Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Biosynthesis of radiolabeled verruculogen by Penicillium simplicissimum.

In surface culture of Penicillium simplicissimum, verruculogen was shown to be biosynthesized from the intact carbon skeletons of tryptophan and proline, isoprenoid derivatives of mevalonic acid, and a methyl group donated by methionine. Selected radiolabeled precursors (1 mCi) pulse-fed at the optimum stage of fermentation yielded verruculogen (specific activity, 5.89 X 10(2) microCi mmol-1) labeled in the prolyl and isoprenyl regions of the molecule and suitable for metabolic studies.     

Transformation and mineralization of halophenols by Penicillium simplicissimum SK9117

The metabolism of monohalophenols by Penicillium simplicissimum SK9117, isolated from a sewage plant was investigated. In submerged cultures, 3-, 4-chlorophenol, and 4-bromophenol were metabolized in the presence of phenol. 3-Chlorophenol was transformed to chlorohydroquinone, 4-chlorocatechol, 4-chloro-1,2,3-trihydroxybenzene, and 5-chloro-1,2,3-trihydroxybenzene. With 4-chlorophenol only 4-chlorocatechol was observed as transient product. A release of chloride ions was not observed. Whereas monobromo-, and monochlorophenols could not support growth as sole carbon and energy source, growth and release of fluoride ions were observed with monofluorophenols as substrates. In presence of phenol, the degradation of all monofluorophenols was enhanced. Substrate and cosubstrate disappeared simultaneously. 3-Fluorophenol and 4-fluorophenol were completely mineralized as shown by the equimolar release of fluoride ions.Parts of the results have been presented at the annual meeting of the VAAM in Stuttgart, Germany, March 1995.     

Transformation and mineralization of halophenols by Penicillium simplicissimum SK9117

The metabolism of monohalophenols by Penicillium simplicissimum SK9117, isolated from a sewage plant was investigated. In submerged cultures, 3-, 4-chlorophenol, and 4-bromophenol were metabolized in the presence of phenol. 3-Chlorophenol was transformed to chlorohydroquinone, 4-chlorocatechol, 4-chloro-1,2,3-trihydroxybenzene, and 5-chloro-1,2,3-trihydroxybenzene. With 4-chlorophenol only 4-chlorocatechol was observed as transient product. A release of chloride ions was not observed. Whereas monobromo-, and monochlorophenols could not support growth as sole carbon and energy source, growth and release of fluoride ions were observed with monofluorophenols as substrates. In presence of phenol, the degradation of all monofluorophenols was enhanced. Substrate and cosubstrate disappeared simultaneously. 3-Fluorophenol and 4-fluorophenol were completely mineralized as shown by the equimolar release of fluoride ions.     

Enzymatic determination of veratryl alcohol

A rapid and sensitive method was developed for the measurement of veratryl alcohol--a secondary metabolite of some lignin degrading fungi. The method is based on the enzymatic oxidation of veratryl alcohol to veratraldehyde by the ligninase of Phanerochaete chrysosporium. The purified enzymes oxidized veratryl alcohol completely to veratraldehyde (75%) and some unidentified products. The enzymatic method was applied to measure veratryl alcohol in the culture filtrates of Chrysosporium pruinosum and it gave the same results as the conventional method involving extraction and separation by high-pressure liquid chromatography. Benefits and limitations of the method are discussed.     

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

Structure of the xylanase from Penicillium simplicissimum.

Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a crystallographic resolution of 1.75 A. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (R(free) = 23%) for data between 10 and 1.75 A. The xylanase folds in an (alpha/beta)8 barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft. In its overall shape, the P. simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.     

Initial steps of phloroglucinol metabolism in Penicillium simplicissimum

The pathway for the aerobic catabolism of 1,3,5-trihydroxybenzene (phloroglucinol) by a new strain of Penicillium was investigated using both in vivo and in vitro cell-free systems. The fungal strain was isolated by enrichment on phloroglucinol and identified as P. simplicissimum (Oud) Thom. It grew optimally at pH 5.5 and 27°C with 119 mM (1.5%w/v) of phloroglucinol in a basal mineral salts medium. Vapours of the crystalline substrate placed in a Petri-plate lid supported the growth of the fungal colonies on the agar surface. Mycelia grown on phloroglucinol accumulated 1,2,4-trihydroxybenzene and resorcinol in the medium. Washed, resting mycelia grown on phloroglucinol, when resuspended in a buffer utilized oxygen in the presence of catechol, resorcinol, pyrogallol and phloroglucinol. A NADPH-dependent reductase in the cell-free extract reduced phloroglucinol to dihydrophloroglucinol. This electron donor could not be replaced by NADH. Resorcinol hydroxylase, phloroglucinol reductase, catechol-1,2-oxygenase, and catechol-2,3-oxygenase were detected in cell-free extracts of mycelia grown on phloroglucinol. The possible steps in the degradation of phloroglucinol are discussed.     

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.     

Purification and properties of lipase from Penicillium simplicissimum.

A Penicillium simplicissimum strain has been found to produce an inducible extracellular lipase. Triolein was the best inducer for the enzyme production with the highest activity being achieved after 48 h of incubation. The purified lipase showed a molecular weight of 56,000 by SDS-PAGE. The enzyme exhibited a high ratio of apolar amino acids. The lipase was stable in the pH range of 5-7 and at 50 degrees C for 15 min. The optimum assay conditions were 37 degrees C and pH 5.0. The enzyme showed a high stability in water immiscible organic solvents. Lipase from P. simplicissimum is nonspecific and hydrolyses each of the three bonds of triacylglycerols.     

Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions

Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics. Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria. However, succinate excretion is also widespread among fungi. In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions. The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)). A strong increase of succinate excretion was observed if the respiratory chain was inhibited. It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle. Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive. A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.     

New Oleanane Triterpene with Three Ketones Produced by Penicillium simplicissimum ATCC 90288

A new oleanane triterpene was isolated from okara fermented with Penicillium simplicissimum ATCC 90288. Its structure was established to be 7β, 15α,24-trihydroxyolean-12-en-3,11,22-trione by spectroscopic techniques and an X-ray crystaliographic analysis.     

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)^–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate. Received: 28 July 1997 / Accepted: 19 November 1997     

Nematicidal alkaloids and related compounds produced by the fungus Penicillium cf. simplicissimum

A new nematicidal alkaloid, peniprequinolone (1), together with the known alkaloids penigequinolones A and B (2a, 2b), 3-methoxy-4-hydroxy-4-(4'-methoxyphenyl)quinolinone (3), and 3-methoxy-4,6-dihydroxy-4-(4'-methoxyphenyl)quinolinone (4), were isolated from Penicillium cf. simplicissimum (Oudemans) Thom. Cyclopenin (5) and a compound (6a/6b) structurally related to cyclopenin also were isolated from the fungus, and their structures were established by spectroscopic analysis. The biological activities of 1, 2, 3, 4, and 5 were examined by a bioassay with root-lesion nematodes.     

Degradation of organophosphonates by Penicillium citrinum

Utilization of organophosphonates as the sole source of phosphorus, carbon or nitrogen by a soil isolate of Penicillium citrinum was studied. Penicillium citrinum was found to utilize 2-aminoethylphosphonic and 2-oxoalkylphosphonic acids as the sole phosphorus source whereas 1-hydroxyalkylphosphonates as well as 1-aminoalkylphosphonates and their dipeptides did not support the growth of the fungus. The mould did not metabolize any of the phosphonates tested, when they served as the sole carbon or nitrogen source.
Penicillium citrinum is perhaps the first mould strain isolated from soil, shown to be capable of organophosphonate degradation.     

Radical intermediates in veratryl alcohol oxidation by ligninase. NMR evidence

Proton nuclear magnetic resonance (NMR) spectra of veratryl alcohol (3,4-dimethoxybenzyl alcohol) were obtained during its oxidation by ligninase. It was observed that a substantial increase in the linewidths of the resonances occurred only in the presence of both the enzyme and hydrogen peroxide. Quenching the reaction by the addition of alkali immediately restored the normal linewidths of the resonances. Furthermore, inversion-recovery experiments showed a decrease in the longitudinal relaxation time of the substrate when the enzyme was actively turning over. Changes in both these NMR parameters are consistent with the generation of radical intermediates during the ligninase-catalysed oxidation of veratryl alcohol.