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Applied Microbiology and Biotechnology

, Volume 100, Issue 12, pp 5401–5414 | Cite as

Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing

  • Timo StresslerEmail author
  • Desirée Leisibach
  • Sabine Lutz-Wahl
  • Andreas Kuhn
  • Lutz Fischer
Biotechnologically relevant enzymes and proteins

Abstract

The industrial manufacturing process of lactose-free milk products depends on the application of commercial β-galactosidase (lactase) preparations. These preparations are often obtained from Kluyveromyces lactis. There is a gene present in the genome of K. lactis which should encode for an enzyme called arylsulfatase (EC 3.1.6.1). Therefore, this enzyme could also be present in β-galactosidase preparations. The arylsulfatase is suspected of being responsible for an unpleasant “cowshed-like” off-flavor resulting from the release of p-cresol from milk endogenous alkylphenol sulfuric esters. So far, no gene/functionality relationship is described. In addition, no study is available which has shown that arylsulfatase from K. lactis is truly responsible for the flavor generation. In this study, we cloned the putative arylsulfatase gene from K. lactis GG799 into the commercially available vector pKLAC2. The cloning strategy chosen resulted in a homologous, secretory expression of the arylsulfatase. We showed that the heretofore putative arylsulfatase has the desired activity with the synthetic substrate p-nitrophenyl sulfate and with the natural substrate p-cresol sulfate. The enzyme was biochemically characterized and showed an optimum temperature of 45–50 °C and an optimum pH of 9–10. Additionally, the arylsulfatase was activated by Ca2+ ions and was inactivated by Zn2+ ions. Moreover, the arylsulfatase was inhibited by p-cresol and sulfate ions. Finally, the enzyme was added to ultra-heat treated (UHT) milk and a sensory triangle test verified that the arylsulfatase from K. lactis can cause an unpleasant “cowshed-like” off-flavor.

Keywords

Arylsulfatase Kluyveromyces lactis pKLAC2 p-Cresol Off-flavor 

Notes

Acknowledgments

The authors would like to thank Wolfgang Claaßen (University of Hohenheim) for his support during the bioreactor cultivation. We also thank Jacob Ewert and Claudia Glück (University of Hohenheim) for their highly appreciated support during the sensory tests. Finally, we thank Veronika Volk (University of Hohenheim) for performing preliminary experiments.

Compliance with ethical standards

Ethical statement

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

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

253_2016_7366_MOESM1_ESM.pdf (403 kb)
ESM 1 (PDF 402 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Timo Stressler
    • 1
    Email author
  • Desirée Leisibach
    • 1
  • Sabine Lutz-Wahl
    • 1
  • Andreas Kuhn
    • 2
  • Lutz Fischer
    • 1
  1. 1.Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme ScienceUniversity of HohenheimStuttgartGermany
  2. 2.Institute of MicrobiologyUniversity of HohenheimStuttgartGermany

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