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. 2002 Sep 1;366(Pt 2):603–611. doi: 10.1042/BJ20020404

Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

Guan-Chiun Lee 1, Li-Chiun Lee 1, Vasyl Sava 1, Jei-Fu Shaw 1
PMCID: PMC1222792  PMID: 12020350

Abstract

The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.

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Selected References

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  1. Bairoch A., Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res. 2000 Jan 1;28(1):45–48. doi: 10.1093/nar/28.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamin S., Pandey A. Candida rugosa lipases: molecular biology and versatility in biotechnology. Yeast. 1998 Sep 15;14(12):1069–1087. doi: 10.1002/(SICI)1097-0061(19980915)14:12<1069::AID-YEA303>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  3. Berman H. M., Westbrook J., Feng Z., Gilliland G., Bhat T. N., Weissig H., Shindyalov I. N., Bourne P. E. The Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235–242. doi: 10.1093/nar/28.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brocca S., Grandori R., Breviario D., Lotti M. Localization of lipase genes on Candida rugosa chromosomes. Curr Genet. 1995 Oct;28(5):454–457. doi: 10.1007/BF00310815. [DOI] [PubMed] [Google Scholar]
  5. Brocca S., Schmidt-Dannert C., Lotti M., Alberghina L., Schmid R. D. Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase. Protein Sci. 1998 Jun;7(6):1415–1422. doi: 10.1002/pro.5560070618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cereghino J. L., Cregg J. M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45–66. doi: 10.1111/j.1574-6976.2000.tb00532.x. [DOI] [PubMed] [Google Scholar]
  7. Diczfalusy M. A., Hellman U., Alexson S. E. Isolation of carboxylester lipase (CEL) isoenzymes from Candida rugosa and identification of the corresponding genes. Arch Biochem Biophys. 1997 Dec 1;348(1):1–8. doi: 10.1006/abbi.1997.0382. [DOI] [PubMed] [Google Scholar]
  8. Ferrer P., Montesinos J. L., Valero F., Solà C. Production of native and recombinant lipases by Candida rugosa: a review. Appl Biochem Biotechnol. 2001 Sep;95(3):221–255. doi: 10.1385/abab:95:3:221. [DOI] [PubMed] [Google Scholar]
  9. Ge L., Rudolph P. Simultaneous introduction of multiple mutations using overlap extension PCR. Biotechniques. 1997 Jan;22(1):28–30. doi: 10.2144/97221bm03. [DOI] [PubMed] [Google Scholar]
  10. Ghosh D., Wawrzak Z., Pletnev V. Z., Li N., Kaiser R., Pangborn W., Jörnvall H., Erman M., Duax W. L. Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase. Structure. 1995 Mar 15;3(3):279–288. doi: 10.1016/s0969-2126(01)00158-7. [DOI] [PubMed] [Google Scholar]
  11. Grochulski P., Bouthillier F., Kazlauskas R. J., Serreqi A. N., Schrag J. D., Ziomek E., Cygler M. Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase. Biochemistry. 1994 Mar 29;33(12):3494–3500. doi: 10.1021/bi00178a005. [DOI] [PubMed] [Google Scholar]
  12. Grochulski P., Li Y., Schrag J. D., Bouthillier F., Smith P., Harrison D., Rubin B., Cygler M. Insights into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem. 1993 Jun 15;268(17):12843–12847. [PubMed] [Google Scholar]
  13. Grochulski P., Li Y., Schrag J. D., Cygler M. Two conformational states of Candida rugosa lipase. Protein Sci. 1994 Jan;3(1):82–91. doi: 10.1002/pro.5560030111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  15. Hernáiz M. J., Rua M., Celda B., Medina P., Sinisterra J. V., Sánchez-Montero J. M. Contribution to the study of the alteration of lipase activity of Candida rugosa by ions and buffers. Appl Biochem Biotechnol. 1994 Mar;44(3):213–229. doi: 10.1007/BF02779658. [DOI] [PubMed] [Google Scholar]
  16. Kaiser R., Erman M., Duax W. L., Ghosh D., Jörnvall H. Monomeric and dimeric forms of cholesterol esterase from Candida cylindracea. Primary structure, identity in peptide patterns, and additional microheterogeneity. FEBS Lett. 1994 Jan 10;337(2):123–127. doi: 10.1016/0014-5793(94)80257-2. [DOI] [PubMed] [Google Scholar]
  17. Kawaguchi Y., Honda H., Taniguchi-Morimura J., Iwasaki S. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature. 1989 Sep 14;341(6238):164–166. doi: 10.1038/341164a0. [DOI] [PubMed] [Google Scholar]
  18. Lee G. C., Tang S. J., Sun K. H., Shaw J. F. Analysis of the gene family encoding lipases in Candida rugosa by competitive reverse transcription-PCR. Appl Environ Microbiol. 1999 Sep;65(9):3888–3895. doi: 10.1128/aem.65.9.3888-3895.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Longhi S., Fusetti F., Grandori R., Lotti M., Vanoni M., Alberghina L. Cloning and nucleotide sequences of two lipase genes from Candida cylindracea. Biochim Biophys Acta. 1992 Jun 15;1131(2):227–232. doi: 10.1016/0167-4781(92)90085-e. [DOI] [PubMed] [Google Scholar]
  20. Lotti M., Grandori R., Fusetti F., Longhi S., Brocca S., Tramontano A., Alberghina L. Cloning and analysis of Candida cylindracea lipase sequences. Gene. 1993 Feb 14;124(1):45–55. doi: 10.1016/0378-1119(93)90760-z. [DOI] [PubMed] [Google Scholar]
  21. Lotti M., Tramontano A., Longhi S., Fusetti F., Brocca S., Pizzi E., Alberghina L. Variability within the Candida rugosa lipases family. Protein Eng. 1994 Apr;7(4):531–535. doi: 10.1093/protein/7.4.531. [DOI] [PubMed] [Google Scholar]
  22. Pernas M. A., López C., Pastrana L., Rúa M. L. Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation. J Biotechnol. 2001 Nov 30;84(2):163–174. doi: 10.1016/s0168-1656(00)00351-5. [DOI] [PubMed] [Google Scholar]
  23. Redondo O., Herrero A., Bello J. F., Roig M. G., Calvo M. V., Plou F. J., Burguillo F. J. Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid p-nitrophenyl esters in mixed micelles with Triton X-100. Biochim Biophys Acta. 1995 Jan 18;1243(1):15–24. doi: 10.1016/0304-4165(94)00112-b. [DOI] [PubMed] [Google Scholar]
  24. Röschlau P., Bernt E., Gruber W. Enzymatische Bestimmung des Gesamt-Cholesterins im Serum. Z Klin Chem Klin Biochem. 1974 Sep;12(9):403–407. [PubMed] [Google Scholar]
  25. Rúa L., Díaz-Mauriño T., Fernández V. M., Otero C., Ballesteros A. Purification and characterization of two distinct lipases from Candida cylindracea. Biochim Biophys Acta. 1993 Feb 13;1156(2):181–189. doi: 10.1016/0304-4165(93)90134-t. [DOI] [PubMed] [Google Scholar]
  26. Svendsen A. Lipase protein engineering. Biochim Biophys Acta. 2000 Dec 29;1543(2):223–238. doi: 10.1016/s0167-4838(00)00239-9. [DOI] [PubMed] [Google Scholar]
  27. Sánchez R., Sali A. Advances in comparative protein-structure modelling. Curr Opin Struct Biol. 1997 Apr;7(2):206–214. doi: 10.1016/s0959-440x(97)80027-9. [DOI] [PubMed] [Google Scholar]
  28. Tang S. J., Shaw J. F., Sun K. H., Sun G. H., Chang T. Y., Lin C. K., Lo Y. C., Lee G. C. Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability. Arch Biochem Biophys. 2001 Mar 1;387(1):93–98. doi: 10.1006/abbi.2000.2235. [DOI] [PubMed] [Google Scholar]
  29. Tang S. J., Sun K. H., Sun G. H., Chang T. Y., Lee G. C. Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli. Protein Expr Purif. 2000 Nov;20(2):308–313. doi: 10.1006/prep.2000.1304. [DOI] [PubMed] [Google Scholar]
  30. Tiss A., Carrière F., Verger R. Effects of gum arabic on lipase interfacial binding and activity. Anal Biochem. 2001 Jul 1;294(1):36–43. doi: 10.1006/abio.2001.5095. [DOI] [PubMed] [Google Scholar]
  31. Valivety R. H., Halling P. J., Peilow A. D., Macrae A. R. Relationship between water activity and catalytic activity of lipases in organic media. Effects of supports, loading and enzyme preparation. Eur J Biochem. 1994 Jun 1;222(2):461–466. doi: 10.1111/j.1432-1033.1994.tb18886.x. [DOI] [PubMed] [Google Scholar]
  32. Waterham H. R., Digan M. E., Koutz P. J., Lair S. V., Cregg J. M. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene. 1997 Feb 20;186(1):37–44. doi: 10.1016/s0378-1119(96)00675-0. [DOI] [PubMed] [Google Scholar]

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