Diamination by N-coupling using a commercial laccase

Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite® II Base, from Novozymes. The amine and the p-hydrobenzoquinone was reacted under mild conditions (at room temperature and at 35 °C) in a reaction vessel open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoquinone. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/or 5-lipoxygenase inhibiting activity.     

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations are expensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

Polymerization of bisphenol A by purified laccase from Trametes villosa

The metabolism of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a highly purified laccase from the basidiomycete Trametes villosa. The enzyme reaction products ranged widely from water-insoluble to -soluble compounds, one of which was previously identified as 4-isopropenylphenol. (1)H NMR and electron-impact mass spectrum analyses showed that one of the insoluble products was a BPA dimer, 5,5'-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2'-diol. Field-desorption mass spectrum analysis revealed BPA oligomers, some of which contained phenol, within the insoluble fraction. These results indicate that the laccase reaction may contain successive BPA polymerization, followed by either the addition of phenol to the formed oligomers or their decomposition to release 4-isopropenylphenol.     

Degradation of bisphenol A by purified laccase from Trametes villosa

Degradation of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a purified laccase from the basidiomycete Trametes villosa. SDS--polyacrylamide gel electrophoresis of the purified laccase gave one single band with a mobility corresponding to MW 65 kDa. The absorption spectrum showed the characteristics of a blue copper protein with a maximum peak at 600 nm. HPLC analysis revealed that 2.2 micromol BPA were degraded by incubation with 1.5 units of the purified laccase in a total volume of 1.0 ml at pH 6.0 and 60 degrees C for 3 h. The enzyme reaction proceeded rapidly without requirement of mediators for the electron transfer. Isolation and identification of several reaction products are in progress, in which one product was identified as 4-isopropenylphenol by a gas chromatography--mass spectrophotometer.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

漆酶高产工程菌构建及漆酶对RBBR的脱色作用

研究提取产漆酶白腐菌 (Fomelignosus)的总RNA ,利用RT PCR克隆到漆酶的cDNA ,并将其克隆到表达载体pGAPZA ,重组质粒经线性化、电激转化PichiapastorisGS115、通过底物显色反应筛选漆酶生产工程菌株 ,在最适培养条件下该菌株产酶活力高达 9 0 3U·mL-1。纯化得到漆酶对RBBR(RemazolbrilliantblueR)有很好的脱色作用 ,该酶的最适脱色pH为 5 0 ,最适脱色温度为 30℃。当溶液中漆酶活力为 1 0U·mL-1,在最适脱色条件下作用12h ,10 0mg·L-1RBBR溶液脱色率可达 90 %以上。     

An unusual spirocyclic isopimarane diterpenoid and other isopimarane diterpenoids from fruiting bodies of Xylaria polymorpha

Two new isopimarane-type diterpenes, spiropolin A (1) and myrocin E (3), were isolated from Xylaria polymorpha together with the known compound, myrocin D (2), in the course of a screening of the fruiting bodies of X. polymorpha. Their structures were determined on the basis of spectroscopic analysis, chemical conversion and X-ray analysis. Spiropolin A (1) restored the growth inhibition caused by the hyperactivated Ca²⁺-signaling in mutant yeast.     

Copper content and other characteristics of purified peach laccase

The laccase from peaches was purified 750 x. The enzyme is a glycoprotein of mol. wt 73 500 containing 2 atm Cu/mol. It is inhibited by diethyldithiocarbamate. Some other characteristics of the enzyme, including its amino acid composition, are described.     

Oxidation of methanol,formaldehyde and formate by catalase purified from methanol-grown Hansenula polymorpha

Catalase has been partially purified from cell-free extracts of methanol-grown Hansenula polymorpha and its peroxidative properties were studied. It was shown that the enzyme is capable of oxidizing methanol, formaldehyde and formate in the presence of hydrogen peroxide. The physiological significance of these reactions in the transduction of energy from the oxidation of methanol in yeasts is discussed.     

漆酶催化苯甲醇氧化制备苯甲醛

以自制的高活性漆酶为催化剂,考察漆酶催化苯甲醇制备苯甲醛的工艺条件(底物浓度、介质体系、溶剂体系、氢受体、酶的用量、通氧方式等)对氧化反应的影响。结果发现:优化反应条件为以2,2,6,6-四甲基哌啶-1-氧基(TEMPO)为介质体系且TEMPO与苯甲醇的摩尔比为1∶4、60 mmol/L的丙酮为氢受体、漆酶比酶活80 U/mL、60mmol/L的苯甲醇,反应体系通O20.5 h后密闭反应36 h,苯甲醛的产率达98%。     

Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 degrees C, respectively. The K(m) value of the enzyme was 55.7 microM, and the V(max) was 0.0541 OD min(-1) U(-1) for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity.     

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

Mediator-assisted selective oxidation of lignin model compounds by laccase from Botrytis cinerea

Laccase from Botrytis cinereacatalyzes benzylic oxidations and cleavage of lignin-related diphenylmethanes. Selective reactions with non-phenolic monomeric or b-1-dimeric model compounds using O 2 and redox mediators can also be carried out. At substrate to mediator ratios of 5:1, 1-hydoxybenzotriazole (HOBT) is 8-20 fold more effective than 2,2-azinobis-3-ethylthiazoline-6-sulfonate (ABTS) for such biotransformations. With unblocked phenols, ring couplings are the dominant endproducts either with or without mediators.     

Selective modulation of NMDA responses by reduction and oxidation

Electrophysiological responses to the glutamate analog N-methyl-D-aspartate (NMDA) measured in three different central neuronal preparations are subject to a novel modulatory mechanism: they are substantially potentiated after exposure to the disulfide reducing agent dithiothreitol, while oxidation with 5-5-dithiobis-2-nitrobenzoic acid decreases the magnitude of the response. Modification of the NMDA response by either oxidation or reduction does not appear to affect the pharmacological properties of the receptor-channel complex. Since we observe that the redox state of the native receptor-channel complex varies widely among neurons, an in vivo mechanism that can strongly regulate NMDA-activated functions by either reduction or oxidation may exist. In addition, these results suggest that it may be possible to design specific redox agents for characterizing the NMDA receptor-channel complex.     

Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.     

Transformation and degradation of the disazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus

The degradation of the disazo dye Chicago Sky Blue 6B by a purified laccase from Pycnoporus cinnabarinus was investigated. Laccase was purified to homogeneity and characterized. The enzyme had a molecular size of 63 kDa as determined by SDS-PAGE and an isoelectric point at pH 3. Amino acid composition and N-terminal amino acid sequence was shown to be similar to other fungal laccases. The purified laccase was stable for 1 h at 60 degrees C and was irreversibly inactivated by sodium azide at 0.1 mM. Laccase was shown to initiate destruction of the chromophore of the disazo dye Chicago Sky Blue, resulting in the formation of two intermediate products with absorption intensities about one order of magnitude lower than the parent molecule. The rate at which the dye was transformed by purified laccase was shown to increase with increasing concentrations of the enzyme.     

A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.     

Stabilities and rates in the laccase/TEMPO-catalyzed oxidation of alcohols

The influence of alcohol, 4-acetylamino,2,2,6,6'-tetramethylpiperidinyloxy (4-acetylamino-TEMPO) and laccase (from Trametes versicolor, TvL) concentration in the aerobic oxidation of furfuryl alcohol was investigated. Studies show that the K _m for 4-acetylamino-TEMPO is around 6.3 mM (V _max=0.18 mM min^-1) using 6.6 U mL^-1 of laccase and a furfuryl alcohol concentration of 140 mM. Under these optimized conditions, the reaction rate is still dependent on the concentration of enzyme in solution. Laccase can be reused, with a residual activity of around 25%. An important conclusion is that laccase is not stable in the presence of oxoammonium salts, presumably due to degradation via oxidation of essential amino acid residues or the glycosyl moieties on the periphery of the enzyme.