## 4 Conclusions  
In this study, we explored strategies to enhance the stability of Antarctic Krill luciferase (LAK) while preserving its catalytic activity. LAK holds significant promise for applications in medical tissue imaging, primarily due to its unique capability to emit violet fluorescence. However, the inherent structural complexity of LAK, characterized by multiple substrate-binding sites and a multifaceted catalytic mechanism, presents challenges for molecular modification. Our findings indicate that the introduction of artificial disulfide bonds can increase the content of \(\alpha\)-helix and \(\beta\)-sheet, subtly alter the secondary structure of LAK, and reduce the overall molecular free energy. These changes collectively contribute to improved protein thermostability. Additionally, the increase in hydrogen bonds within the protein's interior may enhance conformational stability without compromising catalytic performance. The formation of \(\pi\)-alkyl and \(\pi\)-sulfur interactions between the substrate and the catalytic region further strengthens the substrate-binding affinity. Notably, _Mut2_ exhibited a 6.18-fold increase in half-life at 50 \({}^{\circ}\)C and a 2.92-fold enhancement in substrate affinity (_Km_), findings that align with those reported by [PERSON] et al. [30]. Importantly, our results demonstrate that the introduction of disulfide bonds need not be restricted to surface flexible regions. For cold-adapted enzymes, the linker region is also a hotspot for mutation. These regions exhibit greater spatial conformational changes upon temperature increases; stiffening them can significantly improve thermal stability. While the C-terminal domain is crucial for maintaining the enzyme's overall stability, local steric constraints within this region can impede product release, thereby reducing catalytic efficiency. In conclusion, the work presented here indicates that the introduction of artificial disulfide bonds is an effective method for protein engineering. Despite the effectiveness of disulfide bond engineering in improving protein stability, several factors limit its widespread application. First, detailed three-dimensional (3D)  
Figure 7: CD spectroscopy and normalized luminescence spectra of WT and _Muts_. (**a**) CD spectroscopy of WT and _Muts_. (**b**) Normalized luminescence spectra of WT and _Mut2_. The luminescence spectra were measured at 25 \({}^{\circ}\)C.  
structural information of the proteins is essential for designing disulfide bridges using appropriate computational strategies. For enzymes with low sequence homology or those whose structures are difficult to resolve, such as aminopeptides, luciferase, and dehalogenases, selecting optimal mutation sites poses a significant challenge. Second, enzymes that act on complex biomolecular substrates, such as chitol oligosaccharides or carrageenan, possess multiple functional regions, including catalytic active centers, substrate-binding sites, coenzyme-binding sites, and product-release sites. Their intricate catalytic mechanisms mean that single-point mutations often result in detrimental effects or even destabilization. This underscores the need for a deeper understanding of the fine spatial structure of enzyme molecules and a clear elucidation of their catalytic mechanisms. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15073563/s1](https://www.mdpi.com/article/10.3390/app15073563/s1)]([https://www.mdpi.com/article/10.3390/app15073563/s1](https://www.mdpi.com/article/10.3390/app15073563/s1)), Figure S1: The plasmid map of pCold II, Table S1: DNA sequence of LAK. Y.M., writing, idea proposal, experimental data validation; Y.Z., methodology, conceptualization, formal analysis; X.J., software development, submission, and preparation; J.S., review, resources, and editing. All authors have read and agreed to the published version of the manuscript. This work was supported by the National Key Research and Development Program of China (Grant No. 2022 YFC2807505). Not applicable.