Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.     

Cytochrome P450 oxygenases of monoterpene metabolism

The cytochrome P450 monoterpene oxygenases are largely responsible for imparting structural and functional diversity to this family of natural products. In most cases, cytochrome P450-mediated allylic hydroxylation of a parental monoterpene olefin leads to a series of redox transformations and conjugation reactions which yield a family of structurally related derivatives and isomers. An overview is provided of the extant monoterpene oxygenases, with examples mainly from the mint (Lamiaceae) family of essential oil plants, and, where possible, information on the structure, mechanism, localization and regulation of these enzymes is described. The review concludes with a brief assessment of biotechnological applications and a view to future research in this area.     

Melanin biosynthesis in pathogenic species of Sporothrix

Melanins are dark polymers found in the cell wall of pathogenic fungi, including species from the genus Sporothrix that are causative agents of sporotrichosis. In vitro experiments strongly suggest that these pigments are important for fungal virulence and survival in the host. In S. schenckii, melanin biosynthesis occurs via three different common pathways, which generate dihydroxynaphthalene (DHN)-melanin, DOPA-melanin or pyomelanin. Moreover, melanin biosynthesis can be enhanced when the fungus is in contact with some bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Melanin pigments have protective effects against antifungals in this genus. New scanning transmission electron tomography data indicates the accumulation of dark pigments in membrane-bound cytoplasmic organelles (melanosomes) in S. schenckii yeasts. Here, we provide an up to date of review the biosynthesis and role of melanins and discuss its roles on the cell biology and pathogenesis of Sporothrix spp.     

Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.     

Improving the catalytic performance of fungal laccases in monoterpene-based reaction systems

Ternary systems consisting of monoterpenes (α-pinene or d-limonene), tert-butanol and water were used as reaction media to enhance the catalytic performance of laccases from various fungi sources (Trametes versicolor, T. hirsuta and Botrytis cinerea). The enzymes had improved catalytic efficiency (5- to 10-fold) in α-pinene-rich environment, while optimal reaction rates were in high-water content systems (15.5% v/v). The stability of laccases was significantly improved in monoterpene-based systems (up to 90% residual enzyme activity after 24 h at 30°C) in comparison with other non-conventional media. The results indicate that these ternary systems can increase the potential of laccases as catalysts for various oxidations.     

Label-free differentially proteomic analysis of interspecific interaction between white-rot fungi highlights oxidative stress response and high metabolic activity

The laccase production by mycelial antagonistic interaction among white-rot fungi is a very important pathway for lignin degradation research. To gain a better understanding of competitive mechanisms under mycelial antagonistic interaction among three lignin-degrading white-rot basidiomycetes of Trametesversicolor (Tv), Pleurotusostreatus (Po) and Dichomitussqualens (Ds), mycelial morphology and proteins in three co-culture combinations TvPo (Tv cocultivated with Po), PoDs (Po cocultivated with Ds), TvDs (Tv cocultivated with Ds) were compared with corresponding each two mono-cultures. In this study, scanning electron microscopy detection of co-cultures indicated a highly close attachment of fungal hyphae with each other and conidiation could be inhibited under fungal interaction. In addition, a label-free proteomic analysis revealed changes on fungal proteomes existed in their counterpart competitors of co-culture. The maximum number of 1020 differentially expressed proteins (DEPs) were identified in PoDs relative to Po while the minimum number of 367 DEPs were identified in PoDs relative to Ds. Notably, we also found a large number of overexpressed proteins were oxidative stress-related proteins, followed by carbohydrate metabolism-related proteins and energy production-related proteins in all three co-culture combinations compared with control. These results were important for the future exploration of molecular mechanisms underlying lignin-degrading fungal interaction.     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Optimization of laccase production by Botryosphaeria sp. in the presence of veratryl alcohol by the response-surface method

Botryosphaeria sp. produced two laccases (PPO-I and PPO-II) constitutively, whose titers were enhanced by veratryl alcohol. The effect of veratryl alcohol and yeast extract concentration, time of cultivation and agitation speed were evaluated by factorial analysis to select variables for optimizing the production of laccases. Maximal laccase production was determined using a second-order central-composite design and analyzed by the response-surface method. Veratryl alcohol concentration and time of cultivation were the main factors increasing laccase production, while yeast extract had no influence within the range 0.2–2.0% w/v. Response-surface analysis showed that 30.4 mM veratryl alcohol, for 4.5 days at 28°C and 180 rpm, were the optimal conditions to maximize PPO-I production, while conditions for maximal PPO-II production occurred within a range of 28–35 mM veratryl alcohol over a growth period of 4–5.5 days. The model predicted 5.6 U ml⁻¹ for PPO-I, and 0.6–1.0 U ml⁻¹ for PPO-II, which agreed with the experimentally observed results.     

The phenoloxidases of the ascomycete Podospora anserina: The three forms of the major laccase activity

In Podospora anserina three laccase activities (I, II and III) were identified. Present results show the existence of an additional lacaase (an anodic protein; MW 80,000; R_f 0.07). Laccase IV derived from the dissociation at acid pH (4.5) of a protein which showed identical molecular weight (390,000) and R_f (0.1) to the oligomeric laccase I. The recovery of laccase I activity when starting from laccase IV (purified by means of isoelectric focusing) suggests that laccase I itself was the source of laccase IV. In turn, laccase IV gave rise to the laccase III after electrophoresis or dialysis at basic pH (8.5).     

Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0A when a few degrees of data were evaluated on a beam line X11.     

扩环酶的研究进展及其应用前景

扩环酶,也叫脱乙酰氧基头孢菌素Ｃ合成酶,催化青霉素Ｎ扩环生成脱乙酰氧基头孢菌素Ｃ,是头孢菌素生物合成中的关键酶。在真菌中它是一个双功能酶,同时具有扩环酶和羟化酶的活性;而在细菌中扩环和羟化却是由两个独立的酶来承担的。近年来,扩环酶被纯化成均一蛋白,有关它的性质、分子结构以及基因结构等方面的研究都取得了飞速发展,并不断地应用基因工程的技术探索其在抗生素生产上的应用。     

Reduction thermodynamics of the T1 Cu site in plant and fungal laccases

The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV–vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the E ^o′ of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same E ^o′, as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.     

Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterranean coastal area

Laccases (EC 1.10.3.2) are phenoloxidases involved in the transformation of the recalcitrant fraction of organic matter in soil. These enzymes are also able to transform certain aromatic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and are known to be inhibited by chloride ions. This study aims to test the potential of some fungal strains newly isolated from natural environments subjected to high osmotic pressure such as coastal ecosystems, to produce chloride tolerant laccases. Three strains were identified as Chaetomium sp., Xylogone sphaerospora (two Ascomycota), and Coprinopsis sp. (a Basidiomycota) and the laccases produced by these fungi were weakly inhibited by chloride ions compared with previous data from literature. Moreover, we tested their reactivity towards various PAHs which are widespread anthropic pollutants. They were able to transform anthracene to 9,10-anthraquinone and we determine 7.5 eV as the threshold of ionization potential for PAH oxidation by these laccases.     

海藻糖的生产制备及其应用前景

海藻糖是一种广泛分布于细菌、真菌和动植物体内的双糖。在生物体内 ,它不仅作为结构成分和能量物质存在 ,而且在热击和脱水等协迫条件下 ,对生物体和生物大分子起着良好的非特异性保护作用。由于其独特的生物学功能 ,它在食品、分子生物学、医药、化妆品、农业等方面具有广阔的应用前景。简述海藻糖的生产制备、应用研究及其前景展望。     

Testosterone hydroxylase as multibiomarker of effect in evaluating vinclozolin cocarcinogenesis

In this work the modulation of the regio- and stereo-selective hydroxylation of testosterone by vinclozolin was studied in evaluating cocarcinogenic properties. Changes of cytochrome P450-(CYP)-catalysed drug metabolism was investigated in liver, kidney and lung microsomes of Swiss Albino CD1 mice of both sexes after single (625 or 1250 mg kg-1 b.w.) or repeated (daily 750 mg kg-1 b.w. for 3 days) i.p. administrations. Treatment of mice with a single dose of vinclozolin caused in a dose-dependent fashion from 2 1 to 14 1-fold increase in the 7-, 6- and 2-hydroxylations of testosterone in liver. Lower increase in extrahepatic tissues ranging from 2 3 to 8 1-fold for testosterone 6-, 16 -, 2- and 2- hydroxylase activity in the kidney or from 2 2 to 5 1-fold for 6-, 16 -, 16 - and 2- hydroxylase activity in the lung were observed. Repeated treatment with this fungicide did not substantially modify the extent and pattern of induction, the liver being the only tissue responsive (up to 7 6-fold increase, 7-hydroxylation) in both male and female. In the kidney (7-, 6-, 16 -, 2-, 7-hydroxylations) and lung (6-, 7-, 6-, 16 -, 16 - and 2- hydroxylations), a typical sex-dependent induction (up to 9 0-fold, 16 -hydroxylation in the lung, female) was achieved. In general, however, vinclozolin has a complex pattern of induction and suppression of CYP-dependent enzymes, as exemplified from the reduced expression of some hydroxylations depending upon dose, sex and organ considered. For example, after a single administration, 16 -hydroxylation was suppressed in liver (up to 78% loss in male, higher dose), whereas 16 -hydroxylation was reduced in kidney up to 50% in both sexes (at the higher dose). Glutathione S-transferase activity, measured as index of post-oxidative reactions, was markedly increased by vinclozolin in the liver (up to 5 2-fold, female) and kidney (up to 3 9-fold, female) but not in the lung. Because both phase I and phase II reactions were enhanced by vinclozolin treatment in liver and kidney, the ratio between activation/detoxification mechanisms was slightly affected. Conversely, this ratio was shifted toward activating mechanisms in the lung, sustaining, in part, the expression of certain type of tumours tissue-dependent. Taken together, these findings seem to indicate the cotoxic, cocarcinogenic and promoting potential of this fungicide.