Abstract
Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.
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Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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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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FFT, MD, NSF and SM contributed in the laboratory work, analysis of the data and drafted the paper. SMA, RAC and DN gave critical suggestions for designing experimental assays and data analysis. VV and MK designed the work, supervised the study and critically revised the manuscript. All authors have read and approved the final manuscript.
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Tabar, F.F., Valizadeh, V., Keramati, M. et al. Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma. Arch Microbiol 204, 343 (2022). https://doi.org/10.1007/s00203-022-02936-4
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DOI: https://doi.org/10.1007/s00203-022-02936-4