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Exploration and functional expression of homologous lipases of Candida antarctica lipase B

Candida antarctica lipase B의 상동체 효소 탐색과 발현

  • Park, Seongsoon (Department of Chemistry, Center for NanoBio Applied Technology, and Institute of Basic Sciences, Sungshin Women's University)
  • 박성순 (성신여자대학교 화학과)
  • Received : 2015.08.20
  • Accepted : 2015.09.02
  • Published : 2015.09.30

Abstract

Candida (also known as Pseudozyma) antarctica lipase B (CAL-B) has been intensely studied in academic and industrial fields. However, the research related to its homologous enzymes has been rarely reported. In the current investigation, protein sequence similarity search of CAL-B has been conducted and six homologous protein sequences were identified. After the syntheses of their codon-optimized genes, the synthetic genes have been cloned into a periplasmic expression vector to express in Escherichia coli. Among six homologous sequences, four sequences were successfully expressed in E. coli. The hydrolytic activities of the expressed proteins towards 4-nitrophenyl acetate and 4-nitrophenyl butyrate were measured and compared with those of CAL-B to identify whether the expressed proteins work as a hydrolase. It has been revealed that the expressed proteins can hydrolyze the substrates and the specific activities were determined as $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$, which are lower than those of CAL-B. Among these homologous enzymes, Pseudozyma hubeiensis SY62 exhibits the comparable enantioselectivity to that of CAL-B towards the hydrolysis of (${\pm}$)-1-phenylethyl acetate.

Keywords

heterologous expression;homology;lipase

Acknowledgement

Supported by : Sungshin University

References

  1. Andualema, B. and Gessesse, A. 2012. Microbial lipases and their industrial applications: review. Biotechnol. 11, 100-118. https://doi.org/10.3923/biotech.2012.100.118
  2. Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Howe, K.L., Marshall, M., and Sonnhammer, E.L.L. 2002. The Pfam protein families database. Nucleic Acids Res. 30, 276-280. https://doi.org/10.1093/nar/30.1.276
  3. Blank, K., Morfill, J., Gumpp, H., and Gaub, H.E. 2006. Functional expression of Candida antarctica lipase B in Escherichia coli. J. Biotechnol. 125, 474-483. https://doi.org/10.1016/j.jbiotec.2006.04.004
  4. Boekhout, T. 1995. Pseudozyma Bandoni emend. Boekhout, a genus for yeast-like anamorphs of ustilaginales. J. Gen. Appl. Microbiol. 41, 359-366. https://doi.org/10.2323/jgam.41.359
  5. Bornscheuer, U.T. and Kazlauskas, R.J. 2006. Hydrolases in Organic Synthesis. 2nd. Wiley-VCH, Weinheim, Germany.
  6. Bornscheuer, U.T. and Pohl, M. 2001. Improved biocatalysts by directed evolution and rational protein design. Curr. Opin. Chem. Biol. 5, 137-143. https://doi.org/10.1016/S1367-5931(00)00182-4
  7. Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J.P., Christiansen, L., Huge-Jensen, B., Norskov, L., et al. 1990. A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature 343, 767-770. https://doi.org/10.1038/343767a0
  8. Brzozowski, A.M., Derewenda, U., Derewenda, Z.S., Dodson, G.G., Lawson, D.M., Turkenburg, J.P., Bjorkling, F., Huge-Jensen, B., Patkar, S.A., and Thim, L. 1991. A model for interfacial activation in lipases from the structure of a fungal lipaseinhibitor complex. Nature 351, 491-494. https://doi.org/10.1038/351491a0
  9. Carbone, M.N. and Arnold, F.H. 2007. Engineering by homologous recombination: exploring sequence and function within a conserved fold. Curr. Opin. Struct. Biol. 17, 454-459. https://doi.org/10.1016/j.sbi.2007.08.005
  10. Chodorge, M., Fourage, L., Ullmann, C., Duvivier, V., Masson, J.M., and Lefevre, F. 2005. Rational strategies for directed evolution of biocatalysts-application to Candida antarctica lipase B (CALB). Adv. Synth. Catal. 347, 1022-1026. https://doi.org/10.1002/adsc.200505055
  11. Crameri, A., Raillard, S.A., Bermudez, E., and Stemmer, W.P.C. 1998. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288-291. https://doi.org/10.1038/34663
  12. Grochulski, P., Li, Y., Schrag, J.D., Bouthillier, F., Smith, P., Harrison, D., Rubin, B., and Cygler, M. 1993. Insights into interfacial activation from an open structure of Candida rugosa lipase. J. Biol. Chem. 268, 12843-12847.
  13. Grochulski, P., Li, Y., Schrag, J.D., and Cygler, M. 1994. Two conformational states of Candida rugosa lipase. Protein Sci. 3, 82-91.
  14. Gross, R.A., Kumar, A., and Kalra, B. 2001. Polymer synthesis by in vitro enzyme catalysis. Chem. Rev. 101, 2097-2124. https://doi.org/10.1021/cr0002590
  15. Hoegh, I., Patkar, S., Halkier, T., and Hansen, M.T. 1995. Two lipases from Candida antarctica: Cloning and expression in Aspergillus oryzae. Can. J. Bot. 73, 869-875. https://doi.org/10.1139/b95-333
  16. Janes, L.E., Cimpoia, A., and Kazlauskas, R.J. 1999. Proteasemediated separation of cis and trans diastereomers of 2(R,S)-benzyloxymethyl-4(S)-carboxylic acid 1,3-dioxolane methyl ester: intermediates for the synthesis of dioxolane nucleosides. J. Org. Chem. 64, 9019-9029. https://doi.org/10.1021/jo990757c
  17. Jung, S. and Park, S. 2008. Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis. Biotechnol. Lett. 30, 717-722. https://doi.org/10.1007/s10529-007-9591-3
  18. Liu, D., Schmid, R.D., and Rusnak, M. 2006. Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm - a screening system for a frequently used biocatalyst. Appl. Microbiol. Biotechnol. 72, 1024-103. https://doi.org/10.1007/s00253-006-0369-7
  19. Martinelle, M., Holmquist, M., and Hult, K. 1995. On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim. Biophys. Acta 1258, 272-276. https://doi.org/10.1016/0005-2760(95)00131-U
  20. Neylon, C. 2004. Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res. 32, 1448-1459. https://doi.org/10.1093/nar/gkh315
  21. Ollis, D.L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.M., Harel, M., Remington, S.J., and Silman, I. 1992. The alpha/beta hydrolase fold. Protein Eng. 5, 197-211. https://doi.org/10.1093/protein/5.3.197
  22. Rotticci, D. 2003. Understanding and engineering the enantioselectivity of Candida antarctica lipase B towards sec-alcohols, Ph.D. thesis, KTH, Stockholm, Sweden.
  23. Rotticci-Mulder, J.C., Gustavsson, M., Holmquist, M., Hult, K., and Martinelle, M. 2001. Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain. Protein Expr. Purif. 21, 386-392. https://doi.org/10.1006/prep.2000.1387
  24. Schmid, R.D. and Verger, R. 1998. Lipases: interfacial enzymes with attractive applications. Angew. Chem. Int. Ed. 37, 1608-1633. https://doi.org/10.1002/(SICI)1521-3773(19980703)37:12<1608::AID-ANIE1608>3.0.CO;2-V
  25. Takwa, M., Wittrup Larsen, M., Hult, K., and Martinelle, M. 2011. Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide. Chem. Commun. 47, 7392-7394. https://doi.org/10.1039/c1cc10865d
  26. Uppenberg, J., Oehrner, N., Norin, M., Hult, K., Kleywegt, G.J., Patkar, S., Waagen, V., Anthonsen, T., and Jones, T.A. 1995. Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols. Biochem. 34, 16838-16851. https://doi.org/10.1021/bi00051a035
  27. van der Mee, L., Helmich, F., de Bruijn, R., Vekemans, J.A.J.M., Palmans, A.R.A., and Meijer, E.W. 2006. Investigation of lipase-catalyzed ring-opening polymerizations of lactones with various ring sizes: kinetic evaluation. Macromolecules 39, 5021-5027. https://doi.org/10.1021/ma060668j
  28. Verger, R. 1997. Interfacial activation of lipases: facts and artifacts. Trends Biotechnol. 15, 32-38. https://doi.org/10.1016/S0167-7799(96)10064-0
  29. Wu, Q., Soni, P., and Reetz, M.T. 2013. Laboratory evolution of enantiocomplementary Candida antarctica lipase B mutants with broad substrate scope. J. Am. Chem. Soc. 135, 1872-1881. https://doi.org/10.1021/ja310455t
  30. Zhang, N., Suen, W.C., Windsor, W., Xiao, L., Madison, V., and Zaks, A. 2003. Improving tolerance of Candida antartica lipase B towards irreversible thermal inactivation through directed evolution. Protein Eng. 16, 599-605. https://doi.org/10.1093/protein/gzg074

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