真菌漆酶及其介体系统：来源、机理与应用

漆酶是一类含铜氧化酶,广泛分布于植物、真菌、细菌、昆虫中,它们能够高效催化芳香族和非芳香族化合物氧化降解,并最终将分子氧还原为水作为副产物。一些小分子介体能够进一步提高漆酶的降解底物范围、催化效率和稳定性。它们与漆酶构成漆酶/介体系统（laccase mediator systems, LMS）,能够更有效地降解非酚类、多环芳烃类等难降解化合物,在造纸制浆与漂白、染料脱色、环境脱毒等领域有着巨大的应用前景,成为近年来的研究热点之一。对漆酶的来源与功能、真菌漆酶结构与反应机理、介体类型与作用机理、LMS的应用进行了综述,以期为漆酶的应用研究提供参考。     

Bifunctional in vivo role of laccase exploited in multiple biotechnological applications

Laccases are multicopper enzymes present in plants, fungi, bacteria, and insects, which catalyze oxidation reactions together with four electron reduction of oxygen to water. Plant, bacterial, and insect laccases have a polymerizing role in nature, implicated in biosynthesis of lignin, melanin formation, and cuticle hardening, respectively. On the other hand, fungal laccases carry out both polymerizing (melanin synthesis and fruit body formation) as well as depolymerizing roles (lignin degradation). This bifunctionality of fungal laccases can be attributed to the presence of multiple isoforms within the same as well as different genus and species. Interestingly, by manipulating culture conditions, these isoforms with their different induction patterns and unique biochemical characteristics can be expressed or over-expressed for a targeted biotechnological application. Consequently, laccases can be considered as one of the most important biocatalyst which can be exploited for divergent industrial applications viz. paper pulp bleaching, fiber modification, dye decolorization, bioremediation as well as organic synthesis. The present review spotlights the role of fungal laccases in various antagonistic applications, i.e., polymerizing and depolymerizing, and co-relating this dual role with potential industrial significance.

     

Cell thermolysis – A simple and fast approach for isolation of bacterial laccases with potential to decolorize industrial dyes

Laccases belong to multicopper oxidases and are widespread in nature. Currently, mainly fungal laccases are applied in biotechnological processes. One reason for this is that fungal laccases are much better studied. Compared to fungal laccases, bacterial laccases possess some advantageous characteristics like high stability at elevated temperatures and alkaline pH values. Intracellular recombinant expression of bacterial laccases in E. coli makes however downstream processing more complex and time-consuming compared to extracellular expression of fungal enzymes. Here, we demonstrate that cell disruption by cell thermolysis is an efficient and simple method for the isolation and partial purification of recombinant bacterial laccases. Three different laccases, Tth from Thermus thermophilus, CotA from Bacillus subtilis and Ssl1 from Streptomyces sviceus, were used to compare cell disruption by cell thermolysis with sonication and high-pressure homogenization, with and without subsequent heat treatment. Cell thermolysis resulted in high laccase activities per gram of cell wet weight and in the highest specific activities of the laccases. For example, specific activity of Tth laccase after cell thermolysis was 469-fold higher than after sonication. Furthermore, high decolorization activity towards indigo carmine and alizarin red S of these laccases, isolated via cell thermolysis, demonstrate their potential for technical applications.     

Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three-domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.     

The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.     

Fungal laccases - occurrence and properties

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.     

Directed evolution of fungal laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.     

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6-DMP, and non-conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well-studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.     

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 and their occurrence in prokaryotes

Laccases are copper-containing proteins that require O(2) to oxidize phenols, polyphenols, aromatic amines, and different non-phenolic substrates by one-electron transfer, resulting in the formation of reactive radicals. Although their specific physiological functions are not completely understood, there are several indications that laccases are involved in the morphogenesis of microorganisms (e.g., fungal spore development, melanization) and in the formation and/or degradation of complex organic substances such as lignin or humic matter. Owing to their high relative non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. To date, laccases have been found only in eukaryotes (fungi, plants); however, databank searches and experimental data now provide evidence for their distribution in prokaryotes. This survey shows that laccase-like enzymes occur in many gram-negative and gram-positive bacteria. Corresponding genes have been found in prokaryotes that are thought to have branched off early during evolution, e.g., the extremely thermophilic Aquifex aeolicus and the archaeon Pyrobaculum aerophilum. Phylogenetically, the enzymes are members of the multi-copper protein family that have developed from small-sized prokaryotic azurins to eukaryotic plasma proteins.     

Thermotolerant and thermostable laccases

Laccases are phenol-oxidizing, usually four-copper containing metalloenzymes. For industrial and biotechnological purposes, laccases were among the first fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibres. In order to facilitate novel and more efficient bio-catalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability and thermotolerance. This review gives a current overview on the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.     

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。     

Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes

Laccases are oxidoreductases which oxidize a variety of aromatic compounds using oxygen as the electron acceptor and producing water as by-product. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnological applicability. The presence of redox mediators is required for a number of biotechnological applications, providing the oxidation of complex substrates not oxidized by the enzyme alone. The efficiency of laccase–mediator systems to degrade recalcitrant compounds has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compounds related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries.     

Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli

Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria.     

On the Diversity of the Laccase Gene: A Phylogenetic Perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and Other Related Taxa

The present study is the first describing the sequencing of a fragment of the copper-oxidase domain of a laccase gene in the family Botryosphaeriaceae. The aim of this work was to assess the degree of genetic and evolutionary relationships of a laccase gene from Botryosphaeria rhodina MAMB-05 with other ascomycete and basidiomycete laccase genes. The 193-amino acid sequences of the copper-oxidase domain from several different fungi, insects, a plant, and a bacterial species were retrieved from GenBank and aligned. Phylogenetic analyses were performed using neighbor-joining, maximum parsimony, and Bayesian inference methods. The organisms studied clustered into five gene clades: fungi (ascomycetes and basidiomycetes), insects, plants, and bacteria. Also, the topologies showed that fungal laccases of the ascomycetes and basidiomycetes are clearly separated into two distinct clusters. This evidence indicated that B. rhodina MAMB-05 and other closely related ascomycetes are a new biological resource given the biotechnological potential of their laccase genes.     

Fungal laccases as green catalysts for dye synthesis

Laccases from different sources catalyse oxidation of various phenolic and aromatic compounds to products that very often are colourful and may be used as dyes, especially in the textile industry. They catalyse not only catabolic processes such as depolymerisation and degradation but can also carry out various dimerization, oligomerization, and polymerization reactions of some hundred aromatic substrates that synthesize new molecules with valuable functions. Because of their versatile biochemical properties, high protein stability, breadth of substrate spectrum, laccases are the key enzymes having applications in biotechnological processes as eco-friendly biocatalyst. This review refers to the natural abilities of laccases to synthesize colour products with respect to the type of the enzymatic reaction, catalyst characterization and possible use of these colour products as dyestuffs.     

Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent—A comparative study

Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69–80, 2018