Yuan Zheng

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.

Isomaltulose (α-d-glucopyranosyl-1,6-d-fructofuranose) is an important industrial and raw food material, which can be synthesised from the by-products of sugar cane processing through sucrose isomerization conversion. In this study, we constructed a surface display vector of sucrose isomerase from Pantoeadispersa (pSIase) by a glycosylphosphatidylinositol (GPI)-cell wall protein (CWP) anchor signal sequence and successfully displayed pSIase on the cell surface of Yarrowia lipolytica, thereby increasing the conversion efficiency of isomaltulose. The highest activity of the displayed pSIase reached 2910.3 U/g of cell dry weight. Compared with the free pSIase, the displayed enzyme showed good stability at a broad range of temperatures (20–45 °C). The half-life at 40 °C increased from 62 to 141 min and the deactivation constants (k_d) reached 4.91 × 10⁻³ min⁻¹. Using low-cost cane molasses as the substrate, the isomaltulose conversion rate remained at 85% even after 9 batches were processed, which is a highly desired outcome for industrial use.

Objective To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Results Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K _cat/K _m) than that of the original EAG1. Conclusions The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme’s thermostability and catalytic efficiency.

The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.