Lignin degradation by a novel thermophilic and alkaline yellow laccase from Chitinophaga sp.

Laccases (EC 1.10.3.2) are oxidoreductases that belong to the multicopper oxidase subfamily and are classified as yellow/white or blue according to their absorption spectrum. Yellow laccases are more useful for industrial processes since they oxidize nonphenolic compounds in the absence of a redox mediator and stand out for being more stable and functional under extreme conditions. This study aimed to characterize a new laccase that was predicted to be present in the genome of Chitinophaga sp. CB10 - Lac_CB10. Lac_CB10, with a molecular mass of 100.06 kDa, was purified and characterized via biochemical assays using guaiacol as a substrate. The enzyme demonstrated extremophilic characteristics, exhibiting relative activity under alkaline conditions (CAPS buffer pH 10.5) and thermophilic conditions (80-90°C), as well as maintaining its activity above 50% for 5 h at 80°C and 90°C. Furthermore, Lac_CB10 presented a spectral profile typical of yellow laccases, exhibiting only one absorbance peak at 300 nm (at the T2/T3 site) and no peak at 600 nm (at the T1 site). When lignin was degraded using copper as an inducer, 52.27% of the material was degraded within 32 h. These results highlight the potential of this enzyme, which is a novel yellow laccase with thermophilic and alkaline activity and the ability to act on lignin. This enzyme could be a valuable addition to the biorefinery process. In addition, this approach has high potential for industrial application and in the bioremediation of contaminated environments since these processes often occur at extreme temperatures and pH values. IMPORTANCE: The characterization of the novel yellow laccase, Lac_CB10, derived from Chitinophaga sp. CB10, represents a significant advancement with broad implications. This enzyme displays exceptional stability and functionality under extreme conditions, operating effectively under both alkaline (pH 10.5) and thermophilic (80-90°C) environments. Its capability to maintain considerable activity over extended periods, even at high temperatures, showcases its potential for various industrial applications. Moreover, its distinctive ability to efficiently degrade lignin-demonstrated by a significant 52.27% degradation within 32 h-signifies a promising avenue for biorefinery processes. This newfound laccase's characteristics position it as a crucial asset in the realm of bioremediation, particularly in scenarios involving contamination at extreme pH and temperature levels. The study's findings highlight the enzyme's capacity to address challenges in industrial processes and environmental cleanup, signifying its vital role in advancing biotechnological solutions.

Dynamics of the role of LacMeta laccase in the complete degradation and detoxification of malachite green.

Laccases highlight for xenobiotic bioremediation, as well as application in the fine chemical, textile, biofuel and food industries. In a previous work, we described the preliminary characterization of laccase LacMeta, a promising enzyme for the bioremediation of dyes, able to decolorization malachite green (MG), trypan blue, methylene blue. Here we demonstrate that LacMeta is indeed suitable for the complete degradation and detoxification of MG dye, not just for its discoloration, since some works show false positives due to the formation of colorless intermediates such as leucomalachite. The optimal pH and temperature parameters of LacMeta were 5.0 and 50 °C, respectively (MG as substrate). LacMeta was tolerant of up to 10 mmol L- 1 EDTA (82%) and up to 5% (V/V) acetone (91%) and methanol (71%), while SDS promoted severe inhibition. For ions, a high tolerance to cobalt, zinc, manganese, and calcium (10 mmol L- 1) was also observed (> 90%). Even under high-salinity conditions (1 mol L- 1 NaCl), the residual bleaching activity of the dye remained at 61%. Furthermore, the bleaching product of MG did not inhibit the germination of sorghum and tomato seeds and was inert to the vegetative structures of the germinated seedlings. Additionally, this treatment effectively reduced the cytotoxic effect of the dye on microorganisms (Escherichia coli and Azospirillum brasilense), which can be explained by H-NMR spectral analysis results since LacMeta completely degraded the peak signals corresponding to the aromatic rings in the dye, demonstrating extreme efficiency in the bioremediation of the xenobiotic at high concentrations (50 mg L- 1).

Broad thermal spectrum metagenomic laccase with action for dye decolorization and fentin hydroxide treatment.

Laccases are multicopper oxidases that act on various phenolic and non-phenolic compounds, enabling numerous applications including xenobiotic bioremediation, biofuel production, drug development, and cosmetic production, and they can be used as additives in the textile and food industries. This wide range of uses makes these enzymes extremely attractive for novel biotechnology applications. Here, we undertook the kinetic characterization of LacMeta, a predicted as homotrimeric (~ 107,93 kDa) small laccase, and demonstrated that this enzyme performs best at an acidic pH (pH 3-5) towards ABTS as substrate and has a broad thermal spectrum (10-60 °C), which can promote high plastic action potential through dynamic environmental temperature fluctuations. This enzyme showed following kinetic parameters: kcat = 6.377 s-1 ± 0.303, Km = 4.219 mM, and Vmax = 24.43 µM/min (against ABTS as substrate). LacMeta almost completely degraded malachite green (50 mg/mL) in only 2 h. Moreover, the enzyme was able to degrade seven dyes from four distinct classes and it respectively achieved 85% and 83% decolorization of methylene blue and trypan blue with ABTS as the mediator. In addition, LacMeta showed potential for the degradation of two thirds of an agricultural fungicide: fentin hydroxide, thus demonstrating its biotechnological aptitude for bioremediation. The results of this study suggest that LacMeta has potential in textile wastewater treatment and that it could help in the bioremediation of other human/environmental toxins such as pesticides and antibiotic compounds belonging to the same chemical classes as the degraded dyes.

Characterization of a new bifunctional endo-1,4-ß-xylanase/esterase found in the rumen metagenome.

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.