Skip to main content
SpringerLink
Log in

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 104 molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

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

  • Afanassiev V, Sefton M, Anantachaiyong T, Barker G, Walmsley R, Wolfl S (2000) Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances. Mutat Res-gen Tox En 464:297–308

    Article  CAS  Google Scholar 

  • Ardo ML, Sceno D, Huang C-J, Batt CA (2012) Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93:31–39

    Article  Google Scholar 

  • Bahl MI, Hansen LH, Licht TR, Sørensen SJ (2004) In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine. Antimicrob Agents Chemother 48:1112–1117

    Article  PubMed  CAS  Google Scholar 

  • Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R III (1997) Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 15:29–34

    Article  PubMed  CAS  Google Scholar 

  • Goossens KVY, Stassen C, Stals I et al (2011) The N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae binds specifically to mannose carbohydrates. Eukaryot Cell 10:110–117

    Article  PubMed  CAS  Google Scholar 

  • Han ZL, Han SY, Zheng SP, Lin Y (2009) Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface. Appl Microbiol Biotechnol 85:117–126

    Article  PubMed  CAS  Google Scholar 

  • Han SY, Han ZL, Lin Y, Zheng SP (2010) Construction of high efficiency Pichia pastoris surface display system based on Flo1 protein. Prog Biochem Biophys 37(1):1–8

    Google Scholar 

  • Jaeger K, Eggert T (2002) Lipase for biotechnology. Curr Opin Biotechnol 13:390–397

    Article  PubMed  CAS  Google Scholar 

  • Jin Z, Ntwali J, Han SY, Zheng SP, Lin Y (2012) Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor. J Biotechnol 159:108–114

    Article  PubMed  CAS  Google Scholar 

  • Kondo A, Ueda M (2004) Yeast cell-surface display - applications of molecular display. Appl Microbiol Biotechnol 64:28–40

    Article  PubMed  CAS  Google Scholar 

  • Lee SH, Choi JI, Park SJ, Lee SY, Park BC (2004) Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl Environ Microb 70:5074–5080

    Article  CAS  Google Scholar 

  • Li XY, Zhen MC, Lin W, Liu J, Catherine M, Li J, Wang XH (2008) Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods 72:116–123

    Article  PubMed  Google Scholar 

  • Liljeqvist S, Samuelson P, Hansson M, Nguyen TN, Binz H, Ståhl S (1997) Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus. Appl Environ Microb 63:2481–2488

    CAS  Google Scholar 

  • Matsumoto T, Fukuda H, Ueda M et al (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microb 68:4517–4522

    Article  CAS  Google Scholar 

  • Monika B, David R, Francisco V, Pau F (2009) Recent Patents on the Pichia Pastoris expression system: expanding the toolbox for recombinant protein production. Recent Pat Biotechnol 3:192–201

    Google Scholar 

  • Paus EJ, Willey J, Ridge RJ, Legg CR, Finkelman MA, Novitsky TJ, Ketchum PA (2002) Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification. Protein Expres Purif 26:202–210

    Article  CAS  Google Scholar 

  • Samuelson P, Wernerus H, Svedberg M, Ståhl S (2000) Staphylococcal surface display of metal-binding polyhistidyl peptides. Appl Environ Microb 66:1243–1248

    Article  CAS  Google Scholar 

  • Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475

    Article  PubMed  CAS  Google Scholar 

  • Su GD, Huang DF, Han S, Yan Zheng S P, Lin Y (2010) Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis. Appl Microbiol Biotechnol 86:1493–1501

    Article  PubMed  CAS  Google Scholar 

  • Tanaka T, Masunari S, Jun I, Wakamura K, Segawa M, Fukuda H, Kondo A (2010) Displaying non-natural, functional molecules on yeast surfaces via biotin–streptavidin interaction. J Biotechnol 145:79–83

    Article  PubMed  CAS  Google Scholar 

  • Tanino T, Fukuda H, Kondo A (2006) Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol Progr 22:989–993

    Article  CAS  Google Scholar 

  • Tanino T, Ohno T, Aoki T et al (2007) Development of yeast cells displaying Candida antarctica lipaseB and their application to ester synthesis reaction. Appl Microbiol Biotechnol 75:1319–1325

    Article  PubMed  CAS  Google Scholar 

  • Tanino T, Aoki T, Chung W, Watanabe Y, Ogino C, Fukuda H, Kondo A (2009) Improvement of a Candida antarctica lipase B-displaying yeast whole-cell biocatalyst and its application to the polyester synthesis reaction. Appl Microbiol Biotechnol 82:59–66

    Article  PubMed  CAS  Google Scholar 

  • Wang KC, Patel Chirag A, Wang J, Wang JQ, Wang XM, Luo PP, Zhong PY (2010) Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters. J Immunol Methods 354:11–19

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Supported by the grants from National Natural Science Foundation of China (30900017).

Author information

Authors and Affiliations

  1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Higher Education Mega Center, Guangzhou, 510006, People’s Republic of China

    Xing-xiang Liang, Bei-bei Wang, Yu-fei Sun, Ying Lin, Shuang-yan Han, Sui-ping Zheng & Tang-bing Cui

Authors
  1. Xing-xiang Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bei-bei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu-fei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ying Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shuang-yan Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sui-ping Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tang-bing Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Shuang-yan Han or Tang-bing Cui.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liang, Xx., Wang, Bb., Sun, Yf. et al. Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system. Biotechnol Lett 35, 367–374 (2013). https://doi.org/10.1007/s10529-012-1085-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-012-1085-2

Keywords

Search

Navigation