Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing
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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 18.104.22.168). 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.
KeywordsArylsulfatase Kluyveromyces lactis pKLAC2 p-Cresol Off-flavor
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
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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