Skip to main content
SpringerLink
Log in

Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

DMSO:

Dimethyl sulfoxide

ee :

Enantiomeric excess

FT-IR microspectroscopy:

Fourier transform infrared microspectroscopy

KPB:

Potassium phosphate buffer

(±)-MPGM:

(±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester

(−)-MPGM:

(2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester

pNPA:

p-Nitrophenyl acetate

SEM:

Scanning electron microscopy

vvm:

Volume of air per volume of media per minute

References

  1. Keasling, J. D. (2008). Synthetic biology for synthetic chemistry. ACS Chemical Biology, 3, 64–76.

    Article  CAS  Google Scholar 

  2. Jong, H.C., Ki, C.K., Sang, Y.L. (2006) Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science, 61, 876–885.

    Google Scholar 

  3. Ami, D., Natalello, A., Taylor, G., Tonon, G., & Doglia, S. M. (2006). Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy. Biochimica et Biophysica Acta, 1764, 793–799.

    Article  CAS  Google Scholar 

  4. Gonzalez-Montalban, N., García-Fruitós, E., Ventura, S., Aris, A., & Villaverde, A. (2006). The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells. Microbial Cell Factories, 5, 26.

    Article  Google Scholar 

  5. Arie, J. P., Miot, M., Sassoon, N., & Betton, J. M. (2006). Formation of active inclusion bodies in the periplasm of Escherichia coli. Molecular Microbiology, 62, 427–437.

    Article  CAS  Google Scholar 

  6. García-Fruitós, E., González-Montalbán, N., Morell, M., Vera, A., Ferraz, R. M., Arís, A., et al. (2005). Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microbial Cell Factories, 4, 27.

    Article  Google Scholar 

  7. Indu, B., Rajinder, P., Gulam, N. Q., et al. (2008). Lipase enzyme immobilization on synthetic beaded macroporous copolymers for kinetic resolution of chiral drugs intermediates. Process Biochemistry, 43, 321–330.

    Article  Google Scholar 

  8. Gandhi, N. N. (1997). Applications of lipase. Journal of the American Oil Chemists' Society, 74, 621–634.

    Article  CAS  Google Scholar 

  9. Ribeiro, C. M. R., Passaroto, E. N., & Brenelli, E. C. S. (2001). Enzymatic resolution of ethyl 3-hydroxy-3-phenylpropanoate and analogs using hydrolases. Journal of the Brazilian Chemical Society, 12, 742–746.

    Article  CAS  Google Scholar 

  10. Frings, K., Koch, M., & Hartmeier, W. (1999). Kinetic resolution of 1-phenyl ethanol with high enantioselectivity with native and immobilized lipase in organic solvents. Enzyme and Microbial Technology, 25, 303–309.

    Article  CAS  Google Scholar 

  11. Ying-Yang, J., Martin, S., & Hans-Ulrich, B. (2002). The enantioselective hydrogenation of 1-phenyl ethanol and of ketones using Pt and Pd supported on natural polymers. Journal of Molecular Catalysis A: Chemical, 177, 307–308.

    Article  Google Scholar 

  12. Singh, S., & Banerjee, U. C. (2007). Purification and characterization of trans-3-(4-methoxyphenyl) glycidic acid methyl ester hydrolyzing lipase from Pseudomonas aeruginosa. Process Biochemistry, 42, 1063–1068.

    Article  CAS  Google Scholar 

  13. Nagao, T., Sato, M., Nakajima, H., & Kiyomoto, A. (1972). Studies on a new 1,5-benzothiazapine derivative (CRD-401). II. Vasodilator actions. Japanese Journal of Pharmacology, 22, 1–10.

    Article  CAS  Google Scholar 

  14. Hulshof, L.A., Roskan, J.H. (1989) Phenylglycidate stereoisomers, conversion products thereof with e.g. 2-nitrothiophenol and preparation of diltiazem. European Patent Application 343714.

  15. Matsumae, H., Furui, M., & Shibatani, T. (1993). Lipase-catalyzed asymmetric hydrolysis of 3-phenylglycidic acid ester, the key intermediate in synthesis of Diltiazem hydrochloride. Journal of Fermentation and Bioengineering, 75, 93–98.

    Article  CAS  Google Scholar 

  16. Long, Z. D., Xu, J. H., Zhao, L. L., Pan, J., Yang, S., & Hua, L. (2007). Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen. Journal of Molecular Catalysis B: Enzymatic, 47, 105–110.

    Article  CAS  Google Scholar 

  17. Gao, L., Xu, J. H., & Li, X. J. (2004). Optimization of Serratia marcescens lipase production for enantioselective hydrolysis of 3-phenyl glycidic acid ester. Journal Industry Microbiology Biotechnology, 31, 525–530.

    Article  CAS  Google Scholar 

  18. Chin, J. H., Rahman, R. N. Z. A., Salleh, A. B., & Basri, M. (2003). A newly isolated organic solvent-tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. Biochemical Engineering Journal, 15, 147–151.

    Article  Google Scholar 

  19. Diletta, A., Antonino, N., Pietro, G. L., Marina, L., & Silvia, M. D. (2005). Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy. FEBS Letters, 579, 3433–3436.

    Article  Google Scholar 

  20. Lee, S. Y. (1996). High cell-density culture of Escherichia coli. Trends in Biotechnology, 14, 98–105.

    Article  CAS  Google Scholar 

  21. Seo, D. J., Chung, B. H., Hwang, Y. B., & Park, Y. H. (1992). Glucose-limited fed-batch culture of Escherichia coli for production of recombinant human interleukin-2 with the DO-Stat method. Journal of Fermentation and Bioengineering, 74, 196–198.

    Article  CAS  Google Scholar 

  22. Gupta, R., Gupta, N., & Rathi, P. (2004). Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbiology and Biotechnology, 64, 763–781.

    Article  CAS  Google Scholar 

  23. Yuzo, K., Michihiko, K., & Sakayu, S. (2003). A novel lipase from Pseudomonas fluorescens HU380: Gene cloning, overproduction, renaturation-activation, two-step purification, and characterization. Journal of Bioscience and Bioengineering, 96, 242–249.

    Google Scholar 

  24. Zhang, A. J., Gao, R. J., Diao, N. B., Xie, G. Q., et al. (2009). Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963. Journal of Molecular Catalysis B: Enzymatic, 56, 78–84.

    Article  CAS  Google Scholar 

  25. Akatsuka, H., Kawai, E., Omori, K., Komatsubara, S., Shibatani, T., & Tosa, T. (1994). The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide. Journal of Bacteriology, 176, 1949–1956.

    CAS  Google Scholar 

  26. Amada, K., Haruki, M., Imanaka, T., Morikawa, M., & Kanaya, S. (2000). Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp. MIS38. Biochimica et Biophysica Acta, 1478, 201–210.

    Article  CAS  Google Scholar 

  27. Rollof, J., Hedstrom, S. A., & Nilsson-Ehle, P. (1987). Positional specificity and substrate preference of purified Staphylococcus aureus lipase. Biochimica et Biophysica Acta, 921, 370–377.

    Article  CAS  Google Scholar 

  28. Zhao, L. L., Xu, J. H., Zhao, J., Pan, J., & Wang, Z. L. (2008). Biochemical properties and potential applications of an organic solvent tolerant lipase isolated from Serratia marcescens ECU1010. Process Biochemistry, 43, 626–633.

    Article  CAS  Google Scholar 

  29. Shibatani, T., Matsumae, H., Akatsuka, H. (1991) Esterase from Serratia and process for preparing the same. European Patent Application 446771.

  30. Salamone, P. R., & Wodzinski, R. J. (1997). Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens. Applied Microbiology and Biotechnology, 48, 317–324.

    Article  CAS  Google Scholar 

  31. Li, S. X., Pang, H. Y., Lin, K., Xu, J. H., et al. (2011). Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010. Journal of Molecular Catalysis B: Enzymatic, 71, 171–176.

    Article  CAS  Google Scholar 

  32. Henley, J. P., & Sadana, A. (1985). Categorization of enzyme deactivations using a series-type mechanism. Enzyme and Microbial Technology, 7, 50–60.

    Article  CAS  Google Scholar 

  33. Kojima, Y., Yokoe, M., & Mase, T. (1994). Purification and characterization of an alkaline lipase from Pseudomonas fluorescens AK102. Bioscience, Biotechnology, and Biochemistry, 58, 1564–1568.

    Article  CAS  Google Scholar 

  34. Matsumae, H., & Shibatani, T. (1994). Purification and characterization of the lipase from Serratia marcescens Sr41 8000 responsible for asymmetric hydrolysis of 3-phenylglycidic acid esters. Journal of Bioscience and Bioengineering, 77, 152–158.

    CAS  Google Scholar 

  35. Schmidt-Dannert, C., Sztajer, H., Stocklein, W., Menge, U., & Schmid, R. D. (1994). Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochimica et Biophysica Acta, 1214, 43–53.

    Article  CAS  Google Scholar 

  36. Klibanov, A. M. (1997). Why are enzymes less active in organic solvents than in water?. Trends in Biotechnology, 15, 97–101.

    Article  CAS  Google Scholar 

  37. Zaks, A., & Klibanov, A. M. (1988). Enzymatic catalysis in nonaqueous solvents. Journal of Biological Chemistry, 263, 3194–3201.

    CAS  Google Scholar 

  38. Carrió, M. M., Corchero, J. L., & Villaverde, A. (1998). Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria. FEMS Microbiology Letters, 169, 9–15.

    Article  Google Scholar 

  39. Przybycien, T. M., Dunn, J. P., Valax, P., & Georgiou, G. (1994). Secondary structure characterization of beta-lactamase inclusion bodies. Protein Engineering, 7, 131–136.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (no. 20506037) and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic China

    Suxia Li, Kang Lin, Huaiyu Pang & Jianhe Xu

  2. Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Yixin Wu

Authors
  1. Suxia Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kang Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huaiyu Pang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yixin Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jianhe Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Suxia Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, S., Lin, K., Pang, H. et al. Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies. Appl Biochem Biotechnol 169, 612–623 (2013). https://doi.org/10.1007/s12010-012-0028-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-0028-7

Keywords

Search

Navigation