Evaluation of Antibiotic Biodegradation by a Versatile and Highly Active Recombinant Laccase from the Thermoalkaliphilic Bacterium Bacillus sp. FNT.

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, ß-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20-90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL-1) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation.

Recombinant expression and characterization of a new laccase, bioinformatically identified, from the Antarctic thermophilic bacterium Geobacillus sp. ID17.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Heat Shock Tolerance in Deschampsia antarctica Desv. Cultivated in vitro Is Mediated by Enzymatic and Non-enzymatic Antioxidants.

Deschampsia antarctica Desv, is the most successful colonizing species of a cold continent. In recent years due to climate change, the frequency of heat waves has increased in Antarctica, registering anomalous high temperatures during the summer of 2020. However, the populations of D. antarctica are responding positively to these events, increasing in number and size throughout the Antarctic Peninsula. In this work, the physiological and biochemical responses of D. antarctica plants grown in vitro (15 ± 1°C) and plants subjected to two heat shock treatments (23 and 35°C) were evaluated. The results obtained show that D. antarctica grown in vitro is capable of tolerating heat shock treatments; without showing visible damage to its morphology, or changes in its oxidative state and photosynthetic performance. These tolerance responses are primarily mediated by the efficient role of enzymatic and non-enzymatic antioxidant systems that maintain redox balance at higher temperatures. It is postulated that these mechanisms also operate in plants under natural conditions when exposed to environmental stresses.

Antioxidant Responses Induced by UVB Radiation in Deschampsia antarctica Desv.

Deschampsia antarctica Desv. is one of two vascular plants that live in the Maritime Antarctic Territory and is exposed to high levels of ultraviolet-B (UVB) radiation. In this work, antioxidant physiology of D. antarctica was studied in response to UVB induced oxidative changes. Samples were collected from Antarctica and maintained in vitro culture during 2 years. Plants were sub-cultured in a hydroponic system and exposed to 21.4 kJ m-2 day-1, emulating summer Antarctic conditions. Results showed rapid and significant increases in reactive oxygen species (ROS) at 3 h, which rapidly decreased. No dramatic changes were observed in photosynthetic efficiency, chlorophyll content, and level of thiobarbituric acid reactive species (MDA). The enzymatic (superoxide dismutase, SOD and total peroxidases, POD) and non-enzymatic antioxidant activity (total phenolic) increased significantly in response to UVB treatment. These findings suggest that tolerance of D. antarctica to UVB radiation could be attributed to its ability to activate both enzymatic and non-enzymatic antioxidant systems.