Lignin degradeation by white rot fungi

Abstract. The wood-degrading white-rot fungus Phanerochaete chrysosporium , has been the subject of intensive research in recent years and, based upon isolation of the extracellular enzyme ligninase, major advances have now been made toward elucidating the mechanism by which this fungus degrades lignin. From these developments, a model emerges which could explain the process by which wood-degrading fungi in general, attack lignin.     

植物苯丙氨酸解氨酶(PAL)在细胞分化中的作用

烟草、丹参和甜叶菊愈伤组织在分化过程中一般都出现两个PAL活性高峰。第一高峰在培养第一、二、三天中出现;第二高峰在第十一天前后出现。前者在分化或不分化培养基中都存在,似与组织分化无关,后者只在分化条件下才有,似可作为组织启动分化的指示酶。分化程度不同的组织,PAL活性有很大差异,即将或刚分化的组织活性最高,随着分化的进程活性趋于降低,老化的组织甚至丧失活性。PAL活性、木质素合成和管状份子形成之间有着紧密的相关性。     

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

Impact of residue quality on the C and N mineralization of leaf and root residues of three agroforestry species

A laboratory incubation experiment with ¹⁵N labeled root and leaf residues of 3 agroforestry species (Leucaena leucocephala, Dactyladenia barteri and Flemingia macrophylla) was conducted under controlled conditions (25 C) for 56 days to quantify residue C and N mineralization and its relationship with residue quality.No uniform relation was found between the chemical composition of the above and below residues. The leucaena and dactyladenia roots contained more lignin (8 and 26% respectively) and less N (2.0 and 1.0% respectively) than the respective leaves (2 and 13% lignin and 2.9 and 1.4% N, respectively), whereas the differences between the lignin and N contents of the flemingia leaves and roots were not significant (4.6 and 3.0% lignin and 2.63 and 2.68% N, respectively). The leucaena leaves contained more polyphenols than the roots (6.4 and 3.6%), while the polyphenol content of the leaves and roots of the other residues was similar (5.0 and 5.1% for dactyladenia and 4.0 and 3.5% for flemingia).Three patterns of N mineralization could be distinguished. A first pattern, followed by residues producing the highest amounts of CO₂, showed an initial immobilization of soil derived N, followed by a net release of both soil and residue derived N after 7 days of incubation. A second pattern, followed by the flemingia leaf residues which produced intermediate amounts of CO₂ and had an intermediate quality, showed no significant immobilization of soil derived N, and significant mineralization of residue N. A third pattern, followed by both low quality dactyladenia residues, showed a low release of residue derived N and a continued inmobilization of soil derived N.Residue C mineralization was significantly (p<0.05) correlated with the residue lignin content, C-to-N ratio, and polyphenol-to-N ratio. The proportion of residue N mineralized (immobilized) after 56 days of incubation was significantly correlated with the residue N content (p<0.01) and the C-to-N ratio (p<0.05). The relations were quadratic, rather than linear. The ratio of the proportion of residue N mineralized (immobilized) over the proportion of residue C mineralized after 56 days was highly significantly correlated with the lignin content (p<0.01) and C-to-N (p<0.001), lignin-to-N (p<0.01), polyphenol-to-N (p<0.01) and (lignin+polyphenol)-to-N ratios (p<0.01) in a linear way. This indicates that due to the low availability of the residue C, relatively less N is immobilized for the very low quality residues ((lignin+polyphenol)-to-N ratio: 29.7) than for the residues with a relatively higher quality ((lignin+polyphenol)-to-N ratios between 3.3 and 12.5).     

Oxidation and aromatic ring cleavage of 4-methoxy and 3,4-dimethoxycennamic acid by Lentinus edodes

Several products of metabolism and aromatic ring cleavage of 3-methoxy and 3,4-dimethoxycinnamic acid from ligninolytic cultures of Lentinus edodes were isolated and identified.     

Phytochrome-mediated effects on extracellular peroxidase activity, lignin content and bending resistance in etiolated Vicia faba epicotyls

Etiolated Vicia faba seedlings were exposed to continuous red light to investigate whether changes in extracellular peroxidase activity were correlated in time and localization with changes in extension growth and/or lignin content in the subapical region of the epicotyl. Continuous red light: (a) increased extracellular peroxidase activity after a lag of ca 0.5 h, followed by a maximum peak after 2.5 h due to slightly acidic isoforms (pI = 6–6.5, according to isoelectrofocusing gels), a minimum after 4 h and a second maximum after 8 h due to acidic isoforms (pI=4–5), (b) increased lignin content and epicotyl resistance to bending after a lag of ca 4 h, i.e. simultaneously with changes in acidic extracellular peroxidase activity, and (c) reduced extension growth to a stable rate after a lag of ca 1 h, not coinciding with the kinetics of any of the extracellular peroxidase isoforms. These effects of continuous red light were at least partially mediated by phytochrome. Tissue printing and anatomical studies revealed red light effects on extracellular peroxidase activity and lignin content mainly in the outer cortical parenchyma. The results are consistent with the involvement of phyto-chrome-mediated effects on extracellular peroxidases (acidic isoforms) in the transduction chain leading to lignin responses to red light.     

Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of French bean (Phaseolus vulgaris L.)

Antisera raised againstl-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue.l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with-aminooxy--phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.Abbreviations AOPP -aminooxy--phenylpropionic acid - C4H cinnamic acid-4-hydroxylase - CHS chalcone synthase - GRP glycine-rich glycoprotein - HRGP hydroxyproline-rich glycoprotein - Ig immunoglobulin - PAL phenylalanine ammonia-lyase G.P.B. thanks the Agicultural and Food Research Council for support.     

Degradation of the insecticide Hydramethylnon byPhanerochaete chrysosporium

The decomposition of the amidinohydrazone-type insecticide Hydramethylnon (HMN) by soil fungi has been investigated. A simple spectrophotometric method was developed for the estimation of HMN in soil and fungal culture media. HMN was found to be degraded in soil with a half life of 14 to 25 days.Degradation of HMN by the lignolytic fungus,Phanerochaete chrysosporium yielded two major breakdown products;p-(trifluoromethyl)-cinnamic acid (TFCA) andp-(trifluoromethyl)-benzoic acid (TFBA). TFCA was converted to TFBA which was subsequently metabolised via themeta-fission pathway. Fluoride release from HMN could not be detected.Abbreviations BzDAc benzene, dioxane, acetic acid (60: 36: 4) - DCM dichloroethane - DNPH 2,4-dinitro-phenylhydrazine - HMN Hydramethylnon - TDAc toluene, dioxane, acetic acid (90: 30: 1) - TFCA p-(trifluoromethyl)-cinnamic acid - TFBA p-(trifluoromethyl)-benzoic acid - TFP 1,5-bis(trifluoro-p-tolyl)-1,4-pentadien-3-one - VA veratryl alcohol     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Manipulation of quality and mineralization of tropical legume tree prunings by varying nitrogen supply

The effect of N supply on the quality of Calliandra calothyrsus and Gliricidia sepium prunings was studied in a glasshouse over a 7-month growing period. Increasing the concentration of N supplied from 0.625 to 10.0 mM NO₃-N resulted in increased N concentration but decreased polyphenol concentration, protein-binding capacity and C:N ratio of prunings from both species. Lignin concentration was not consistently altered by the N treatment. Mineralization of N from the prunings was measured over a 14-week period under controlled leaching and non-leaching conditions. The results indicated a strong interaction between legume species and concentration of N supply in their influence on N mineralization of the prunings applied to the soil. Differences in the %N mineralized were dictated by the quality of the prunings. The (lignin + polyphenol):N ratio was the pruning quality factor which could be used most consistently and accurately to predict N mineralization of the legume prunings incubated under leaching conditions, and the relationship was best described by a linear regression. Under non-leaching conditions, however, the protein-binding capacity appeared to be the most important parameter in determining the patterns of N release from the prunings studied. The relationship between the N mineralization rate constant and the protein-binding capacity was best described by a negative exponential function, y=0.078 exp(–0.0083x). The present study also indicated that the release of N from legume prunings containing a relatively high amount of polyphenol could be enhanced by governing the N availability conditions under which the plant is grown, for example whether or not it is actively fixing nitrogen. Estimates of pruning N mineralization after 14 weeks with the difference method averaged 6% (leaching conditions) and 22% (nonleaching conditions) more than with the ¹⁵N method for all legume prunings studied. The recovery of pruning by maize (4–38%) was well correlated with the % pruning N mineralized suggesting that incubation data closely reflect the pruning N value for a given catch crop under non-leaching conditions.