# Inactivation of Thioglucidase from _Sinapis alba_ (White Mustard) Seed by Metal Salts
## 2 Results  
### Preliminary Experiments with Commercial Thioglucidase  
A preliminary experiment relied on analyzing volatile glucosinolate breakdown products in aqueous system. To select the most effective agent for the inactivation of thioglucidase, the volatile products were determined by GC/MS system. The peak area represents the total ion current (TIC). As shown in Figure 1a not all of the investigated metal salts were effective at blocking myrosinase, resulting in a similar amount of obtained isothiyanates as in the control test (H\({}_{2}\)O). Manganese ions proved to be completely ineffective at blocking the enzymatic reaction for both glucosinolates, glucotropeaolin, and synigrin. The adequate molar concentration of calcium and sodium ions and saturated solution of sodium chloride exhibited inhibition effect for synigrin, nevertheless they were ineffective in the case of blocking glucotropeaolin hydrolysis. The maximum efficiency in inhibition of enzyme system was noticed for iron ions (Fe\({}^{2+}\) and Fe\({}^{3+}\)), silver ions, saturated solution of calcium chloride, and anhydrous sodium sulfate. The use of these salts did not produce volatile isothiyanates for both glucotropeaolin and synigrin. However, some of the potential enzyme-blocking agents produced volatile nitriles, for example iron(II) and saturated calcium chloride. Except for these salts, copper(II) ions show the ability to produce a considerable amount of nitriles (Figure 1b). Previous preliminary experiments show that silver ions were one of the most effective methods to inactivate myrosinase enzymatic activity resulting in no volatile products derived from glucosinolate. Figure 2a shows silver ions dependent inhibition of sinigrin hydrolysis curve where concentration is presented as a logarithm function. The same experiment was performed on glucotropeaolin glucosinolate (Figure 2b). The average peak areas were measured in extracted-ion chromatogram  
Figure 1: Volatile products of glucosinolates breakdown in miscellaneous enzyme inactivation media; sat.—saturated solution; anh.—anhydrorous; a, b, c, d, e—the result of statistical analysis (one-way ANOVA Duncan test as post-hoc), values followed by the same letter in a Duncan grouping are not significantly different, the subscript number and letter color are corresponding to the chart legend (\(\alpha\) = 0.05); (n = 3); (a) Isothiocyanates (isothiocyanatomethylbenzene derived from glucotropaeolin and 3-isothiocyanatoprop-1-ene is a product from sinigrin); (b) Nitriles (2-phenylacetonitrile from glucotropaeolin and but-3-enenenitrile from sinigrin). mode (EIC) for products of enzymatic hydrolysis: 3-isothiocyanatoprop-1-ene (_m_/_z_ = 99.2) and for isothiyanato methylbenzene (_m_/_z_ = 91.2). The silver ions were an effective inhibition factor for the inactivation of thioglucidase for both glucosinolate sinigrin and glucotropeaolin. The lowest concentration of silver salt, which is effective in blocking thioglucidase enzyme activity was 5 \(\times\) 10\({}^{-5}\) mol/L. Results obtained for the lowest investigated concentration of silver salt (5 \(\times\) 10\({}^{-20}\) mol/L) equals average peak areas measured for the control sample (water, enzyme, sinigrin, or glucotropeaolin). Results obtained from the first experiment indicated that the favored volatile products for some enzyme-blocking agents (metal salts) are nitriles. An investigation of the interaction between glucosinolate and inactivation agent to investigate non enzymatic reaction pathway was performed. To this stage, metal salts were selected with the best ability in blocking enzymatic hydrolysis of glucosinolates. In that part of the study, volatile compounds after the addition of glucosinolate to an aqueous solution of metal salts without enzyme (thioglucidase from _Sinapis alba_) addition were measured. The control sample was water and glucosinolate (sinigrin potassium salt or glucotropaeolin potassium salt) in which no volatiles were produced.