Applied Microbiology and Biotechnology

, Volume 75, Issue 3, pp 549–555 | Cite as

Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties

  • Michiko Kato
  • Jun Fuchimoto
  • Takanori Tanino
  • Akihiko Kondo
  • Hideki Fukuda
  • Mitsuyoshi Ueda
Biotechnologically Relevant Enzymes and Proteins

Abstract

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by α-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T 1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60°C for mCALB and 44°C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.

Keywords

Candida antarctica lipase B Mutation Yeast cell surface engineering Substrate specificity 

Notes

Acknowledgements

This work was partially supported by the Research and Development Program for New Bio-industry Initiatives and the Ministry of Education, Science, Sports and Culture, Japan, a grant-in-aid for Scientific Research on Priority Areas and Research.

References

  1. Agterberg M, Adriaanse H, Lankhof H, Meloen R, Tommassen J (1990) Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants: immunogenicity of epitopes of foot-and-mouth disease virus. Vaccine 8:85–91CrossRefGoogle Scholar
  2. 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
  3. Benhar I (2001) Biotechnological applications of phage and cell display. Biotechnol Adv 19:1–33CrossRefGoogle Scholar
  4. Blank K, Morfill J, Gumpp H, Gaub HE (2006) Functional expression of Candida antarctica lipase B in Eschericha coli. J Biotechnol 125:474–483CrossRefGoogle Scholar
  5. Charbit A, Molla A, Saurin W, Hofnung M (1988) Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria. Gene 70:181–189CrossRefGoogle Scholar
  6. Fujita Y, Takahashi S, Ueda M, Tanaka A, Okada H, Morikawa Y, Kawaguchi T, Arai M, Fukuda H, Kondo A (2002) Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl Environ Microbiol 68:5136–5141CrossRefGoogle Scholar
  7. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  8. Kato M, Kuzuhara Y, Maeda H, Shiraga S, Ueda M (2006) Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A. Appl Microbiol Biotechnol 72:1229–1237CrossRefGoogle Scholar
  9. Kirk O, Christensen MW (2002) Lipases from Candida antarctica: unique biocatalysts from a unique origin. Org Process Res Dev 6:446–451CrossRefGoogle Scholar
  10. Kobori H, Sato M, Osumi M (1992) Relationship of actin organization to growth in the two forms of the dimorphic yeast Candida tropicalis. Protoplasma 167:193–204CrossRefGoogle Scholar
  11. Kondo A, Ueda M (2004) Yeast cell-surface display—applications of molecular display. Appl Microbiol Biotechnol 64:28–40CrossRefGoogle Scholar
  12. Kondo A, Shigechi H, Abe M, Uyama K, Matsumoto T, Takahashi S, Ueda M, Tanaka A, Kishimoto M, Fukuda H (2002) High-level production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell surface glucoamylase. Appl Microbiol Biotechnol 58:291–296CrossRefGoogle Scholar
  13. Martinell M, Holmquist M, Hult K (1995) On the interfacial activation of Candida antarctica lipase A and lipase B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta 1258:272–276Google Scholar
  14. Murai T, Ueda M, Atomi H, Shibasaki S, Kamasawa N, Osumi M, Kamaguchi T, Arai M, Tanaka A (1997) Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 48:499–503CrossRefGoogle Scholar
  15. Murai T, Ueda M, Kamaguchi T, Arai M, Tanaka A (1998) Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing β-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. Appl Environ Microbiol 64:4857–4861Google Scholar
  16. Narita J, Okano K, Tateno T, Tanino T, Sewaki, T, Sung MH, Fukuda H, Kondo A (2006) Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion. Appl Microbiol Biotechnol 70:564–572CrossRefGoogle Scholar
  17. Patkar S, Bjorkling F, Zundell M, Schulein M, Svendsen A, Heldt-Hansen HP, Gormsen E (1993) Purification of two lipases from Candida antarctica and their inhibition by various inhibitors. Indian J Chem 32B:76–80Google Scholar
  18. Rotticci D, Norin T, Hult K, Martinell M (2000) An active-site titration method for lipases. Biochim Biophys Acta 1483:132–140Google Scholar
  19. Sharma R, Chisti Y, Banerjee C (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662CrossRefGoogle Scholar
  20. Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa M, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by the fluorometric and confocal laser scanning microscopic analysis. Appl Microbiol Biotechnol 55:471–475CrossRefGoogle Scholar
  21. Shiraga S, Ueda M, Takahashi S, Tanaka A (2002) Construction of the combinatorial library of Rhizopus oryzae lipase mutated in the lid domain by displaying on yeast cell surface. J Mol Catal B Enzym 17:167–173CrossRefGoogle Scholar
  22. Shiraga S, Ishiguro M, Fukami H, Nakao M, Ueda M (2005a) Creation of Rhizopus oryzae lipase having a unique oxyanion hole by combinatorial mutagenesis in the lid domain. Appl Microbiol Biotechnol 68:779–785CrossRefGoogle Scholar
  23. Shiraga S, Kawakami M, Ishiguro M, Ueda M (2005b) Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents. Appl Environ Microbiol 71:4335–4338CrossRefGoogle Scholar
  24. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317CrossRefGoogle Scholar
  25. 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
  26. Tajima M, Nogi Y, Fukasawa T (1985) Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1:67–77CrossRefGoogle Scholar
  27. Takahashi S, Ueda M, Atomi H, Beer HD, Bornscheuer UT, Schmid RD, Tanaka A (1998) Extracellular production of active Rhizopus oryzae lipase by Saccharomyces cerevisiae. J Ferment Bioeng 86:164–168CrossRefGoogle Scholar
  28. Ueda M (2004) Future direction of molecular display by yeast-cell surface engineering. J Mol Catal B Enzym 28:139–143CrossRefGoogle Scholar
  29. Uppenberg J, Hansen MT, Patkar S, Jones TA (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 2:293–308CrossRefGoogle Scholar
  30. 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
  31. Zou W, Ueda M, Tanaka A (2002) Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library. Appl Microbiol Biotechnol 58:806–812CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Michiko Kato
    • 1
  • Jun Fuchimoto
    • 1
  • Takanori Tanino
    • 2
  • Akihiko Kondo
    • 2
  • Hideki Fukuda
    • 3
  • Mitsuyoshi Ueda
    • 1
  1. 1.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Department of Chemical Science and Engineering, Faculty of EngineeringKobe UniversityKobeJapan
  3. 3.Division of Molecular Science, Graduate School of Science and TechnologyKobe UniversityKobeJapan

Personalised recommendations