Expression Of Manganese Peroxidase Research Articles

Production of active manganese peroxidase in Escherichia coli by co-expression of chaperones and in vitro maturation by ATP-dependent chaperone release

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn2+ to Mn3+, a diffusible and strong non-specific oxidant capable of attacking bulky phenolic substrates. Therefore, MnP is indispensable in the polymer and paper industries. Previous attempts of MnP expression in Escherichia coli resulted in the formation of inclusion bodies which required invitro refolding. Aiming to investigate the bacterial production of MnP, we have revealed an interesting mechanism underlying chaperone-assisted maturation of this enzyme to its active form. Since we previously found that invitro expression of MnP in E.coli system depends on disulfide bond isomerase DsbC, we chose SHuffle T7 Express, an E. coli constitutively expressing DsbC, as the host for invivo expression of MnP. Initially, only a low amount of the enzyme was present in the soluble fraction, with no detectable peroxidase activity. Co-expression of MnP with different chaperone revealed that DnaK, DnaJ, and GrpE contributed the most to the solubility improvement, however, remained in a complex with the MnP, preventing the enzyme to assume its active conformation. We resolved this by invitro maturation, involving incubation of the MnP-chaperone complex with hemin, ATP, and ATP regeneration system. While ATP enables the chaperones to finish the refolding cycle and release the MnP in its correctly folded form, hemin supports the formation of the holo-enzyme with fully recovered peroxidase activity. We believe that the findings of this paper will serve as an important clue for establishing the bacterial production of fungal peroxidases in the future.

Tiamulin removal by wood-rot fungi isolated from swine farms and role of ligninolytic enzymes

Tiamulin (TIA) is a widely used antibiotic in Thai swine farms and persists in swine manure storage. Significant amounts remain in swine manure after composting, posing risks related to the spread of the compound and the evolution of resistant genes when this compost is used as a fertilizer. Therefore, it is necessary to enhance TIA degradation. This study successfully purified TIA-degrading fungi, which have a high capacity for treating antibiotic-contaminated manure. Twelve strains were collected from swine farms and preliminarily examined for their ability to degrade recalcitrant compounds using brilliant green and crystal violet dyes at 0.05% (w/v). Three isolated fungi and three white-rot fungal strains–namely, Lasiodiplodia sp. F1, Fusarium sp. F5, Galactomyces sp. F8, Verticillium sp., Trametes versicolor, and Trametes hirsuta AK4–capable of developing considerable decolorized zones on both dyes exhibited their abilities to degrade 10 mg l−1 of TIA over a period of 12 days: 93.2 ± 0.0%, 82.4 ± 10.2%, 73.8 ± 5.6%, 89.3 ± 0.0%, 86.1 ± 0.0%, and 66.8 ± 4.8%, respectively. Lasiodiplodia sp. F1, Verticillium sp., and Trametes versicolor were able to remove TIA most efficiently (>85%) and have the potential to be applied to swine waste for the elimination of antibiotic substances. Manganese peroxidase (MnP) was predominantly produced; MnP expression was induced by tiamulin, suggesting its main role in TIA degradation.

Effects of saline-alkaline stress on benzo[a]pyrene biotransformation and ligninolytic enzyme expression by Bjerkandera adusta SM46.

Benzo[a]pyrene (BaP) accumulates in marine organisms and contaminated coastal areas. The biotreatment of waste water using saline-alkaline-tolerant white rot fungi (WRF) represents a promising method for removing BaP under saline-alkaline conditions based on WRF's ability to produce ligninolytic enzymes. In a pre-screening for degradation of polycyclic aromatic hydrocarbons of 82 fungal strains using Remazol brilliant blue R, Bjerkandera adusta SM46 exhibited the highest tolerance to saline-alkaline stress. Moreover, a B. adusta culture grown in BaP-containing liquid medium exhibited resistance to salinities up to 20 g l(-1). These conditions did not inhibit fungal growth or the expression of manganese peroxidase (MnP) or lignin peroxidase (LiP). The degradation rate also became higher as salinity increased to 20 g l(-1). Fungal growth and enzyme expression were inhibited at a salinity of 35 g l(-1). These inhibitory effects directly decreased the degradation rate (>24%). The presence of MnSO4 as an inducer improved the degradation rate and enzyme expression. MnP and LiP activity also increased by seven- and fivefold, respectively. SM46 degraded BaP (38-89% over 30 days) in an acidic environment (pH 4.5) and under saline-alkaline stress conditions (pH 8.2). Investigating the metabolites produced revealed BaP-1,6-dione as the main product, indicating the important role of ligninolytic enzymes in initializing BaP cleavage. The other metabolites detected, naphthalene acetic acid, hydroxybenzoic acid, benzoic acid, and catechol, may have been ring fission products. The wide range of activities observed suggests that B. adusta SM46 is a potential agent for biodegrading BaP under saline conditions.

Expression of manganese peroxidase by Lentinula edodes and Lentinula boryana in solid state and submerged system fermentation

The production of ethanol from lignocellulosic biomass is referred as a second generation biofuel, whose processing is one of the most promising technologies under development. There are few available studies on the use of enzymes produced by fungi as active for the biodegradation of lignocellulosic biomass. However, the manganese peroxidase (MnP) enzyme presents high potential to degrade lignin and the basidiomycetes are the major producers of this oxidase. Thus, this study aimed at evaluating the ability of fungi Lentinula edodes and Lentinula boryana to produce this enzyme when cultivated in submerged fermentation system (SS) and also in solid-state fermentation system (SSF) containing Eucalyptus benthamii sawdust with or without corn cob meal. In the SS the greatest MnP expression occurred on the 25th day, being of 70 UI.L-1 for L. boryana and of 20 UI.L-1 for L. edodes. In the SSF, the best results were obtained on the 10th day for L. edodes, while for L. boryana it happened between the 20th and the 25th days, despite both species presented values close to 110 UI.L-1. Therefore, the results indicated that the studied fungi express the enzyme of interest and that its production is enhanced when cultivated in solid system.

Site-directed mutagenesis of manganese peroxidase from Phanerochaete chrysosporium in an in vitro expression system

An enzymatically active fungal manganese peroxidase (MnP) from Phanerochaete chrysosporium was synthesized in an in vitro coupled transcription–translation system. The synthesized MnP had the expected molecular mass (43,000 Da) and catalyzed the oxidation of 2,6-dimethoxyphenol (DMP) in the presence of hydrogen peroxide and Mn(II). A distal arginine residue (Arg 42) of the peroxide binding pocket and the potential N-glycosylation site (Asn 131) was site-directed mutagenized and corresponding mutant enzymes were also in vitro synthesized. Activities of the mutant enzymes towards 2,6-DMP were not significantly compromised although their dynamic characteristics were obviously different from the wild-type enzyme. The effect of the mutations was explained by using a computer-based three-dimensional modeling. These results demonstrated that in vitro expression of MnP provided a convenient and efficient system for characterization of fungal peroxidases.

Degradation of β-5 lignin model dimers by Ceriporiopsis subvermispora

The degradation of two β-5 lignin model dimers by Ceriporiopsis subvermispora was investigated. The β-5 phenolic dimer was rapidly degraded by the fungus while the non-phenolic dimer remained untouched after 21 days of incubation under limited- and non-limited nitrogen conditions. Fungal degradation of phenolic compounds was a function of manganese concentration. When manganese was omitted from the medium the degradation of the phenolic compounds was much lower. Depletion of manganese was shown to strongly influence manganese peroxidase (MnP) production whereas laccase titer did not change. The β-5 phenolic compound was degraded in vitro by using a crude enzyme filtrate from C. subvermispora. Enzymatic degradation was enhanced both by Tween 80 and Tween 20. Vanillic acid was the predominant aromatic degradation product detected by GC/MS and LC/MS following fungal and enzymatic degradation.The addition of fully methylated β-5 compound in fungal liquid cultures almost completely inhibited MnP expression. Nevertheless, crude MnP activity from C. subvermispora was not affected in vitro by the fully methylated β-5 compound indicating that in vivo some of the steps involved in its production were blocked. Preliminary results showed that crude MnP, in the presence of Tween 80, was able to oxidize the fully methylated β-5 compound. In contrast, degradation in the presence of Tween 20 was very limited.

Truncated-gene reporter system for studying the regulation of manganese peroxidase expression.

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of Phanerochaete chrysosporium is regulated by Mn, heat shock (HS), and H2O2 at the level of gene transcription. We have constructed a homologous gene reporter system to further examine the regulation of two mnp genes, mnp1 and mnp2, encoding individual MnP isozymes. Internal deletions of 234 and 359 bp were made within the coding regions of the mnp1 and mnp2 genes, respectively. The truncated mnp genes were subcloned into the shuttle vector pOGI18, which includes the Schizophylum commune ade5 gene as a selectable marker, and transformed into a P. chrysosporium Ade1 auxotrophic mutant. Northern-blot analysis of purified Ade+ transformants demonstrated that both of the truncated mnp genes were regulated in a manner similar to the endogenous mnp genes with respect to nitrogen limitation and induction by Mn, HS, and H2O2.

Regulation of manganese peroxidase gene transcription by hydrogen peroxide, chemical stress, and molecular oxygen

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by H2O2 and various chemicals, including ethanol, sodium arsenite, and 2,4-dichlorophenol, as well as by Mn(II) and heat shock. Northern (RNA) blot analysis demonstrates that the addition of 1.0 mM H2O2 to 5-day-old cultures grown in the absence of Mn results in the appearance of mnp mRNA within 15 min. Higher levels of mnp mRNA are obtained with simultaneous induction by Mn and H2O2 than with H2O2 alone. Although neither MnP activity nor associated protein is detectable in H2O2-induced cultures grown in the absence of Mn, simultaneous induction with Mn and H2O2 results in a 1.6-fold increase in MnP activity compared with the MnP activity resulting from Mn induction alone. In the presence of Mn, purging of low-nitrogen cultures with 100% O2, in contrast to incubation under air, results in an increase in the accumulation of mnp mRNA and a 13-fold increase in MnP activity on day 5. However, in contrast to the effects of H2O2 and heat shock, O2 purging of Mn-deficient cultures results in negligible accumulation of mnp mRNA.

Heat Shock Induction of Manganese Peroxidase Gene Transcription in Phanerochaete chrysosporium

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of Phanerochaete chrysosporium is regulated by heat shock at the level of gene transcription. Nitrogen limitation and manganous ion [Mn(II)] previously have been shown to regulate mnp gene transcription. Northern (RNA) blot analysis demonstrates that 45 degrees C heat shock results in the accumulation of mnp mRNA, even in cells grown in the absence of Mn. Heat shock induces mnp gene transcription in 4- or 5-day-old cells, and mnp mRNA is detectable after 15 min at 45 degrees C. Maximum accumulation of mnp mRNA is observed 1 to 2 h after transfer of cultures to 45 degrees C. Two hours after heat shock-induced cultures grown in the absence of Mn are transferred back to 37 degrees C, mnp mRNA is no longer detectable. Higher levels of mnp mRNA are obtained with simultaneous induction by Mn and heat shock than by either treatment alone. Neither MnP enzyme activity nor protein is detectable in heat-shocked cultures grown in the absence of Mn. However, higher MnP activity is found in the extracellular medium of cultures induced by both heat shock and Mn than in the medium of cultures induced by Mn alone. These results suggest that the putative heat shock elements found in the promoter region of the mnp genes are physiologically functional and that Mn may be required for a posttranscriptional step of MnP production under heat shock conditions.

Manganese peroxidase gene transcription in Phanerochaete chrysosporium: activation by manganese

The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, we demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 microM. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but not from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

Characterization of a cDNA Encoding a Manganese Peroxidase, from the Lignin-degrading Basidiomycete Phanerochaete Chrysosporium

A cDNA clone of a manganese peroxidase (MnP) from Phanerochaete chrysosporium was isolated and characterized. The cDNA contains 1314 nucleotides excluding the poly(A) tail and the coding region has 68% G + C content. The deduced mature MnP protein contains 357 amino acids and is preceded by a 21-amino acid leader sequence. The experimentally determined N-terminal sequence of the purified MnP-1 protein, pI = 4.9, corresponds to the deduced N-terminal sequence of the gene. The Mr of the mature MnP-1 deduced from the cDNA is 37,439, which is approximately 81.4% of the experimentally determined molecular weight. The difference is due to glycosylation and a single potential N-glycosylation site with the general sequence Asn-X-Thr/Ser is present in the deduced MnP-1 sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine, and the distal arginine are all conserved and regions flanking these residues display homology with other peroxidases. Northern blot analysis indicates that MnP expression is controlled by nutrient nitrogen at the level of transcription. Southern blot hybridization analysis suggests that MnP-1 is a member of a family of MnP genes.