title: Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing.
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 Ca(2+) ions and was inactivated by Zn(2+) 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.
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany. t.stressler@uni-hohenheim.de.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Desirée
authors.initials: D
authors.lastname: Leisibach
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lutz-Wahl
authors.affiliation: Institute of Microbiology, University of Hohenheim, Garbenstr. 30, 70599, Stuttgart, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuhn
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000818:Animals; D001192:Arylsulfatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003408:Cresols / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007218:Industrial Microbiology; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics; D007785:Lactose / Q000032:analysis / Q000378:metabolism; D008892:Milk / Q000737:chemistry* / Q000378:metabolism; D009578:Nitrobenzenes / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D003408:Cresols; D009578:Nitrobenzenes; D013463:Sulfuric Acid Esters; C023379:4-nitrophenyl sulfate; C032538:4-cresol; C408690:4-cresol sulfate; D001192:Arylsulfatases; D001616:beta-Galactosidase; D007785:Lactose
keywords: Arylsulfatase; Kluyveromyces lactis; Off-flavor; p-Cresol; pKLAC2