The isolation and characterization of lipase from Carica papaya latex using zwitterion sodium lauroyl sarcosinate as agent

Dang Minh Nhat; Phan Thi Viet  Ha

doi:10.5219/1164

Authors

Dang Minh Nhat The University of Da Nang ”“ University of Science and Technology, Faculty of Chemical Engineering, Department of Food Technology, 54 Nguyen Luong Bang, Da Nang City, Viet Nam, Tel.: +84 913 486 813 https://orcid.org/0000-0001-6515-1879
Phan Thi Viet Ha Duy Tan University, Faculty of Natural Sciences, 03 Quang Trung, Da Nang City, Viet Nam, Tel.: +84 935 133 255 https://orcid.org/0000-0001-7686-6343

DOI:

https://doi.org/10.5219/1164

Keywords:

lipase, papaya, enzyme isolation, enzyme purification

Abstract

Most of industrial lipases are derived from microbial sources, following by a wide variety of plants. Among plant lipases, lipase from Carica papaya latex has been the focus of intense and growing research due to low cost, easy acceptance by consumers and its unique characteristics. This enzyme has been successfully applied for lipid modification and synthesis of some organic compounds. However, research for its molecular structure has been limited due to the difficulty to isolate the enzyme from the latex matrix. In this study, we suggested a modified approach using sodium lauroyl sarcosinate to solubilize the latex, then the protein was precipitated by ammonium sulphate. We also carried out the characterization of the lipase obtained from Carica papaya latex. The results showed that freeze-drying the fresh latex could improve significantly lipase activity of latex powder in comparison with sun-drying or oven-drying. The zwitterion sodium lauroyl sarcosinate could solubilize nearly 50% of the latex and the achieved supernatant exhibited great lipase activity. There was no need to use an organic solvent to delipidate the latex prior to solubilization with sodium lauroyl sarcosinate due to possible denaturation of enzymes. The proteins which were fractionally precipitated with 50 - 60%, 60 - 70% and 70 - 80% ammonium sulphate saturation showed lipolytic activity. The fraction from 50 - 60% saturation with the greatest mass was subjected to ion exchange chromatography, SDS electrophoresis and kinetic parameter determination. The results showed the presence of two proteins with molecular mass ranging from 35 kDa to 55 kDa and both presented lipase activity. The K_m and V_max of the lipase fraction from 50 - 60% saturation was 1.12 mM and 1.2 x 10^-6 mM.min^-1.mL^-1 respectively. So, the freeze-drying of papaya latex could help to preserve its lipase activity and the usage of sodium lauroyl sarcosinate could improve the isolation of the lipase from the papaya latex and pave the way for research on the molecular structure of Carica papaya latex lipases.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...

References

Abdelkafi, S., Ogata, H., Barouh, N., Fouquet, B., Lebrun, R., Pina, M., Scheirlinckx, F., Villeneuve, P., Carrière, F. 2009. Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex. Biochimica et Biophysica Acta, vol. 1791, p. 1048-1056. https://doi.org/10.1016/j.bbalip.2009.06.002 DOI: https://doi.org/10.1016/j.bbalip.2009.06.002

Abdelkafi, S., Barouh, N., Fouquet, B., Fendri, I., Pina, M., Scheirlinckx, F., Villeneuve, P., Carrière, F. 2011. Carica papaya Lipase: A Naturally Immobilized Enzyme with Interesting Biochemical Properties. Plant Foods Hum Nutr., vol. 66, p. 34-40. https://doi.org/10.1007/s11130-010-0206-0 DOI: https://doi.org/10.1007/s11130-010-0206-0

AOAC 927.05. Moisture Determination.

AOAC 425.06. Ash Determination.

AOAC 2011.04. Protein Determination.

AOAC 948.15. Lipid Determination.

Azarkan, M., El Moussaoui, A., van Wuytswinkel, D., Dehon, G., Looze, Y. 2003. Fractionation and purification of the enzymes stored in the latex of Carica papaya. Journal of Chromatography B, vol. 790, p. 229-238. https://doi.org/10.1016/S1570-0232(03)00084-9 DOI: https://doi.org/10.1016/S1570-0232(03)00084-9

Burgess, R. 2009, Protein Precipitation Techniques. In Burgess, R., Deutscher, M. Guide to Protein Purification. Cambridge, USA : Academic Press, p. 332-342. ISBN 9780080923178. https://doi.org/10.1016/S0076-6879(09)63020-2 DOI: https://doi.org/10.1016/S0076-6879(09)63020-2

Campillo, A. G., Tovar, M. A. 2013. Recent advances and applications of the lipolytic activity of Carica papaya latex. Journal of Molecular Catalysis B: Enzymatic, vol. 90, p. 49-60. https://doi.org/10.1016/j.molcatb.2013.01.015 DOI: https://doi.org/10.1016/j.molcatb.2013.01.015

Casas, G. L., Duquesne, S., Bordes, F., Sandoval, G., Marty, A. 2012. Lipase: An Overview. In Sandoval G. Lipase and Phospholipase-Methods and Protocols. New Jersey, USA : Humana Press, p. 3-30. ISBN 978-1-61779-599-2. https://doi.org/10.1007/978-1-61779-600-5_1 DOI: https://doi.org/10.1007/978-1-61779-600-5_1

Darvishi, F., Destain, J., Nahvi, I. 2012. Effect of additives on freeze-drying and storage of Yarrowia lipolytica Lipase. Appl. Biochem. Biotechnol., vol. 168, p. 1101-1107. https://doi.org/10.1007/s12010-012-9844-z DOI: https://doi.org/10.1007/s12010-012-9844-z

Gururaj, P., Ramalingam, S., Devi, N. G., Gautam, P. 2016. Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07. Braz. J. Microbiol., vol. 47, no. 3, p. 647-657. https://doi.org/10.1016/j.bjm.2015.04.002 DOI: https://doi.org/10.1016/j.bjm.2015.04.002

Holme, P. D. J., Peck, H. 1998. Analytical Biochemistry. 3rd ed. Harlow, England : Pearson Education Limited, 507 p. ISBN-0 582 29438-X.

Kambourova, M., Mandeva, N., Derekova, A. 2003. Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7. Journal of Molecular Catalysis B: Enzymatic, vol. 22, no. 5-6, p. 307-313. https://doi.org/10.1016/S1381-1177(03)00045-6 DOI: https://doi.org/10.1016/S1381-1177(03)00045-6

Lazreg, H. A., Mosbah, H., Fekih, A., Kenani, A. 2014. Purification and biochemical characterization of lipase from Tunisian Euphorbia peplus latex. J. Am. Oil Chem. Soc., vol. 91, p. 943-951. https://doi.org/10.1007/s11746-014-2444-z DOI: https://doi.org/10.1007/s11746-014-2444-z

Macalood, S. J., Vicente, J. H., Boniao, D. R., Gorospe, G. J., Roa, C. E. 2013. Chemical Analysis of Carica papaya L. Crude Latex. American Journal of Plant Sciences, vol.4, no. 10, p. 1941-1948. https://doi.org/10.4236/ajps.2013.410240 DOI: https://doi.org/10.4236/ajps.2013.410240

de María, P. D. D., José, V. S., Tsai, S. W., Andrés, R. A. 2006. Carica papaya lipase (CPL): An emerging and versatile biocatalyst. Biotechnology Advances, vol. 24, p. 493-499. https://doi.org/10.1016/j.biotechadv.2006.04.002 DOI: https://doi.org/10.1016/j.biotechadv.2006.04.002

Minaev, M., Makhova, A. A. 2019. Recombinant metalloprotease as a perspective enzyme for meat tenderization. Potravinarstvo Slovak Journal of Food Sciences, vol. 13, no. 1, 628-633. https://doi.org/10.5219/1087 DOI: https://doi.org/10.5219/1087

Okunwaye, T., Obibuzor, U. J., Okogbenin, A. E. 2015. Purification and biochemical properties of lipase from raphia palm fruit mesocarp. African Journal of Biochemistry Research, vol. 9, no. 5, p. 73-80. https://doi.org/10.5897/AJBR2015.0818 DOI: https://doi.org/10.5897/AJBR2015.0818

Palacios, D., Busto, D. M., Ortega, N. 2014. Study of a new spectrophotometric end-point assay for lipase activity determination in aqueous media. LWT - Food Science and Technology, vol. 55, no. 2, p. 536-542. https://doi.org/10.1016/j.lwt.2013.10.027 DOI: https://doi.org/10.1016/j.lwt.2013.10.027

Paques, F. W., Pio, T. F., Carvalho, P. de O., Macedo, G. A. 2008. Characterization of the lipase from Carica papaya residues. Braz. J. Food Technol, vol. 11, no. 1, p. 20-27.

Tripathi, R., Singh, J., Barti, K. R., Thakur, S. I. 2014. Purification and characterization of lipase from Microbacterium sp. and its application in biodiesel production. Energy Procedia, vol. 54, p. 518-529. https://doi.org/10.1016/j.egypro.2014.07.293 DOI: https://doi.org/10.1016/j.egypro.2014.07.293