Activity of myrosinase from Sinapis alba seeds immobilized into Ca-polygalacturonate as a simplified model of soil-root interface mucigel

Abstract

Ca-polygalacturonate is a demethoxylated component of pectins which are constitutive of plant root mucigel. In order to define the role of root mucigel in myrosinase immobilization and activity at root level, a myrosinase enzyme which had been isolated from Sinapis alba seeds was immobilized into Capolygalacturonate. The activity profile for the immobilized and free enzyme was evaluated using the pH-Stat method as a function of time, temperature, and pH. The Michaelis-Menten kinetic parameters change between the immobilized (V max =127±13 U mg −1 protein; K M =6.28±0.09mM) and free (V max =17±1 U mg −1 protein; K M =0.96±0.01mM) forms of myrosinase, probably due to conformational changes involving the active site as a consequence of enzyme immobilization. Immobilized enzyme activity evaluated as a function of different substrates gave the highest value with nasturtin, the glucosinolate that is typical of several brassicaceae plant roots containing the glucosinolatemyrosinase defensive system. No feedback regulation mechanism was found in the presence of an excess of enzymatic reaction products (i.e. allyl isothiocyanate or sulphate). The high enzyme immobilization yield into Ca-polygalacturonate and its activity preservation under different conditions suggest that the enzyme released by plants at root level could be entrapped in root mucigel in order to preserve its activity.

Synthesis and Biochemical Evaluation of an Artificial, Fluorescent Glucosinolate (GSL)

“…In the absence of myrosinase no formation of 10 was observed (Figure A). Substrate conversion followed Michaelis–Menten kinetics with K m =888±143 μ m and a k cat of 37.9 s −1 (Figure ), that is, values within the range determined with natural GSLs …”

Selection and microencapsulation of myrosinase enzyme from broccoli sprouts of different varieties and characteristics evaluation

“…At 65 C, the free enzyme activity was less than half of the When the temperature arrived at 75 C, the enzyme activity was decreased to 62% of the maximum enzyme activity. Another study reported a suitable temperature range of 50-60 C for immobilized MYR in Ca-polygalacturonate (Braschi et al, 2011). The improved heat resistance of the enzyme may due to that the MYR was adsorbed by SA and cross-linking with glutaraldehyde, after which enhanced the firmness of the enzyme configuration.…”

“…for MYR in immobilized Ca-polygalacturonate (Braschi et al, 2011). Increased suitable temperature and pH range for MYR would be beneficial for supplying encapsulated MYR as exogenous enzyme accompanied with GLs to promote SF production in vivo or vitro.…”

“…In normal, GLs in cell are separated from MYR (Bhat, Kaur, Khajuria, Vyas, & Vyas, 2015). Only when the cells are broken, they will be combined and then react, resulting in the production of SF and other metabolites (Braschi, Leoni, Cinti, Palmieri, & Gessa, 2011; Okunade, Ghawi, Methven, & Niranjan, 2015). However, when endogenous MYR was used to catalyze GLs, the miscellaneous enzyme, mainly epithiospecifier protein (ESP), in the raw material would cause most of the substrate breakdown to by‐product nitriles, which not only led to a significant reduction of SF yield, but also increased the separation difficulty (Chhajed, Misra, Tello, & Chen, 2019).…”

“…A very early report showed that the enzyme was encapsulated on granular nylon 6.6 with the crosslinking technique for industrialized producing pure isothiocyanates (Leoni, Iori, & Palmieri, 2000). Another report defined the role of root mucigel in MYR immobilization and activity at root level by immobilized MYR into Capolygalacturonate (Braschi et al, 2011). However, encapsulated MYR used as exogenous enzyme accompanied with GLs to promote SF production in vivo or vitro has yet not been reported.…”