Laccases as green and versatile biocatalysts: from lab to enzyme market—an overview

Laccases are multi-copper oxidase enzymes that catalyze the oxidation of different compounds (phenolics and non-phenolics). The scientific literature on laccases is quite extensive, including many basic and applied research about the structure, functions, mechanism of action and a variety of biotechnological applications of these versatile enzymes. Laccases can be used in various industries/sectors, from the environmental field to the cosmetics industry, including food processing and the textile industry (dyes biodegradation and synthesis). Known as eco-friendly or green enzymes, the application of laccases in biocatalytic processes represents a promising sustainable alternative to conventional methods. Due to the advantages granted by enzyme immobilization, publications on immobilized laccases increased substantially in recent years. Many patents related to the use of laccases are available, however, the real industrial or environmental use of laccases is still challenged by cost–benefit, especially concerning the feasibility of producing this enzyme on a large scale. Although this is a compelling point and the enzyme market is heated, articles on the production and application of laccases usually neglect the economic assessment of the processes. In this review, we present a description of laccases structure and mechanisms of action including the different sources (fungi, bacteria, and plants) for laccases production and tools for laccases evolution and prediction of potential substrates. In addition, we both compare approaches for scaling-up processes with an emphasis on cost reduction and productivity and critically review several immobilization methods for laccases. Following the critical view on production and immobilization, we provide a set of applications for free and immobilized laccases based on articles published within the last five years and patents which may guide future strategies for laccase use and commercialization.

Separator-less Amino Acid-powered Biofuel Cell Through an Artificial Multi-enzyme Cascade Reaction

Objective This study was aimed at constructing a highly stable one-compartment enzymatic biofuel cell (EFC) without a separator through a multi-enzyme cascade reaction pathway. Results A separator-less EFC composed of a multi-enzyme cascade anode containing four dehydrogenases from a thermophile and a cathode devised using a multi-copper oxidase mutant with enhanced enzyme activity from a hyperthermophile was developed. To fabricate an EFC without a separator, redox mediators utilized in the enzymatic cascade reaction were also immobilized on the anode. In the presence of the fuel 100 mM L -proline, the separator-less EFC with four thermophilic dehydrogenase-modified anode achieved a maximum power density of 11.3 υW/cm 2 at 37°C, which was 1.6-fold higher than that of a similar EFC fabricated with a one enzyme-modified anode. The separator-less EFC composed of a multi-enzyme modified anode maintained approximately 57% of the load current at 0.3 V measured on the first day of EFC fabrication, even after 4 days.Conclusion Efficient L-proline electric generation utilizing a separator-less EFC composed of a multi-enzyme modified anode through a multi-step fuel oxidation reaction and a highly stable multi-copper oxidase mutant-modified cathode was successfully achieved over a long period. The long-term stability of the separator-less EFC can facilitate its application as an efficient power source for implantable medical devices requiring continuous operation.

Technological Parameters, Anti-Listeria Activity, Biogenic Amines Formation and Degradation Ability of L. plantarum Strains Isolated from Sheep-Fermented Sausage

The aim of this work was to identify and characterize, from a technological and safety point of view, the lactic acid bacteria (LAB) isolated from traditional sheep-fermented sausage. First, LABs were identified then were screened for some technological parameters such as acidifying and growth ability, proteolytic and lipolytic activity and for antimicrobial activity. Finally, biogenic amine production and degradation abilities were also evaluated. This research reveals the predominance of Lactiplantibacillus (L.) plantarum on LAB community. Almost all L. plantarum strains were active against Listeria monocytogenes strains (inhibition zone diameters > 1 cm). None of the tested strains were positive in histidine (hdcA), lysine (ldc) and tyrosine (tyrdc) decarboxylase genes and only one (L. plantarum PT9-2) was positive to the agmatine deiminase (agdi) gene. Furthermore, given the positive results of the sufl (multi-copper oxidase) gene detection, all strains showed a potential degradation ability of biogenic amines.

A Putative Lignin Copper Oxidase from Trichoderma reesei

The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.