Multiple lignin peroxidases of Phanerochaete chrysosporium INA-12.

Nine proteins with lignin peroxidase activity were separated from cultures of Phanerochaete chrysosporium INA-12 in glycerol as carbon source and non-nitrogen limited. Four lignin peroxidase isozymes (4, 5, 8, 9) were purified and characterized. Although differences in kinetic parameters could be shown, antibody reaction showed homology between isozymes. However, thermal stability studied, peptide mapping results, and N-terminal sequence analyses established a higher degree of homology between isozymes 4/5 and 8/9 types. Protein characterization and kinetic data indicate that lignin peroxidase isozymes 4, 5, 8, and 9 differ from described isozymes in strain BKM. The higher specific activity of lignin peroxidase isozymes in cultures with glycerol than in nitrogen-starved cultures accounts for the higher lignin peroxidase activity obtained in these conditions.     

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.     

Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium.

Two types of glycosylated peroxidases are secreted by the white-rot fungus Phanerochaete chrysosporium, lignin peroxidase (LiP) and manganese peroxidase (MnP). The thermal stabilities of recombinant LiPH2, LiPH8, and MnPH4, which were expressed without glycosylation in Escherichia coli, were lower than those of corresponding native peroxidases isolated from P. chrysosporium. Recovery of thermally inactivated recombinant enzyme activities was higher than with that of the thermally inactivated native peroxidases. Removal of N-linked glycans from native LiPH8 and MnPH4 did not affect enzyme activities or thermal stabilities of the enzymes. Although LiPH2, LiPH8, and MnPH4 contained O-linked glycans, only the O-linked glycans from MnPH4 could be removed by O-glycosidase, and the glycan-depleted MnPH4 exhibited essentially the same activity as nondeglycosylated MnPH4, but thermal stability decreased. Periodate-treated MnPH4 exhibited even lower thermal stability than O-glycosidase treated MnPH4. The role of O-linked glycans in protein stability was also evidenced with LiPH2 and LiPH8. Based on these data, we propose that neither N- nor O-linked glycans are likely to have a direct role in enzyme activity of native LiPH2, LiPH8, and MnPH4 and that only O-linked glycans may play a crucial role in protein stability of native peroxidases.     

1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.     

Haloperoxidase activity of Phanerochaete chrysosporium lignin peroxidases H2 and H8.

Monochlorodimedone (MCD), commonly used as a halogen acceptor for haloperoxidase assays, was oxidized by hydrogen peroxide in the presence of lignin peroxidase isoenzymes H2 and H8. When oxidized, it produced a weak absorption band with an intensity that varied with pH. This absorbance was used as a simple method for the product analysis because it disappeared when MCD was brominated or chlorinated. We assessed the activity of the lignin peroxidases for oxidation of bromide by measuring the bromination of MCD, the formation of tribromide, the bromide-mediated oxidation of glutathione, and the bromide-mediated catalase-like activity. We analyzed the reaction products of MCD and the halide-mediated oxidation of glutathione when bromide was replaced by chloride. These enzymes demonstrated no significant activity for oxidation of chloride. Unlike other peroxidases, the lignin peroxidases exhibited similar pH-activity curves for the iodide and bromide oxidations. The optimum pH for activity was about 2.5. Surprisingly, this pH dependence of lignin peroxidase activity for the halides was nearly the same in the reactions with hydrogen donors, such as hydroquinone and guaiacol. The results suggested that protonation of the enzymes with pKa approximately 3.2 is necessary for the catalytic function of lignin peroxidases, irrespective of whether the substrates are electron or hydrogen donors. These unique reaction profiles of lignin peroxidases are compared to those of other peroxidases, such as lactoperoxidase, bromoperoxidase, chloroperoxidase, and horseradish peroxidase. Isozyme H2 was more active than isozyme H8, but isozyme H8 was more stable at very acidic pH.     

Phosphorylation of lignin peroxidases from Phanerochaete chrysosporium. Identification of mannose 6-phosphate

Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K32PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K32PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed 32P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.     

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.     

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.     

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.     

Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.     

Cultivation of Phanerochaete chrysosporium and production of lignin peroxidases in submerged stirred tank reactors

Production of lignin peroxidases by Phanerochaete chrysosposorium in a submerged stirred tank reactor is affected by certain critical parameters, some of which have been investigated in the present paper. These factors are: inoculum, pellet size, certain organic compounds such as polypropylene glycol or polyethylene glycol, culture conditions and composition.A rich inoculum results in formation of small pellets, fast depletion of glucose, and no production of lignin peroxidase. Reduced inoculum size prolongs the development of the culture followed by an active so-called secondary phase. The activity of the culture, however, is just enough to decolorize the blue color of Remazol dye but not strong enough to show extracellular lignin peroxidase. The presence of polypropylene glycol (PPG), polyethylene glycol (PEG) or hexadecane in the culture activates the culture towards lignin peroxidase production. The favorable effect of PPG exists only in cultures made up with tap water and reduced inoculum size at pH 4.5. Trace elements but not vitamins may be left out of the medium without impairing lignin peroxidase-producing ability. The use of desalinated water leads not only to the absence of lignin peroxidase production but also to retardation in growth of the fungi, emphasizing the need for a systematic investigation of the culture medium. The experiments were conducted in a 42 l stirred tank reactor and scaled up to 300 l reactor. Constant impeller tip speed and constant gas flow rate are not sufficient criteria for upscaling of this system.     

New pathway for degradation of sulfonated azo dyes by microbial peroxidases of Phanerochaete chrysosporium and Streptomyces chromofuscus.

Pathways for the degradation of 3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid (I) and 3-methoxy-4-hydroxyazobenzene-4'-sulfonamide (II) by the manganese peroxidase and ligninase of Phanerochaete chrysosporium and by the peroxidase of Streptomyces chromofuscus have been proposed. Twelve metabolic products were found, and their mechanisms of formation were explained. Preliminary oxidative activation of the dyes resulted in the formation of cationic species, making the molecules vulnerable to the nucleophilic attack of water. Two types of hydrolytic cleavage were observed. Asymmetric splitting gave rise to quinone and diazene derivatives, while symmetric splitting resulted in the formation of quinone monoimine and nitroso derivatives. These unstable intermediates underwent further redox, oxidation, and hydrolytic transformation, eventually furnishing 11 organic products and ammonia.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

粉刺侧孢霉产木质素过氧化物酶酶活性与pH值的关系

研究了粉刺侧孢霉 (Phanerochaetechrysosporium)在以微晶纤维素为C源时 ,木素过氧化酶与 pH值的关系 .试验采用 37～ 39°C下液体振荡培养 ,藜芦醇为产酶诱导剂 ,测试不同起始 pH值所得木素过氧化物酶活性 .结果表明 ,起始 pH值 6.0所得酶活最高达 0 .1 2 6U·ml- 1 ,说明以微晶纤维素代替葡萄糖为C源 ,其最佳 pH值相对较高 .菌丝球直径与酶活呈正相关 .     

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