Skip to main content
SpringerLink
Log in

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
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Anderson EM, Larsson KM, Kirk O (1998) One biocatalyst—many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal Biotransform 16:181–204

    Article  CAS  Google Scholar 

  • Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408

    Article  CAS  Google Scholar 

  • 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 96:13703–13708

    Article  CAS  Google Scholar 

  • Bornscheuer UT, Kazlauskas RJ (1999) Hydrolases in organic synthesis: regio- and stereolelective biotransformations. Wiley-VCH, Weinheim

  • 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 72:248–254

    Article  CAS  Google Scholar 

  • 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 347:1022–1026

    Article  CAS  Google Scholar 

  • 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 43:802–809

    Article  Google Scholar 

  • Goldstein J, Pollitt NS, Inouye M (1990) Major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A 87:283–287

    Article  CAS  Google Scholar 

  • 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 12:185–188

    Article  CAS  Google Scholar 

  • Hoegh I, Patkar S, Halkier T, Hansen MT (1995) Two lipases from Candida antarctica: cloning and expression in Aspergillus oryzae. Can J Bot 73:869–875

    Article  Google Scholar 

  • Invitrogen (2002) pPICZ(A, B, and C. Pichia expression vectors for selection on Zeozin and purification of secreted, recombinant proteins. 1–36

  • 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 320:1–10

    Article  CAS  Google Scholar 

  • 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 64:280–286

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • 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) 11:187–193

    Google Scholar 

  • 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 23:338–347

    Article  CAS  Google Scholar 

  • Liao HH (1991) Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli. Protein Expr Purif 2:43–50

    Article  CAS  Google Scholar 

  • Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

    Article  CAS  Google Scholar 

  • 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 1258:272–276

    Article  Google Scholar 

  • 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 278:19102–19110

    Article  CAS  Google Scholar 

  • 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 64:1694–1699

    Article  CAS  Google Scholar 

  • Nishihara K, Kanemori M, Yanagi H, Yura T (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 66:884–889

    Article  CAS  Google Scholar 

  • Novagen (1998) pET-32a-c(+) vectors Manual 1–2

  • Novagen (2004) Competent cells User protocol TB009 Rev. F0104 1–23

  • Novy R, Berg J, Yaeger K, Mierendorf R (2004) pET TRX fusion system for increased solubility of proteins expressed in E.coli. Novagen product information

  • 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 28:705–712

    Article  CAS  Google Scholar 

  • 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 93:95–101

    Article  CAS  Google Scholar 

  • 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 272:15661–15667

    Article  CAS  Google Scholar 

  • 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 1–61

  • 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 1–74

  • 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 21:386–392

    Article  CAS  Google Scholar 

  • 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 1–183

  • Schmid RD, Verger R (1998) Lipasen: Grenzflächen-Enzyme mit attraktiven Anwendungen. Angew Chem Int Ed 37:1608–1633

    Article  Google Scholar 

  • 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 17:133–140

    Article  CAS  Google Scholar 

  • TaKaRa (2003a) Cold shock expression system pCold DNA, Manual, pp 1–9

  • TaKaRa (2003b) Chaperone plasmid set manual, pp 1–7

  • 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 2:293–308

    Article  CAS  Google Scholar 

  • 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 16:599–605

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    D. Liu, R. D. Schmid & M. Rusnak

Authors
  1. D. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. D. Schmid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Rusnak
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Rusnak.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, D., Schmid, R.D. & Rusnak, M. Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm—a screening system for a frequently used biocatalyst. Appl Microbiol Biotechnol 72, 1024–1032 (2006). https://doi.org/10.1007/s00253-006-0369-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0369-7

Keywords

Search

Navigation