Abstract
Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12–302 ss] was not further active which could be due to the large truncation. However, T344 [8–339 ss], T380 [8–339 ss] and T380 [12–302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25–90 °C and pH 6–9.5. Interestingly, remaining enzyme activity of T344 [8–339 ss], T380 [8–339 ss] and T380 [12–302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8–339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors wish to express their deep gratitude to all who provided support during the course of this research. This project was financially supported by Pasteur Institute of Iran.
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M.R. (Ph.D. student) contributed in the laboratory work, analysis of the data and drafted the paper. A.A., S.P. and S.M. helped with the experimental assays, R.A.C. gave critical suggestions for designing enzyme engineered forms. V.V. designed the work, supervised the study and critically revised the manuscript and N.D. helped with data analysis and critical reading of the manuscript.
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Rouhani, M., Valizadeh, V., Aghai, A. et al. Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability. World J Microbiol Biotechnol 38, 17 (2022). https://doi.org/10.1007/s11274-021-03195-z
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DOI: https://doi.org/10.1007/s11274-021-03195-z