Thermal and pressure stability of myrosinase enzymes from black mustard (Brassica nigra L. W.D.J. Koch. var. nigra), brown mustard (Brassica juncea L. Czern. var. juncea) and yellow mustard (Sinapsis alba L. subsp. maire) seeds

Abstract

This study investigates the effects of temperature and pressure on inactivation of myrosinase extracted from black, brown and yellow mustard seeds. Brown mustard had higher myrosinase activity (2.75 un/mL) than black (1.50 un/mL) and yellow mustard (0.63 un/mL). The extent of enzyme inactivation increased with pressure (600-800 MPa) and temperature (30-70° C) for all the mustard seeds. However, at combinations of lower pressures (200-400 MPa) and high temperatures (60-80 °C), there was less inactivation. For example, application of 300 MPa and 70 °C for 10 min retained 20%, 80% and 65% activity in yellow, black and brown mustard, respectively, whereas the corresponding activity retentions when applying only heat (70° C, 10 min) were 0%, 59% and 35%. Thus, application of moderate pressures (200-400 MPa) can potentially be used to retain myrosinase activity needed for subsequent glucosinolate hydrolysis.

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

“…Velocities rapidly dropped at higher temperatures, reaching complete enzymatic inactivation between 75–85 °C. These optimal temperatures are supported by other accounts on thioglucosidase activity, which show variations based on species of origin: Brevicoryne brassicae , optimal temperature 35–45 °C [32], Carica papaya , optimal temperature 40–50 °C [30], mustard ( Sinapis ) variants have demonstrated relatively higher thermal stability [31,40]. …”

“…For reactions conducted at temperatures above 65 °C, it was noted that [Gluc] t decreased early in a trial, then appeared to stagnate; nonlinear regression of these data were poorly fit to the Lambert W (x). Previous accounts have noted thermal stability of Sinapis thioglucosidase activity up to this temperature range: Sinapis negra , 60 °C [31], Sinapis hirta , 60 °C [40]. …”

“…Studies describing the pH-dependence of myrosinase catalytic activity on natural substrates have noted a maximum efficiency at pH 6.5 for enzyme isolated from Sinapis alba [29] and pH 6.5–8.5 for Carica papaya [30]. Similarly, thioglucohydrolases demonstrate an Arrhenius-type increase in the rate of catalysis of natural glucosinolates between 0–40 °C, an optimum temperat ure range between 40–55 °C, and complete thermal inactivation by 80 °C [30,31,32]. If the ca talytic mechanism of myrosinase on non-natural glucosinolate substrates parallels the mechanism for natural substrates, it would be expected that similar dependence patterns on pH, temperature, and their effects on the ratio of organic product distribution (e.g.…”

“…The validated HPLC approach is ideally suited for these enzymological studies, as it offers the possibility of simultaneously and independently evaluating glucosinolate and any resultant organic product(s), a feature which has broader methodological considerations and applications [19]. Initial rates ( V 0 ) for glucosinolate hydrolysis and product formation would be obtained from nonlinear reaction progress curves fit with the Lambert W (x) [19,33]; these elucidated V 0 would provide explicit rate information, an advantage over many previous accounts which solely reported relative rates [31,32]. …”

Supplementation of the Diet by Exogenous Myrosinase via Mustard Seeds to Increase the Bioavailability of Sulforaphane in Healthy Human Subjects after the Consumption of Cooked Broccoli

“…The cooked portions were rapidly cooled on ice. Myrosinase activity in cooked broccoli portions was then assayed as described and adapted by Okunade et al . Briefly, the ground material is extracted on ice with buffer (Tris‐HCl 0.2 m , pH 7.5 containing EDTA 0.5 m m , dithiothreitol 1.5 m m and 0.4 g polyvinylpolypyrrolidone) and the protein in the filtered supernatant is precipitated with ammonium sulfate.…”