Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

The white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>: conditions for the production of lignin-degrading enzymes

Investigating optimal conditions for lignin-degrading peroxidases production by Phanerochaete chrysosporium (P. chrysosporium) has been a topic for numerous researches. The capability of P. chrysosporium for producing lignin peroxidases (LiPs) and manganese peroxidases (MnPs) makes it a model organism of lignin-degrading enzymes production. Focusing on compiling and identifying the factors that affect LiP and MnP production by P. chrysosporium, this critical review summarized the main findings of about 200 related research articles. The major difficulty in using this organism for enzyme production is the instability of its productivity. This is largely due to the poor understanding of the regulatory mechanisms of P. chrysosporium responding to different nutrient sources in the culture medium, such as metal elements, detergents, lignin materials, etc. In addition to presenting the major conclusions and gaps of the current knowledge on lignin-degrading peroxidases production by P. chrysosporium, this review has also suggested further work, such as correlating the overexpression of the intra and extracellular proteins to the nutrients and other culture conditions to discover the regulatory cascade in the lignin-degrading peroxidases production process, which may contribute to the creation of improved P. chrysosporium strains leading to stable enzyme production.     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

Oligomers of 4-chloroaniline are intermediates formed during its biodegration by Phanerochaete chrysosporium

Abstract Lignin peroxidase H2 (LP-H2) from Phanerochaete chrysosporium oxidized 4-chloroaniline to form several oligomers. Included among the compounds identified were: 4,4'-dichloroazobenzene, 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil and 2-amino-5-(4-chloroanilino) benzoquinone-di-4-chloroanil. In contrast to results by other, we showed that oligomers of 4-chloroaniline were also formed by the fungus in vivo. It was also demonstrated that, although these potentially toxic intermediates are made, they are also degraded.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Regulation of intracellular cyclic AMP levels in the white-rot fungus Phanerochaete chrysosporium during the onset of idiophasic metabolism

Adenylate cyclase activity in Phanerochaete chrysosporium was present in cell fractions sedimenting at 1,000xg, 15,000xg, and in the 150,000xg supernatant. A small amount of activity in the 1,000xg pellet could be solubilised by treatment with Triton X-100, and the enzyme in all fractions required an ATP-Mn²⁺ substrate. Adenylate cyclase activity in the 150,000xg pellet was low (0.003 nmol/mg protein·min) and may have resulted from contamination by other fractions. Highest adenylate cyclase specific activity (0.37 nmol/mg protein ·min) was recorded in the 150,000xg supernatant at the onset of idiophasic metabolism. During this growth phase, adenylate cyclase activity also increased in the 1,000xg pellet and was maximally 4.5-fold greater than that in primary phase cultures. No significant cAMP-phosphodiesterase activity could be detected during growht in any of the cell fractions or in the growth medium with either Mn²⁺, Mg²⁺, or Ca²⁺ as added cations. The extracellular cAMP concentration increased logarithmically during primary growth; however, in cultures in idiophasic metabolism cAMP levels remained constant and relatively low. We suggest that excretion into the medium is the principal means by which intracellular cAMP levels are decreased in P. chrysosporium.Abbreviation EB extraction buffer     

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5′-adenosyl)-l-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.     

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 .     

Methanogenic bacteria as a key factor involved in changes of town gas stored in an underground reservoir

Growth of Phanerochaete chrysosporium in a nitrogen-limited medium buffered with sodium acetate, instead of the commonly used 2,2-dimethylsuccinate (DMS), resulted in quantitative and qualitative differences in the production of various extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) involved in lignin degradation. The results indicate that production of LIPs and MNPs can be selectively enhanced by manipulation of culture conditions. Partial N-terminal analyses of the major LIPs and MNPs have made it possible to assign a specific protein to the specific genes and cDNAs that have been reported recently. The LIPs and MNPs differed widely in their ability to decolorize various dyes that are known to be degraded by the lignin degrading enzyme system of P. chrysosporium.     

培养于天然冷杉木片的黄孢原毛平革菌木质素过氧化物酶基因表达的RT-PCR分析

利用RT-PCR方法分析了生长于冷杉木片上的黄孢原毛平革菌木质素过氧化物酶基因lipA2(GLG3)、lipC1(GLG2)、lipC2(GLG5)、lipD2(GLG1)、lipE(LPO811)的表达。结果发现在不同的培养时间里仅有特定的基因表达,在第2周时仅有lipA2(GLG3)基因表达,在第4周时未检测到任何基因的表达,在第6周时lipD2(GLG1)和lipC1(GLG2)基因表达,在第8周时仅有lipA2(GLG3)基因表达。这些结果说明,在冷杉木片上培养的黄孢原毛平革菌的lip基因表达具有明显的时间特异性,并且与限定培养基中得到的结果明显不同。     

根癌农杆菌介导的白腐丝状真菌——黄孢原毛平革菌的转化

利用农杆菌介导的方法成功地对黄孢原毛平革菌（Phanerochaete chrysosporium）进行了遗传转化。将含有潮霉素磷酸转移酶融合基因的双元质粒pCH61300转入根癌农杆菌（Agrobacterium tumefaciens）208中,然后用该转化菌分别感染黄孢原毛平革菌的分生孢子和原生质体,获得16株可能的转化子,经复筛,共获得6株潮霉素抗性水平为100μg/mL的稳定转化子,分生孢子和原生质体的转化频率没有明显差别。PCR检测结果显示,抗性基因已导入黄孢原毛平革菌细胞中;Southern杂交表明,TDNA以单拷贝形式整合到黄孢原毛平革菌基因组中。其中的一个转化子菌落形态与原野生型菌株相比有所不同,菌丝稀薄,分生孢子较少。利用分生孢子转化更为简便易行,无需特殊的设备和制备原生质体,此方法为深入开展该菌的遗传转化研究奠定了基础。     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

Novel heme-containing enzyme possibly involved in lignin degradation by the white-rot fungus Junghuhnia separabilima

Abstract The white-rot fungus Junghuhnia separabilima (Pouz.)Ryv, showed high levels of laccase production in cultures supplemented with veratric acid. Laccase, lignin peroxidase and an unknown peroxidase were separated from the extracellular culture fluid using anion-exchange FPLC. Three laccase species, three lignin peroxidases and a novel heme-containing protein were characterized by gel electrophoresis and isoelectric focusing. The new hemoprotein has a molecular mass of 44 kDa, isoelectric point of 3,4 and pH optimum of 5.5 for oxidation of o -dianisidine in the presence of H₂O₂. However it oxidised diaminobenzidine and guaiacol in the absence of H₂O₂. Veratryl alcohol and phenol red were not substratesfor this enzyme with or without addition of H₂O₂ and Mn(II). In addition the enzyme did not produce H₂O₂.     

Mineralization of biphenyl and PCBs by the white rot fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium is unique in its ability to totally degrade a wide variety of recalcitrant pollutants. We have investigated the degradation of biphenyl and two model chlorinated biphenyls, 2,2',4,4'-tetrachlorobiphenyl and 2-chlorobiphenyl by suspended cultures of P. chrysosporium grown under conditions that maximize the synthesis of lignin-oxidizing enzymes. Radiolabeled biphenyl and 2'-chlorobiphenyl added to cultures at concentrations in the range 260 nM to 8.8 muM were degraded extensively to CO(2) within 30 days. In addition, from 40% to 60% of the recovered radioactivity was found in water-soluble compounds. A correlation between the rate of degradation and the synthesis of ligninases or Mn-dependent peroxidases could not be observed, indicating that yet unknown enzymatic system may be resonsible for the initial oxidation of PCBs. The more heavily chlorinated PCB congener, 2,2',4,4'-tetrachlorobiphenyl was converted to CO(2) less readily; approximately 9% and 0.9% mineralization was observed in cultures incubated with 40 nM and 5.3 muM, respectively. Overall, our results indicate that P. chrysosporium is a promising organism for the treatment of wastes contaminatd with lightly and moderately chlorinated PCBs. (c) 1992 John Wiley & Sons, Inc.     

Lignin peroxidase is involved in the biobleaching of manganese-less oxygen-delignified hardwood kraft pulp by white-rot fungi in the solid-fermentation system

Biobleaching of manganese-less oxygen-delignified hardwood kraft pulp (E-OKP) by the white-rot fungi Phanerochaete sordida YK-624 and P. chrysosporium was examined in the solid-state fermentation system. P. sordida YK-624 possessed a higher brightening activity than P. chrysosporium, increasing pulp brightness by 13.4 points after seven days of treatment. In these fermentation systems, lignin peroxidase (LiP) activity was detected as the principle ligninolytic enzyme, and manganese peroxidase and laccase activities were scarcely detected over the course of treatment of E-OKP by either fungus. Moreover, a linear relationship between brightness increase and cumulative LiP activity was observed under all tested culture conditions with P. sordida YK-624 and P. chrysosporium. These results indicated that LiP is involved in the brightening of E-OKP by both white-rot fungi.     

Effect of Organic Acids on Reactions Catalyzed by Manganese Peroxidase from Phanerochaete chrysosporium

The effect of several organic acids on the oxidation of Mn(II) catalyzed by manganese peroxidase was studied. Reactivities of manganese peroxidase and chemically prepared Mn(III) organic acid complexes towards phenolic compounds were compared. If lactate appears to be the best complexant for manganese peroxidase activity, chemically prepared Mn(III)—lactate complex is a less effective oxidant towards phenolic compounds than other Mn(III)—complexes. Our results agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. Malonate and lactate seem to be the most attractive complexants for practical applications of manganese peroxidase and were used in enzymatic treatment of hardwood kraft pulp. Bleaching of kraft pulp was studied and after alkaline extraction, a significant decrease of kappa number was measured. The bleaching was enhanced in lactate buffer.