Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine
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- 作者：Christian Kaiser ; Steffen Uhlig ; Torsten Gerlach ; Martina Kö ; rner ; Kirsten Simon ; Kirstin Kunath ; Kristina Florschü ; tz ; Keith Baronian ; Gotthard Kunze
- 关键词：Arxula adeninivorans ; Endocrine disruption ; Estrogen receptor ; Reporter assay ; Urine ; Yeast assay
- 刊名：Science of the Total Environment
- 出版年：2010
- 出版时间：1 November 2010
- 年：2010
- 卷：408
- 期：23
- 页码：6017-6026
- 全文大小：915 K
文摘
A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6–25 h assay period with detection and determination limits and EC50 values for 17β-estradiol of 2.8 ng L− 1, 5.9 ng L− 1, 33.2 ng L− 1 (nAES-P) and 3.1 ng L− 1, 6.7 ng L− 1 and 39.4 ng L− 1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.