Evaluation of Fluorescence-Based Screening Assays for the Detection and Quantification of Silyl Hydrolase Activity

TL;DR

Researchers developed a new fluorescence-based method to detect and measure silyl hydrolase enzyme activity using a stable and effective substrate called MycoF.

Contribution

The novel contribution is the development of a stable fluorogenic substrate, MycoF, for high-throughput and quantitative detection of silyl hydrolase activity.

Findings

MycoF showed good differentiation between enzyme-catalyzed and background hydrolysis in aqueous media.

MycoF enabled detection of enzyme activity in cell lysates and identified positive hits in high-throughput screening.

Michaelis–Menten parameters were successfully determined using MycoF in kinetic assays.

Abstract

This study reports the development of fluorometric assays
for the
detection and quantification of silyl hydrolase activity using silicatein
as a model enzyme. These assays employed a series of organosilane
substrates containing either mycophenolate or umbelliferone moieties,
which become fluorescent upon hydrolysis of a scissile Si–O
bond. Among these substrates, the mycophenolate-derived molecule MycoF,
emerged as the most promising candidate due to its relative stability
in aqueous media, which resulted in good differentiation between the
enzyme-catalyzed and uncatalyzed background hydrolysis. The utility
of MycoF was also demonstrated in the detection of enzyme activity
in cell lysates and was found to be capable of qualitative identification
of positive “hit” candidates in a high-throughput format.
These fluorogenic substrates were also suitable for use in quantitative
kinetic assays, as demonstrated by the acquisition of their Michaelis–Menten
parameters.

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