Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper

The white-rot fungus Trametes pubescens MB 89 has been identified as an outstanding, although not-yet-described, producer of the industrially important enzyme laccase. Extracellular laccase formation could be greatly stimulated by the addition of Cu(II) to a simple, glucose-based culture medium. Using optimum Cu concentrations (1.5-2.0 mM), maximum values for laccase activity of approximately 65 U/ml were obtained. The synthesis of the laccase protein depended on the presence of Cu in the medium as shown by Western blot analysis. Copper had to be supplemented during the exponential phase of growth for its maximal effect; addition during the stationary phase, during which laccase activity is predominantly formed, resulted in markedly reduced laccase productivity. As was shown by X-ray microanalysis of T pubescens mycelia obtained from a laboratory fermentation, Cu was rapidly taken up by the fungal biomass. A possible explanation for this stimulatory effect of Cu on laccase biosynthesis could be a role for this enzyme activity in melanin synthesis. The stimulatory effect of Cu on laccase synthesis was also effective for several other basidiomycetes and hence could be used as a simple method to boost the production of this enzyme.     

The role and mechanism of diacylglycerol-protein kinase C1 signaling in melanogenesis by Cryptococcus neoformans

The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes a life-threatening meningoencephalitis by expression of virulence factors such as melanin, a black pigment produced by the cell wall-associated enzyme laccase. In previous studies (Heung, L. J., Luberto, C., Plowden, A., Hannun, Y. A., and Del Poeta, M. (2004) J. Biol. Chem. 279, 21144-21153) we proposed that the sphingolipid enzyme inositol-phosphoryl ceramide synthase 1 (Ipc1) regulates melanin production through the generation of diacylglycerol (DAG), which was found to activate in vitro protein kinase C1 (Pkc1). Here, we investigated the molecular mechanisms by which DAG regulates Pkc1 in vivo and the effect of this regulation on laccase activity and melanin synthesis. To this end we deleted the putative DAG binding C1 domain of C. neoformans Pkc1 and found that the C1 deletion abolished the activation of Pkc1 by DAG. Deletion of the C1 domain repressed laccase activity and, consequently, melanin production. Finally, we show that these biological effects observed in the C1 deletion mutant are mediated by alteration of cell wall integrity and displacement of laccase from the cell wall. These studies define novel molecular mechanisms addressing Pkc1-laccase regulation by the sphingolipid pathway of C. neoformans, with important implications for understanding and targeting the Ipc1-Pkc1-laccase cascade as a regulator of virulence of this important human pathogen.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.     

How the interaction of Listeria monocytogenes and Acanthamoeba spp. affects growth and distribution of the food borne pathogen

Listeria monocytogenes is a foodborne opportunistic pathogen capable to switch from an environmental saprophyte to a potentially fatal human pathogen. The fact that the pathogen maintains the genes suitable for an elaborate infectious process indicates that these genes are required to survive in the environment. However, no environmental host reservoir for L. monocytogenes has been identified so far. The similarity of free-living, bacteria-scavenging amoebae to macrophages led to the hypothesis that protozoa may represent the missing link in the ecology and pathology of L. monocytogenes. Consequently, numerous studies have been published reporting on the potential of Acanthamoeba spp. to serve as host for a variety of pathogenic bacteria. However, the data on the interaction of L. monocytogenes with Acanthamoeba spp. are inconsistent and relatively little information on the impact of this interaction on growth and distribution of the foodborne pathogen is currently available. Hence, this review focuses on the interaction of L. monocytogenes and Acanthamoeba spp. affecting survival and growth of the foodborne pathogen in natural and man-made environments, in order to highlight the potential impact of this interplay on food safety and human health.     

Laccase activity tests and laccase inhibitors

Sulfhydryl organic compounds described as laccase inhibitors: dithiothreitol, thioglycolic acid, cysteine, diethyldithiocarbamic acid, and sodium azide were tested for their activity toward laccase of Trametes versicolor in different test systems utilising 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2, 6-dimethoxyphenol as enzyme substrates. Only sodium azide acted as a true laccase inhibitor and showed no significant interference with the enzyme tests. All other substances did not significantly inhibit the laccase activity and the previously reported inhibitory effects result from the reductions of the reaction products such as ABTS radical cation and diquinone or subsequent non-enzymatic interactions during substrate oxidation. The latter apparently forms a complex with unreacted ABTS displaying varied spectral characteristics and resulting in an underestimation of enzyme activity.     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.     

影响白腐真菌AH28-2菌株漆酶合成的因子和发酵条件

White-rot fungus AH28-2, a newly isolated strain, produced effectively laccase by induction when grown on a synthetic medium. Aromatic compounds of low molecular weight had an inducing influence on laccase production and its isoenzyme compositions. The using of o-toluidine or syringic acid had the best inducing effect. Cu2+ concentration in medium had distinguished effect on laccase production. Enzyme activity was notably increased by Cu2+ and reached the maximum when Cu2+ final concentration was 5 mumol/L. Mn2+ inhibited the synthesis of laccase. Carbon and nitrogen limitation were not beneficial to laccase synthesis, while high nutrient organic medium was beneficial to the growth of cell and the synthesis of laccase. Using cellobiose as the sole carbon source, the highest level enzyme activity reached 82,923. 7 u/L under the condition of optimum fermentation with ABTS as substrate. This enzyme activity was 2.9-fold higher compared to the reported data on international references in recent years.     

Azo Dye Biodecolorization Enhanced by Echinodontium taxodii Cultured with Lignin

Lignocellulose facilitates the fungal oxidization of recalcitrant organic pollutants through the extracellular ligninolytic enzymes induced by lignin in wood or other plant tissues. However, available information on this phenomenon is insufficient. Free radical chain reactions during lignin metabolism are important in xenobiotic removal. Thus, the effect of lignin on azo dye decolorization in vivo by Echinodontium taxodii was evaluated. In the presence of lignin, optimum decolorization percentages for Remazol Brilliant Violet 5R, Direct Red 5B, Direct Black 38, and Direct Black 22 were 91.75% (control, 65.96%), 76.89% (control, 43.78%), 43.44% (control, 17.02%), and 44.75% (control, 12.16%), respectively, in the submerged cultures. Laccase was the most important enzyme during biodecolorization. Aside from the stimulating of laccase activity, lignin might be degraded by E. taxodii, and then these degraded low-molecular-weight metabolites could act as redox mediators promoting decolorization of azo dyes. The relationship between laccase and lignin degradation was investigated through decolorization tests in vitro with purified enzyme and dozens of aromatics, which can be derivatives of lignin and can function as laccase mediators or inducers. Dyes were decolorized at triple or even higher rates in certain laccase–aromatic systems at chemical concentrations as low as 10 µM.     

Structural and functional responses of leaf‐associated fungal communities to chemical pollution in streams

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The role of laccase in prostaglandin production by Cryptococcus neoformans

Recently, it has been demonstrated that the opportunistic fungal pathogen Cryptococcus neoformans can synthesize authentic immunomodulatory prostaglandins. The mechanism by which this takes place is unclear as there is no cyclooxygenase homologue in the cryptococcal genome. In this study, we show that cryptococcal production of both PGE₂ and PGF_2α can be chemically inhibited by caffeic acid, resveratrol and nordihydroguaiaretic acid. These polyphenolic molecules are frequently used as inhibitors of lipoxygenase enzymes; however, blast searches of the cryptococcal genome were unable to identify any homologues of mammalian, plant or fungal lipoxygenases. Next we investigated cryptococcal laccase, an enzyme known to bind polyphenols, and found that either antibody depletion or genetic deletion of the primary cryptococcal laccase ( lac1 Δ) resulted in a loss of cryptococcal prostaglandin production. To determine how laccase is involved, we tested recombinant laccase activity on the prostaglandin precursors, arachidonic acid (AA), PGG₂ and PGH₂. Using mass spectroscopy we determined that recombinant Lac1 does not modify AA or PGH₂, but does have a marked activity toward PGG₂ converting it to PGE₂ and 15-keto-PGE₂. These data demonstrate a critical role for laccase in cryptococcal prostaglandin production, and provides insight into a new and unique fungal prostaglandin pathway.     

Optimization of some variables that affect the synthesis of laccase by Pleurotus ostreatus

The influence of initial pH, concentration of yeast extract, inducer, type of enzyme releaser and buffer system on the composition of a medium for laccase production by Pleurotus ostreatus DM-1513 was investigated. A 2⁵ full factorial experimental design was initially employed to evaluate the effects of these variables on the enzyme synthesis. Data analysis showed that low pH and high yeast extract concentration values, as well as the absence of both an inducer and a buffer system, had positive effects on the secreted enzyme levels, whereas the type of enzyme releaser did not have a significant effect. The highest levels of laccase activity (489–540?U/l) were obtained in optimization experiments using media with initial pH between 6.0 and 6.5 and yeast extract concentrations of 0–0.25%     

Direct and up-close views of plant cell walls show a leading role for lignin-modifying enzymes on ensuing xylanases

Background

A key barrier that limits the full potential of biological processes to create new, sustainable materials and fuels from plant fibre is limited enzyme accessibility to polysaccharides and lignin that characterize lignocellulose networks. Moreover, the heterogeneity of lignocellulosic substrates means that different enzyme combinations might be required for efficient transformation of different plant resources. Analytical techniques with high chemical sensitivity and spatial resolution that permit direct characterization of solid samples could help overcome these challenges by allowing direct visualization of enzyme action within plant fibre, thereby identify barriers to enzyme action.

Results

In the current study, the high spatial resolution (about 30 nm) of scanning transmission X-ray microscopy (STXM), and the detection sensitivity (ppm) of time-of-flight secondary ion mass spectrometry (ToF-SIMS), were harnessed for the first time to investigate the progression of laccase, cellulase and xylanase activities through wood samples, and to evaluate complementary action between lignin-modifying and polysaccharide-degrading enzymes. In particular, complementary insights from the STXM and ToF-SIMS analyses revealed the key role of laccase in promoting xylanase activity throughout and between plant cell walls.

Conclusions

The spatial resolution of STXM clearly revealed time-dependent progression and spatial distribution of laccase and xylanase activities, whereas ToF-SIMS analyses confirmed that laccase promoted protein penetration into fibre samples, leading to an overall increase in polysaccharide degradation. Spectromicroscopic visualizations of plant cell wall chemistry allowed simultaneous tracking of changes to lignin and polysaccharide contents, which provides new possibilities for investigating the complementary roles of lignin-modifying and carbohydrate-active enzymes.

     

Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes

Three white-rot fungi displayed a wide diversity in their response to supplemented aromatic compounds. Pyrogallol stimulated Cerrena unicolor laccase and manganese peroxidase (MnP) synthesis in synthetic medium 2.5- and 2-fold, respectively, whereas 2,4,6-trinitrotoluene (TNT) brought about a 2.8-fold increase in laccase yield by Trametes versicolor in submerged fermentation of ethanol production residue. No effect of the tested aromatic compounds on enzyme secretion by Ganoderma lucidum in mannitol-containing medium was detected. Nevertheless, G. lucidum is a potent producer of laccase in submerged fermentation of wheat bran and enzyme synthesis can be further increased by supplementation of medium with an appropriate inducer. The structure and the concentration of aromatic compounds play an important role in the regulation of enzyme synthesis. The supplementation of synthetic medium with 0.03–0.3 mM TNT or hydroquinone increased the differential rate of laccase synthesis by C. unicolor from 1,267 to 3,125–8,630 U mg biomass^?1 day^?1. Moreover, the same aromatic compound may function as either an inducer or a repressor, depending on the fungus and enzyme studied. Thus, hydroquinone increased 3-fold T. versicolor laccase activity decreasing 2- and 8-fold the yields of MnP and endoglucanase, respectively.     

Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969

Laccase is a widespread group of multi-copper enzymes which can catalyze the oxidation of a variety of organic compounds, with concomitant reduction of molecular oxygen to water. It has a wide application in industrial processes, particularly in renewable bio-energy industry. In this study, Pleurotus ostreatus strain 10969 with high yield of laccase, previously isolated from edible fungus growing on Juncao, was applied for optimization of fermentation media and growth parameters for the maximal enzyme production through response surface methodology and further characterization of the laccase activity. The results show that glucose and Mg²⁺ are the key ingredients for laccase production with the optimum concentration of 0.0988 g/mL and 7.3 mmol/L respectively. Compared to the initial medium, the highest laccase yield observed is approximately increased by 2.5 times under the optimized conditions. Extracellular laccase was then purified and its characters were analyzed. The molecular weight of the laccase is about 40 kDa, and the optimum pH and temperature for its activity is 4.0 and 50 °C with the corresponding Km and Vmax of 0.31 mmol/L and 303.25 mmol/min respectively. DTT, β-mercaptoethanol and NaN₃ nearly inhibit all activity of the laccase, as well as the metal ions especially Ag⁺. In summary, our results will facilitate the utilization of plant lignin in biomass energy industry.     

Platelet-activating factor acetylhydrolase increases during macrophage differentiation. A novel mechanism that regulates accumulation of platelet-activating factor

Monocytes and macrophages produce bioactive lipids, such as platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), that mediate inflammation. These cells synthesize PAF following their activation, but not constitutively. Previous studies have demonstrated that PAF accumulation is regulated by the activity of the synthetic enzymes. We observed that the accumulation of PAF in stimulated human monocytes decreased by 90% as they differentiated into macrophages. There was no decrease in the activities of the synthetic enzymes; however, the activity of the degradative enzyme, PAF acetylhydrolase, increased 260-fold. The increase in PAF acetylhydrolase activity appeared to result from a net increase in the synthesis of a new enzyme. These studies demonstrate a novel mechanism in which an increase of the degradative enzyme regulates the accumulation of PAF. This may be an important mechanism by which macrophages modulate inflammatory responses.