Evaluation of the environmental conditions for the continuous production of lignin peroxidase by Phanerochaete Chrysosporium in fixed-bed bioreactors

A new system to produce lignin peroxidase (LiP) continuously by Phanerochaete chrysosporium is described. A fixed-bed bioreactor with a pulsing device was used as the optimal bioreactor configuration. Addition of veratryl alcohol (1 mM), tryptophan (1 mM), no Mn²⁺ addition, low glucose addition rate (60–70 mg l^–1 h) and an atmosphere of O₂ gave maximum LiP activities of 700 U l^–1, which are higher than those previously reported.     

Utilisation of lignocellulosic wastes for lignin peroxidase production by semi-solid-state cultures of Phanerochaete chrysosporium

In the present work, the production of ligninolytic enzymes by semi-solid-statecultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725),employing different lignocellulosic wastes as support, was investigated. Thewaste materials employed were grape seeds, wheat straw and wood shavings.Maximum lignin peroxidase activities of 1620 ± 123 U/l, 364 ± 35 U/l and 571 ± 42 U/l were attained, respectively. Nevertheless, lowmanganese-dependent peroxidase activities were found, being insignificantin the grape seed cultures. Moreover, the in vivo decolourisation of a model dye compound, the polymeric dye Poly R-478 (polyvinylamine sulfonateanthrapyridone), by the above-mentioned cultures was monitored to assessthe degrading capability of the extracellular liquid secreted by such cultures.The percentage of biological decolourisation attained by grape seed and woodshaving cultures was around 74% and 63%, respectively, whereas it was ratherlow (40%) in the wheat straw ones.     

Optimization of lignin peroxidase production and stability by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> using sewage-treatment-plant sludge as substrate in a stirred-tank bioreactor

A laboratory-scale study was carried out to produce lignin peroxidase (ligninase) by white rot fungus (Phanerochaete chrysosporium) using sewage-treatment-plant (STP) sludge as the major substrate. The optimization was done using full-factorial design (FFD) with agitation and aeration as the two parameters. Nine experiments indicated by the FFD were fermented in a stirred-tank bioreactor for 3 days. A second-order quadratic model was developed using the regression analysis of the experimental results with the linear, quadratic, and interaction effects of the parameters. Analysis of variance (ANOVA) showed a high coefficient of determination (R ²) value of 0.972, thus indicating a satisfactory fit of the quadratic model with the experimental data. Using statistical analysis, the optimum aeration and agitation rates were determined to be 2.0 vvm and 200 rpm, respectively, with a maximum activity of 225 U l⁻¹ in the first 3 days of fermentation. The validation experiment showed the maximum activity of lignin peroxidase was 744 U l⁻¹ after 5 days of fermentation. The results for the tests of the stability of lignin peroxidase showed that the activity was more than 80% of the maximum for the first 12 h of incubation at an optimum pH of 5 and temperature of 55°C.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica

The cDNA encoding for lignin peroxidase of Phanerochaete chrysosporium was expressed in the Pichia methanolica under the control of the alcohol oxidase (AUG1) promoter which was followed by either the lignin peroxidase leader peptide of Phanerochaete chrysosporium or the Saccharomyces cerevisiae alpha-factor signal peptide. Both peptides efficiently directed the secretion of lignin peroxidase from the recombinant yeast cell. The extracellular lignin peroxidase activity in two recombinants was 932 U l(-1) and 1933 U l(-1). The purity of the recombinant product was confirmed by SDS-PAGE.     

Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 ± 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/5, with significantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 ± 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultrafiltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output.     

Effect of culture conditions on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium (ATCC 20696) and separation of its lignin peroxidase

The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted. After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10 g, ammonium tartrate 0.11 g, Tween-80 0.5 g, MnSO₄ 7 mg, and veratryl alcohol 0.3 g in 10 mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture. Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with 60% (NH₄)₂SO₄ yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1 and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes are 38 kDa and 40 kDa, respectively.     

Nitrogen regulation of lignin peroxidase and manganese-dependent peroxidase production is independent of carbon and manganese regulation in Phanerochaete chrysosporium

In this study, a N-deregulated mutant (der8-5) of Phanerochaete chrysosporium was used as a tool to investigate the interrelationships between N, C, and Mn(II) regulation of LIP and MNP production in this organism. The results showed that LIP and MNP production by der8-5 was blocked in excess C medium but not in excess N medium. Furthermore, LIP and MNP production in this organism was subject to Mn(II) regulation regardless of the fact whether it is grown in low N medium or in high N medium. These and other results indicate that N regulation of LIP and MNP production in P. chrysosporium is independent of C and Mn(II) regulation.Abbreviations LIP lignin peroxidase - MNP manganese-dependent peroxidase - WT wild-type - der8-5 nitrogen-deregulated mutant     

Decolorization of melanin by lignin peroxidase from<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Melanin was decolorized by lignin peroxidase fromPhanerochaete chrysosporium. This decolorization reaction showed a Michaelis-Mentens type relationship between the decolorization rate and concentration of two substrates: melanin and hydrogen peroxide. Kinetic constants of the decolorization reaction were 0.1 OD₄₇₅/min (V _max) and 99.7 mg/L (K _m) for melanin and 0.08 OD₄₇₅/min (V _max) and 504.9 μM (K _m) for hydrogen peroxide, respectively. Depletion of hydrogen peroxide interrupted the decolorization reaction, indicating the essential requirement of hydrogen peroxide. Pulsewise feeding of hydrogen peroxide continued the decolorizing reaction catalyzed by lignin peroxidase. These results indicate that enzymatic decolorization of melanin has applications in the development of new cosmetic whitening agents.     

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.     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Influence of primary and secondary proteases produced by free or immobilized cells of the white-rot fungus Phanerochaete chrysosporium on lignin peroxidase activity

Primary and secondary extracellular proteases produced by free or immobilized cells of the white-rot fungus Phanerochaete chrysosporium have been studied in relation to lignin peroxidase (LiP) decay. Proteases produced during primary metabolism exhibited a maximum activity on day 2; they could totally inactivate LiP activity and partially fragment LiP. Proteases produced during secondary metabolism did not inactivate or decay LiP.These proteases most likely are aspartic- and thiol-proteases.     

Impact of pellet size on growth and lignin peroxidase activity of Phanerochaete chrysosporium

We investigated the influence of pellet size on the growth and lignin peroxidase (LiP) productivity of Phanerochaete chrysosporium. Different pellet sizes were obtained by varying the vessel diameter under constant shaking conditions. Under these varying conditions the pellet size was in the range of 2–18 mm, while the number of pellets in a single vessel varied from around 1,200 in the Erlenmeyer flask to around 6 in the narrowest vessel. A correlation between the final pellet size and the shear rate was obtained, demonstrating that the pellet size is mainly affected by hydrodynamics. The growth of large pellets was described by a cubic growth model. Despite different pellet sizes, LiP activity appeared in all vessels, but the onset of LiP activity showed a delay based upon the pellet size, while maximal LiP activities varied by only 15%, being around 850 U/l.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.     

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, C and C, to yield a C-aldehyde structure, which was further oxidized to the C-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between C and C, and oxidation of the C-alcohol to an aldehyde group. Further oxidation of C to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific C-C epoxidation followed by non-enzymatic hydrolysis of the epoxide.Abbreviations TLC thin layer chromatography - LDA lithium diisopropyl amide - DDQ 2,3-dichloro-5,6-dicyanobenzoquinone - MS mass spectrometry - UV ultraviolet spectroscopy     

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.     

A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve ¹⁴CO₂ from ¹⁴C ring, side chain and 3-O-¹⁴C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve ¹⁴CO₂ from a variety of lignin model compounds including ¹⁴C-4-methoxy labeled veratrylglycerol--guaiacyl (V) ether, -¹⁴C-guaiacylglycerol--guaiacyl ether (VI), as well as 1-(¹⁴C-4-methoxy, 3-methoxyphenyl)1,2 propene (III) and 1-(¹⁴C-4-methoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H₂O₂ to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including -aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.Abbreviations GLC gas liquid chromatography - TMSi trimethylsilyl - MS mass spectrometry - LDS lignin degrading system