Abstract
Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g−1 wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg−1 protein.
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Acknowledgements
We are grateful to Südchemie (München, Germany) for financial support and to Dr. Dirk Albrecht from the Division of Microbial Physiology and Molecular Biology of the Ernst-Moritz-Arndt-University Greifswald for the MALDI-TOF mass spectrometry analysis.
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Durban, M.A., Silbersack, J., Schweder, T. et al. High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis . Appl Microbiol Biotechnol 74, 634–639 (2007). https://doi.org/10.1007/s00253-006-0712-z
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DOI: https://doi.org/10.1007/s00253-006-0712-z