Abstract
Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4− is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.
Highlights
The glucosinolate-myrosinase-isothiocyanate system is a widely distributed chemical defense system of the Brassicales [1]
Seeing the limitations of the above, we aimed to develop a sensitive, straightforward on-gel assay for myrosinase that can be used to detect bands on native PAGE gels or SDS-PAGE gels, after a simple washout protocol
Our approach was to detect the release of H+, a side product of the myrosinase catalyzed glucosinolate hydrolysis (Figure 1)
Summary
The glucosinolate-myrosinase-isothiocyanate system is a widely distributed chemical defense system of the Brassicales [1]. As the volatile isothiocyanates are highly bioactive molecules that are at the same time beneficial to human consumers, the system is of high scientific and industrial interest [2]. The plants biosynthesize the glucosinolate precursors, which come in contact with their activation enzyme myrosinase under some circumstances, usually when tissue damage occurs [1]. The default products are isothiocyanates, or, in vivo in the presence of so called specifier proteins, other less toxic volatiles can be formed. The activity itself is shown to be present in various Brassicaceae plants [3,4], Molecules 2018, 23, 2204; doi:10.3390/molecules23092204 www.mdpi.com/journal/molecules
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