A Simple Method for On-Gel Detection of Myrosinase Activity

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.

Near Infrared-II Photothermal and Colorimetric Synergistic Sensing for Intelligent Onsite Dietary Myrosinase Profiling

“…As Figure A,B and Figure S11 show, when Sin was present at a concentration of 0.75 mM, Myr was detectable in the range of 0–345 mU/mL, and its linear detection range in the absorption spectrum was 0 to 172.5 mU/mL with an LOD of 2.96 mU/mL. Comparatively, our method shows superiority in sensitivity with an LOD lower than that of previously reported work. , Additionally, the linear relationship between temperature and the logarithm of Myr concentration was also exhibited by the orange line in Figure B with a similar trend as indicated previously . These results further validate that the method could be employed to Myr analysis in real samples by both temperature and absorbance variations.…”

“…However, the deficiency in specificity and the requirement of sophisticated instrumentation are always the challenging concerns. Some new methods such as immunological methods could detect denatured Myr with no nutritional value and may lead to false-positive signals, , and on-gel detection requires extensive gel preparations, which are time-consuming, labor-intensive, and not suitable for onsite Myr tests. , Consequently, it is highly desired to develop a reliable, simple, interference-free, and rapid method for real-time Myr profiles in complex dietary samples.…”

The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers

“…Toluidine blue has been used to stain leaf epidermal peels, stem epidermis, leaves, roots, myrosinase-containing myrosin cells (myrosin grains) [ 7 , 11 , 36 , 37 , 38 , 39 ], and to detect myrosinase [ 40 , 41 ]. In earlier studies, we showed that myrosin cells from semi-thin sections of transgenic B. napus seeds lacking myrosinase appeared empty or as empty holes after staining with toluidine blue [ 37 , 42 ].…”

Variability of Bioactive Glucosinolates, Isothiocyanates and Enzyme Patterns in Horseradish Hairy Root Cultures Initiated from Different Organs

“…When the plant tissues are damaged (e.g., by crushing), the myrosinase comes in contact with the GLSs, resulting in the release of bioactive ITCs, nitriles, thiocyanates, epithionitriles, or oxazolidines, depending on the reaction conditions, the substrate, and the presence/absence of specifier proteins [1,2,3,14]. ITCs are the main products of this reaction at a pH range of 5–8 and at 20–45 °C [3,15].…”