Production of Phanerochaete chrysosporium lignin peroxidase

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.     

Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium

Two cDNA clones encoding lignin peroxidase isozymes from Phanerochaete chrysosporium have been isolated and characterized. One of the clones, lambda ML-4, encodes isozyme H8 as does the previously reported clone lambda ML-1 [Tien, M. and Tu, C.-P.D. Nature 326 (1987) 520-523; 328, 742]. Our data are consistent with lambda ML-1 and lambda ML-4 being allelic variants. The other clone, lambda ML-5, encodes a homologous isozyme. We have also isolated the genomic clone corresponding to lambda ML-4 cDNA. Conserved residues thought to be essential for peroxidase function were identified in the predicted amino acid sequences of both cDNA clones. Northern blot analyses indicate that these isozymes are expressed during secondary metabolism, appearing on day 4 of growth and increasing on days 5 and 6.     

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.     

Oxidation of aromatic sulfides by lignin peroxidase from Phanerochaete chrysosporium.

The reaction of H2O2 with 4-substituted aryl alkyl sulfides (4-XC6H4SR), catalysed by lignin peroxidase (LiP) from Phanerochaete chrysosporium, leads to the formation of sulfoxides, accompanied by diaryl disulfides. The yields of sulfoxide are greater than 95% when X = OMe, but decrease significantly as the electron donating power of the substituent decreases. No reaction is observed for X = CN. The bulkiness of the R group has very little influence on the efficiency of the reaction, except for R = tBu. The reaction exhibits enantioselectivity (up to 62% enantiomeric excess with X = Br, with preferential formation of the sulfoxide with S configuration). Enantioselectivity decreases with increasing electron density of the sulfide. Experiments in H218O show partial or no incorporation of the labelled oxygen into the sulfoxide, with the extent of incorporation decreasing as the ring substituents become more electron-withdrawing. On the basis of these results, it is suggested that LiP compound I (formed by reaction between the native enzyme and H2O2), reacts with the sulfide to form a sulfide radical cation and LiP compound II. The radical cation is then converted to sulfoxide either by reaction with the medium or by a reaction with compound II, the competition between these two pathways depending on the stability of the radical cation.     

Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.     

Removal of hazardous compounds by lignin peroxidase from Phanerochaete chrysosporium

Phenolic compounds, which are present in many industrial wastewaters, have become a cause for worldwide concern due to their persistence, toxicity and health risks. Enzymatic approaches to remove phenol have been tried for some years as they have several advantages compared with the conventional methods. This paper reports some studies on the use of the white rot fungus Phanerochaete chrysosporium which produces the enzyme lignin peroxidases for the removal of phenol, chlorophenol, and dyes. Batch studies in Erylenmeyer flasks showed complete removal of phenol (500 2 10^х kg/m³) in 30 h. It was also seen that phenol has a significant inhibitory effect on the biomass growth and the enzyme synthesis if added in the early stages of the growth. However, phenol was effectively removed when added after attaining the maximum enzyme activity. 90% of the dyes were removed in about three days, whereas only 62% of the added 4-chlorophenol was removed in about ten days.     

Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus

The cDNA clone lambda ML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus, Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the lambda ML-1 DNA encodes the lignin peroxidase H8.     

Continuous production of lignin peroxidase by Phanerochaete chrysosporium

Phanerochaete chrysosporium spores were immobilized both in agarose and agar gel beads, and used for the production of lignin peroxidase in repeated batch cultures on carbon-limited medium both with 0.5 g l⁻¹ glucose and without glucose. Veratryl alcohol was used as an activator of enzyme production. The biocatalyst was more stable in agarose gel with the maximum activity of 245 U l⁻¹ obtained in a 70 h batch. The biocatalyst could be used for at least 12 batches on the glucose medium with a gradual decrease in lignin peroxidase activity after the sixth batch. Further, mycelium pellets grown on carbon-limited medium were employed both in vertical and horizontal column reactors for the continuous production of lignin peroxidase. The bioreactor produced lignin peroxidase for at least 20 days in the horizontal system at 49 h residence time, with a maximum activity of 95 U l⁻¹.     

Studies on compound I formation of the lignin peroxidase from Phanerochaete chrysosporium

Ligninase, isolated from the wood-destroying fungus Phanerochaete chrysosporium, catalyzes the oxidation of lignin and lignin-related compounds. Ligninase reacts with H2O2 to form the classical peroxidase intermediates Compounds I and II. We have determined the activation energy of ligninase Compound I formation to be 5.9 kcal/mol. The effect of pH and ionic strength on the rate of ligninase Compound I formation was studied. In contrast to all other peroxidases, no pH effect was observed. This is despite homology of active-site amino acids residues (Tien, M., and Tu, C.-P. D. (1987) Nature 326, 520-523) which are proposed to affect the pH profile of Compound I formation. Ligninase Compound I formation can also be supported by organic peroxides. The second-order rate constants with the organic peroxides are lower, suggesting that H2O2 is the preferred substrate.     

Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura+ transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.     

Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium.

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

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 .     

Optimisation of lignin peroxidase production from Phanerochaete chrysosporium using response surface methodology

The ligninolytic white rot fungus Phanerochaete chrysosporium, holds good promise as a biological treatment tool due to its ability to produce the lignin peroxidase enzyme which has the potential to degrade a wide variety of hazardous compounds. The effective application of this technique requires optimisation of the process variables to maximise the enzyme production. Response surface methodology was applied to determine the effects of glucose, ammonium tartarate and ferrous sulphate and their mutual interactions on lignin peroxidase production. With a view to simultaneously reducing the number of experiments and obtaining more information on the mutual interactions between the variables, a 2³ full-factorial central composite experimental design was adopted. The experimental data were fitted to a second order polynomial equation using multiple regression analysis and also analysed by appropriate statistical methods. Solving the regression equation using the multi-stage monte-carlo optimisation techniques, the optimum process conditions for enhanced production of lignin peroxidase were obtained as: glucose 0.9728 kg/m³, ammonium tartarate 0.288 kg/m³, and ferrous sulphate 0.097 kg/m³.     

Influence of lignin peroxidase concentration and localisation in lignin biodegradation by Phanerochaete chrysosporium

Summary The lignin mineralization rate in cultures of Phanerochaete chrysosporium increases with lignin peroxidase concentration up to 20 nkat ml^–1. At higher concentrations the rate of lignin mineralization decreases with increasing lignin peroxidase concentration. The amount of mycelium is not a limiting factor for lignin mineralization at high exocellular lignin peroxidase in association with the mycelium as pellets and no free exocellular enzyme induce a lignin mineralization rate equivalent to cultures reconstituted with washed pellets supplemented with 15 nkat ml^–1 of exogenous free enzyme. These results show that although lignin degradation by lignin peroxidase seems to be facilitated when lignin peroxidase is localised on the surface of the mycelium, free exocellular lignin peroxidase can also efficiently enhance mineralization of lignin by P. chrysosporium.     

Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium

Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.