Cold-adapted alginate lyases have unique advantages for alginate oligosaccharide (AOS) preparation and brown seaweed processing. Robust and cold-adapted alginate lyases are urgently needed for industrial applications. In this study, a cold-adapted alginate lyase-producing strain Vibrio sp. W2 was screened. Then, the gene ALYW201 was cloned from Vibrio sp. W2 and expressed in a food-grade host, Yarrowia lipolytica. The secreted Alyw201 showed the activity of 64.2 U/mL, with a molecular weight of approximate 38.0 kDa, and a specific activity of 876.4 U/mg. Alyw201 performed the highest activity at 30 °C, and more than 80% activity at 25–40 °C. Furthermore, more than 70% of the activity was obtained in a broad pH range of 5.0–10.0. Alyw201 was also NaCl-independent and salt-tolerant. The degraded product was that of the oligosaccharides of DP (Degree of polymerization) 2–6. Due to its robustness and its unique pH-stable property, Alyw201 can be an efficient tool for industrial production.

Alginate lyase possesses wide application prospects for the degradation of brown algae and preparation of alginate oligosaccharides, and its degradation products display a variety of biological activities. Although many enzymes of this type have been reported, alginate lyases with unique properties are still relatively rare. In the present work, an alginate lyase abbreviated as Alyw203 has been cloned from Vibrio sp. W2 and expressed in food-grade Yarrowia lipolytica. The Alyw203 gene consists of an open reading frame (ORF) of 1,566 bp containing 521 amino acids, of which the first 17 amino acids are considered signal peptides, corresponding to secretory features. The peak activity of the current enzyme appears at 45°C with a molecular weight of approximately 57.0 kDa. Interestingly, Alyw203 exhibits unique heat recovery performance, returning above 90% of its initial activity in the subsequent incubation for 20 min at 10°C, which is conducive to the recovery of current enzymes at low-temperature conditions. Meanwhile, the highest activity is obtained under alkaline conditions of pH 10.0, showing outstanding pH stability. Additionally, as an alginate lyase independent of NaCl and resistant to metal ions, Alyw203 is highly active in various ionic environments. Moreover, the hydrolyzates of present enzymes are mainly concentrated in the oligosaccharides of DP1–DP2, displaying perfect product specificity. The alkali suitability, heat recovery performance, and high oligosaccharide yield of Alyw203 make it a potential candidate for industrial production of the monosaccharide and disaccharide.

The voltage-dependent cardiac sodium channel plays a key role in cardiac excitability and conduction and it is the drug target of medically important. However, its atomic- resolution structure is still lack. Here, we report a modeled structure of Na_v1.5 pore domain in closed state. The structure was constructed by Rosetta-membrane homology modeling method based on the template of eukaryotic Na_v channel Na_vPaS and selected by energy and direct coupling analysis (DCA). Moreover, this structure was optimized through molecular dynamical simulation in the lipid membrane bilayer. Finally, to validate the constructed model, the binding energy and binding sites of closed-state local anesthetics (LAs) in the modeled structure were computed by the MM-GBSA method and the results are in agreement with experiments. The modeled structure of Na_v1.5 pore domain in closed state may be useful to explore molecular mechanism of a state-dependent drug binding and helpful for new drug development.

The first luciferase from Antarctic krill (LAK) was cloned and successfully expressed in Escherichia coli BL21(DE3). LAK exhibits the unique ability to emit bright violet fluorescence at an emission wavelength of 350 nm, which represents the lowest reported bioluminescence wavelength for luciferases. However, its low thermal stability poses a limitation to its broader application. In this study, we employed a rational design approach to introduce three pairs of artificial disulfide bonds into LAK. Circular dichroism (CD) analysis revealed that the introduction of artificial disulfide bonds resulted in a significant increase in the secondary structural content of α-helices and β-sheets compared to the wild-type (WT) enzyme. However, these modifications did not influence the emission spectrum. Among the resultant mutant strains, two exhibited markedly enhanced thermal stability. Notably, Mut2 demonstrated a 6.18-fold increase in half-life at 50 °C. Molecular docking studies indicated that D-fluorescein can form additional hydrogen bonds with surrounding amino acid residues (A323, T347, and K534). The docking energies between the enzyme and substrate for WT and Mut2 were −19.5 kcal/mol and −23.4 kcal/mol, respectively, thereby establishing strong interactions within the catalytic pocket region. These interactions likely contribute to a 2.92-fold improvement in substrate affinity, as evidenced by a reduced Michaelis–Menten constant (Km). Our thermal stability and catalytic activity analyses revealed that the linker region between the N- and C-domains plays a crucial role in the overall stability of the enzyme. Furthermore, the C-terminus of LAK does not participate in substrate-binding and catalysis; its local excessive rigidity was found to restrict the release of the AMP product, thereby negatively impacting catalytic activity. These findings offer new insights into the mutagenesis of luciferases and pave the way for the further optimization of LAK for various biotechnological applications.