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.     

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.     

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.     

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.     

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.     

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.     

Production of Phanerochaete chrysosporium lignin peroxidase under various culture conditions

Lignin peroxidase production by the white-rot fungus Phanerochaete chrysosporium is markedly influenced by the buffer system employed. In immobilized P. chrysosporium cultures with carbon-limited glucose medium, the use of acetate buffer resulted in higher lignin peroxidase activities than tartrate. With acetate as the buffer in shake-flask cultures a 20% to over 100% improvement in lignin peroxidase production was obtained as compared to tartrate-buffered systems. Of trace elements, Cu²⁺, Mn²⁺ and Zn²⁺ seemed to have the greatest influence on lignin peroxidase production. Furthermore, an increase in the Cu²⁺ and Zn²⁺ concentrations resulted in considerably higher ligninase activities. Although it has been shown previously that high manganese levels repress ligninase production, for maximum ligninase production the presence of some Mn²⁺ appeared to be necessary. The concentration of phosphorus had surprisingly little effect on ligninase production. Highest lignin peroxidase activities were obtained with lower phosphorus concentrations, but reasonably high activities were obtained within the whole studied phosphorus range of 0.12–4.60 g l^–1. Diammonium tartrate alone was a better nitrogen source than a mixture of diammonium tartrate, proteose peptone and yeast extract. The addition of solid manganese (IV) oxide to 3-day-old immobilized biocatalyst cultures increased the maximum ligninase activity obtained by about one-third. Correspondence to: S. Linko     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

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.     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Aspergillus niger

The lignin peroxidase (isoenzyme H8) of Phanerochaete chrysosporium was expressed in Aspergillus niger under the control of plant nopaline synthase (NOS) promoter and terminator. H8 mRNA was produced in this heterologous system. Western blot analysis showed that the recombinant protein was of size similar to the native H8. The extracellular lignin peroxidase activity in these primary constructs was positive, though weak (1.12 nKat mg^–1).     

Genetic factors influencing lignin peroxidase activity in Phanerochaete chrysosporium ME446

Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP-mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase-active state within the idiophasic mycelial mat and may be the mating-type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.     

In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

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).     

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 .