Journal
JOURNAL OF MICROBIOLOGY
Volume 51, Issue 3, Pages 359-366Publisher
MICROBIOLOGICAL SOCIETY KOREA
DOI: 10.1007/s12275-013-2627-6
Keywords
purification; characterization; Bacillus subtilis; anti-fungal protease; self-formed adaptor PCR
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Funding
- National Basic Research Program of China [2011CB100503]
- National Department of Public Benefit Research Foundation of the Ministry of Agriculture of China [201103004]
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A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30-60% (NH4)(2)SO4 precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOF (TM) Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two beta-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 mu g/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0-10.6 and 70A degrees C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na+, Fe3+, Cu2+, Mg2+ (5 mmol/L each) and H2O2 (2%, v/v). However, protease activity was activated by Ca2+, K+, Mn2+ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).
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