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

Publikations-Art

Zeitschriftenbeitrag (peer-reviewed)

Autoren

Stressler, T.; Reichenberger, K.; Glück, C.; Leptihn, S.; Pfannstiel, J.; Swietalski, P.; Kuhn, A., Seitl; I.; Fischer, L.

Erscheinungsjahr

2018

Veröffentlicht in

Applied Microbiology and Biotechnology

Verlag

Springer

DOI

10.1007/s00253-018-8828-5

Kluyveromyces lactisis 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. Inthe 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. Thebest 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 DSM70799 (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 showedfull 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 packingand 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