<Emphasis Type="Italic">Bacillus pumilus</Emphasis>laccase: a heat stable enzyme with a wide substrate spectrum

Background  

Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product.     

Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes – What for?

Genome analyses revealed in various basidiomycetes the existence of multiple genes for blue multi-copper oxidases (MCOs). Whole genomes are now available from saprotrophs, white rot and brown rot species, plant and animal pathogens and ectomycorrhizal species. Total numbers (from 1 to 17) and types of mco genes differ between analyzed species with no easy to recognize connection of gene distribution to fungal life styles. Types of mco genes might be present in one and absent in another fungus. Distinct types of genes have been multiplied at speciation in different organisms. Phylogenetic analysis defined different subfamilies of laccases sensu stricto (specific to Agaricomycetes), classical Fe2+-oxidizing Fet3-like ferroxidases, potential ferroxidases/laccases exhibiting either one or both of these enzymatic functions, enzymes clustering with pigment MCOs and putative ascorbate oxidases. Biochemically best described are laccases sensu stricto due to their proposed roles in degradation of wood, straw and plant litter and due to the large interest in these enzymes in biotechnology. However, biological functions of laccases and other MCOs are generally little addressed. Functions in substrate degradation, symbiontic and pathogenic intercations, development, pigmentation and copper homeostasis have been put forward. Evidences for biological functions are in most instances rather circumstantial by correlations of expression. Multiple factors impede research on biological functions such as difficulties of defining suitable biological systems for molecular research, the broad and overlapping substrate spectrum multi-copper oxidases usually possess, the low existent knowledge on their natural substrates, difficulties imposed by low expression or expression of multiple enzymes, and difficulties in expressing enzymes heterologously.     

真核生物来源漆酶的异源表达研究进展

漆酶属于多铜氧化酶家族中的一种,广泛存在于昆虫、植物、真菌和细菌中。由于其作用的底物范围较广,因此在纺织、制浆、食品以及木质素的降解等方面有广阔的应用前景。但是自然界中的漆酶存在表达量和酶活低、高温易失活等问题,限制了它的应用。对漆酶进行大量高效的异源表达,是解决这一问题的有效途径。近年来,越来越多不同来源的漆酶基因被克隆,并在不同宿主中异源表达。但这些大多局限于实验室研究,还未达到工业化生产的水平。笔者对真核生物来源漆酶的异源表达研究进展进行综述,重点介绍了真核生物来源的漆酶在不同表达系统中的异源表达情况以及在酵母细胞中表达漆酶时提高表达量和酶活性能的方法,以期为研究者们提供参考。     

Activity of tocopherol oxidase in Phaseolus coccineus seedlings

In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ? β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.     

Expression of industrially relevant laccases: prokaryotic style

Laccases are a class of multi-copper oxidases (MCOs) that catalyze the one-electron oxidation of four equivalents of a reducing substrate, with the concomitant four-electron reduction of dioxygen to water. They can catalyze a multitude of reactions, including the degradation of polymers and oxidative coupling of phenolic compounds, positioning them as significant industrial enzymes. Although fungal laccases are well known and well characterized, only recently has in silico biology led to rapid advances in the discovery, characterization and engineered expression of prokaryotic laccases. We describe the recent burgeoning of prokaryotic laccases, their catalytic properties, structural features and molecular evolution, vis-à-vis fungal laccases where possible. Special focus is given to the application of laccases to the emerging cellulosic biofuel industry.     

Engineering and Applications of fungal laccases for organic synthesis

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.     

Laccases: A Useful Group of Oxidoreductive Enzymes

Using enzymes as decontaminating agents has received great attention. One of the most promising groups of enzymes, laccases, are used to decontaminate phenol-polluted systems and for bio technological applications. Higher plants and fungi, mostly wood-rotting fungi, are the main producers of laccases, but bacterial laccases also have been found. Belonging to the class of phenoloxidases, laccases catalyze the polymerization of several phenolic substances to polymeric products. In addition, they have transformed lignin and lignin-related compounds, showing a very broad substrate specificity. Specific compounds acting as protein-synthesis inducers historically have been used to improve the production of the enzyme. Recent success in fungal molecular and cellular engineering technology has contributed to significantly increase the industrial production of recombinant laccase. Kinetic (Michaelis-Menten parameters, optimum pH, kcat) and stability properties of laccases may vary according to the source of the enzymes. Laccases are used in a variety of applications, such as to remove toxic compounds from aquatic and terrestrial systems, to produce and treat beverages, as analytical tools, and as biosensors to estimate the quantity of phenols in natural juices or the presence of other enzymes. Laccases have been used successfully in immobilized form as well as dissolved in organic solvents.     

Bacterial laccases

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are polyphenol oxidases (PPO) that catalyze the oxidation of various substituted phenolic compounds by using molecular oxygen as the electron acceptor. The ability of laccases to act on a wide range of substrates makes them highly useful biocatalysts for various biotechnological applications. To date, laccases have mostly been isolated and characterized from plants and fungi, and only fungal laccases are used currently in biotechnological applications. In contrast, little is known about bacterial laccases, although recent rapid progress in the whole genome analysis suggests that the enzymes are widespread in bacteria. Since bacterial genetic tools and biotechnological processes are well established, so developing bacterial laccases would be significantly important. This review summarizes the distribution of laccases among bacteria, their functions, comparison with fungal laccases and their applications.     

Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences

A phylogenetic analysis of more than 350 multicopper oxidases (MCOs) from fungi, insects, plants, and bacteria provided the basis for a refined classification of this enzyme family into laccases sensu stricto (basidiomycetous and ascomycetous), insect laccases, fungal pigment MCOs, fungal ferroxidases, ascorbate oxidases, plant laccase-like MCOs, and bilirubin oxidases. Within the largest group of enzymes, formed by the 125 basidiomycetous laccases, the gene phylogeny does not strictly follow the species phylogeny. The enzymes seem to group at least partially according to the lifestyle of the corresponding species. Analyses of the completely sequenced fungal genomes showed that the composition of MCOs in the different species can be very variable. Some species seem to encode only ferroxidases, whereas others have proteins which are distributed over up to four different functional clusters in the phylogenetic tree.     

Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme

Background  

Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts.     

LccA,an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ± 10 mU·ml⁻¹) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U·mg⁻¹ with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO₄. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent K_m values for SGZ, bilirubin, and ABTS were 35, 236, and 670 μM, with corresponding k_cat values of 22, 29, and 10 s⁻¹, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h.Multicopper oxidases (MCOs) are a family of enzymes that include laccases (p-diphenol: dioxygen oxidoreductases; EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), ferroxidases (EC 1.16.3.1), bilirubin oxidases (EC 1.3.3.5), and other enzyme subfamilies (27, 65). MCOs couple the oxidation of organic and/or inorganic substrates to the four-electron reduction of molecular oxygen to water. These enzymes often have four Cu atoms classified into type 1 (T1), type 2 (T2), and type 3 (T3) centers, in which a mononuclear T1 center on the surface of the enzyme provides long-range intramolecular one-electron transfer from electron-donating substrates to an internal trinuclear T2-T3 center formed by a T2 Cu coordinated with a T3 Cu pair. The T2-T3 cluster subsequently reduces dioxygen to water.Enzymes of the laccase subfamily oxidize a broad range of compounds, including phenols, polyphenols, aromatic amines, and nonphenolic substrates, by one-electron transfer to molecular oxygen and thus have a wide variety of applications from biofuels to human health. The best-known application is the use of a laccase from the lacquer tree Rhus vernicifera in paint and adhesives for more than 6,000 years in East Asia (29). Laccases have also been used in the delignification of pulp, bleaching of textiles and carcinogenic dyes, detoxification of water and soils, removal of phenolics from wines, improving adhesive properties of lignocellulosic products, determination of bilirubin levels in serum, and transformation of antibiotics and steroids (60). In addition, laccases have demonstrated potential for use in biosensors, bioreactors, and biofuel cells (61).Laccases, once thought to be restricted to eukaryotes (fungi, plants, and insects), appear to be widespread in bacteria (10). Laccase-like MCOs are now known to have numerous biological roles in bacteria, including sporulation, electron transport, pigmentation, metal (copper, iron, and manganese) homeostasis, oxidation of phenolate-siderophores, phenoxazinone synthesis, cell division, and morphogenesis (9). In contrast to the widespread occurrence of laccases in bacteria and eukaryotes, only a few MCOs have been identified in archaea, and this is based only on genome sequences (e.g., the hyperthermophilic crenarchaeote Pyrobaculum aerophilum and the halophilic euryarchaeotes Haloferax volcanii and Halorubrum lacusprofundi). Most archaea with sequenced genomes, however, are anaerobes. Since MCOs reduce molecular oxygen to water, this likely accounts for the limited number of MCOs among archaea.Many archaea thrive under harsh environmental conditions, including high temperature, extreme pH, and/or low water activity. Thus, they have many biochemical and physiological properties that are ideal for industrial applications. Here, we report the identification of a highly thermostable and salt/solvent-tolerant laccase (LccA) from the halophilic archaeon H. volcanii that catalyzed the oxidation of a wide variety of phenolic compounds. LccA was readily secreted and purified from the culture broth as a blue multicopper oxidase that was glycosylated and processed by the removal of 31 amino acid residues from its N terminus.     

Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds

1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.     

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

A Novel Lentinula edodes Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better “green” applications. To address the issue, this study began with cloning the laccase family of Lentinula edodes. Three laccases perfectio sensu stricto (Lcc4A, Lcc5, and Lcc7) were then expressed from Pichia pastoris, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on “green” applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of L. edodes laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient “green” applications.     

Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH₂). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH₂ extended the reactions catalyzed by these enzymes to the production of H₂O₂, the oxidation of Mn²⁺, the reduction of Fe³⁺, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

Evolutionary trace analysis at the ligand binding site of laccase

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.     

Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL^-1 compared to activities of 0.13 ± 0.028 UL^-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.     

The selective inhibition of ortho- and para-diphenol oxidases

Ortho- and para-diphenol oxidases (DPO's) are often distinguished by substrate specificity tests which are not always unequivocal. This paper suggests that they may be differentiated by their response patterns to certain inhibitors and activators. In general o-DPO's (‘catecholases’) are inhibited by substituted cinnamic acids (cinnamic, p-coumaric and ferulic), or polyvinylpyrrolidone (PVP) and may be activated by anionic detergents. By contrast p-DPO's (‘laccases’) are unaffected by cinnamic acids and PVP but are inhibited by cationic detergents such as cetyltrimethylammonium bromide (CTAB). Thus in crude extracts these enzymes may be clearly distinguished by a simple combination of substrate and inhibitor specificity tests.