Heterologous laccase production and its role in industrial applications

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry.     

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.     

Extracellular laccase from Monilinia fructicola: isolation,primary characterization and application

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5–5.0 and 3.0–5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.     

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.     

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.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

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。     

Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

Development of new laccases by directed evolution: functional and computational analyses

Laccases are blue multicopper oxidases that couple the four-electron reduction of oxygen with the oxidation of a broad range of aromatic substrates. These fungal enzymes can be used for many applications such as bleaching, organic synthesis, bioremediation, and in laundry detergents. Laccases from Pleurotus ostreatus have been successfully heterologously expressed in yeasts. The availability of established recombinant expression systems has allowed the construction of mutated, "better performing" enzymes through molecular evolution techniques. In the present work, random mutagenesis experiments on poxc and poxa1b cDNAs, using error prone PCR (EP-PCR) have been performed. By screening a library of 1100 clones the mutant 1M9B was selected, it shows a single mutation (L112F) leading to an enzyme more active but less stable with respect to the wild-type enzyme (POXA1b) in all the analyzed conditions. This mutant has been used as a template for a second round of EP-PCR. From this second generation library of 1200 clones, three mutants have been selected. Properties of the four mutants, 1M9B screened from the first library, and 1L2B, 1M10B, and 3M7C from the second library, were analyzed. The better performing mutant 3M7C presents, besides L112F, only one substitution (P494T) responsible both for the significantly increased stability and for the exhibited higher activity of this mutant. Molecular dynamics simulations have been performed on three-dimensional models of POXA1b, 1M9B, and 3M7C, and hypotheses on the structure-function relationships of these proteins have been formulated.     

Molecular characterization of a blue-copper laccase, TILA, of Aspergillus nidulans

Laccases are blue-copper enzymes, which oxidize phenolic substrates and thereby reduce molecular oxygen. They are widespread within fungi and are involved in lignin degradation or secondary metabolism such as pigment biosynthesis. Many fungi contain several laccases, not all of whose functions are known. In Aspergillus nidulans one, yA, is expressed during asexual development and converts a yellow precursor to the green pigment. We identified a second laccase gene, which encodes a 66.3-kDa protein 37.6% identical to laccase I of A. nidulans. The protein harbors an N-terminal secretion signal, and three characteristic copper-binding centers. The enzyme localizes at the growing hyphal tip. The gene was therefore named tilA (=tip laccase). Deletion or overexpression of the gene had no discernible phenotype under laboratory conditions.     

Laccases: blue enzymes for green chemistry

Laccases are oxidoreductases belonging to the multinuclear copper-containing oxidases; they catalyse the monoelectronic oxidation of substrates at the expense of molecular oxygen. Interest in these essentially "eco-friendly" enzymes--they work with air and produce water as the only by-product--has grown significantly in recent years: their uses span from the textile to the pulp and paper industries, and from food applications to bioremediation processes. Laccases also have uses in organic synthesis, where their typical substrates are phenols and amines, and the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. Here, we provide a brief discussion of this interesting group of enzymes, increased knowledge of which will promote laccase-based industrial processes in the future.     

Specificities of a chemically modified laccase from Trametes hirsuta on soluble and cellulose-bound substrates

Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently attached onto a laccase from Trametes hirsuta. Different molecular weights of the synthetic polymer were tested in terms of adsorption behaviour and retained laccase activity. Covalent attachment of PEG onto the laccase resulted in enhanced enzyme stability while with increasing molecular weight of attached PEG the substrate affinity for the laccase conjugate decreased. The activity of the modified laccases on fibre bound dye was drastically reduced decreasing the adsorption of the enzyme on various fabrics. Compared to the 5 kDa PEG laccase conjugate (K/S value 47.60) the K/S value decreased much more (47.96–46.35) after the treatment of dyed cotton fabrics with native laccase.     

Partial purification and characterization of the novel halotolerant and alkalophilic laccase produced by a new isolate of Bacillus subtilis LP2

Laccases (benzenediol: oxygen oxidoreductases, [EC1.10.3.2] are mostly known as members of the blue multicopper oxidase family that are used in very different industrial applications: textile, pulp and paper, food, cosmetics industries, bioremediation process, biosensor, biofuel and organic synthesis. Stability against the extreme conditions is an important property and it makes laccase suitable for several industrial processes. Laccase should have salt resistance to be used in textile dye degradation because the textile wastewaters include dyes with high concentrations of salts, especially NaCl. Bacterial laccases are preferable to be used for bioremediation process due to their high stability to extremely salt contaminated and alkalophilic environment. Bacillus subtilis LP2 was identified as a source of alkali-tolerant, salt resistant laccase. Laccase showed activity over a wide pH (4–10) and temperature (30–80?°C) range. Maximum laccase activity was observed as 140.4?U/mg (umol/min*mg) at pH 8 and 50?°C with the substrate guaiacol. Stability of laccase was determined as 60% and 20% after incubation of the enzyme for different time intervals of 20 and 40?min at 50?°C and pH 8. SDS (10?mM) and EDTA (5?mM) decreased laccase activity from 100% to 0% and 56%, respectively. Despite the other inhibitors, NaCI increased the activity of laccase to 167% at 500?mM concentration. Laccase from Bacillus subtilis LP2 barely showed the activity on the substrates vanillin and L-tyrosine. These results clearly show that laccase from Bacillus subtilis LP2 has high potential to be used for several applications in textile industry.     

真菌漆酶异源表达研究进展

由于漆酶能够氧化芳香类化合物和其它一些非芳香类有机物,具有广泛的底物特异性,因此在纸浆漂白、纺织品染料脱色、有毒废弃物的去除、生物修复和生物传感器等方面具有巨大的应用潜力。但是缺少大量廉价的酶源供应阻碍了漆酶商业化的应用,解决这个问题的一个主要方法就是通过漆酶的异源表达来获得大量的漆酶。综述了真菌漆酶在酵母表达系统和丝状真菌表达系统中表达的研究结果,着重总结了影响漆酶异源表达的因素和提高漆酶表达的策略。     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.     

Laccase: new functions for an old enzyme

Laccases occur widely in fungi; they have been characterized less frequently in higher plants. Here we have focused on more recent reports on the occurrence of laccase and its functions in physiological development and industrial utility. The reports of molecular weights, pH optima, and substrate specificity are extremely diverse. Conclusive proof of the occurrence of laccase in a tissue must demonstrate that the enzyme be able to oxidize quinol with concomitant uptake of oxygen. Laccase is involved in the pigmentation process of fungal spores, the regeneration of tobacco protoplasts, as fungal virulence factors, and in lignification of cell walls and delignification during white rot of wood. Commercially, laccases have been used to delignify woody tissues, produce ethanol, and to distinguish between morphine and codeine. A very wide variety of bioremediation processes employ laccase in order to protect the environment from damage caused by industrial effluents. Research in recent years has been intense, much of it elicited by the wide diversity of laccases, their utility and their very interesting enzymology.     

一株产漆酶土壤真菌F-5的分离及土壤修复潜力

漆酶因可氧化许多种有机污染物,在土壤污染修复方面的应用潜力受到广泛重视。筛选具有较高漆酶活性的土壤真菌,可以为污染土壤修复提供生物资源。通过培养基中愈创木酚颜色反应,从土壤中筛选获得1株真菌菌株F-5。18S rRNA基因序列显示该菌株属于巨座壳科(Family Magnaporthaceae)。单因素试验和正交试验结果显示,蔗糖和蛋白胨分别是最有利于该菌产漆酶的碳源和氮源。在适当培养条件下,真菌F-5培养液酶活性可达4033U/L,表现出该菌具有较强的产漆酶能力。在多环芳烃(PAHs)污染土壤的生物修复中,真菌F-5可使土壤中苯并(a)芘、二苯并(a,h)蒽等高环、高毒性多环芳烃降解,并使土壤多环芳烃毒性当量大幅降低。因此,真菌F-5适合修复PAHs污染土壤。     

Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.