Applied Microbiology and Biotechnology

, Volume 72, Issue 5, pp 1024–1032

Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm—a screening system for a frequently used biocatalyst

Applied Genetics and Molecular Biotechnology

Abstract

In this paper, we report for the first time the functional expression of lipase B from the yeast Candida antarctica (CalB) in the Escherichia coli cytoplasm. The enzyme possessing three disulfide bonds was functionally expressed in the strain Origami B. Expression under the control of a lac promoter yielded 2 U mg−1, whereas expression of a thioredoxin–CalB fusion protein yielded 17 U mg−1. The native enzyme was most efficiently expressed under control of the cspA promoter (11 U mg−1). Coexpression of different chaperones led to a strong increase in active protein formation (up to 61 U mg−1). A codon-optimized synthetic variant of calb did not show significant effects on functional protein yield. Functional CalB expression was not only achieved in shake flasks but also in microtiter plate scale. Therefore, this CalB expression system is suitable for high-throughput applications, including the screening of large gene libraries as those derived from directed evolution experiments.

References

  1. Anderson EM, Larsson KM, Kirk O (1998) One biocatalyst—many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal Biotransform 16:181–204CrossRefGoogle Scholar
  2. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408CrossRefGoogle Scholar
  3. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A 96:13703–13708CrossRefGoogle Scholar
  4. Bornscheuer UT, Kazlauskas RJ (1999) Hydrolases in organic synthesis: regio- and stereolelective biotransformations. Wiley-VCH, WeinheimGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Chodorge M, Fourage L, Ullmann C, Duvivier V, Masson JM, Lefèvre F (2005) Rational strategies for directed evolution of biocatalysts—application to Candida antarctica lipase B (CALB). Adv Synth Catal 347:1022–1026CrossRefGoogle Scholar
  7. De la Torre F, Garcia-Gutierrez A, Crespillo R, Canton FR, Avila C, Canovas FM (2002) Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties. Plant Cell Physiol 43:802–809CrossRefGoogle Scholar
  8. Goldstein J, Pollitt NS, Inouye M (1990) Major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A 87:283–287CrossRefGoogle Scholar
  9. Hale RS, Thompson G (1998) Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. Protein Expr Purif 12:185–188CrossRefGoogle Scholar
  10. Hoegh I, Patkar S, Halkier T, Hansen MT (1995) Two lipases from Candida antarctica: cloning and expression in Aspergillus oryzae. Can J Bot 73:869–875Google Scholar
  11. Invitrogen (2002) pPICZ(A, B, and C. Pichia expression vectors for selection on Zeozin and purification of secreted, recombinant proteins. 1–36Google Scholar
  12. Jurado P, Ritz D, Beckwith J, de Lorenzo V, Fernandez LA (2002) Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli. J Mol Biol 320:1–10CrossRefGoogle Scholar
  13. Kim MH, Kim HK, Lee JK, Park SY, Oh TK (2000) Thermostable lipase of Bacillus stearothermophilus: high-level production, purification, and calcium-dependent thermostability. Biosci Biotechnol Biochem 64:280–286CrossRefGoogle Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  15. LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 11:187–193CrossRefGoogle Scholar
  16. Levy R, Weiss R, Chen G, Iverson BL, Georgiou G (2001) Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones. Protein Expr Purif 23:338–347CrossRefGoogle Scholar
  17. Liao HH (1991) Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli. Protein Expr Purif 2:43–50CrossRefGoogle Scholar
  18. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538Google Scholar
  19. Martinelle M, Holmquist M, Hult K (1995) On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta 1258:272–276Google Scholar
  20. Mutsuda M, Michel KP, Zhang X, Montgomery BL, Golden SS (2003) Biochemical properties of CikA, an unusual phytochrome-like histidine protein kinase that resets the circadian clock in Synechococcus elongatus PCC 7942. J Biol Chem 278:19102–19110CrossRefGoogle Scholar
  21. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699Google Scholar
  22. Nishihara K, Kanemori M, Yanagi H, Yura T (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 66:884–889CrossRefGoogle Scholar
  23. Novagen (1998) pET-32a-c(+) vectors Manual 1–2Google Scholar
  24. Novagen (2004) Competent cells User protocol TB009 Rev. F0104 1–23Google Scholar
  25. Novy R, Berg J, Yaeger K, Mierendorf R (2004) pET TRX fusion system for increased solubility of proteins expressed in E.coli. Novagen product informationGoogle Scholar
  26. Nthangeni MB, Patterton H, van Tonder A, Vergeer WP, Litthauer D (2001) Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. Enzyme Microb Technol 28:705–712CrossRefGoogle Scholar
  27. Patkar S, Vind J, Kelstrup E, Christensen M, Svendsen A, Borch K, Kirk O (1998) Effect of mutations in Candida antarctica B lipase. Chem Phys Lipids 93:95–101CrossRefGoogle Scholar
  28. Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:15661–15667CrossRefGoogle Scholar
  29. Rotticci D (2000) Understanding and engineering the enantioselectivity of Candida antarctica Lipase B towards sec-alcohols. Department of Chemistry, Organic Chemistry Stockholm Royal institute of Technology, pp 1–61Google Scholar
  30. Rotticci-Mulder JC (2003) Expression and mutagenesis studies of Candida antarctica lipase B Department of Biotechnology, Royal Institute of Technology Stockholm AlbaNova University Centre, pp 1–74Google Scholar
  31. Rotticci-Mulder JC, Gustavsson M, Holmquist M, Hult K, Martinelle M (2001) Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain. Protein Expr Purif 21:386–392CrossRefGoogle Scholar
  32. Rusnak M (2004) Untersuchungen zur enzymatischen Enantiomerentrennung von Glykolethern und Etablierung neuer Methoden des synthetischen. Shufflings Institute of Technical Biochemistry Stuttgart University of Stuttgart, pp 1–183Google Scholar
  33. Schmid RD, Verger R (1998) Lipasen: Grenzflächen-Enzyme mit attraktiven Anwendungen. Angew Chem Int Ed 37:1608–1633CrossRefGoogle Scholar
  34. Suen WC, Zhang N, Xiao L, Madison V, Zaks A (2004) Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel 17:133–140CrossRefGoogle Scholar
  35. TaKaRa (2003a) Cold shock expression system pCold DNA, Manual, pp 1–9Google Scholar
  36. TaKaRa (2003b) Chaperone plasmid set manual, pp 1–7Google Scholar
  37. Uppenberg J, Hansen MT, Patkar S, Jones A (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 2:293–308CrossRefGoogle Scholar
  38. Zhang N, Suen WC, Windsor W, Xiao L, Madison V, Zaks A (2003) Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein Eng 16:599–605CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Institute of Technical BiochemistryUniversity of StuttgartStuttgartGermany

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