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.
2. George-Okafor UO, Odibo FJC. Screening and optimal protease production by Bacillus sp. Sw-2 using low cost substrate medium. Res J Microbiol 2012; 7: 327-336.
3. Qian G, Wang Y, Liu Y, Xu F, He YW, Du L, et al. Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Appl Environ Microbiol 2013; 79: 6604-6616.
4. Ohara T, Makino K, Shinagawa H, Nakata A, Norioka S, Sakiyama F. Cloning nucleotide sequence and expression of Achromobacter lyticus protease I gene. J Biol Chem 1989; 264: 20625-20631.
5. Glazer AN, Nikaido H (1995). Microbial enzymes. In: Glazer AN, Nikaido H (eds) Microbial Biotechnology, Freeman and Co, New York, pp. 24-263.
6. Jekel PA, Weijer WJ, Beintema JJ. Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Anal Biochem 1983; 134: 347-354.
7. Demain AL, Adrio JL. Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation. Prog Drug Res 2008; 65: 251, 253-89.
8. Bose JL. Chemical and UV mutagenesis. Methods Mol Biol 2016; 1373:111-115.
9. Kodym A, Afza R. Physical and chemical mutagenesis. Methods Mol Biol 2003; 236:189-204.
10. Chibani HR, Fellahi SO, Chibani AB. Enhancement of protease production by Bacillus sp. and Micrococcus variants induced by UV mutagenesis. Int J Environ Agric Biotechnol 2017; 2: 2348-2353.
11. Basavaraju S, Kathera C, Jasti PK. Induction of alkaline protease production by Bacillus mutants through U.V. irradiation. Int J Pharm Sci Rev Res 2014; 26: 78-83.
12. Rani M, Prasad NN, Sambasivarao KR. Optimization of cultural conditions for the production of alkaline protease from a mutant Aspergillus Flavus AS2. Asian J Exp Biol Sci 2012; 3: 565-576.
13. Kuhlman PA, Chen R, Alcantara J, Szarka S. Rapid purification of Lys-C from Lysobacter enzymogenes cultures: A sequential chromatography technique. Bioprocess Int 2009; 7:28-38.
14. Zarif BR, Azin M. Increasing the bioethanol yield in the presence of furfural via mutation of a native strain of Saccharomyces cerevisiae. Afr J Microbiol Res 2011; 5: 651-656.
15. Ghazi S, Sepahy AA, Azin M, Khaje K, Khavarinejad R. UV mutagenesis for the overproduction of xylanase from Bacillus mojavensis PTCC 1723 and optimization of the production condition. Iran J Basic Med Sci 2014; 17: 844-853.
16. Yokota K, Furusawa N, Abe T, Takenaka S. Application of casein agar plate method for the determination of protease activity. Eisei Kagaku 1988; 34: 241-247.
17. Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 1938; 22: 79-89.
18. Keay L, Wildi BS. Proteases of the genus Bacillus. I. Neutral proteases. Biotechnol Bioeng 1970; 12:179-212.
19. Wang XC, Zhao HY, Liu G, Cheng XJ, Feng H. Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4. Genet Mol Res 2016; 15(2): gmr7831.
20. Jekel PA, Weijer JW, Beintema JJ. Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Anal Biochem 1983; 134: 347-354.
21. Karn N, Karn SK. Evaluation and characterization of protease production by Bacillus sp. Induced by UV-mutagenesis. Enz Eng 2014; 3:1.
22. Bommasamudram J, Devappa S. Strain improvement through mutagenesis and optimization of protease production by Aspergillus terreus CJS-127 using jatropha Seed Cack as Substrate. J Microbiol Biotechnol Food Sci 2017; 7: 174-180.
23. Taguchi S, Ozaki A, Momose H. Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Appl Environ Microbiol 1998; 64:492-495.
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