Strategy for effective inhibition of arylsulfatase/β-glucuronidase to prevent deconjugation of sulfate and glucuronide conjugates in wastewater during sample collection and storage.

Arylsulfatase and β-glucuronidase are two important enzymes that are responsible for deconjugation of estrogen conjugates. It is important to keep estrogen conjugates intact during sample collection and storage, while the effective inhibition conditions for arylsulfatase and β-glucuronidase remain unknown. To elucidate these conditions, inhibition experiments were performed by adding several inhibitors or by introducing extreme pH conditions. This work confirms that arylsulfatase and β-glucuronidase can tolerate some extremes, including high concentrations of mercury dichloride, ethanol, and EDTA, while low pH (<3) or high pH (>11) can effectively inhibit their activities. The high tolerance of arylsulfatase and β-glucuronidase for mercury dichloride explains why estrogen conjugates in wastewater samples were deconjugated, even in the extremely unfavorable condition with a high concentration of mercury dichloride. Although low pH (<3) can effectively inhibit arylsulfatase/β-glucuronidase, deconjugation of sulfate conjugates by acid hydrolysis readily occurs; thus, a high pH of 11 is an appropriate storage condition for the effective inhibition of arylsulfatase/β-glucuronidase. This appropriate storage condition was confirmed and validated with diluted and sterilized activated sludge samples in which arylsulfatase/β-glucuronidase inhibition was effective for 48 h at room temperature and with a high pH of 11. The developed appropriate storage condition for effective inhibition of arylsulfatase/β-glucuronidase has wide application potential not only for estrogen conjugates but also for all conjugates of other organic micropollutants.