Claudia Rodríguez-Almazán

Laccases belong to the multicopper oxidase family (MCO). Compared with fungal lac-cases, bacterial laccases have high stability and activity under extreme pH and temperature conditions. Because of these characteristics, bacterial laccases have become highly impactful in industrial processes. In the present work, we engineered the Thermus thermophilus HB27 laccase (Tth-Lac) by directed evolution. The library had a size of 1.2 x10 ⁵ colonies, and we screened mutants by malachite green decolorization on solid medium. The results showed that Q36H mutant exhibited higher catalytic activity than Tth-laccase (WT) at 90 °C. Both enzymes showed similar pH profiles, but the activity at 90 °C of Q36H mutant was X-fold higher than that of the wild-type enzyme. In contrast, the kinetic constants were similar for both enzymes. In the voltammetric responses, at 61.5 °C, the Q36H mutant showed increased current, and the T1 copper potential shifted by 100 mV relative to the tth-Lac. These results suggest that the Q36H mutation has high potential for industrial applications because replacing a glutamine (Q) with a histidine (H) at position 36 enhances the enzyme’s structural stability, allowing it to function efficiently under extreme temperature conditions.

Thermus thermophilus HB27 laccase (Tth-Lac) is a thermostable enzyme that contains a β-hairpin (Ala292-Gln307) covering the substrate entrance. We analyzed the role of this β-hairpin in the enzymatic activity of Tth-Lac through three β-hairpin mutants: two variants without the β-hairpin (C1Tth-Lac and C2Tth-Lac) and one with a partially modified β-hairpin (P1Tth-Lac). Enzymatic activity was assayed with different substrates with and without copper. C1Tth-Lac showed a higher dependency on copper, increasing its activity by 1600-fold for syringaldazine (SGZ). All mutants presented a higher activity than Tth-Lac with phenolic substrates in the presence of copper. The position of the signal associated with CuT2 also changed, as shown in EPR spectra. Elucidation of the crystal structure of P1Tth-Lac mutant (PDB: 9CPM) showed that the partial deletion of the β-hairpin did not significantly affect the overall tertiary structure compared to the wild-type (PDB: 2xu9) nor the coordination of the four internally bound Cu atoms. Higher B-factors of the residues downstream of the deletion indicate increased flexibility (Q307, G308, P309, S310) that were otherwise more ordered in the Tth-Lac structure. Redox potential experiments on platinum electrodes have shown that all proteins have high redox potential, a finding that could have significant implications in the field of protein research.

The bioprospecting of sea anemone tissues and secretions has revealed that they are natural libraries of polypeptides with diverse biological activities that can be utilized to develop of biotechnological tools with potential medical and industrial applications. This study conducted a proteomic analysis of crude venom extracts from Anthopleura dowii Verrill, 1869, and Lebrunia neglecta Duchassaing & Michelotti, 1860. The obtained data allowed us to identify 201 polypeptides, of which 39% were present in both extracts. Among the obtained sequences, hydrolase-type enzymes, oxidoreductases, transferases, heat shock proteins, adhesion proteins, and protease inhibitors, among others, were identified. Interaction analysis and functional annotation indicated that these proteins are primarily involved in endoplasmic reticulum metabolic processes such as carbon metabolism and protein processing. In addition, several proteins related to oxidative stress were identified, including superoxide dismutase, peroxiredoxins, thioredoxin, and glutathione oxidase. Our results provide novel information on the polypeptide composition of the crude venom extract from sea anemones, which can be utilized to develop molecules for therapeutic tools and industrial applications.

Chronic inflammatory diseases, such as cancer, diabetes mellitus, stroke, ischemic heart diseases, neurodegenerative conditions, and COVID-19 have had a high number of deaths worldwide in recent years. The accurate detection of the biomarkers for chronic inflammatory diseases can significantly improve diagnosis, as well as therapy and clinical care in patients. Graphene derivative materials (GDMs), such as pristine graphene (G), graphene oxide (GO), and reduced graphene oxide (rGO), have shown tremendous benefits for biosensing and in the development of novel biosensor devices. GDMs exhibit excellent chemical, electrical and mechanical properties, good biocompatibility, and the facility of surface modification for biomolecular recognition, opening new opportunities for simple, accurate, and sensitive detection of biomarkers. This review shows the recent advances, properties, and potentialities of GDMs for developing robust biosensors. We show the main electrochemical and optical-sensing methods based on GDMs, as well as their design and manufacture in order to integrate them into robust, wearable, remote, and smart biosensors devices. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers with improved sensitivity, reaching limits of detection from the nano to atto range concentration.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 ± 0.3 µg/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications.

Sea anemone venom contains a complex and diverse arsenal of peptides and proteins of pharmacological and biotechnological interest, however, only venom from a few species has been explored from a global perspective to date. In the present study, we identified the polypeptides present in the venom of the sea anemone Anthopleura dowii Verrill, 1869 through a transcriptomic and proteomic analysis of the tentacles and the proteomic profile of the secreted mucus. In our transcriptomic results, we identified 261 polypeptides related to or predicted to be secreted in the venom, including proteases, neurotoxins that could act as either potassium (K⁺) or sodium (Na⁺) channels inhibitors, protease inhibitors, phospholipases A2, and other polypeptides. Our proteomic data allowed the identification of 156 polypeptides—48 exclusively identified in the mucus, 20 in the tentacles, and 88 in both protein samples. Only 23 polypeptides identified by tandem mass spectrometry (MS/MS) were related to the venom and 21 exclusively identified in the mucus, most corresponding to neurotoxins and hydrolases. Our data contribute to the knowledge of evolutionary and venomic analyses of cnidarians, particularly of sea anemones.

Article Info Keywords Anthopleura; pore-forming protein; Venom; lung carcinoma; sea anemone