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bioRxiv · 10.1101/2025.10.09.681370

StabilizeIT: An Automated Workflow for Protein Stabilization

Kutnowski, N.·Budic, Y.·Alon, N.·Chalik, M.·Levin, I.·Lapidoth, G.·Zimmerman, L.
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Abstract

The industrial application of enzymes is often hampered by poor stability and low expression yields. While computational tools can predict stabilizing mutations, many are bound by restrictive licenses that hinder their broader adoption. To address this, we developed StabilizeIT, a powerful, open-access webserver for enhancing protein stability and expression. StabilizeIT integrates a pipeline of curated open-source tools such as ProteinMPNN, AlphaFold2 and SaProt with our state-of-the-art model, SolvIT, which accurately predicts heterologous expression titers in E. coli. This unique combination allows for the simultaneous optimization of melting temperature (Tm) and solubility. The pipeline exhibits remarkable speed, generating dozens of high-quality candidates with predicted high titers and increased stability in under an hour, streamlining the path to experimental validation. To demonstrate its efficacy, StabilizeIT was used to engineer multiple enzymes in our novel biosynthetic pathway for Hyaluronic Acid. The resulting variants showed greatly enhanced thermal stability and expression, proving the pipelines real-world utility. StabilizeIT is now available to the community, offering an accessible and validated solution to accelerate the development of robust proteins for diverse applications. The webserver is freely available at https://stabilizeit.enzymit.com

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Kutnowski, N., Budic, Y., Alon, N., Chalik, M., Levin, I., Lapidoth, G., Zimmerman, L.. 2025-10-10. StabilizeIT: An Automated Workflow for Protein Stabilization. https://doi.org/10.1101/2025.10.09.681370

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