Impact of Leucine 278 Residue on Fatty Acid Length Specificity of <i>Candida antarctica</i> Lipase B
- 1 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China
- 2 State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
- 3 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China
- 4 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China
- 5 State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
- 6 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China
- 7 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China
Abstract
Structural analysis of Candida antarctica lipase B (CALB) indicates that side chain of leucine at 278 site lies above the entrance of the catalytic pocket, which prognosticates its potential role on substrate specificity of the enzyme. To verify this presumption, shortened side chain of glycine or proline was rational designed and mutants were constructed by site-directed mutagenesis method. The colorimetric assay using p -nitrophenyl esters of fatty acids with various chain-lengths was used to study the substrate preference of lipases. Results indicated that L278G or L278P mutations both induced the drift of substrate specificity of CALB from p -nitrophenyl caprylate ( p NP-C8) to longer carbon chain length of p -nitrophenyl caprate ( p NP-C10). Meanwhile, V max value of two mutants to p NP-C10 was both higher than that of wild-type. Docking results also indicated that shortened side chain of glycine or proline residues substitution at this site could get rid of the space block present above the catalytic pocket, and made longer chain substrate ( p NP-C10) enter into the catalytic pocket easier. The modulation of specificity observed allowed for building substrate binding model and opened new possibilities for designing ligand specific lipases.
- Juhl, P.B., Doderer, K., Hollmann, F., Thum, O. and Pleiss, J. (2010) Engineering of Candida antarctica Lipase B for Hydrolysis of Bulky Carboxylic Acid Esters. Journal of Biotechnology, 150, 474-480. http://dx.doi.org/10.1016/j.jbiotec.2010.09.951
- Park, C.G., Kwon, M.A., Song, J.K. and Kim, D.M. (2011) Cell-Free Synthesis and Multifold Screening of Candida antarctica Lipase B (CalB) Variants after Combinatorial Mutagenesis of Hot Spots. Biotechnology Progress, 27, 47-53. http://dx.doi.org/10.1002/btpr.532
- Kim, S.Y., Sohn, J.H., Pyun, Y.R., Yang, I.S., Kim, K.H. and Choi, E.S. (2007) In Vitro Evolution of Lipase B from Candida antarctica Using Surface Display in Hansenula polymorpha. Journal of Microbiology and Biotechnology, 17, 1308-1315.
- Xie, Y., An, J., Yang, G., Wu, G., Zhang, Y., Cui, L. and Feng, Y. (2014) Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site. The Journal of Biological Chemistry, 289, 7994-8006. http://dx.doi.org/10.1074/jbc.M113.536045
- Marton, Z., Léonard-Nevers, V., Syrén, P.-O., Bauer, C., Lamare, S., Hult, K., Tranc, V. and Graber, M. (2010) Mutations in the Stereospecificity Pocket and at the Entrance of the Active Site of Candida antarctica Lipase B Enhancing Enzyme Enantioselectivity. Journal of Molecular Catalysis. B, Enzymatic, 65, 11-17. http://dx.doi.org/10.1016/j.molcatb.2010.01.007
- Santarossa, G., Lafranconi, P.G., Alquati, C., DeGioia, L., Alberghina, L., Fantucci, P. and Lotti, M. (2005) Mutations in the “Lid” Region Affect Chain Length Specificity and Thermostability of a Pseudomonas fragi Lipase. FEBS Letters, 579, 2383-2386. http://dx.doi.org/10.1016/j.febslet.2005.03.037
- Yang, J., Koga, Y., Nakano, H. and Yamane, T. (2002) Modifying the Chain-Length Selectivity of the Lipase from Burkholoderia cepacia KWI-56 through in Vitro Combinatorial Mutagenesis in the Substrate-Binding Site. Protein Engineering, 15, 147-152. http://dx.doi.org/10.1093/protein/15.2.147
- Liu, L., Gao, C.L., Lan, D.M., Yang, B. and Wang, Y.H. (2012) Molecular Basis for Substrate Selectivity of a Mono- and Diacylglycerol Lipase from Malassezia globosa. Biochemical and Biophysical Research Communications, 424, 285-289. http://dx.doi.org/10.1016/j.bbrc.2012.06.108
- Larsen, M.W., Bornscheuer, U.T. and Hult, K. (2008) Expression of Candida antarctica Lipase B in Pichia pastoris and Various Escherichia coli Systems. Protein Expression and Purification, 62, 90-97. http://dx.doi.org/10.1016/j.pep.2008.07.012