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

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.

Increment in protease activity of Lysobacter enzymogenes strain by ultra violet radiation.

BACKGROUND AND OBJECTIVES: Increasing the amount of protease from microbial sources is in the focus of attention. Random mutagenesis by physical methods like ultraviolet (UV) radiation is a cost effective and convenient procedure for strain improvement. Therefore, in the present study attempts were made to investigate the effect of UV radiation on Lysobacter enzymogenes in order to increase its protease activity. MATERIALS AND METHODS: UV mutagenesis was induced in L. enzymogenes fresh culture at the distance of 20 cm from light source for different exposure times of 70, 90, 150 and 200 seconds. The mutated isolates were randomly cultured from the nutrient agar medium to casein agar plate, as a selective medium. The primary screening was performed by observing hydrolysis of casein in the plate and the secondary screening was carried out on skim milk agar on the basis of zone of hydrolysis using bacterial supernatants. Quantification of protease activity was done by Anson's method using tyrosine as standard. RESULTS: UV radiation resulted in obtaining 12 mutants out of 100 examined L. enzymogenes strains with increased protease activity. The mutant M2, at 90s exposure time was selected as the best mutant bacterium which produced 1.96 fold more protease over the parent strain. CONCLUSION: Random mutation by UV radiation is a simple and convenient method to increase the protease activity of Lysobacter enzymogenes. Furthermore, it seems that the middle time of exposure to UV, 90 s, was the best time because it can induce mutagenesis but did not hamper the bacteria growth and viability.