Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability

• Published in: World journal of microbiology & biotechnology 2022-01, Vol.38 (1), p.17, Article 17
• Main Authors: Rouhani, Maryam, Valizadeh, Vahideh, Aghai, Atousa, Pourasghar, Sogol, Molasalehi, Sara, Cohan, Reza Ahangari, Norouzian, Dariush
• Format: Article
• Language: English
• Subjects: Analogs; Applied Microbiology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biomedical and Life Sciences; Biotechnology; Calorimetry; Differential scanning calorimetry; Disulfide bonds; E coli; Environmental conditions; Environmental Engineering/Biotechnology; Enzymatic activity; Enzyme activity; Enzyme Stability; Enzymes; Escherichia coli - metabolism; Genetic Engineering; Hydrogen-Ion Concentration; Industrial Microbiology; Life Sciences; Microbiology; Mutagenesis; Mutagenesis, Site-Directed; Original Paper; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; pH effects; Protease; Proteinase; Proteins; Proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Room temperature; Temperature; Thermal stability
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-021-03195-z

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. Graphical abstract