D. Schlosser

Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood

Trametes versicolor was shown to produce extracellular laccase during surface cultivation on glucose, wheat straw and beech wood. Growth on both wheat straw and beech wood led to an increase as high as 3.5-fold in extracellular laccase activity, in comparison with growth on glucose. The corresponding yields in fungal biomass reached only about 20% of the value obtained on glucose. Manganese peroxidase activity␣appeared during growth on wheat straw and beech wood. Mycelia grown on glucose, wheat straw and beech wood also showed intracellular laccase activities, monitored with 2,6-dimethoxyphenol, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) and 3,4-dihydroxyphenylalanine (l-DOPA). Assaying intracellular laccase with 2,6-dimethoxyphenol, syringaldazine and l-DOPA showed the maximum oxidation rates to be at pH values different from those producing maximum oxidation rates with extracellular laccase. In each case most of the total laccase activity was recovered from the culture filtrates. Growth on wheat straw and beech wood led to increased values for both extra- and intracellular laccase activities, based on fungal dry weight, in comparison with growth on glucose.

Novel enzymatic oxidation of Mn2+ to Mn3+ catalyzed by a fungal laccase

Fungal laccases are extracellular multinuclear copper‐containing oxidases that have been proposed to be involved in ligninolysis and degradation of xenobiotics. Here, we show that an electrophoretically homogenous laccase preparation from the white rot fungus Trametes versicolor oxidized Mn2+ to Mn3+ in the presence of Na‐pyrophosphate, with a K m value of 186 μM and a V max value of 0.11 μmol/min/mg protein at the optimal pH (5.0) and a Na‐pyrophosphate concentration of 100 mM. The oxidation of Mn2+ involved concomitant reduction of the laccase type 1 copper site as usual for laccase reactions, thus providing the first evidence that laccase may directly utilize Mn2+ as a substrate.

Degradation of 2-chlorophenol and formation of 2-chloro-1,4-benzoquinone by mycelia and cell-free crude culture liquids of Trametes versicolor in relation to extracellular laccase activity.

The white‐rot fungus Trametes versicolor was used to study the influence of extracellular laccase activity on the degradation of 2‐chlorophenol (2‐CP) and the formation of metabolites under conditions, characterized by the absence of other phenol‐oxidizing enzymes. 2‐CP enhanced the production of extracellular laccase by fungal mycelia. The formation of the metabolite 2‐chloro‐1,4‐benzoquinone (2‐ClBQ) was found to be correlated with extracellular laccase activity. In cell‐free crude culture liquids laccase was responsible for the oxidation of 2‐CP. In contrast to this, the disappearance of 2‐CP caused by the entire organism did not correlate with extracellular laccase activity. The primary oxidative step during the degradation of this compound can thus only partially be attributed to extracellular laccase; indicating the involvement of cell‐bound processes.

Steroid hydroxylation with free and immobilized cells of Penicillium raistrickii in the presence of β-cyclodextrin

Free and Ca-alginate-immobilized cells of Penicillium raistrickii i 477 were used for 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione. The product formation in the presence of β-cyclodetrin, in comparison with reactions carried out in the presence of methanol. Application of β-cyclodextrin led to increasing solubility of the steroid substrate. The fungus was able to utilize β-cyclodextrin as a carbon source.

Involvement of cytochrome P-450 in the 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

The steroid 15α-hydroxylase ofPenicillium raistrickii I 477 is inducible

SummaryIt could be shown that the 15α-hydroxylase ofPenicillium raistrickii i 477 is inducible by the substrate 13-ethylgon-4-en-3, 17-dione as well as other 3,17-ketosteroids. The influence of inducer concentration, time of this induction and the kind of steroid inducer is described and discussed.

Immobilization ofPenicillium raistrickii spores and mycelium growth for 15α-hydroxylation of 13-ethylgon-4-en-3,17-dione

Spores ofPenicillium raistrickii immobilized by gel entrapment, microencapsulation and surface adsorption were allowed to grow into mycelial form, in order to accomplish hydroxylation of 13-ethylgon-4-en-3,17-dione at the 15α-position. The hydroxylating activity of both free and immobilized cells at different pH and substrate concentration as well as under starvation conditions was studied. The productivity of cells immobilized by several techniques is compared.

Microbial Hydroxylation of Androsta-1, 4-diene-3, 17-dione

Abstract The possibility for hydroxylations of androsta-1, 4-diene-3, 17-dione (ADD, II) was studied using different strains of the fungi Penicillium raistrickii, Aspergillus ochraceus, Curvularia lunata and Beauveria bassiana. The isolated products were 15α-hydroxy-ADD (III, P. raistrickii), 11α-hydroxy-ADD (IV, A. ochraceus, B. bassiana), 14-hydroxy-ADD (V, C. lunata) and 7α, 11α-dihydroxy-ADD (VI, B. bassiana). Yields were observed up to 70%. The structures were elucidated by 1H-NMR, 13C-NMR, 2d-NMR, MS and UV/VIS.