Laccase–luminol chemiluminescence system: an investigation of substrate inhibition

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase–luminol system, determine its kinetic parameters, and analyze the effects of protein and OHˉ concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase–luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase–luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.     

变色栓菌漆酶基因的克隆与序列分析

本研究以PCR扩增的方法,从变色栓(菌Trametes versicolor)的基因组DNA中扩增出了一预期大小的DNA片断,并将其克隆到了pUCM-T载体上。经筛选、酶切、PCR鉴定,序列分析,证明该片断为变色栓菌漆酶基因的克隆。该基因的开放阅读框由1560个核苷酸组成,编码一个由519个氨基酸组成的多肽。与GeneBank中发表的Laccase基因(AY081188)序列比较发现,编码的氨基酸序列同源性为96%,而核苷酸序列的同源性为92%。     

Recombinant Laccase: I. Enzyme cloning and characterization

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white‐rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET‐28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl‐β‐d‐thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. J. Cell. Biochem. 114: 599–605, 2013. © 2012 Wiley Periodicals, Inc.     

真菌漆酶的酶活测定方法评价

目前真菌漆酶酶活的测定方法多样,没有统一的标准,致使不同研究之间的漆酶酶活无法进行比较分析,也造成漆酶产品在酶活质量意义上的混乱。因此,对测定真菌漆酶酶活的各种不同的分光光度法进行了综述和比较分析,认为采用ABTS法作为测定漆酶酶活的方法较具合理性和科学性,建议作为漆酶酶活测定的统一方法。     

Sequence analysis and homology modeling of laccase from Pycnoporus cinnabarinus

Industrial effluents of textile, paper, and leather industries contain various toxic dyes as one of the waste material. It imparts major impact on human health as well as environment. The white rot fungus Pycnoporus cinnabarinus Laccase is generally used to degrade these toxic dyes. In order to decipher the mechanism of process by which Laccase degrade dyes, it is essential to know its 3D structure. Homology modeling was performed in presented work, by satisfying Spatial restrains using Modeller Program, which is considered as standard in this field, to generate 3D structure of Laccase in unison, SWISSMODEL web server was also utilized to generate and verify the alternative models. We observed that models created using Modeller stands better on structure evaluation tests. This study can further be used in molecular docking techniques, to understand the interaction of enzyme with its mediators like 2, 2-azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and Vanillin that are known to enhance the Laccase activity.     

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.     

Factors affecting the activation of pulps with laccase

Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new functional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reactions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals.     

Production of lignin peroxidase and laccase by Phlebia radiata

Summary A cultivation method using carrierbound mycelium was developed for the production of lignin-modifying enzymes by Phlebia radiata. Laccase and lignin peroxidase were produced in batch and semi-continuous cultivations. Laccase activity was clearly enhanced by veratryl alcohol. The presence of both veratryl alcohol and Tween 80 was required for lignin peroxidase production in submerged cultivations. During the course of the semi-continuous cultivations production of lignin peroxidase activity increased fourfold compared with static cultivations.     

A disposable Laccase–Tyrosinase based biosensor for amperometric detection of phenolic compounds in must and wine

An amperometric biosensor for the detection of polyphenols in wine has been developed immobilizing the two enzymes Tyrosinase and Laccase on graphite screen printed electrodes modified with ferrocene. Different immobilization procedures have been carried out, the sensor operational parameters have been optimized, determining the best conditions and the analytical method for the analysis of samples. The biosensor has been then tested with real samples, using wines and musts supplied by Astra, experimental winery, in Imola (Italy). The biosensor gave good results when employed for wine analysis, showing a good agreement with the spectrophotometric data obtained with the Folin-Ciocalteu test, the official method for polyphenols’ analysis in wine. On the other hand, the measurements on musts and wines recently bottled, were seriously affected by the presence of an high level of free sulphur dioxide. SO₂ is the likely responsible for enzyme activity inhibition on the sensor. Further studies are currently proceeding to find out the most suitable conditions to obtain results not influenced by the presence of sulphur dioxide.     

Proton NMR Spectra of 2,4-Dinitrophenyl Derivatives of Amino Acids

During the decay of wood by the typical white rot fungus Coriolus versicolor, Laccase III was the most abundantly secreted phenol oxidase. In this study, we proposed a possibility of the intermediate degradation steps of polymeric lignin by a purified Laccase III using synthetic [β-¹³C] and [β-¹⁴C]lignin (DHP). When the [β-¹⁴C]DHP was incubated with Laccase III, the water-soluble degradation product formed was about 8% of the applied [β-¹⁴C]DHP. The enzymic attack of Laccase III catalyzed the cleavage of the intermonomer linkages in the side chain structure of the polymeric lignin. In polymeric lignin metabolism by this fungus, laccase activity was closely related to the accumulation of water-soluble degradation products.     

真菌漆酶性质、分子生物学及其应用研究进展

漆酶是一种含铜的多酚氧化酶。目前发现多种生物能够产生漆酶,包括植物、真菌、昆虫和细菌等,其中,以真菌中的白腐真菌研究最多。由于漆酶在生物漂白、农作物秸秆利用以及环境污染处理等方面具有广阔的应用前景,漆酶研究受到越来越多的关注。同时,随着分子生物学技术的发展,漆酶研究已经深入到基因水平,多种漆酶基因已经成功获得克隆,一些漆酶基因也实现了异源表达。现针对真菌漆酶的生物学性质、分子生物学及其应用的研究进展进行了概括总结,并对其前景进行了展望。     

Reexamination of the mechanisms of oxidative transformation of the insect cuticular sclerotizing precursor, 1,2-dehydro-N-acetyldopamine

1,2-dehydro-N-acetyldopamine (dehydro NADA) is an important catecholamine derivative formed during the sclerotization of insect cuticle. Earlier we have reported that tyrosinase-catalyzed oxidation of dehydro NADA produces a reactive quinone methide imine amide that forms adducts and cross-links through its side chain, thereby accounting for sclerotization reactions. Recently, laccase has also been identified as a key enzyme associated with sclerotization. Hence, we re-examined oxidation of dehydro NADA by tyrosinase and laccase using high performance liquid chromatography – tandem mass spectrometry. Tyrosinase-catalyzed oxidation of dehydro NADA not only generated dimers as reported earlier, but also generated significant amounts of oligomers. The course of laccase-catalyzed oxidation of dehydro NADA significantly differed from the tyrosinase reaction kinetically and mechanistically. Laccase failed to produce any detectable quinone or quinone methide as the primary two-electron oxidation product. Since laccases are known to generate primarily semiquinones as the initial products, lack of accumulation of two-electron oxidation products indicated that laccase reaction is primarily occurring via free radical coupling mechanism. Consistent with this proposal, laccase-catalyzed oxidation of dehydro NADA, resulted in the production of largely dimeric products and failed to produce any significant amount of oligomeric materials. These studies call for radical coupling as yet another major mechanism for sclerotization of insect cuticle.     

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants in wastewater. Laccase of the white-rot fungus Coriolopsis polyzona has been successfully immobilized on fumed silica nanoparticles using a novel method. It consists in the sorption of the enzyme to amino-modified silica nanoparticles and the subsequent covalent cross-linking using glutaraldehyde as a homobifunctional linker. The so-produced nanoparticulate material has been characterized by means of scanning electron microscopy and Brunauer–Emmett–Teller surface area analysis revealing modifications of the surface structure and area during the coupling procedure. Laccase immobilization on spherical nanoparticles produced according to the method of St?ber has been shown to be much less efficient than on fumed silica nanoparticles. Long-term stability assays revealed that the novel developed method allows a drastic stabilization of the enzyme. In real wastewater, 77% of the laccase activity remained on the nanoparticles over 1 month, whereas the activity of free laccase dropped to 2.5%. The activity loss on the nanoparticles resulted from partial inactivation of the immobilized enzymes and additional release into the surrounding solution with subsequent fast inactivation of the free enzymes, since almost no activity was found in the supernatants.     

Degradation of trilinolein by laccase enzymes

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.     

Loss of Cytochrome c Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of Azospirillum lipoferum

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.     

Applicability of Coriolopsis rigida for Biodegradation of Polycyclic Aromatic Hydrocarbons

Laccase was produced by Coriolopsis rigida using barley bran as substrate in solid-state fermentation (SSF) and also by submerged fermentation (SmF). The best results were obtained in SSF with twice the amount of laccase production. Laccase could be produced from repeated batch cultures of SSF over 30 days. The laccase degraded several polycyclic aromatic hydrocarbons (PAHs) in vivo and in vitro. The addition of an effective mediator, 1-hydroxybenzotriazol (50 µM), during in vitro treatment increased the degradation rate.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

真菌漆酶及其应用

漆酶是一种含铜多酚氧化酶,在白腐菌中普遍存在,少数低等真菌和植物中也产生,多为分泌型糖蛋白。至少20种漆酶得到了分离和纯化。该酶是一种氨基酸残基在500个左右的单体酶,一般都为酸性蛋白,含有4个铜离子,形成3个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的漆酶基因被克隆,许多来源的基因都是以家族形式存在于染色体上的。已研究的漆酶基因中都含有10个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊序列的存在与否决定了该酶的表达形式-诱导型或组成型,诱导型菌株的调控序列中含有一段受酚类化合物作用的序列,而不含有该序列的酶基因则都是组成型表达的。漆酶在S.cerevisiae、Trichodermareesei、A.oryzaeTATAamylase和Pichiapasti等异源表达系统中有成功表达的报道。漆酶的应用集中在以下几方面:漆酶参与的有机合成;生物检测;有毒化合物的消除;工业废水处理;纸浆的生物漂白;等等。     

Enzymatic scarification of Anacamptis morio (Orchidaceae) seed facilitates lignin degradation,water uptake and germination

• The seed coat of many species contains hydrophobic lignins, and in soil the action of microbial ligninases may contribute to release from dormancy. Laboratory use of ligninases to stimulate germination is promising because of the specific action on the seed coat, whereas chemical scarification agents may also corrode the embryo. We hypothesised that exposure of Anacamptis morio (Orchidaceae) seeds to fungal laccase would stimulate germination, and that the mechanism involves lignin degradation and increased imbibition.
• Germination capacity in vitro was quantified with 1 U filter‐sterilised laccase added to agar medium following autoclaving, compared to a 10% bleach solution (standard bleach surface sterilisation/scarification method used in orchid seed sowing). Lignin degradation was quantified using an optical method (phloroglucinol‐HCl staining) combined with image analysis, following experimental pre‐treatments involving immersion in laccase solution, distilled water (negative control) or bleach (positive control). Water uptake after experimental treatments was quantified as the proportion of seeds exhibiting visible uptake of an aqueous fluorochrome under UV excitation.
• Laccase stimulated a doubling of germination in vitro with respect to bleach surface sterilisation/scarification alone, from 23.7 to 49.8% (P = 0.007). Laccase and bleach methods both significantly decreased the optical signal of phloroglucinol (for laccase, to 79.9 ± 1.3% of controls; anova : F = 10.333, P = 0.002). Laccase resulted in a modest but highly significant (P < 0.0001) increase in water uptake with respect to the control (11.7%; cf 99.4% for bleach).
• Laccase scarification can stimulate germination of A. morio through a mechanism of targeted seed coat degradation. The results demonstrate the potential of this relatively non‐invasive enzymatic scarification technique.