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Applied Microbiology and Biotechnology

, Volume 100, Issue 22, pp 9529–9541 | Cite as

Characterization of a novel enzyme—Starmerella bombicola lactone esterase (SBLE)—responsible for sophorolipid lactonization

  • Katarzyna Ciesielska
  • Sophie L. K. W. Roelants
  • Inge N. A. Van Bogaert
  • Stijn De Waele
  • Isabel Vandenberghe
  • Sara Groeneboer
  • Wim Soetaert
  • Bart DevreeseEmail author
Biotechnologically relevant enzymes and proteins

Abstract

We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of K m . We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser194 in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.

Keywords

Starmerella (Candida) bombicola Sophorolipids Lactonization Lactone esterase Natural product synthesis Enzyme 

Notes

Compliance with ethical standards

Funding

This work was financially supported by European Union’s Seventh Framework Program Biosurfing project (PR 289219). KC and BD are also indebted to the Belgian Federal Science Policy agency for the support in the framework of the ESA-PRODEX project Bioreactor and the IAP network iPROS (P7/44).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7633_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1075 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Katarzyna Ciesielska
    • 1
  • Sophie L. K. W. Roelants
    • 2
  • Inge N. A. Van Bogaert
    • 2
  • Stijn De Waele
    • 1
  • Isabel Vandenberghe
    • 1
  • Sara Groeneboer
    • 1
  • Wim Soetaert
    • 2
  • Bart Devreese
    • 1
    Email author
  1. 1.Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and MicrobiologyGhent UniversityGhentBelgium
  2. 2.Laboratory of Industrial Biotechnology and BiocatalysisGhent UniversityGhentBelgium

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