized manganese peroxidase.

BACKGROUND: In view of compliance with increasingly stringent environmental legislation, an eco-friendly treatment technology of industrial dyes and effluents is a major environmental challenge in the color industry. In present study, a promising and eco-friendly entrapment approach was adopted to immobilize purified manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum IBL-05 on Ca-alginate beads. The immobilized MnP was subsequently used for enhanced decolorization and detoxification of textile reactive dyes). RESULTS: MnP isolated from solid-state culture of G. lucidum IBL-05, presented highest immobilization yield (83.9 %) using alginate beads prepared at optimized conditions of 4 % (w/v) sodium alginate, 2 % (w/v) Calcium chloride (CaCl2) and 0.5 mg/ml enzyme concentration. Immobilization of MnP enhanced optimum temperature but caused acidic shift in optimum pH of the enzyme. The immobilized MnP showed optimum activity at pH 4.0 and 60 °C as compared to pH 5.0 and 35 °C for free enzyme. The kinetic parameters K(m) and V(max) of MnP were significantly improved by immobilization. The enhanced catalytic potential of immobilized MnP led to 87.5 %, 82.1 %, 89.4 %, 95.7 % and 83 % decolorization of Sandal-fix Red C4BLN, Sandal-fix Turq Blue GWF, Sandal-fix Foron Blue E2BLN, Sandal-fix Black CKF and Sandal-fix Golden Yellow CRL dyes, respectively. The insolubilized MnP was reusable for 7 repeated cycles in dye color removal. Furthermore, immobilized MnP also caused a significant reduction in biochemical oxygen demand (BOD) (94.61-95.47 %), chemical oxygen demand (COD) (91.18-94.85 %), and total organic carbon (TOC) (89.58-95 %) of aqueous dye solutions. CONCLUSIONS: G. lucidum MnP was immobilized in Ca-alginate beads by entrapment method to improve its practical effectiveness. Ca-alginate bound MnP was catalytically more vigorous, thermo-stable, reusable and worked over wider ranges of pH and temperature as compared to its free counterpart. Results of cytotoxicity like hemolytic and brine shrimp lethality tests suggested that Ca-alginate immobilized MnP may effectively be used for detoxification of dyes and industrial effluents.

High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli.

Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 µg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 µM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).

Nutrient media optimization for simultaneous enhancement of the laccase and peroxidases production by coculture of Dichomitus squalens and Ceriporiopsis subvermispora.

Coculturing of two white-rot fungi, Dichomitus squalens and Ceriporiopsis subvermispora, was explored for the optimization of cultivation media for simultaneous augmentation of laccase and peroxidase activities by response surface methodology (RSM). Nutrient parameters chosen from our previous studies with the monocultures of D. squalens and C. subvermispora were used to design the experiments for the cocultivation study. Glucose, arabinose, sodium nitrate, casein, copper sulfate (CuSO4 ), and manganese sulfate (MnSO4 ) were combined according to central composite design and used as the incubation medium for the cocultivation. The interaction of glucose and sodium nitrate resulted in laccase and peroxidase activities of approximately 800 U/g protein. The addition of either glucose or sodium nitrate to the medium also modifies the impact of other nutrients on the ligninolytic activity. Both enzyme activities were cross-regulated by arabinose, casein, CuSO4 , and MnSO4 as a function of concentrations. Based on RSM, the optimum nutrient levels are 1% glucose, 0.1% arabinose, 20 mM sodium nitrate, 0.27% casein, 0.31 mM CuSO4 , and 0.07 mM MnSO4 . Cocultivation resulted in the production of laccase of 1,378 U/g protein and peroxidase of 1,372 U/g protein. Lignin (16.9%) in wheat straw was degraded by the optimized enzyme mixture.

Investigation of photo-assisted and crude peroxidase mediated transformations of chlorinated phenols (CPs) from spiked and industrial wastewaters: identification of reaction products.

This work focused on photo-assisted crude peroxidase mediated transformations of chlorinated phenols (CPs) from spiked and industrial wastewaters and the identification of reaction products formed. Garden radish Raphanus sativus was the source of crude peroxidase. No chlorine bearing compounds were detected by gas chromatography-high resolution mass spectrometry analysis. Under identical test conditions, the concentrations of 4-chlorophenol and 2,4-dichlorophenol were demoted to zero from 514 mg/L, 652 mg/L and that of 2,4,6-trichlorophenol and pentachlorophenol were reduced to 18 mg/L and 37 mg/L from 790 mg/L and 1066 mg/L, respectively (high-pressure liquid chromatography analysis). Chloride ion release profiles also showed a progressively increasing trend. A neat chemical oxygen demand removal to the extent of 63-79% was achieved in the case of spiked wastewater sample and to the extent of 77% for industrial wastewaters. A hypothesis reaction scheme was also suggested to comprehend the mechanism of degradation reactions.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

Production of hydroxylated polybrominated diphenyl ethers from bromophenols by bromoperoxidase-catalyzed dimerization.

Hydroxylated polybrominated diphenyl ethers (HO-PBDEs) are emerging endocrine-disrupting compounds that are widely present in the marine environment. The origin of HO-PBDEs is generally attributed to metabolism of PBDEs and natural production in the environment. However, it is unclear how HO-PBDEs are produced naturally. Here we report the formation of HO-PBDEs from simple bromophenols (BPs) [e.g., 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP)] under the catalysis of bromoperoxidase (BPO) isolated from the common marine red alga Corallina officinalis. Experiments at room temperature showed that BPO readily catalyzes the conversion of 2,4-DBP and 2,4,6-TBP to HO-PBDEs in the presence of Br(-) and H2O2. From analysis of the original forms and their corresponding methylated derivatives, the reaction products were tentatively identified as 2'-HO-BDE-121 and 4'-HO-BDE-121. The formation of HO-PBDEs was likely resulted from the coupling of bromophenoxy radicals generated by the oxidation of BPs via BPO-mediated processes. The presence of Br(-) in the reaction favored the conversion. The production of HO-PBDEs was found to be pH-dependent, and a higher yield was obtained at pH 6.5. In view of the abundance of BPs and C. officinalis in the marine environment, bioconversion of BPs mediated by BPO may be a potential route for the natural production of HO-PBDEs.

New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes.

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.

Purification and characterization of peroxidase from avocado (Persea americana Mill, cv. Hass).

BACKGROUND: Avocado (Persea americana Mill, cv. Hass) fruit ranks tenth in terms of the most important products for Mexico. Avocado products are quite unstable due to the presence of oxidative enzymes such as polyphenol oxidase and peroxidase. The present study is to characterize the activity of purified avocado peroxidase from avocado in order to ascertain the biochemical and kinetic properties and their inhibition conditions. RESULTS: Purification was performed by Sephacryl S 200 HR gel filtration chromatography and its estimated molecular weight was 40 kDa. The zymogram showed an isoelectric point of 4.7. Six substrates were tested in order to ascertain the affinity of the enzyme for these substrates. The purified peroxidase was found to have low Km (0.296 mM) and high catalytic efficiency (2688 mM(-1) s(-1)) using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), optimum activity being reached at 51°C, pH 3.8. The addition of dithiothreitol, ß-mercaptoethanol, ascorbic acid, sodium azide, L-cysteine and Tween-20 had high inhibitory effects, while metals ions such as Cu(+), Fe(2+) and Mn(2+) had weak inhibitory activity on purified avocado peroxidase. CONCLUSION: The purified avocado peroxidase exhibits high inhibition (Ki = 0.37 µM) with 1.97 µM n-propyl gallate using ABTS as substrate at 51°C, pH 3.8 for 10 min.

Genomic and proteomic dissection of the ubiquitous plant pathogen, Armillaria mellea: toward a new infection model system.

Armillaria mellea is a major plant pathogen. Yet, no large-scale "-omics" data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms.

Decolorization applicability of sol-gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum.

BACKGROUND: An eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents. RESULTS: A large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol-gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the permissible limits. CONCLUSIONS: In conclusion, G. lucidum MnP was immobilized by sol-gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.