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Purification and bio-chemical characterization of a solvent-tolerant and highly thermostable lipase of Bacillus licheniformis strain SCD11501

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

The lipase of Bacillus licheniformis SCD11501 was purified with a fold purification of 10.6 and a yield of 8.4 % by ion-exchange chromatography on a DEAE-cellulose column. The purified enzyme was found to be electrophoretically pure by denaturing SDS-PAGE and possessed a Mr of approximately 35 kDa. A single band ~70 kDa was observed in native-PAGE confirming that the lipase of B. licheniformis appeared to be a holozyme that comprises two identical subunits of ~35 kDa in SDS-PAGE. Thus the lipase appeared to be a homodimeric protein which was an interesting observation. The purified lipase was optimally active at 55 °C and pH 9.5 and showed maximum hydrolytic activity towards a relatively longer C-chain length p-NPP substrate. The lipase was stimulated only in the presence of Fe2+ while the presence of the chelating agent(s) inhibited its activity. All surfactants (Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80 and SDS) had an inhibitory effect on lipase activity. The purified lipase also retained good activity in the presence of both hydrophobic (n-hexane) as well as hydrophilic (DMSO) solvents that indicated its usefulness in promoting both hydrolytic as well as synthetic reactions. The extracellular lipase was highly thermostable as it showed a half-life of 150 min at 55 °C and 60 min at 75 °C. The K m and V max of purified lipase of B. licheniformis SCD11501 was found to be 0.43 mM and 2.27 µmol/ml/min, respectively for hydrolysis of p-NPP.

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References

  1. Nagar M, Dwivedi SK, Shrivastava D (2013) A review on industrial application in microbial lipases. Int J Pharma Res Sci 2:631–641

    Google Scholar 

  2. Nikola M, Nedeljko M, Jovanka F, Zorica KJ, Melina KK (2012) Efficient immobilization of lipase from Candida rugosa by entrapment into poly (N-isopropylacrylamide-coitaconic acid) hydrogels under mild conditions. Polym Bull 69:47–52

    Google Scholar 

  3. Bajaj A, Lohan P, Jha PN, Mehrotra R (2010) Biodiesel production through lipase catalyzed transesterification: an overview. J Mol Catal B-Enzym 62:9–14

    Article  CAS  Google Scholar 

  4. Sharma S, Dogra P, Chauhan GS, Kanwar SS (2014) Synthesis of alkyl coumarate esters by lipase of Bacillus licheniformis SCD11501. J Mol Catal B Enzym 101:80–86

    Article  CAS  Google Scholar 

  5. Durmaz E, Kuyucak S, Sezerman UO (2013) Modifying the catalytic preference of tributyrin in Bacillus thermocatenulatus lipase through in silico modeling of enzyme substrate complex. Protein Eng Des Sel 26:325–333

    Article  CAS  PubMed  Google Scholar 

  6. Saxena RK, Misra S, Rawat I, Gupta P, Dutt K, Parmar VS (2011) Production of 1, 3 regiospecific lipase from Bacillus sp. RK-3: it’s potential to synthesize cocoa butter substitute. Malays J Microbiol 7:41–48

    CAS  Google Scholar 

  7. Kanimozhi K, Devairrakam EGWJ, Kumar DJ (2013) Decolorization of leather effluent by lipase producing Bacillus sp. Acad Indus Res 1:813–815

    Google Scholar 

  8. Stathopoulou PM, Savvides AL, Karagouni AD, Hatzinikolaou DG (2013) Unraveling the lipolytic activity of thermophilic bacteria isolated from a volcanic environment. BioMed Res Int. doi:10.1155/2013/703130

    PubMed  PubMed Central  Google Scholar 

  9. Saxena RK, Ghosh PK, Gupta R, Davinson WS, Bradoo S, Gulati R (1999) Potential biocatalysis and future industry. Curr Sci 77:110–115

    Google Scholar 

  10. Peng R, Lin J, Wei D (2011) Synthesis of butyl acetate in n-heptane by the recombinant CS-2 lipase immobilized on kieselguhr. Afr J Food Sci Technol 2:59–66

    Google Scholar 

  11. Almeida VM, Branco CRC, Assis SA, Vieira IJC, Filho RB, Branco A (2012) Synthesis of naringin 6″-ricinoleate using immobilized lipase. Chem Cent J 6:41–48

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sharma S, Kanwar SS (2014) Organic solvent tolerant lipases and applications. Sci World J. doi:10.1155/625258

    Google Scholar 

  13. Sellek GA, Chaudhuri JB (1999) Biocatalysis in organic media using enzymes from extremophiles-negatively charged groups artificially introduced into -chymotrypsin provide additional activation and stabilization effects. Enzym Microbiol Technol 25:471–482

    Article  CAS  Google Scholar 

  14. Inoue A, Horikoshi KA (1989) Pseudomonas thrives in high concentrations of toluene. Nature 338:264–266

    Article  CAS  Google Scholar 

  15. Chakraborty K, Raj RP (2008) An extra-cellular alkaline metallolipase from Bacillus licheniformis MTCC 6824: purification and biochemical characterization. Food Chem 109:727–736

    Article  CAS  PubMed  Google Scholar 

  16. Annamalai N, Elayaraja S, Vijayalakshmi S, Balasubramanian T (2011) Thermostable, alkaline tolerant lipase from Bacillus licheniformis using peanut oil cake as a substrate. Afr J Biochem Res 5:176–181

    CAS  Google Scholar 

  17. Sharma CK, Kanwar SS (2012) Purification of a novel thermophilic lipase from B. licheniformis MTCC-10498. ISCA J Biol Sci 1:43–48

    Google Scholar 

  18. Winkler UK, Stuckmann M (1979) Glucogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Scopes RK (1994) Protein purification, principles and practice, 3rd edn. Springer, New York, pp 7–101

  20. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement by folin- phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  21. Mahadevan GD, Neelagund SE (2012) Thermostable lipase from Geobacillus sp. Iso5: bioseparation, characterization and native structural studies. J Basic Microbiol. doi:10.1002/jobm.201200656

    Google Scholar 

  22. Gayathri VR, Peruma P, Mathew LP, Prakash B (2013) Screening and molecular characterization of extracellular lipase producing Bacillus species from coconut oil mill soil. Int J Sci Technol 2:502–509

    Google Scholar 

  23. Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R (2005) Production, purification and characterization of lipase from thermophilic and alkalophilic Bacillus coagulans BTS-3. Protein Expr Purif 41:38–44

    Article  CAS  PubMed  Google Scholar 

  24. Begam MS, Pradeep FS, Pradeep BV (2012) Production, purification, characterization and applications of lipase from Serratia marcescens MBB05. Asian J Pharm Clin Res 5:237–245

    Google Scholar 

  25. Larbidaouadi K, Benattouche Z, Abbouni B (2014) Screening selection identification production and optimization of bacterial lipase isolated from industrial rejection of gas station. J Chem Pharma Res 6:455–459

    CAS  Google Scholar 

  26. Gokbulut AA, Arslanoglu A (2013) Purification and biochemical characterization of an extracellular lipase from psychrotolerant Pseudomonas fluorescens KE38. Turk J Biol 37:538–546

    Article  CAS  Google Scholar 

  27. Do H, Lee JH, Kwon MH, Song HE, An JY, Eom SH, Lee SG, Kim HJ (2013) Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea. Acta Cryst 4:920–924

    Google Scholar 

  28. Zouaoui B, Bouziane A, Ghalem BR (2012) Production, optimization and purification of lipase from Pseudomonas aeruginosa. Afr J Microbiol Res 6:4417–4423

    CAS  Google Scholar 

  29. Mohammed HJ (2013) Physicochemical factors affected the partial purified lipase activity of Acinetobacter baumannii “local isolates”. Iraqi J Pharm Sci 22:82–89

    Google Scholar 

  30. Castro-Ochoa LD, Rodriguez-Gomez C, Valerio-Alfaro G, Ros RO (2005) Screening, purification and characterization of the thermo-alkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzym Microb Technol 37:648–654

    Article  CAS  Google Scholar 

  31. Daoud L, Kamoun J, Ali MB, Jallouli R, Bradai R, Mechichi T, Gargouri Y, Ali YB, Aloulou A (2013) Purification and biochemical characterization of a halotolerant Staphylococcus sp. extracellular lipase. Int J Biol Macromol 57:232–237

    Article  CAS  PubMed  Google Scholar 

  32. Kavitha M, Shanthi C (2013) Isolation and characterization of cold active lipase producing Pseudomonas sp. 4 from marine samples of Tamil Nadu coast. Res J Biotech 8:57–62

    CAS  Google Scholar 

  33. Ghori MI, Iqbal MJ, Hameed A (2011) Characterization of a novel lipase from Bacillus sp. isolated from tannery wastes. Braz J Microbiol 42:22–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work is financially supported by an annual grant sanctioned by Department of Biotechnology, Ministry of Science and Technology, Government of India to Department of Biotechnology, Himachal Pradesh University, Shimla, India. Further, the authors have no conflict of interest among themselves or with the parent institution.

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  1. Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171 005, India

    Shivika Sharma & Shamsher S. Kanwar

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  1. Shivika Sharma
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  2. Shamsher S. Kanwar
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Correspondence to Shivika Sharma.

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Sharma, S., Kanwar, S.S. Purification and bio-chemical characterization of a solvent-tolerant and highly thermostable lipase of Bacillus licheniformis strain SCD11501. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 87, 411–419 (2017). https://doi.org/10.1007/s40011-015-0612-z

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  • DOI: https://doi.org/10.1007/s40011-015-0612-z

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