Laccase-like Multicopper Oxidase Research Articles

Functional screening pipeline to uncover laccase-like multicopper oxidase enzymes that transform industrial lignins

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.

Heterologous expression, purification, and characterization of thermo- and alkali-tolerant laccase-like multicopper oxidase from Bacillus mojavensis TH309 and determination of its antibiotic removal potential.

Laccases or laccase-like multicopper oxidases have great potential in bioremediation to oxidase phenolic or non-phenolic substrates. However, their inability to maintain stability in harsh environmental conditions and against non-substrate compounds is one of the main reasons for their limited use. The gene (mco) encoding multicopper oxidase from Bacillus mojavensis TH309 were cloned into pET14b( +), expressed in Escherichia coli, and purified as histidine tagged enzyme (BmLMCO). The molecular weight of the enzyme was about 60kDa. The enzyme exhibited laccase-like activity toward 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The highest enzyme activity was recorded at 80°C and pH 8. BmLMCO showed a half-life of ~ 305, 99, 50, 46, 36, and 20min at 40, 50, 60, 70, 80, and 90°C, respectively. It retained more than 60% of its activity after pre-incubation in the range of pH 5-12 for 60min. The enzyme activity significantly increased in the presence of 1mM of Cu2+. Moreover, BmLMCO tolerated various chemicals and showed excellent compatibility with organic solvents. The Michaelis constant (Km) and the maximum velocity (Vmax) values of BmLMCO were 0.98mM and 93.45µmol/min, respectively, with 2,6-DMP as the substrate. BmLMCO reduced the antibacterial activity of cefprozil, gentamycin, and erythromycin by 72.3 ± 1.5%, 79.6 ± 6.4%, and 19.7 ± 4.1%, respectively. This is the first revealing shows the recombinant production of laccase-like multicopper oxidase from any B. mojavensis strains, its biochemical properties, and potential for use in bioremediation.

Oxidative degradation of polyethylene by two novel laccase-like multicopper oxidases from Rhodococcus opacus R7.

The production of synthetic plastics, especially polyethylene, has reached a crucial level, making highly challenging plastic waste management. This paper aims to elucidate the role of two novel laccase-like multicopper oxidases in polyethylene oxidative degradation based on transcriptomic data from Rhodococcus opacus R7 grown on polyethylene. The purification of the recombinant proteins, LMCO2 and LMCO3 belonging respectively to the three- and two-domains laccase families, showed that they are endowed with different functional features. LMCO2 showed the highest enzyme activity at 65 °C with an optimal pH of 7.0, while LMCO3 exhibited the highest activity at 80 °C at acidic pH. LMCOs showed activity on both phenolic (i.e., 2,6-DMP) and non-phenolic (i.e., ABTS) compounds and they were thermostable.The oxidative activity of LMCO2 and LMCO3 on untreated low-density polyethylene was assessed by combining Fourier transform infrared spectroscopy and gas chromatography coupled with mass spectrometry analyses. Oxidation of PE was recorded within a short time range (24-48 h) and revealed previously not described patterns of alkyl compounds and oxygenated products including ketones, alcohols, and carboxylic acids. Structural analysis of LMCO2 and LMCO3 together with density functional theory calculations allowed to identify structural and electronic elements presumably involved in the oxidation of polyethylene.

Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role.

Multicopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure-function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.

A Genome-Wide Identification and Expression Pattern of LMCO Gene Family from Turnip (Brassica rapa L.) under Various Abiotic Stresses.

Laccase-like multi-copper oxidases (LMCOs) are a group of enzymes involved in the oxidation of numerous substrates. Recently, these enzymes have become extremely popular due to their practical applications in various fields of biology. LMCOs generally oxidize various substrates by linking four-electron reduction of the final acceptor, molecular oxygen (O2), to water. Multi-copper oxidases related to laccase are extensively distributed as multi-gene families in the genome sequences of higher plants. The current study thoroughly investigated the LMCO gene family (Br-Lac) and its expression pattern under various abiotic stresses in B. rapa L. A total of 18 Br-Lac gene family members located on five different chromosomes were identified. Phylogenetic analysis classified the documented Br-Lac genes into seven groups: Group-I (four genes), Group-II (nine genes), Group-III (eight genes), Group-IV (four genes), Group-V (six genes), and Group-VI and Group-VII (one gene each). The key features of gene structure and responsive motifs shared the utmost resemblance within the same groups. Additionally, a divergence study also assessed the evolutionary features of Br-Lac genes. The anticipated period of divergence ranged from 12.365 to 39.250 MYA (million years ago). We also identified the pivotal role of the 18 documented members of the LMCO (Br-lac) gene family using quantitative real-time qRT-PCR. Br-Lac-6, Br-Lac-7, Br-Lac-8, Br-Lac-16, Br-Lac-17, and Br-Lac-22 responded positively to abiotic stresses (i.e., drought, heat, and salinity). These findings set the stage for the functional diversity of the LMCO genes in B. rapa.

Gut microbiome of mealworms (Tenebrio molitor Larvae) show similar responses to polystyrene and corn straw diets

BackgroundSome insects can degrade both natural and synthetic plastic polymers, their host and gut microbes play crucial roles in this process. However, there is still a scientific gap in understanding how the insect adapted to the polystyrene (PS) diet from natural feed. In this study, we analyzed diet consumption, gut microbiota responses, and metabolic pathways of Tenebrio molitor larvae exposed to PS and corn straw (CS).ResultsT. molitor larvae were incubated under controlled conditions (25 ± 1 °C, 75 ± 5% humidity) for 30 days by using PS foam with weight-, number-, and size-average molecular weight (Mw, Mn, and Mz) of 120.0, 73.2, and 150.7 kDa as a diet, respectively. The larvae exhibited lower PS consumption (32.5%) than CS (52.0%), and these diets had no adverse effects on their survival. The gut microbiota structures, metabolic pathways, and enzymatic profiles of PS- and CS-fed larvae showed similar responses. The gut microbiota of larvae analysis indicated Serratia sp., Staphylococcus sp., and Rhodococcus sp. were associated with both PS and CS diets. Metatranscriptomic analysis revealed that xenobiotics, aromatic compounds, and fatty acid degradation pathways were enriched in PS- and CS-fed groups; laccase-like multicopper oxidases, cytochrome P450, monooxygenase, superoxidase, and dehydrogenase were involved in lignin and PS degradation. Furthermore, the upregulated gene lac640 in both PS- and CS-fed groups was overexpressed in E. coli and exhibited PS and lignin degradation ability.ConclusionsThe high similarity of gut microbiomes adapted to biodegradation of PS and CS indicated the plastics-degrading ability of the T. molitor larvae originated through an ancient mechanism that degrades the natural lignocellulose.C96ehShQjSBPouDCMhmhGsVideo Graphical

Degradation of polyethylene by Klebsiella pneumoniae Mk-1 isolated from soil

The massive accumulation of polyethylene (PE) in the natural environment has caused persecution to the ecological environment. At present, the mechanism of microbial degradation of PE remains unclear, and the related enzymes for degrading PE need to be further explored. In this study, a strain of Klebsiella pneumoniae Mk-1 which can effectively degrade PE was obtained from the soil. The degradation performance of the strains was evaluated by weight loss rate, SEM, ATR/FTIR, WCA, and GPC. The key gene of PE degradation in the strain was further searched, which may be the laccase-like multi-copper oxidase gene. Then, the laccase-like multi-copper oxidase gene (KpMco) was successfully expressed in E.coli and its laccase activity was verified, which reached 85.19 U/L. The optimum temperature and pH of the enzyme are 45 °C and 4.0, respectively; it shows good stability at 30–40 °C and pH 4.5–5.5; Mn2+ and Cu2+ can activate the enzyme effect. After the enzyme was applied to the degradation of PE film, it was found that the laccase-like multi-copper oxidase did have a certain degradation effect on PE film. This study provides new strain and enzyme gene resources for the biodegradation of PE, thereby promoting the process of PE biodegradation.

Characterization of laccase gene StLAC6 and its involvement in the pathogenicity and peroxisome function in Setosphaeria turcica

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.

Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody “hog fuel” pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

Soil management and compost amendment are the main drivers of carbon sequestration in rainfed olive trees agroecosystems: An evaluation of chemical and biological markers

Traditional olive groves are considered at high risk of erosion and desertification due to low organic matter inputs and scarce vegetation cover coupled to highly intensity tillage, leading to a further decrease in soil organic carbon contents. In a context of climate change, carbon sequestration through waste management in the framework of a circular economy becomes of primary importance for rainfed Mediterranean agricultural soils. To unveil the mechanisms of carbon sequestration in those agroecosystems, we evaluated the effect of the application of two composts (olive waste “alperujo” and biosolid) at two different doses coupled to the sustainable soil management allowing spontaneous vegetation growing on the soil surface.With this aim, solid state nuclear magnetic resonance was used to trace the evolution of the chemical composition of compost-derived and soil-derived organic matter. Successively, the activity of soil microbial community after the compost addition was monitored by measuring two enzymatic activities (β-glucosidase and dehydrogenase) and soil CO2 respiration. Lastly, the adaptation of microbial communities was quantified using the genes encoding soil enzymes associated with C cycling -glucosidase and laccase-like multicopper oxidases. The results of this study show that biosolid compost has a higher potential for C storage at mid-term (24 to 30 months after first application) than alperujo compost, yielding up to 50% net C sequestration in the topsoil in addition to the compost-derived C applied. We attribute these results to biosolid compost chemical composition, closer to that of the native organic matter, which led to a less abrupt adaptation of soil microbial functioning (lower flushes of water soluble C, lower number of copies of genes encoding enzymatic activities) to the new C source, if compared to alperujo compost. The results encourage the reduction of soil perturbation and the use of organic amendments instead of mineral fertilizers to increase carbon storage and improve sustainability of olive groves in the Mediterranean agroecosystems.

Enzymatic Oxidation of Ca-Lignosulfonate and Kraft Lignin in Different Lignin-Laccase-Mediator-Systems and MDF Production

Laccase-mediator-oxidized lignin offers replacement for conventional chemical binders to produce fiberboards. Compared to the previously reported laccase–mediator system (LMS), a lignin-laccase-mediator-system (LLMS) has an advantage in that it requires much shorter fiber-enzyme incubation time due to significantly increased redox reactions. However, the cost of regularly applying laccase on an industrial scale is currently too high. We have employed CcLcc5 from cultures of the basidiomycete Coprinopsis cinerea as a novel basi-laccase (a CAZy subfamily AA1_1 laccase) in medium-density fiberboard (MDF) production, in comparison to the commercial formulation Novozym 51003 with recombinantly produced asco-laccase MtL (a CAZy subfamily AA1_3 laccase-like multicopper oxidase from the ascomycete Myceliophthora thermophila). With the best-performing natural mediator 2,6-dimethoxyphenol (DMP), unpurified CcLcc5 was almost as good as formulated Novozym 51003 in increasing the molecular weight (MW) of the technical lignins tested, the hydrophilic high-MW Ca-lignosulfonate and the hydrophobic low-MW kraft lignin (Indulin AT). Oxygen consumption rates of the two distantly related, poorly conserved enzymes (31% sequence identity) with different mediators and lignosulfonate were also comparable, but Indulin AT significantly reduced the oxidative activity of Novozym 51003 unlike CcLcc5, regardless of the mediator used, either DMP or guaiacol. Oxygen uptake by both laccases was much faster with both technical lignins with DMP than with guaiacol. In case of lignosulfonate and DMP, 20–30 min of incubation was sufficient for full oxygen consumption, which fits in well in time with the usual binder application steps in industrial MDF production processes. LLMS-bonded MDF was thus produced on a pilot-plant scale with either crude CcLcc5 or Novozym 51003 at reduced enzyme levels of 5 kU/kg absolutely dry wood fiber with lignosulfonate and mediator DMP. Boards produced with CcLcc5 were comparably good as those made with Novozym 51003. Boards reached nearly standard specifications in internal bond strength (IB) and modulus of rupture (MOR), while thickness swelling (TS) was less good based on the hydrophilic character of lignosulfonate. LLMS-bonded MDF with Indulin AT and DMP performed better in TS but showed reduced IB and MOR values.

Efficient Degradation of Aflatoxin B1 and Zearalenone by Laccase-like Multicopper Oxidase from Streptomyces thermocarboxydus in the Presence of Mediators.

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.

Discovery of two novel laccase-like multicopper oxidases from Pleurotus citrinopileatus and their application in phenolic oligomer synthesis

BackgroundLaccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited.ResultsIn the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity.ConclusionsOverall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.

Involvement of laccase-like enzymes in humic substance degradation by diverse polar soil bacteria.

Humic substances (HS) in soil are widely distributed in cold environments and account for a significant fraction of soil's organic carbon. Bacterial strains (n=281) were isolated at 15°C using medium containing humic acids (HA), a principal component of HS, from a variety of polar soil samples: 217 from the Antarctic and 64 from the Arctic. We identified 73 potential HA-degrading bacteria based on 16S rRNA sequence similarity, and these sequences were affiliated with phyla Proteobacteria (73.9%), Actinobacteria (20.5%), and Bacteroidetes (5.5%). HA-degrading strains were further classified into the genera Pseudomonas (51 strains), Rhodococcus (10 strains), or others (12 strains). Most strains degraded HA between 10 and 25°C, but not above 30°C, indicating cold-adapted degradation. Thirty unique laccase-like multicopper oxidase (LMCO) gene fragments were PCR-amplified from 71% of the 73 HA-degrading bacterial strains, all of which included conserved copper-binding regions (CBR) I and II, both essential for laccase activity. Bacterial LMCO sequences differed from known fungal laccases; for example, a cysteine residue between CBR I and CBR II in fungal laccases was not detected in bacterial LMCOs. This suggests a bacterial biomarker role for LMCO to predict changes in HS-degradation rates in tundra regions as global climate changes. Computer-aided molecular modeling showed these LMCOs contain a highly-conserved copper-dependent active site formed by three histidine residues between CBR I and CBR II. Phylogenetic- and modeling-based methods confirmed the wide occurrence of LMCO genes in HA-degrading polar soil bacteria and linked their putative gene functions with initial HS-degradation processes.

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System.

SUMMARYResearch backgroundIn recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil.Experimental approachWe used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates.Results and conclusionsProfile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol.Novelty and scientific contributionOur results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes.

FTacV study of electroactive immobilized enzyme/free substrate reactions: Enzymatic catalysis of epinephrine by a multicopper oxidase from Thermothelomyces thermophila

In the present work, a kinetic analysis is made concerning the reaction of an electroactive immobilized enzyme with a free substrate, based on a Michaelis–Menten scheme. The proposed kinetic equations are investigated numerically for conditions describing large amplitude fast Fourier transform alternating current voltammetry (FTacV), under different reaction states (transient or steady state for the reaction intermediate as well as quasi or complete reversibility of the electrochemical step). The dependence of two chief observables that occur from the analysis of the results of the method, that is, the maximum of the harmonics and the potential shift of the corresponding dominant peaks, on substrate concentration is presented. The FTacV method is applied experimentally for an immobilized laccase-like multicopper oxidase from Thermothelomyces thermophila, TtLMCO1, and its reaction with epinephrine. From the experimental findings it is shown that the intrinsic characteristics of the system do not lead to the extraction of the desired kinetic data although indications on the relation between the kinetic constants is revealed. Finally, the response of the third harmonic for the first additions of epinephrine at subnanomolarity range can be exploited for the detection of epinephrine at rather low concentrations.

Biodegradation of polyethylene microplastic particles by the fungus Aspergillus flavus from the guts of wax moth Galleria mellonella

Polyethylene (PE) products are widely used in daily life, agriculture, and industry because of their convenience and economic value. However, PE is one of the polymer materials remarkably resistant to degradation. Current methods of plastic waste disposal pose a threat to the environment and produce microplastic particles (MPP), which becomes a global environmental concern because of its accumulation. In this study, a PE-degrading fungus Aspergillus flavus named PEDX3, was isolated from the gut contents of wax moth Galleria mellonella. The results indicated that high-density polyethylene (HDPE) MPP was degraded into the MPP with a lower molecular weight by strain PEDX3 after 28 days incubation. In addition, Fourier Transform - Infrared Spectroscopy (FT-IR) results showed the appearance of carbonyl groups and ether groups of MPP, which also validated the degradation of PE. Furthermore, the potential degradation enzymes were investigated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Finally, two laccase-like multicopper oxidases (LMCOs) genes, AFLA_006190 and AFLA_053930, displayed up-regulated expression during the degradation process, which may be the candidate PE-degrading enzymes. These results have demonstrated that the A. flavus strain PEDX3 has an ability to degrade microplastic particles and the two PE-degrading enzymes provide a promising application for the PE MPP remediation.

Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition.

The aim of this study is to determine the key laccase-encoding gene in the life cycle of Morchella importuna SCYDJ1-A1, and to characterize the biochemical properties of the laccase. Two laccase-like multicopper oxidase (LMCO) genes were identified in the genome of M. importuna SCYDJ1-A1 as putative laccase-encoding genes. The two genes, belonging to Auxiliary Activity family 1 subfamily 3, were named as MiLacA and MiLacB. Phylogenetic analysis of deduced amino acid sequences showed that MiLacA is closest to a LMCO of M. importuna 22J1, while MiLacB had low similarity with known Morchella LMCOs. Real-time quantitative PCR results showed that MiLacA was expressed at much higher levels than MiLacB throughout the entire course of artificial cultivation. MiLacA was overexpressed in Pichia pastoris as a recombinant protein. Biochemical characterization of the purified enzyme showed that MiLacA simultaneously possessed laccase and polyphenol-oxidase activities. MiLacA could be strongly inhibited by Fe2+, which is unusual. The optimum pH was four and optimum temperature was 60°C. The enzyme retained over 74% of the laccase activity after 16-h incubation at 60°C, which means that its thermostability is at the forefront among the currently known laccases. Our findings may help to elucidate how the laccase of M. importuna is involved in decaying lignin in plant litter, and could also provide a candidate thermostable laccase for potential industrial application.

The Altered Expression of microRNA408 Influences the Arabidopsis Response to Iron Deficiency.

MicroRNAs contribute to the adaptation of plants to varying environmental conditions by affecting systemic mineral nutrient homeostasis. Copper and iron deficiencies antagonistically control the expression of Arabidopsis thaliana microRNA408 (miR408), which post-transcriptionally regulates laccase-like multicopper oxidase family members LAC3, LAC12, and LAC13. In this work, we used miR408 T-DNA insertion mutants (408-KO1 and 408-KO2) and a previously characterized transgenic line overexpressing miR408 (35S:408-14) to explore how miR408 influences copper- and iron-dependent metabolism. We observed that the altered expression of miR408 diminished plant performance and the activation of the iron-regulated genes under iron-deficient conditions. Consistently with the low expression of the miR408-target laccases, we showed that the vascular bundle lignification of the 35S:408-14 plants diminished. The decrease in the phenoloxidase and ferroxidase activities exhibited by wild-type plants under iron deficiency did not occur in the 408-KO1 plants, probably due to the higher expression of laccases. Finally, we observed that the hydrogen peroxide levels under iron starvation were altered in both the 408-KO1 and 35S:408-14 lines. Taken together, these results suggest that Arabidopsis plants with modified miR408 levels undergo multiple deregulations under iron-deficient conditions.

Identification of the laccase-like multicopper oxidase gene family of sweet cherry (Prunus avium L.) and expression analysis in six ancient Tuscan varieties

Laccase-like multicopper oxidases (LMCOs) are versatile enzymes used as biocatalysts performing the oxidation of different substrates of industrial relevance, with or without the intervention of a mediator. They have attracted a lot of interest for biotechnological applications in light of their eco-friendliness: they indeed oxidize the substrate(s) by coupling the four electron reduction of the final acceptor, molecular oxygen (O2), to water. Plant LMCOs represent a still poorly studied, important class of oxidoreductases controlling e.g. the post-harvest quality of fruits and enabling the tailoring of designer energy crops. We here sought to identify the LMCOs in Prunus avium L., whose fruits are rich in bioactive molecules, but are also highly perishable. The goal was to analyze them using bioinformatics (phylogenetic and in silico structural analyses) and to perform a targeted expression study on a subset of genes in six ancient varieties from Tuscany, all threatened by genetic erosion. These sweet cherry varieties contain higher amount of bioactive molecules, as compared to commercial counterparts. The results shown demonstrate strikingly different gene expression patterns in the six ancient varieties (‘Benedetta’, ‘Carlotta’, ‘Crognola’, ‘Maggiola’, ‘Morellona’, ‘Moscatella’) belonging to the Tuscan Regional Bank of Germplasm, as compared to a widely used commercial one (‘Durone’). The motivation of this study is the economic importance of P. avium and the involvement of LMCOs in post-harvest fruit parameters, like color. The results presented pave the way to follow-up researches on LMCOs of sweet cherry exploring post-harvest fruit parameters (e.g. anthocyanin stability responsible for pericarp browning and the preservation of the appealing red color), as well as developmental processes, like stony pit formation.