RESUMO
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.
RESUMO
BACKGROUND: Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment. OBJECTIVE: This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation. METHOD: In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed. RESULTS: According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity. CONCLUSION: SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.