Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis

Abstract

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon−oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis−Menten kinetic parameters for silicatein.

Evaluation of a fluorescence–based screening assay for the detection of silyl hydrolase activity

“…As compound 1 gave the best differentiation between the enzymatic and non-enzymatic reactions, the effect of pH on assays involving this substrate was further investigated, since this experimental parameter can have a significant effect on the fluorescence intensity and enzyme activity. Here, it was found that the best enzyme activity (as evidenced by the highest net reaction rate) occurred at pH 8.5 (Figure 2), which was in agreement with the previous studies 17 . In general, the non-enzymatic rate of hydrolysis was broadly similar across the pH range with only a slight increase towards the higher pH levels.…”

“…To investigate the utility of these substrates, enzyme assays were carried out using Silα, fused with trigger factor at the N-terminal and a Strep-tag II affinity tag at the C-terminal (henceforth referred to as TF-Silα-Strep), as the model enzyme. Initially, the assays were carried out under the same conditions as the previously reported 4-nitrophenoxy substrates 16,17 . Negative control reactions were also carried out where the enzyme was omitted, to evaluate the stability of each substrate towards aqueous (non-enzymatic) background hydrolysis.…”

“…The fluorometric assay design was based on methods previously reported for the spectrophotometric assays 16,17,25 . Firstly, stock solutions (5 mM) of each fluorogenic substrate…”

“…Silyl ether substrates encompassing the 4-nitrophenol derivatives have been developed for continuous assays by UV-vis spectrophotometry 16,17 , whereby hydrolysis of the Si-O bond results in the release of highly absorbing nitrophenolate ions. However, these anions absorb in the ~400 nm region (i.e., in yellow region of the visible spectrum), which also overlaps with the typical absorption spectra and colour of crude cell supernatants 18 , so these reagents are therefore unsuitable for screening experiments involving such cell lysates.…”

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

“…Silyl ether substrates encompassing the 4-nitrophenol derivatives have been developed for continuous assays by UV–vis spectrophotometry, , whereby hydrolysis of the Si–O bond results in the release of highly absorbing nitrophenolate ions. However, these anions absorb in the ∼400 nm region (i.e., in the yellow region of the visible spectrum), which also overlaps with the typical absorption spectra and color of crude cell supernatants, so these reagents are therefore unsuitable for screening experiments involving such cell lysates.…”