Abstract
Lipase from Aneurinibacillus thermoaerophilus strain HZ (HZ lipase) represents the first member of subfamily I.9 true lipases. The resultant of unique characteristics and structural features of HZ lipase has affirmed that subfamily I.9 is located between mesophilic and thermostable lipases. In advance to clone and express the HZ lipase gene, protein solubility of fusion HZ lipase was predicted and analyzed using different software. Then, to overexpress the target gene, high-level expression was performed in a prokaryotic system using different strains and vectors, and production conditions were optimized. HZ lipase was expressed under the control of strong and chemically inducible T7 promoter for high-level expression. It was fused to Trx-, His- and S-tags to solubilize the protein and also to specify and accelerate the purification procedure. The high amount of the HZ lipase protein was obtained as the soluble form (72.5 U/mL) using IPTG final concentration of 0.025 mM after 8 h induction at 30ºC. The expression was analyzed by SDS-PAGE and presence of His-tag was confirmed by Western blotting of protein. As the HZ lipase is the only member of subfamily I.9 that yet cloned and overexpressed, this procedure could be applied to the other close members.
Similar content being viewed by others
References
Hasan, F., Shah, A.A., and Hameed, A., Enzyme Microb. Technol., 2006, vol. 39, no. 2, pp. 235–251.
Frikha, F., Ladjimi, M., Gargouri, Y., and Miled, N., FEMS Microbiol. Lett., 2008, vol. 286, no. 2, pp. 207–221.
DeLucas, L.J., Drug Discov. Today, 2001, vol. 6, no. 14, pp. 734–744.
Masomian, M., Rahman, R.N.Z.R.A., Salleh, A.B., and Basri, M., Process Biochem., 2013, vol. 48, no. 1, pp. 169–175.
Masomian, M., Rahman, R.N.Z.R.A., Salleh, A.B., and Basri, M., PLoS ONE, 2016, vol. 11, no. 3, p. e0149851.
Magnan, C.N., Randall, A., and Baldi, P., Bioinformatics, 2009, vol. 25 no. 17, pp. 2200–2207.
Smialowski, P., Doose, G., Torkler, P., Kaufmann, S., and Frishman, D., FEBS J., 2012, vol. 279, no. 12, pp. 2192–2200.
Hirose, S., and Noguchi, T., Proteomics, 2013, vol. 13, no. 9, pp. 1444–1456.
Agostini, F., Cirillo, D., Livi, C.M., Ponti, R.D., and Tartaglia, G.G., Bioinformatics, 2014, vol. 30, no. 20, pp. 2975–2977.
Sambrook, J., and Russell, D.W., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2001.
Kwon, D.Y., and Rhee, J.S., J. Am. Oil Chem. Soc., 1986, vol. 63, no. 1, pp. 89–92.
Laemmli, U.K., Nature, 1970, vol. 227, no. 5259, pp. 680–685.
Martini, V.P., Glogauer, A., Müller–Santos, M., Iulek, J., de Souza, E.M., Mitchell, D.A., Pedrosa, F.O., and Krieger, N., Microb. Cell Fact., 2014, vol. 13, no. 1, pp. 1–14.
Preeti, A., Hemalatha, D., Rajendhran, J., Mullany, P., and Gunasekaran, P., Indian J. Microbiol., 2014, vol. 54, no. 3, pp. 284–292.
Davis, E.O., Dullaghan, E.M., and Rand, L., J. Bacteriol., 2002, vol. 184, no. 12, pp. 3287–3295.
Makrides, S.C., Microbiol. Rev., 1996, vol. 60, no. 3, pp. 512–538.
Studier, F.W., Protein Expr. Purif., 2005, vol. 41, no. 1, pp. 207–234.
Valdez–Cruz, N.A., Caspeta, L., Pérez, N.O., Ramírez, O.T., and Trujillo–Roldán, M.A., Microb. Cell Fact., 2010, vol. 9, no. 1, pp. 18.
Sorensen, H.P., and Mortensen, K.K., J. Biotechnol., 2005a, vol. 115, no. 2, pp. 113–128.
Denizci, A.A., Kazan, D., Abeln, E.C.A., and Erarslan, A., J. Appl. Microbiol., 2004, vol. 96, no. 2, pp. 320–327.
Rahman, R.N.Z.R.A., Hamid, T.H.T.A., Eltaweel, M.A., Basri, M., and Salleh, A.B., J. Biotechnol., 2008, vol. 136, no. pp. S335.
Leow, T.C., Rahman, R.N.Z.R.A., Basri, M., and Salleh, A.B., Biosci. Biotechnol. Biochem., 2004, vol. 68, no. 1, pp. 96–103.
Sinchaikul, S., Sookkheo, B., Phutrakul, S., Pan, F.–M., and Chen, S.-T., Protein Expr. Purif., 2001, vol. 22, no. 3, pp. 388–398.
Shariff, F.M., Rahman, R.N.Z.R.A., Basri, M., and Salleh, A.B., Int. J. Mol. Sci., 2011, vol. 12, no. 5, pp. 2917–2934.
Author information
Authors and Affiliations
Corresponding author
Additional information
The article is published in the original.
Rights and permissions
About this article
Cite this article
Masomian, M., Rahman, R.N.Z.R.A., Salleh, A.B. et al. Solubility Analysis, Cloning and Functional Overexpression of the Lipase from Aneurinibacillus thermoaerophilus strain HZ, the First Member of True Lipases Subfamily I.9. Appl Biochem Microbiol 54, 269–276 (2018). https://doi.org/10.1134/S0003683818030109
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0003683818030109