Myrosinase immobilized on Ca-polygalacturonate hydrogel: enzyme efficiency as a function of soil characteristics

Myrosinase (MYR) from Sinapis alba (EC 3.2.1.147) was immobilized on a network of Ca-polygalacturonate hydrogel, which shows a composition and morphology similar to the mucigel present at the soil-root interface. The influence of pH, temperature, and time of storage on immobilized enzyme activity was investigated using sinigrin as a substrate. The immobilized MYR shows an activity which is 40% lower than the free enzyme, an optimum temperature at 55°C and an optimum pH in the range 5-7, similarly to the free enzyme. We observed that Ca-polygalacturonate preserved the activity of the immobilized enzyme more than two months. The Michaelis-Menten parameters, Vmax and Km, were determined for the free and immobilized MYR. The Vmax increased from 60 U mg-1 protein in free form to 127 U mg-1 protein in immobilized form, whereas the Km was much more increased: from 0,17 mM (for the free enzyme) up to 1,55 mM. Similarly to the behaviour of other immobilized enzymes, the kinetic parameters of MYR immobilization induces a higher specific activity together with a lower substrate affinity. MYR activity was also studied as a function of different substrates: sinigrin, gluconasturtin, sinalbin, and epi-progroitrin. It is important to mention that the highest activity of immobilized enzyme on the mucigel network was recorded using as a substrate the gluconasturtin, the glucosinolate found in roots of Brassicaceae. The activity of immobilized enzyme was also tested as a function of soil water potential in three soils differently characterized in organic matter content. At the field capacity (2,0 pF), the MYR activity was better preserved in soils poor of organic matter for two weeks, whereas, at the wilting point (4,2 pF), the activity was very quickly lost in all soils. The immobilization of MYR on Ca-polygalacturonate hydrogel preserved the enzymatic activity with time only in soils with low organic matter content. These results indicate not only that the soil water status is an important factor for MYR protection, but also that mucigel maintains the enzyme in the hydrated form at the soil-root interface. On the other hand, the soil organic matter content, owing to its hydrophilic properties, strongly competes with the mucigel for soil water content thus reducing the maintenance of the enzymatic activity.