# Inactivation of Thioglucidase from _Sinapis alba_ (White Mustard) Seed by Metal Salts
## 3 Discussion  
The composition of Brassica vegetable tissue usually promotes the formation of isothiyanates [19]. Glucosinolates are prone to hydrolysis by myrosinase and as a result of degradation various volatile compounds can be obtained, which are strictly dependent on the conditions (e.g., pH value, availability of iron(II) (Fe\({}^{2+}\)) and the presence of specific proteins that interact with myrosinase) [20; 21]. Several heavy metals were reported to exhibit inactivation of enzymatic reaction on different types of enzymes that are used especially in biosensors studies [22]. Saturated sodium chloride and calcium chloride solutions are known to inactivate enzyme system and they are commonly used in flavor study [23; 24]. An alternative method to stop the enzyme reaction is to remove water by using anhydrous sodium sulfate [25]. However, myrosinase has so far been poorly studied, particularly concerning the volatile analysis and flavor study. Results of the preliminary experiments showed that the addition of metal salts may have different consequences such as decreasing or stopping of enzyme activity, change of breakdown pathways, or salting-out effect. Silver and iron(III) ions are enzyme reaction inhibitors as heavy metals. Silver ions completely block the action of myrosinase, even at a concentration of \(5\times 10^{-5}\) mol/L, regardless of the glucosinolate used. Their action can be explained by the conformational change of myrosinase protein. Anhydrous sodium sulfate is also effective because it removes water without which the enzyme is inactive. A saturated solution of sodium chloride is a valuable inhibitor concerning aliphatic glucosinolates however, in the case of aromatic glucosinolates it is ineffective. A slight inhibitory effect with a similar tendency was observed in equimolar solutions of sodium and calcium ions. This effect can be explained by changing the enzyme's active center, which blocks the enzyme's activity only in relation to a specific substrate--the group of aliphatic glucosinolates. The increased average peak area of isothiyanates in samples with additional manganese salt is related to the salting-out effect. It is well known that iron(II) ions as well as the acidic environment promote the formation of nitriles, which is explained by the formation of a complex with glucosinolate. Copper also forms considerable amounts of nitriles because, like iron(II), it is a transition metal that willingly forms supramolecular structures. What is more, both the aqueous solution of iron(II) and copper(II) are acidic (pH \(<\) 7), because their salts are derived from strong acids and transition metals. Nevertheless, the previously tested aqueous solution of manganese chloride does not change the reaction pathway to nitriles, which suggests that when using transition metal salt ions, the formation of the complex is a more important factor than the pH value of the reaction medium. Interestingly, a saturated sodium chloride solution indeed blocked the possibility of obtaining isothiyanates, but nitriles were recorded instead. The use of saturated sodium chloride solution to inactivate the enzyme may falsely increase the proportion of nitriles in the analysis of volatile compounds directly from vegetables in the sample. Metal salts which do not form any volatile compounds such as isothiyanates and nitriles from the glucosinolate can be considered as a suitable blocking agent. An experiment with glucosinolate and selected metal salts was executed to show that nitriles can be formed without the enzyme presence. The highest amount of nitriles was obtained when iron(II) ions were used, which is consistent with current scientific reports [26; 27]. The presence of nitriles was also noted in samples with copper ions and a saturated sodium chloride solution. The absence of the enzyme in the samples tested suggests that the degradation of glucosinolates can be non-enzymatic.