In situ fabrication of aloe-like Au-ZnO micro/nanoarrays for ultrasensitive biosensing of catechol.

Currently, the large-scale and controllable fabrication of nanostructures on substrates remains a great challenge for further practical applications. In this work, a novel 3D aloe-like Au-ZnO nanocomposite was designed for in situ synthesis on an ITO substrate, achieving real-time detection of trace catechol (CC) in water. A seed-assisted hydrothermal approach was proposed to control the crystal distribution and growth direction to build a ZnO aloe-like architecture. To eliminate the natural weak conductivity of ZnO, Au nanoparticles were further deposited on all ZnO arrays to construct Au-ZnO micro/nanostructures. The synergetic effects derived from the aloe-like ZnO with a large specific area and Au nanoparticles with high conductivity resulted in both high electrocatalysis and fast electron transfer in enzymatic reactions. After laccase immobilization, the as-prepared biosensor exhibited specific recognition of catechol among other dihydroxybenzenes and phenol with an ultrahigh sensitivity of 131 µA mM-1, as well as an extremely wide linear range from 75 nM to 1100 µM and an ultralow detection limit of 25 nM. In addition, in the detection of real lake samples, this biosensor showed satisfactory anti-interference ability and provided reliable assay results.

[Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)].

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Laccase-Mediated Grafting on Biopolymers and Synthetic Polymers: A Critical Review.

Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. In recent decades this technique has also been employed to modify fibres with a polysaccharide backbone, such as cellulose or chitosan, to infer colouration, antimicrobial activity or antioxidant activity to the material. The scope of this approach has been further widened by researchers, who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. In all cases, the methodology relies on one- or two-electron oxidation of the surface functional groups or of the graftable molecule in solution. However, similar results can very often be achieved through simple deposition, even after extensive washing. This unintended adsorption of the active substance could have an adverse effect on the durability of the applied coating. Differentiating between actual covalent binding and adsorption is therefore essential, but proves to be challenging. This review not only covers excellent research on the topic of laccase-mediated grafting over the last five to ten years, but also provides a critical comparison to highlight either the lack or presence of compelling evidence for covalent grafting.

Laccase Fermentation of Clove Extract Increases Content of Dehydrodieugenol, Which Has Neuroprotective Activity against Glutamate Toxicity in HT22 Cells.

Enzyme fermentation is a type of food processing technique generally used to improve the biological activities of food and herbal medicines. In this study, a Syzygii Flos (clove) extract was fermented using laccase derived from Trametes versicolor (LTV). The fermented clove extract showed greater neuroprotective effects against glutamate toxicity on HT22 than the non-fermented extract did. HPLC analysis revealed that the eugenol (1) and dehydrodieugenol (2) contents had decreased and increased, respectively, after fermentation. The content of 2 peaked at 1 h after fermentation to 103.50 ± 8.20 mg/gex (not detected at zero time), while that of 1 decreased to 79.54 ± 4.77 mg/gex (185.41 ± 10.16 mg/gex at zero time). Compound 2 demonstrated promising HT22 neuroprotective properties with inhibition of Ca2+ influx, the overproduction of intracellular reactive oxygen species, and lipid peroxidation. In addition, LTV showed the best fermentation efficacy compared with laccases derived from Pleurotus ostreatus and Rhus vernicifera.

Two-Electron Reduction versus One-Electron Oxidation of the Type 3 Pair in the Multicopper Oxidases.

Multicopper oxidases (MCOs) utilize an electron shuttling Type 1 Cu (T1) site in conjunction with a mononuclear Type 2 (T2) and a binuclear Type 3 (T3) site, arranged in a trinuclear copper cluster (TNC), to reduce O2 to H2O. Reduction of O2 occurs with limited overpotential indicating that all the coppers in the active site can be reduced via high-potential electron donors. Two forms of the resting enzyme have been observed in MCOs: the alternative resting form (AR), where only one of the three TNC Cu's is oxidized, and the resting oxidized form (RO), where all three TNC Cu's are oxidized. In contrast to the AR form, we show that in the RO form of a high-potential MCO, the binuclear T3 Cu(II) site can be reduced via the 700 mV T1 Cu. Systematic spectroscopic evaluation reveals that this proceeds by a two-electron process, where delivery of the first electron, forming a high energy, metastable half reduced T3 state, is followed by the rapid delivery of a second energetically favorable electron to fully reduce the T3 site. Alternatively, when this fully reduced binuclear T3 site is oxidized via the T1 Cu, a different thermodynamically favored half oxidized T3 form, i.e., the AR site, is generated. This behavior is evaluated by DFT calculations, which reveal that the protein backbone plays a significant role in controlling the environment of the active site coppers. This allows for the formation of the metastable, half reduced state and thus the complete reductive activation of the enzyme for catalysis.

Mechanism of the reduction of the native intermediate in the multicopper oxidases: insights into rapid intramolecular electron transfer in turnover.

The multicopper oxidases (MCOs) are the family of enzymes that catalyze the 4-electron reduction of O2 to H2O coupled to the four 1-electron oxidations of substrate. In the catalytic cycle electrons are transferred intramolecularly over ∼13 Šfrom a Type 1 (T1) Cu site that accepts electrons from substrate to a trinuclear Cu cluster (TNC) where O2 is reduced to H2O at rapid rates consistent with turnover (560 s(-1)). The oxygen reduction mechanism for the MCOs is well-characterized, whereas the rereduction is less understood. Our initial study of Rhus vernicifera Laccase (Heppner et al. J. Am. Chem. Soc. 2013, 135, 12212) experimentally established that the native intermediate (NI), the species formed upon O-O bond cleavage, is reduced with an IET rate >700 s(-1) and is the catalytically relevant fully oxidized form of the enzyme, rather than the resting state. In this report, we present kinetic and spectroscopic results coupled to DFT calculations that evaluate the mechanism of the 3 e(-)/3 H(+) reduction of NI, where all three catalytically relevant intramolecular electron transfer (IET) steps are rapid and involve three different structural changes. These three rapid IET processes reflect the sophisticated mechanistic control of the TNC to enable rapid turnover. All three IET processes are fast due to the associated protonation of the bridging oxo and hydroxo ligands, generated by O-O cleavage, to form water products that are extruded from the TNC upon full reduction, thereby defining a unifying mechanism for oxygen reduction and rapid IET by the TNC in the catalytic cycle of the MCOs.

Quantum dots as optical labels for ultrasensitive detection of polyphenols.

Considering the fact that polyphenols have versatile activity in-vivo, its detection and quantification is very much important for a healthy diet. Laccase enzyme can convert polyphenols to yield mono/polyquinones which can quench Quantum dots fluorescence. This phenomenon of charge transfer from quinones to QDs was exploited as optical labels to detect polyphenols. CdTe QD may undergo dipolar interaction with quinones as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, "turn-off" fluorescence method was applied for ultrasensitive detection of polyphenols by using laccase. We observed proportionate quenching of QDs fluorescence with respect to polyphenol concentration in the range of 100 µg to 1 ng/mL. Also, quenching of the photoluminescence was highly efficient and stable and could detect individual and total polyphenols with high sensitivity (LOD-1 ng/mL). Moreover, proposed method was highly efficient than any other reported methods in terms of sensitivity, specificity and selectivity. Therefore, a novel optical sensor was developed for the detection of polyphenols at a sensitive level based on the charge transfer mechanism.

Laccase versus laccase-like multi-copper oxidase: a comparative study of similar enzymes with diverse substrate spectra.

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term "laccase-like multi-copper oxidase" (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis.

KEY MESSAGE: The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied. Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant-aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K m and K cat values were 7.90 mM and 52.31 s(-1), respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe(126) is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.

Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

Mechanism of monolignol biotransformation by Rhus laccases in water-miscible organic solutions.

Several important monolignols such as coniferyl alcohol were catalyzed using Rhus laccase (RL) from Rhus vernicifera in a water/acetone solution. The enzymatic mechanism is discussed in detail. Sites 6, ß, and phenolic oxygen were the main active sites of phenylpropanoid compounds, which were first oxidized by the enzyme and then radicalized. RL was also responsible for lignin biosynthesis, especially in the early stage.

Intramolecular electron transfer in laccases.

Rate constants and activation parameters have been determined for the internal electron transfer from type 1 (T1) to type 3 (T3) copper ions in laccase from both the fungus Trametes hirsuta and the lacquer tree Rhus vernicifera, using the pulse radiolysis method. The rate constant at 298 K and the enthalpy and entropy of activation were 25 ± 1 s(-1), 39.7 ± 5.0 kJ·mol(-1) and -87 ± 9 J·mol(-1) ·K(-1) for the fungal enzyme and 1.1 ± 0.1 s(-1), 9.8 ± 0.2 kJ·mol(-1) and -211 ± 3 J·mol(-1) ·K(-1) for the tree enzyme. The initial reduction of the T1 site by pulse radiolytically produced radicals was direct in the case of T. hirsuta laccase, but occured indirectly via a disulfide radical in R. vernicifera. The equilibrium constant that characterizes the electron transfer from T1 to T3 copper ions was 0.4 for T. hirsuta laccase and 1.5 for R. vernicifera laccase, leading to full reduction of the T1 site occurring at 2.9 ± 0.2 electron equivalents for T. hirsuta and 4 electron equivalents for R. vernicifera laccase. These results were compared with each other and with those for the same process in other multicopper oxidases, ascorbate oxidase and Streptomyces coelicolor laccase, using available structural information and electron transfer theory.

Evaluation of the potential of fungal and plant laccases for active-packaging applications.

Laccases from Trametes versicolor (TvL), Myceliophthora thermophila (MtL), and Rhus vernicifera (RvL) were investigated with regard to their potential utilization as oxygen scavengers in active packages containing food susceptible to oxidation reactions. The substrate selectivity of the laccases was investigated with a set of 17 reducing substrates, mainly phenolic compounds. The temperature dependence of reactions performed at low temperatures (4-31 °C) was studied. Furthermore, the laccases were subjected to immobilization in a latex/clay matrix and drying procedures performed at temperatures up to 105 °C. The results show that it is possible to immobilize the laccases with retained activity after dispersion coating, drying at 75-105 °C, and subsequent storage of the enzyme-containing films at 4 °C. TvL and, to some extent, MtL were promiscuous with regard to their reducing substrate, in the sense that the difference in activity with the 17 substrates tested was relatively small. RvL, on the other hand, showed high selectivity, primarily toward substrates resembling its natural substrate urushiol. When tested at 7 °C, all three laccases retained >20% of the activity they had at 25 °C, which suggests that it would be possible to utilize the laccases also in refrigerated food packages. Coating and drying resulted in a remaining enzymatic activity ranging from 18 to 53%, depending on the drying conditions used. The results indicate that laccases are useful for active-packaging applications and that the selectivity for reducing substrates is an important characteristic of laccases from different sources.

Effects of organic solvents on the activity of free and immobilised laccase from Rhus vernicifera.

Rhus laccase (RL) was covalently immobilised onto chitosan, and the effects of immobilisation on pH optimum, enzyme activity, thermostability, and re-use evaluated, using either N,N-dimethyl-p-phenylenediamine or 2,6-dimethoxyphenol as substrate. Immobilisation greatly enhanced enzyme thermostability, resulted in negligible loss of activity, and showed excellent re-use potential, with >80% relative activity retained after 15 cycles in aqueous solvent. Immobilised Rhus laccase (I-RL) was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With water-immiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity. With water-miscible organic solvents, in general a water content of ∼20-50% (v/v) was required to achieve activity using free RL, whereas with I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents. Kinetic investigations showed that the rate of substrate disappearance generally followed a pseudo-first-order law, and for evaluated water-immiscible organic solvents rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain obtained results.

Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin.

The electrochemical studies of laccase-mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases.

Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites.

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.

An alkali-stable enzyme with laccase activity from entophytic fungus and the enzymatic modification of alkali lignin.

Mycelia Sterilia YY-5, an entophytic fungus, was isolated from Rhus chinensis Mill and its extracellular enzyme had a higher laccase activity (MS-Lac). After been purified by anion exchange and gel filtration chromatography, MS-Lac, which had a molecular mass of 45 kDa, was found to be an alkali-stable enzyme with an optimum pH of 10.0 and capable of retaining 80% activity after incubation for 72 h with syringaldazine as substrate. It was also found that syringaldazine had a higher affinity than 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as substrate for MS-Lac, which was determined in sodium phosphate buffer (pH 6.0, 0.1M) at 30 degrees C. Meanwhile, the lignin modification, catalyzed by MS-Lac, indicated that it could oxidize the phenolic hydroxyl, side chain substituent or carbonyl group of spruce alkali lignin in cetyltrimethylammonium bromide (CTAB) reversed micelles (20 mM, pH 6.0, W/O=40) and steam-exploded wheat straw alkali lignin in NaOH solution (20 mM, pH 10.0).

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate.

Multicopper oxidases (MCOs) catalyze the 4e(-) reduction of O(2) to H(2)O. The reaction of the fully reduced enzyme with O(2) generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the O(2)-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a micro(3)-oxo ligand in NI. Notably, the one azide-bound NI (NI(Az)) exhibits spectral features very similar to those of NI, in which the micro(3)-oxo ligand in NI has been replaced by a micro(3)-bridged azide. Comparison of the spectral features of NI and NI(Az), combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the rate-limiting step involves structural rearrangement of the micro(3)-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover.

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase.

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6A, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through pi-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.