Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)₂]PF₆ and Ru(bpy)₂CO₃ under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)₂]PF₆ and 3 for Ru(bpy)₂CO₃. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Synthesis and structural characterization of a novel phenoxazinone dye by use of a fungal laccase

Laccase, isolated from Cerrena unicolor, is able to transform 3-amino-4-hydroxybenzensulfonic acid into a water soluble phenoxazine dye with an extinction coefficient (ɛ) of 8600 M⁻¹ cm⁻¹. The dye has been characterized using a variety of different analytic and spectroscopic techniques like UV–vis spectroscopy, HPLC (High Performance Liquid Chromatography), ESI/MS (Electrospray Ionization Mass Spectrometry) and the following NMR experiments: ¹H, ¹³C, TOCSY (Total Correlation Spectroscopy), HSQC (Heteronuclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Coherence) showing the structure of 2-amino-3-oxo-3H-phenoxazine-8-sulfonic acid. The advantages of the presented biocatalytic system, in alignment with chemical system to obtain Curie_22, are eco-sustainability and one step performance.     

A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms

Extracellular laccase (benzenediol: oxygen oxidoreductase EC 1.10.3.2) from the lignin-degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.     

Asymmetric diphenol formation by a fungal laccase.

A laccase isolated from the fungus Rhizoctonia praticola catalyzed the cross-coupling of two differently halogenated phenols. When 2,4-dichlorophenol and 4-bromo-2-chlorophenol were incubated together with the enzyme, three dimers were formed and isolated by thin-layer chromatography. The molecular weights of these compounds were determined by mass spectrometry as 322, 410, and 366, which correspond with the respective dimers of each of the phenols and with a hybrid formed from both, tentatively assigned the structure 3,3',5'-trichloro-5-bromo-2,2'-diphenol. Gas chromatography-mass spectrometry analysis of these products and of their methylated derivatives lent support to these structural assignments.     

Asymmetric diphenol formation by a fungal laccase.

A laccase isolated from the fungus Rhizoctonia praticola catalyzed the cross-coupling of two differently halogenated phenols. When 2,4-dichlorophenol and 4-bromo-2-chlorophenol were incubated together with the enzyme, three dimers were formed and isolated by thin-layer chromatography. The molecular weights of these compounds were determined by mass spectrometry as 322, 410, and 366, which correspond with the respective dimers of each of the phenols and with a hybrid formed from both, tentatively assigned the structure 3,3',5'-trichloro-5-bromo-2,2'-diphenol. Gas chromatography-mass spectrometry analysis of these products and of their methylated derivatives lent support to these structural assignments.     

真菌漆酶的结构与功能

漆酶是一种含铜的多酚氧化酶,能催化氧化酚类和芳香类化合物,同时伴随4个电子的转移,并将分子氧还原成水。漆酶结构的解析是阐明其催化作用机理、了解蛋白质结构与功能关系的基础。综述近年来对真菌漆酶蛋白结构及其功能研究的进展。     

Extraction and purification of laccase by employing a novel rhamnolipid reversed micellar system

Biosurfactant-based reversed micellar extraction (RME) is an innovative method for separation and purification of biomolecules. In this study, rhamnolipid (RL), a kind of biosurfactant, was firstly adopted to form a novel reversed micellar system for extracting and purifying laccase from Coriolus versicolor crude extract. Several significant factors affecting both forward and backward extraction processes were studied. The appropriate conditions for forward extraction process were: 3.3 mM RL, 50 mM KCl, pH 5.5 and extracting time 40 min. As regards backward extraction process, 250 mM KCl, pH 7.0 and extracting time 40 min were suggested. The corresponding activity recovery (AR) and purification fold (PF) were 92.7% and 4.79, respectively. Electrophoresis analysis indicated that the laccase was successfully purified. After this reversed micellar system was reused three times, the AR and PF declined to 70.8% and 4.35, respectively, indicating that the reversed micellar system could be reused. Comparisons results of synthetic surfactant-based RME and RL-based RME further verified the superiority of RL.     

Development of a fungal transformation system based on selection of sequences with promoter activity.

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.     

Development of a novel DNA chip based on a bipolar semiconductor microchip system

We have applied an integrated circuit photodiode array (PDA) chip system to a DNA chip. The PDA chip system, constructed using conventional bipolar semiconductor technology, acts as a solid transducer surface as well as a two-dimensional photodetector. DNA hybridization was performed directly on the PDA chip. The target DNA, the Bacillus subtilis sspE gene, was amplified by polymerase chain reaction (PCR). The 340-bp PCR product was labeled using digoxigenin (DIG). A silicon nitride layer on the photodiode was treated with poly-L-lysine to immobilize the DNA on the surface of the photodiode detection elements. Consequently, the surface of the photodiode detector became positively charged. An anti-DIG-alkaline phosphatase conjugate was reacted with the hybridized DIG-labeled DNA. A color reaction was performed based on the enzymatic reaction between nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining solution and a DNA complex containing antibodies. A blue precipitate was formed on the surfaces of the photodiode detection elements. Successful quantitative analysis of the hybridized PCR products was achieved from the light absorption properties of the blue enzymatic reaction product that was produced after a series of reaction processes. Our DNA chip system avoids the complicated optical alignments and light-collecting optical components that are usually required for an optical DNA chip device. As a result, a simple, compact, portable and low-cost DNA chip is achieved. This system has great potential as an alternative system to the conventional DNA reader.     

Development of a novel probiotic delivery system based on microencapsulation with protectants

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host’s gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F₁F₀-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.     

Stable expression of a fungal laccase protein using transplastomic tobacco

Laccase catalyzes the oxidation of various phenolic compounds that can be used in a wide range of industrial applications such as waste detoxification and the textile industry. In the present study, we generated transplastomic tobacco plants to develop a reliable commercial source of laccase production. The stability of the laccase protein in the transgenic plants was increased by using the enhancer sequence from green fluorescent protein, resulting in three independent lines with high levels of laccase accumulation (up to 2?% of total protein); significant laccase activity, however, was not detected. Interestingly, the transplastomic lines showed slightly retarded vegetative growth, with a light green leaf color in comparison with the control, which may be attributable to copper deficiency induced by ligand chelation by abundantly produced laccase. These results suggest that the tobacco chloroplast is an efficient system for the mass production of laccase protein, but further studies are needed to obtain active enzyme.     

Comparative study of substrates of fungal laccase

Coriolus versicolor, Pycnoporus cinnabarinus and Pycnoporus coccineus were grown under conditions to produce extracellular laccase. Prior to estimating enzyme activity, culture fluids were pretreated with catalase to destroy hydrogen peroxide and hence minimize peroxidase activity which might interfere with laccase determinations. Similar trends in enzyme assay were shown when colour reagents contained either syringaldazine or 3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolone hydrasone as laccase substrates. Use of 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as laccase substrate showed a different trend which was attributed to peroxidatic activity of the catalase using hydrogen peroxide generated by fungal oxidases. Peroxidatic activity was not observed with the other substrates.     

Autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of "active" and "resting" forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the "resting" enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned.     

Dehalogenation of chlorinated hydroxybiphenyls by fungal laccase

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by Pycnoporus cinnabarinus. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C---C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified.     

Use of laccase as a novel, versatile reporter system in filamentous fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

Incapacitating effects of fungal coinfection in a novel pathogen system

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd‐susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community‐level declines.     

Reactions of nitric oxide with tree and fungal laccase

The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.     

Production of a novel laccase from Paraphoma Sp

By using a laccase-secretion indicator for screening laccase-producing microorganisms, a novel laccase-producing strain was isolated and identified as Paraphoma sp. strain GZS18, it produced increased laccase and mycelia at 34?°C. Further investigations showed that the production of laccase by Paraphoma sp. GZS18 was greatly enhanced by less toxic inducers copper sulphate and methyl orange. Copper sulphate and methyl orange were added into the cultivation medium at 12 and 60?h, respectively, and the maximum laccase production was obtained. Through Plackett–Burman design and response surface methodology, we obtained the optimum production conditions as follows: methyl orange, 39.90?μM; addition time of copper sulphate, 11.95?h; addition time of methyl orange, 51.40?h. Under the above conditions, the experimental value of laccase production was 12,250.76?U/L. The extracellular laccase from Paraphoma sp. GZS18 was purified to homogeneity, which showed a molecular mass of 75?kDa. N-terminal amino acid sequences was AX^aVSVASREMT.