Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.     

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.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.     

Tween 80 enhanced TNT mineralization by Phanerochaete chrysosporium

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT.L-1. The presence of 1% (w/v) Tween 80, at 20 mg.L-1 TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth of P. chrysosporium without any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80.     

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.     

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

Bioelectrocatalytic reduction of H₂O₂ 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 H₂O₂ 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 H₂O₂ 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.     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase

Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis. Oxidation of Mn2+ was practically unaffected. However, several mutations modified the oxidation kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reduction of VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme-product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant.     

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.     

Tween 80 effect on bacteriocin synthesis by Lactococcus lactis subsp. cremoris J46

E. HUOT. C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. Sorbitan polyoxyethylene monooleate (Tween 80) suppressed bacteriocin cell adhesion. Within the range 0–1% (v/v), there was an increase in bacteriocin production in regulated (pH 5.5 or 6.0) batch cultures with increasing Tween 80 concentration. For example, at pH 5.5 and in the presence of 1% Tween 80, bacteriocin production was about fourfold higher than in its absence. However, further increase in Tween 80 concentration did not result in a significant modification of the bacteriocin titre. It was shown that the increase was not linked to an activating effect of the surfactant on preformed enzyme, to an increase of bacteriocin availability or to a sensitization of the target cell, demonstrating that Tween 80 promoted bacteriocin production.     

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol Brilliant Blue R

Lignin peroxidase (LiP) produced by Trametes versicolor decolorizes Remazol Brilliant Blue R (RBBR) in the presence as well as in the absence of veratryl alcohol (VA). VA enhances and stabilizes the RBBR-decolorization rates by lignin peroxidase. RBBR has better substrate reactivity than VA for LiP. RBBR is also decolorized directly by LiP and competitively inhibits VA oxidation by LiP. In the presence of higher concentrations of RBBR (i) RBBR decolorization rates improve, (ii) veratryl aldehyde appears after a lag and (iii) VA oxidation rates decrease. The lag is due to consumption of VA cation radical (VA⁺) generated upon LiP-catalyzed VA oxidation, during RBBR oxidation. That may result in the formation of compound III in the absence of VA⁺ and contributes to the inhibitory influence of RBBR on LiP activity.     

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.     

Cleavage of nonphenolic beta-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium.

Purified manganese peroxidase (MnP) from Phanerochaete chrysosporium oxidizes nonphenolic beta-1 diarylpropane lignin model compounds in the presence of Tween 80, and in three- to fourfold lower yield in its absence. In the presence of Tween 80, 1-(3',4'-diethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (I) was oxidized to 3,4-diethoxybenzaldehyde (II), 4-methoxyacetophenone (III) and 1-(3',4'-diethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (IV), while only 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) were detected when the reaction was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the alpha position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4-diethoxybenzaldehyde, 4-methoxyacetophenone remained constant, while the yield of the alpha-keto dimeric product (IV) decreased by approximately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the alpha-keto dimeric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18O2, both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of 18O. Finally, MnP oxidized the substrate 1-(3',5'-dimethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3',5'-dimethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP-catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring.     

Toxicity of phenanthrene dissolved in nonionic surfactant solutions to Pseudomonas putida P2

The fraction in which direct contact occurs between micellar-phase phenanthrene and the bacterial cell surface was estimated by measuring the toxicity of nonionic surfactant (Tween 80 and Triton X-100) solutions to the phenanthrene-degrading bacterium, Pseudomonas putida P2. Cell viability of completely dissolved phenanthrene decreased by 30% at concentrations greater than 0.3 mg L(-1), which is equal to approximately one third of its solubility. Both nonionic surfactants had no effect on cell viability up to 5 g L(-1). Cell viability increased with increasing surfactant concentration at a fixed phenanthrene concentration, due to the decreased concentration of aqueous-pseudophase phenanthrene and the reduced fraction of direct contact. The fraction of direct contact was c. 20% or more below 3 g L(-1) of Triton X-100. The fraction of direct contact for Tween 80 was estimated to be lower than Triton X-100.     

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca²⁺ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H₂O₂ and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k_cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (K_m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.     

Parameter optimization for production of ligninolytic enzymes using agro-industrial wastes by response surface method

Lignin and manganese peroxidase (LiP, MnP) and laccase production by Phanerocheate chrysosporium was optimized by response surface methodology for brewery waste and apple pomace. The effect of moisture, copper sulphate, and veratryl alcohol (VA) concentrations on enzyme production was studied. Moisture and VA had significant positive effect on MnP and LiP production and the viability of P. chrysosporium (p < 0.05) and copper sulphate produced a negative effect. However, moisture and copper sulphate had a significant positive (p < 0.05) effect on laccase production, but VA had an insignificant positive effect (p < 0.05). Higher values of MnP, LiP and viability of P. chrysosporium on apple pomace (1287.5 U MnP/gds (units/gram dry substrate), 305 U LiP/gds, and 10.38 Log 10 viability) and brewery waste (792 U MnP/gds and 9.83 Log 10 viability) were obtained with 80% moisture, 3 mmol/kg VA, and 0.5 mmol/kg copper. LiP production in brewery waste (7.87 U/gds) was maximal at 70% moisture, 2 mmol/kg VA, and 1 mmol/kg copper. Higher production of laccase in apple pomace (789 U/gds) and brewery waste (841 U/gds) were obtained with 80% moisture, 3 mmol/kg VA, and 1.5 mmol/kg copper. Thus, moisture along with VA and copper sulphate was pertinent for the production of ligninolytic enzymes and increased cell viability.     

Estimation of direct-contact fraction for phenanthrene in surfactant solutions by toxicity measurement

The toxicity of solutions containing nonionic surfactants Tween 80, Brij 35 and/or phenanthrene to Pseudomonas putida ATCC 17484 was investigated. The fraction of direct contact between micellar-phase phenanthrene and bacterial cell surface was estimated by using the toxicity data and a mathematical model. The mathematical model was used to calculate phenanthrene concentration in the micellar phase and aqueous pseudophase separately. The first-order death rate constant increased from 0.088+/-0.016 to 0.25+/-0.067 h(-1) when the phenanthrene concentration was increased from 0 to 5.17 x 10(-6)M (equals water solubility). The intrinsic toxicity of surfactant was higher in Brij 35 than in Tween 80. When phenanthrene concentration was increased to 9.7 x 10(-5)M in surfactant solutions, the death rate constant increased to 1.8 +/- 0.024 and 0.41 +/- 0.088 h(-1) for 8.4 x 10(-4)M Brij 35 and 7.6 x 10(-4)M Tween 80. The direct-contact fraction was 0.083 and 0.044 for Brij 35 and Tween 80, respectively, under these conditions using exponential model. The toxicity increased with increasing phenanthrene concentration at a fixed surfactant concentration. The toxicity decreased with increasing the surfactant concentration at a fixed phenanthrene concentration due to decreased contact of bacteria with phenanthrene present in the interior of surfactant micelles.     

Polycyclic aromatic hydrocarbon oxidation by the white-rot fungus Bjerkandera sp. strain BOS55 in the presence of nonionic surfactants

The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures.