A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity

Applied Microbiology and Biotechnology volume 102, pages2709–2721(2018)Cite this article

Abstract

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as β-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of β-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant “cowshed-like” off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

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Acknowledgements

The authors would like to thank Wolfgang Claaßen (University of Hohenheim) for his support during the bioreactor cultivations. We also thank Susanne Herr and Sabine Lutz-Wahl (University of Hohenheim) for their support during the construction of the arylsulfatase variants. In addition, many thanks to Veronika Volk and Desirée Leisibach (University of Hohenheim) for performing preliminary experiments.

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Affiliations

  1. Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany

    Timo Stressler, Katrin Reichenberger, Claudia Glück, Paul Swietalski, Ines Seitl & Lutz Fischer

  2. Institute of Microbiology, University of Hohenheim, Garbenstr. 30, 70599, Stuttgart, Germany

    Sebastian Leptihn & Andreas Kuhn

  3. Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, August-von-Hartmann-Str. 3, 70599, Stuttgart, Germany

    Jens Pfannstiel

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  1. Timo Stressler
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  2. Katrin Reichenberger
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  3. Claudia Glück
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  9. Lutz Fischer
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Correspondence to Timo Stressler.

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Stressler, T., Reichenberger, K., Glück, C. et al. A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity. Appl Microbiol Biotechnol 102, 2709–2721 (2018). https://doi.org/10.1007/s00253-018-8828-5

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Keywords

  • Arylsulfatase
  • Kluyveromyces lactis
  • Amino acid exchange
  • p-nitrophenyl sulfate
  • p-cresol sulfate
  • Formylglycine
  • Secondary structure determination