In silico study on the effects of Glucosamine group on Thermal Stability of Yarrowia lipolytica Lip۲ lipase

Yarrowia lipolytica is recognized as a promising host for heterologous protein production due to its high secretion capacity. Among its enzymes, lipase plays a crucial role in diverse industrial applications, including detergent, food, cosmetic, pharmaceutical, and environmental sectors. Lip۲, the only extracellular lipase of Y. lipolytica, is a glycosylated enzyme composed of ۳۰۱ amino acids and several disulfide bonds. Its crystal structure reveals two N-glycosylation sites located at N۱۱۳ and N۱۳۴. Lip۲ exhibits catalytic activity at low temperatures (around ۵ °C), shows optimal performance at ۳۷ °C, and rapidly loses activity above ۵۰ °C. To explore the influence of sugar moieties on the thermodynamic stability of Lip۲, the complete glycosylated structure was examined through molecular dynamics (MD) simulations. The simulations were carried out using GROMACS ۵.۱.۴ with the CHARMM۳۶M force field at three functional temperatures (۳۰۰ K, ۳۱۰ K, and ۳۳۳ K) for a total production run of ۴۰ ns. The results demonstrated that the glycosylated Lip۲ exhibited higher temperature-dependent structural fluctuations, while the deglycosylated form showed enhanced thermostability upon temperature increase. Further analysis revealed that glycosylation affects not only the residues adjacent to the glycosylation sites but also induces conformational changes in distant regions of the protein. This leads to increased residue flexibility and a higher radius of gyration, which was supported by root-mean-square deviation (RMSD) analysis. Additionally, hydrogen bond analysis indicated that the non-glycosylated form maintained a higher number and longer lifetimes of hydrogen bonds across all temperatures, suggesting a more stable folding pattern. In conclusion, the overall stability of the glycosylated Lip۲ decreases compared to its non-glycosylated counterpart, potentially due to complex molecular interactions. This in silico study provides new insights into the thermodynamic behavior of Lip۲ in aqueous environments and establishes a foundation for future studies on the independent effects of glucosamine groups on enzyme stability.

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Keywords: Yarrowia lipolytica ، Lip۲ lipase ، Glycosylation ، Thermal stability