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Applied Microbiology and Biotechnology

, Volume 103, Issue 9, pp 3761–3771 | Cite as

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols

  • Yu Ji
  • Shohana Islam
  • Alan M. Mertens
  • Daniel F. Sauer
  • Gaurao V. Dhoke
  • Felix Jakob
  • Ulrich SchwanebergEmail author
Biotechnologically relevant enzymes and proteins
  • 232 Downloads

Abstract

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s−1) and catalytic efficiency (kcat/KM = 0.41 μM−1 s−1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two–fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Keywords

Directed evolution Sulfotransferase Catechols 4-Aminoantipyrine p-Nitrophenyl sulfate 

Notes

Acknowledgements

The authors thank Dr. Kilian E. C. Smith and David Kämpfer for helping with the MS measurement and Prof. Dr. Jun Okuda for the NMR support.

Funding

This research was funded by the China Scholarship Council (CSC) (No. 201608080082) and German Federal Ministry of Education and Research (BMBF) under the projects “FuPol” (Functionalization of Polymers [FKZ: 031A227F]) alliance and IBÖ-03: BioSulfa-Effiziente Sulfatierung von Biomolekülen [FKZ: 031B0255].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not provide any research involving human participants and/or animal experiments.

Supplementary material

253_2019_9688_MOESM1_ESM.pdf (650 kb)
ESM 1 The electrical supplementary material includes optimization of screening parameters, ESI-MS, and NMR data. (PDF 650 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Lehrstuhl für BiotechnologieRWTH Aachen UniversityAachenGermany
  2. 2.DWI – Leibniz-Institut für Interaktive Materialien e.V.AachenGermany

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