A research team has successfully enhanced the thermostability of Carboxypeptidase A (CPA), a crucial enzyme with significant potential in the food and pharmaceutical industries, through the innovative use of disulfide bonds. This development promises to expand the enzyme's use in processes that require high-temperature conditions, such as degrading ochratoxin A (OTA) and reducing the bitterness of peptides in food.
Carboxypeptidase A (CPA) is widely used in the food and pharmaceutical industries for its ability to hydrolyze toxic ochratoxin A (OTA) into non-toxic compounds and remove bitterness from peptides in food processing. However, CPA's natural mesophilic nature limits its function at higher temperatures, restricting its application in industrial settings where elevated heat is common. Enzyme thermostability is essential for extending operational temperatures, improving efficiency, and reducing production costs. Previous efforts to enhance enzyme thermostability have shown that protein engineering, particularly the introduction of disulfide bonds, can significantly stabilize enzymes for industrial use.
A study (DOI: 10.48130/fia-0024-0017) published in Food Innovation and Advances on 25 June 2024, marks a significant leap forward in enzyme engineering.
The research utilized a rational design strategy to enhance the thermostability of Carboxypeptidase A (CPA) by introducing disulfide bonds. First, flexible regions of CPA were identified using B-factor analysis, which characterizes thermal motion in proteins, and RMSF values, reflecting local conformational flexibility. Through a comprehensive analysis of these data, ten highly flexible regions of CPA were selected as targets for rigidification via disulfide bonds. The DbD and MODIP programs predicted potential residue pairs for introducing disulfide bonds, and after conservativeness analysis, two mutants, D93C/F96C and K153C/S251C, were selected.
Homology models of the mutants were built using SWISS-MODEL, and MD simulations evaluated their conformational stability. The results showed that both mutants exhibited lower RMSD values, indicating enhanced thermostability compared to the wild-type (WT) CPA. The study demonstrated that the mutants exhibited increased optimal operating temperatures—10°C higher than the wild-type—and maintained their activity for extended periods at 65°C. Additionally, the mutants’ half-inactivation temperatures (T5015) increased by 8.5°C and 11.4°C, further demonstrating their improved heat resistance. While the D93C/F96C mutant exhibited improved enzymatic activity and thermostability, K153C/S251C displayed a slight reduction in activity due to a trade-off between stability and catalytic efficiency. Structural analysis showed that the introduction of disulfide bonds increased α-helix content, and surface charge redistribution contributed to the mutants' enhanced stability.
According to the study's lead researcher, Dr. Zhihong Liang, "Enhancing CPA's thermostability opens up many opportunities for its application in the food and pharmaceutical industries. By introducing disulfide bonds, we have been able to significantly increase CPA's resistance to higher temperatures without compromising its catalytic activity, which is a crucial advancement for industrial processes."
This breakthrough in enhancing CPA’s thermostability through the introduction of disulfide bonds marks a major leap forward in enzyme engineering. The research not only provides insights into CPA's molecular mechanisms but also lays the groundwork for its broader use in industrial applications, particularly in high-temperature environments. As the industry moves towards more efficient and sustainable processes, thermostable enzymes like CPA will be key drivers of innovation.
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References
DOI
10.48130/fia-0024-0017
Original Source URL
https://maxapress.com/article/doi/10.48130/fia-0024-0017
Funding information
the National Key Research and Development Program of China (No. 2023YFD1301000);
the Shandong Province Natural Science Foundation (No. ZR202102260301).
About Food Innovation and Advances
Food is essential to life and relevant to human health. The rapidly increasing global population presents a major challenge to supply abundant, safe, and healthy food into the future. The open access journal Food Innovation and Advances (e-ISSN 2836-774X), published by Maximum Academic Press in association with China Agricultural University, Zhejiang University and Shenyang Agricultural University, publishes high-quality research results related to innovations and advances in food science and technology. The journal will strive to contribute to food sustainability in the present and future.