Colorimetric Determination of Sulfate via an Enzyme Cascade for High-Throughput Detection of Sulfatase Activity.

Abstract

High-throughput screening (HTS) methods have become decisive for the discovery and development of new biocatalysts and their application in numerous fields. Sulfatases, a broad class of biocatalysts that hydrolyze sulfate esters, are involved in diverse relevant cellular functions (e.g., signaling and hormonal regulation) and are therefore gaining importance, particularly in the medical field. Additionally, various technical applications have been recently devised. One of the major challenges in the field of enzyme development is the sensitive and high-throughput detection of the actual product of the biocatalyst of interest without the need for chromophore analogues. Addressing this issue, a colorimetric assay for sulfatases was developed and validated for detecting sulfate through a two-step enzymatic cascade, with a linear detection range of 3.3 (limit of detection) up to 250 μM. The procedure is compatible with relevant compounds employed in sulfatase reactions, including cosolvents, cations, and buffers. The assay was optimized and performed as part of a 96-well screening workflow that included bacterial growth, heterologous sulfatase expression, cell lysis, sulfate ester hydrolysis, inactivation of cell lysate, and colorimetric sulfate determination. With this procedure, the activity of an aryl and an alkyl sulfatase could be confirmed and validated. Overall, this assay provides a simple and fast alternative for screening and engineering sulfatases from DNA libraries (e.g., using metagenomics) with medical or synthetic relevance.