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

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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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  1. Carreno FLG, Dimes LE, Haard NF (1993) Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal Biochem 214:65–69

  2. Castro GR (1999) Enzymatic activities of proteases dissolved in organic solvents. Enzyme Microb Technol 25:639–782

  3. Chand S, Mishra P (2003) Research and application of microbial enzymes-India’s contribution. Adv Biochem Eng Biotechnol 85:95–124

  4. Cruden DL, Wolfram JH, Rogers RD, Gibson DT (1992) Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium. Appl Environ Microbiol 58:2723–2729

  5. Estrada P, Sanchez-muniz R, Acebal C, Arche R, Castillon MP (1991) Characterization and optimization of immobilized polyphenol oxidase in low-water organic solvents. Biotechnol Applied Biochem 14:12–20

  6. Fersht A (1985) Enzyme structure and mechanism. Freeman, San Francisco

  7. Fujiwara N, Masui A, Imanaka T (1993) Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. J Biotechnol 30:245–256

  8. Furth AJ (1980) Removing unbound detergent from hydrophobic proteins. Anal Biochem 109:207–215

  9. Guagliardi A, Manco G, Rossi M, Bartolucci S (1989) Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents. Eur J Biochem 183:25–30

  10. Gupta A, Roy I, Patel RK, Singh SP, Khare SK, Gupta MN (2005) One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp. J Chromatogr A 1075:103–108

  11. Huheey JE (1972) Chemistry: Principles of structure and reactivity. Harper and Row, New York

  12. Hun CJ, Rahman RNA, Salleh AB, Basri M (2003) A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. Biochem Eng J 15:147–151

  13. Isken S, de Bont JA (1998) Bacteria tolerant to organic solvents. Extremophiles 2:229–238

  14. Jönsson A, Adlercreutz P, Mattiasson B (1996) Temperature effects on protease catalyzed acyl transfer reactions in organic media. J Mol Catal B: Enzym 2:43–51

  15. Ke T, Klibanov AM (1998) On enzymatic activity in organic solvents as a function of enzyme history. Biotechnol Bioeng 57:746–750

  16. Khmelnitsky YL, Levashov AV, Klyachko NL, Martinek K (1988) Engineering biocatalytic systems in organic media with low water content. Enzyme Microb Technol 10:710–724

  17. Kidd RD, Sears P, Huang DH, Witte K, Wong CH, Farber GK (1999) Breaking the low barrier hydrogen bond in a serine protease. Protein Sci 8:410–417

  18. Kim K, Lee S, Lee K, Lim D (1998) Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73. J Bacteriol 180:3692–3696

  19. Klibanov AM (1986) Enzymes that work in organic solvents. Chem Tech 16:354–359

  20. Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409:241–245

  21. Kwon OH, Imanishi Y, Ito Y (1999) Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media. Biotechnol Bioeng 66:265–270

  22. Laane C, Boeren S, Hilhorst R, Veeger C (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 30:81–87

  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

  25. Matsumoto M, de Bont JA, Isken S (2002) Isolation and characterization of the solvent-tolerant Bacillus cereus strain R1. J Biosci Bioeng 94:45–51

  26. Meos H, Haga M, Aavishkar A, Schuster M, Jakubke HD (1993) Single-step synthesis of kyotorphin in frozen solutions by chymotrypsin. Tetrahedron Asymmetry 7:1559–1564

  27. Moriya K, Yanigitani S, Usami R, Horikoshi K (1995) Isolation and some properties of an organic solvent tolerant marine bacterium degrading cholesterol. J Mar Biotechnol 2:131–133

  28. Norling B (1986) The effect of anionic detergents on the ATPase activity of isolated F1 from the thermophilic bacterium PS3. Biochem Biophys Res Commun 136:899–905

  29. Ogino H, Yasui K, Shiotani T, Ishihara T, Ishikawa H (1995) Organic solvent-tolerant bacterium which secretes an organic solvent-stable proteolytic enzyme. Appl Environ Microbiol 61:4258–4262

  30. Ogino H, Yasui K, Watanabe F, Ishikawa H (1996) An organic solvent-tolerant bacterium and its organic solvent-stable protease. Ann N Y Acad Sci 799:311–317

  31. Ogino H, Uchiho T, Yokoo J, Kobayashi R, Ichise R, Ishikawa H (2001) Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability. Appl Environ Microbiol 67:942–947

  32. Pinkart HC, Wolfram JW, Rogers R, White DC (1996) Cell envelope changes in solvent-tolerant and solvent-sensitive Pseudomonas putida strains following exposure to o-xylene. Appl Environ Microbiol 62:1129–1132

  33. Ramos JL, Duque E, Huertas MJ, Haidour A (1995) Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons. J Bacteriol 177:3911–3916

  34. Reslow M, Adlercreutz P, Mattiasson B (1987) Organic solvents for bioorganic synthesis. Appl Microbiol Biotechnol 26:1–8

  35. Sareen R, Bornscheuer U, Mishra P (2004a) A microtiter plate assay for the determination of the synthetic activity of protease. Anal Biochem 333:193–195

  36. Sareen R, Bornscheuer U, Mishra P (2004b) Synthesis of kyotorphin precursor by an organic solvent stable protease from Bacillus licheniformis RSP-09–37. J Mol Catal B Enzym 32:1–5

  37. Segel IH (1975) Enzyme kinetics. Wiley, New York

  38. Strongin AY, Izotova LS, Abramov ZT, Gorodetsky DI, Ermakova LM, Baratova LA, Belyanova LP, Strepanov VM (1978) Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins. J Bacteriol 133:1401–1411

  39. Towatana NH, Painupong A, Suntinanalert P (1999) Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719. J Biosc Bioeng 87:581–587

  40. You L, Arnold FH (1996) Directed evolution of subtilisin E in Bacillus subtilis to enhance activity in aqueous dimethyl formamide. Protein Eng 9:77–83

  41. Zaks A, Klibanov AM (1985) Enzyme-catalyzed processes in organic solvents. Proc Natl Acad Sci U S A 82:3192–3196

  42. Zaks A, Klibanov AM (1988) Enzymatic catalysis in nonaqueous solvents. J Biol Chem 263:3194–3201

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