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Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

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Abstract

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with α-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50°C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-l-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.

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Acknowledgments

Ritu Sareen would like to thank the financial support provided by Council of Scientific and Industrial research, New Delhi for senior research fellowship. This work was partially supported by a grant from Ministry of Human Resource Development, Government of India to one of the authors (P.M.).

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Correspondence to Prashant Mishra.

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Sareen, R., Mishra, P. Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37. Appl Microbiol Biotechnol 79, 399–405 (2008). https://doi.org/10.1007/s00253-008-1429-y

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Keywords

  • Purification
  • Organic solvent tolerance
  • Transesterification
  • Protease
  • Bacillus licheniformis