Engineering catalytic efficiency of thermophilic lipase from Geobacillus zalihae by hydrophobic residue mutation near the catalytic pocket


In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues; Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.

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Wahab, R. , Basri, M. , Rahman, M. , Rahman, R. , Salleh, A. and Chor, L. (2012) Engineering catalytic efficiency of thermophilic lipase from Geobacillus zalihae by hydrophobic residue mutation near the catalytic pocket. Advances in Bioscience and Biotechnology, 3, 158-167. doi: 10.4236/abb.2012.32024.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Ventura, S., Vega, M.C., Lacroix, E., Angrand, I., Spagnolo, L. and Serrano, L. (2002) Conformational strain in the hydrophobic core and its implications for protein folding and design. Nature: Structural Biology, 9, 485-493. doi:10.1038/nsb799
[2] Kim, D.E., Gu, H. and Baker, D. (1998) The sequences of small proteins are not extensively optimized for rapid folding by natural selection. Proceedings of the National Academy of Science USA, 95, 4981-4986. doi:10.1073/pnas.95.9.4982
[3] Demetrius, L. (2002) Thermodynamics and kinetics of protein folding an evolutionary perspective. Journal of Theoretical Biology, 217, 397-411. doi:10.1006/jtbi.2002.3006
[4] Saraboji, K., Michael Gromiha, M. and Ponnuswamy, M.N. (2005) Relative importance of secondary structure and solvent accessibility to the stability of protein mutants. A case study with amino acid properties and energetic on T4 and human lysozymes. Computational Biology and Chemistry, 29, 25-35. doi:10.1016/j.compbiolchem.2004.12.002
[5] Rose, G.D., Fleming, P.J., Banawar, J.R. and Maritan A. (2006) A backbone-based theory of protein folding. Proceedings of the National Academy of Science USA, 45, 16623-16633. doi:10.1073/pnas.0606843103
[6] Yutani, K., Ogasahara, K., Tsujita, T. and Yoshinobu, S. (1987) Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase α subunit. Proceedings of the National Academy of Science USA, 84, 4441-4444. doi:10.1073/pnas.84.13.4441
[7] Leow, T.C., Rahman, R.N.Z.R.A., Basri, M. and Salleh, A.B. (2007) High temperature crystallization of thermostable T1 lipase. Crystal Growth Design, 7, 406-410. doi:10.1021/cg050506z
[8] Leow, T.C., Rahman, R.N.Z., Basri, M. and Salleh, A.B. (2007) A thermoalkaliphilic lipase of Geobacillus sp. T1. Extremophiles, 11, 527-535. doi:10.1007/s00792-007-0069-y
[9] Matsumura, H., Yamamoto, T., Leow, T.C., Mori, T., Basri, M., Rahman, R.N.Z., Inoue, T., Inoue, T., Kai, Y. and Salleh, A.B. (2008) Novel cation-π interaction revealed by crystal structure of thermoalkalophilic lipase. Proteins: Structure, Function and Bioinformatics, 70, 592-598. doi:10.1002/prot.21799
[10] Carrasco-López, C., Godoy, C., de las Rivas, B., Fernández-Lorente, G., Palomo, J.M., Guisan, G.M., Fernández-Lorente, R., Martinez-Ripoll, M. and Hermoso, J.A. (2009) Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements. Journal of Biological Chemistry, 284, 4365-4372.
[11] Rother, K., Hildebrand, P.W., Goede, A., Gruening, B. and Preissner, R. (2009) Voronoia: Analyzing packing in protein structures. Nucleic Acids Reviews, 37, D393-D395. doi:10.1093/nar/gkn769
[13] Kwon, D.K. and Rhee, J.S. (1986) A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. Journal of American Oil Chemistry Society, 63, 89-92. doi:10.1007/BF02676129
[14] Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3
[15] Ishikawa, K., Nakamura, H., Morikawa, K. and Kanaya, S. (1993). Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core. Biochemistry, 32, 6171-6178. doi:10.1021/bi00075a009
[16] Zhang, W.M. and Lei, X.G. (2008) Cumulative improvements of thermostability and pH-activity profile of Aspergillus niger PhyA phytase by site-directed mutagenesis. Applied Microbiology and Biotechnology, 77, 1033- 1040. doi:10.1007/s00253-007-1239-7
[17] Eyal, E., Najmanovich, R., Edelman, M. and Sobolev, V. (2003) Protein side-chain rearrangement in regions of point mutations. Protein Structure, Function, and Bioinformatics, 50, 272-282. doi:10.1002/prot.10276
[18] Eriksson, A.E., Baase, W.A., Zhang, X-J., Heinz, D.W., Blaber, M., Baldwin, E.P. and Matthews, B.W. (1992) Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science, 255, 178-183. doi:10.1126/science.1553543
[19] Ventura, S. and Serrano, L. (2004) Designing protein from the inside out. Proteins Structure, Function and Bioinformatics, 56, 1-10. doi:10.1002/prot.20142
[20] Fu, D., Li, Z.-Y., Huang, H.-Q., Yuan, T., Shi, P., Luo, H., Meng, K., Yang, P. and Yao, B. (2011) Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site. Applied Environmental Microbiology, 90, 1295-1302.
[21] Seo, H.S., Koo, Y.J., Lim, J.Y., Song, J.T., Kim, C.H., Kim, J.K., Lee, J.S. and Choi, Y.D. (2000) Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Applied Environmental Microbiology, 66, 2484-2490. doi:10.1128/AEM.66.6.2484-2490.2000
[22] Kim, S.-H., Pokhrel, S. and Yoo, Y.-J. (2008) Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase. Journal of Molecular Catalysis B: Enzymatic, 55, 130-136. doi:10.1016/j.molcatb.2008.02.006
[23] Laane, C., Boeren, S., Hilhorst, R. and Veeger, C. (1987) Optimization of biocatalysis in organic media. In: Laane, C., Tramper, J. and Lilly, M.D., Eds., Biocatalysis in Organic Media, Elsevier Science Publishers, Amsterdam, 65-84.
[24] Klibanov, A.M. (2001) Improving enzymes by using them in organic solvent. Nature, 409, 241-246. doi:10.1038/35051719
[25] Klibanov, A.M. (1997) Why are enzymes less active in organic solvents than in water? Trends in Biotechnology, 15, 77-83. doi:10.1016/S0167-7799(97)01013-5
[26] Dandavate, V., Jinjala, J., Keharia, H. and Madamwar, D. (2009) Production, partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis. Bioresource Technology, 100, 3374-3381. doi:10.1016/j.biortech.2009.02.011
[27] Tanford, C. (1961) Physical chemistry of macromolecules. John Wiley and Sons, New York.
[28] Valstar, A. (2000) Protein-surfactant interactions. Ph.D. Thesis, Uppsala University, Uppsala.
[29] Schmidt-Dannert, C., Rua, M.L., Atomi, H. and Schmid, R.D. (1996) Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning nucleotide sequence, purification and some properties. Biochimie and Biophysica Acta, 1301, 105-114.
[30] Eltaweel, M.A., Rahman, R.N.Z., Basri, M. and Salleh, A.B. (2005) An organic-solvent stable lipase from Bacillus sp. strain 42. Analytical Biochemistry, 53, 187-192.
[31] Leow, T.C. (2005) Molecular studies, characterization and structure elucidation of a thermostable lipase from Geobacillus sp. Ph.D. Thesis, Universiti Putra Malaysia, Serdang.
[32] Hermoso J., Pignolo, D., Kerfelec, B. and Crenon, I. (1996) Lipase activation by non-ionic detergents. The crystal structure of porcine lipase-colipase-tetraethylene glycol monooctyl ether complex. Journal of Biological Chemistry, 271, 18007-18016. doi:10.1074/jbc.271.30.18007
[33] Ruiz-Pena, M., Oropesa-Nuriez, R., Pons, T., Louro, S.R. and Perez-Gamatges, A. (2010) Physico-chemical studies of molecular interactions between nonionic surfactants and bovine serum albumin. Colloids and Surfaces B: Bio- interfaces, 75, 282-289. doi:10.1016/j.colsurfb.2009.08.046
[34] Glusker, J.P., Katz, A.K. and Bock, C.W. (1999) Metal ions in biological systems. Rigaku Journal, 16, 8-16.
[35] Rahman, R.N.Z., Baharum, S.N., Basri, M. and Salleh, A.B. (2005) High yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Analytical Biochemistry, 341, 267-274. doi:10.1016/j.ab.2005.03.006
[36] Don, H., Ga, S., Han, S.P. and Cao, S.G. (1999) Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media. Biotechnology and Applied Biochemistry, 30, 251-156.

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