A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity
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 220.127.116.11). 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.
KeywordsArylsulfatase Kluyveromyces lactis Amino acid exchange p-nitrophenyl sulfate p-cresol sulfate Formylglycine Secondary structure determination
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.
Compliance with ethical standards
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.
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