Katabolismus von 4′,6-dihydroxyauron in pflanzlichen zellsuspensionskulturen

Cell suspension cultures of Glycine max, Phaseolus aureus, Cicer arietinum and Petroselinum hortense were shown to catabolize (α - ¹⁴C) - 4′,6-dihydroxyaurone as measured by ¹⁴CO₂ production and isolation of (¹⁴C)-p-hydroxybenzoic acid. Aurone catabolism in plants is thus comparable with the degradation of chalcones flavanones and flavonols because in all cases the B-ring is liberated as a substituted benzoic acid.     

Solubilization and Mineralization of Lignin by White Rot Fungi

The white rot fungi Lentinula edodes, Phanerochaete chrysosporium, Pleurotus sajor-caju, Flammulina velutipes, and Schizophyllum commune were grown in liquid media containing ¹⁴C-lignin-labelled wood, and the formation of water-soluble ¹⁴C-labelled products and ¹⁴CO₂, the growth of the fungi, and the activities of extracellular lignin peroxidase, manganese peroxidase, and laccase were measured. Conditions that affect the rate of lignin degradation were imposed, and both long-term (0- to 16-day) and short-term (0- to 72-h) effects on the production of the two types of product and on the activities of the enzymes were monitored. The production of ¹⁴CO₂-labelled products from the aqueous ones was also investigated. The short-term studies showed that the different conditions had different effects on the production of the two products and on the activities of the enzymes. Nitrogen sources inhibited the production of both products by all species when differences in growth could be discounted. Medium pH and manganese affected lignin degradation by the different species differently. With P. chrysosporium, the results were consistent, with lignin peroxidase playing a role in lignin solubilization and manganese peroxidase being important in subsequent CO₂ production.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded cellulose faster with organic nitrogen sources than with NH₄Cl. Simple and complex nitrogen sources added at the time of inoculation to N-limited cultures of P. chrysosporium, with glucose as carbon/energy source, transiently stimulated degradation of synthetic [¹⁴C]lignin to ¹⁴CO₂. The same nitrogen sources added 5 days after inoculation, when the cultures were entering secondary metabolism, delayed ¹⁴CO₂ production. The various N sources affected synthetic lignin degradation in defined medium differently than lignin degradation in aspen wood.     

Peroxidase-mediated chlorophyll degradation in horticultural crops

One of the symptoms of senescence in harvested horticultural crops is the loss of greenness that comes with the degradation of chlorophyll (Chl). With senescence, peroxidase, which is involved in Chl degradation, increased greatly in stored horticultural crops. C13²-hydroxychlorophyll a, an oxidized form of Chl a, is formed in vitro through Chl oxidation by peroxidase. Peroxidase mediates Chl degradation in the presence of phenolic compounds such as p-coumaric acid and apigenin, which have a hydroxyl group at the p-position. Apparently, not all phenolic compounds are able to degrade Chl in this system, and their effectiveness appears to depend on their molecular configuration. In peroxidase-mediated Chl degradation, peroxidase oxidizes the phenolic compounds with hydrogen peroxide and forms phenoxy radical; then, the phenoxy radical oxidizes Chl and its derivatives to colorless low molecular weight compounds through the formation of C13²-hydroxychlorophyll a,a fluorescent Chl catabolite and a bilirubin-like compound as an intermediate. In addition to the phenoxy radical, superoxide anion, which is formed in the peroxidase-catalyzed reaction, might be involved in Chl oxidation. Moreover, Chl degradation by peroxidase seems to occur in the chloroplast and/or the vacuole. The involvement of peroxidase in Chl degradation in senescing horticultural crops is also discussed.     

Biosynthesis of piperlongumine

The incorporation of l-[U-¹⁴C]lysine and l-[U-¹⁴C]phenylalanine into piperlongumine has been demonstrated in Piper longum. The subsequent stepwise degradation to methyl-(3,4,5-trimethoxyphenyl)-propanoate and δ-aminovaleric acid revealed that the C₆-C₃ moiety of the alkamide arises from phenylalanine; the heterocyclic ring is biosynthesised from lysine. It has also been shown that dl-[2-¹⁴C]tyrosine and [2-¹⁴C]sodium acetate are poor precursors of piperlongumine.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Oxidative coupling of tyrosine and ferulic acid residues: Intra- and extra-protoplasmic occurrence,predominance of trimers and larger products,and possible role in inter-polymeric cross-linking

I discuss the range of oxidative phenolic coupling products formed from the tyrosine residues of cell wall glycoproteins and from the feruloyl residues of wall polysaccharides possibly by the action of peroxidases and/or laccases. In the cases of both tyrosine- and ferulate-coupling, the coupling products are not confined to dimers but include trimers and probably higher oligomers, which are sometimes predominant. Thus, some previous assays, in which specifically dimers were monitored, will have underestimated the extent of phenolic coupling. The possibility is discussed that some of the phenolic coupling products, in both glycoproteins and polysaccharides, are inter-polymeric and that they may therefore act as cross-links in the cell wall. The limitations in the evidence for this hypothesis are stressed. The sub-cellular site of oxidative phenolic coupling is discussed. In-vivo radiolabelling of cultured maize cells with [¹⁴C]cinnamate has shown that, especially in young, rapidly growing cultures, much oxidative coupling of feruloyl-arabinoxylans occurs within the endomembrane system, before secretion of the polysaccharides into the cell wall. Appreciable feruloyl coupling within the cell wall depended on the supply of H₂O₂ and on culture age. The situation with tyrosine coupling in glycoproteins is also debated. Although peroxidase activity has long been known to occur in the endomembrane system, the recent finding of intraprotoplasmic feruloyl coupling provided the first evidence that peroxidases (and/or laccases) may act in this sub-cellular location in vivo. I draw attention to the distinction between peroxidase action (in vivo) and activity (assayed in vitro), and to the unknown origin of H₂O₂ within the endomembrane system.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Lignocellulose Degradation during Solid-State Fermentation: Pleurotus ostreatus versus Phanerochaete chrysosporium

Lignocellulose degradation and activities related to lignin degradation were studied in the solid-state fermentation of cotton stalks by comparing two white rot fungi, Pleurotus ostreatus and Phanerochaete chrysosporium. P. chrysosporium grew vigorously, resulting in rapid, nonselective degradation of 55% of the organic components of the cotton stalks within 15 days. In contrast, P. ostreatus grew more slowly with obvious selectivity for lignin degradation and resulting in the degradation of only 20% of the organic matter after 30 days of incubation. The kinetics of ¹⁴C-lignin mineralization exhibited similar differences. In cultures of P. chrysosporium, mineralization ceased after 18 days, resulting in the release of 12% of the total radioactivity as ¹⁴CO₂. In P. ostreatus, on the other hand, 17% of the total radioactivity was released in a steady rate throughout a period of 60 days of incubation. Laccase activity was only detected in water extracts of the P. ostreatus fermentation. No lignin peroxidase activity was detected in either the water extract or liquid cultures of this fungus. 2-Keto-4-thiomethyl butyric acid cleavage to ethylene correlated to lignin degradation in both fungi. A study of fungal activity under solid-state conditions, in contrast to those done under defined liquid culture, may help to better understand the mechanisms involved in lignocellulose degradation.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Studies on flavonoid metabolism. Biosynthesis of (+)-[14C]catechin by the plant Uncaria gambir Roxb

1. The formation of (+)-[¹⁴C]catechin has been demonstrated in Uncaria gambir after the administration of ¹⁴CO₂ and [1-¹⁴C]acetate. 2. By alkaline degradation to phloroglucinol and protocatechuic acid it has been shown that administration of ¹⁴CO₂ resulted in equal labelling of the A and B rings of catechin, whereas [1-¹⁴C]-acetate gave rise to labelling largely in the A ring. 3. Incorporation of ¹⁴C from both ¹⁴CO₂ and [1-¹⁴C]acetate into (+)-catechin was greater in young than in older leaves.     

Metabolism of estradiol by uterine peroxidase: Nature of the water-soluble products

The nature of the water-soluble products formed by incubating labelled estradiol with uterine peroxidase in the presence of H₂O₂ and tyrosine was examined by two-dimensional thin-layer chromatography and high voltage electrophoresis. It was shown that the steroid and amino acid were associated in a 1:2 or 1:3 ratio and evidence was provided by ³H-exchange for the interaction of tyrosine with ring A of estradiol at C-2 and C-4. The possible role of estrogen-induced peroxidase in the uterus in vivo is discussed.     

Content of carotenoids and the state of tissue antioxidant enzymatic complex in bivalve mollusc Anadara inaequivalvis Br.

This work deals with studies on the content of carotenoids, the state of antioxidant (AO) enzymatic complex, and the intensity of lipid peroxidation in tissues (hepatopancreas, gill, and foot) of the Black Sea bivalve mollusc Anadara inaequivalvis. Tissues with a high content of the pigment have been established to have a low activity of the key AO enzymes: superoxide dismutase, catalase, and glutathione reductase, as well as an elevated content of reduced glutathione (R ² = 0.81–0.97). The differences of the recorded activities between the tissues reached 1.7–2.9 times (p < 0.05–0.01). At increased concentrations (more than 2.5 mg · 100 g^?1 tissue), carotenoids show an insignificant pro-oxidant effect manifested in a rise of glutathione peroxidase activity. The competitive interrelations between these molecular complexes for the same kinds of reactive oxygen species (O₂, OH^·, and ¹O₂) are discussed.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

Biodegradation of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Ring Cleavage Product 4-Nitro-2,4-Diazabutanal by Phanerochaete chrysosporium

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO₂ and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH₂NHNO₂, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium. A soil sample taken from an ammunition plant contained RDX (342 μmol kg⁻¹), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 μmol kg⁻¹), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 μmol kg⁻¹), and traces of NDAB (3.8 μmol kg⁻¹). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ± 22 μmol kg⁻¹) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N₂O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N₂O. To determine C stoichiometry, we first generated [¹⁴C]NDAB in situ by incubating [¹⁴C]RDX with strain DN22, followed by incubation with the fungus. The production of ¹⁴CO₂ increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies.     

A fluorometric method for the kinetic assay of indole-3-acetic acid oxidase.

The oxidation of IAA by peroxidase (1) and by more specific oxidases (2) leads to the formation of products which may have physiological activity (3, 4). The colorimetric estimation of IAA oxidation products involving reaction with p-dimethylaminocinnamaldehyde (DMACA) is reported to be more sensitive than other end point determinations such as the Salkowski and Ehrlich procedures which monitor the disappearance of IAA (5). These methods are end point procedures and, as such, are awkward and time consuming and present difficulties in obtaining kinetic data and measuring lag times. IAA oxidation has also been monitored by measuring ¹⁴CO₂ released from [1-¹⁴C] IAA (6) and uv spectral shifts during oxidation of IAA were reported by Meudt (3). The present paper reports a new procedure for the assay of horseradish peroxidase catalyzed oxidation of IAA. The assay procedure is based on the continuous measurement of a fluorescent product of the reaction.     

α-Acid degradation by suspension culture cells of humulus lupulus

In a suspension culture of Humulus lupulus hop α-acids could not be detected. However, the culture was shown to have an in vivo ability to degrade exogenous α-acids and related compounds. It is shown that this is due to peroxidase with a rate constant for α-acid degradation of 2.7 × 10⁴/M · sec.