# Inactivation of Thioglucidase from _Sinapis alba_ (White Mustard) Seed by Metal Salts
## 2 Results
### Study of the Effect of Metal Salts on the Native Enzyme in Kohlrabi Tissue  
There is no doubt that plant tissue is a different environment than water, that is why the behavior of glucosinolates (absent in kohlrabi and used previously in the model experiment) was investigated in a model matrix in which this class of compounds occur naturally. The same batches of kohlrabi were used for established volume (3 mL) of aqueous metal salts solutions or sufficient amount of anhydride sodium sulfate salt. The control sample was water and glucosinolate (sinigrin potassium salt or glucotropaeolin potassium salt) without any metal salt addition. Compared to the model experiment, a larger volume of salt solution was used--allowing the complete immersion of plant tissue in the aqueous solution and an adequately increased amount of glucosinolate. It was observed that kohlrabi samples after extraction and analysis differed depending on the metal salt used for the analysis. Results of GC/MS analyses are shown in Figure 4.  
The main volatiles derived from glucosinolates in various environmental conditions in the kohlrabi matrix are presented in Table 1. But-3-enemitrile was formed only in samples with addition of synigrin with the presence of iron(II) and copper(II) ions. The most abundant peak for 3-isothiocyanatoprop-1-ene was recorded in control sample (H\({}_{2}\)O), low amounts of this isothiamate were also noted for a saturated solution of calcium chloride and anhydrous sodium sulfate. Iron(III) and silver ions caused the complete blockage of the enzyme that hydrolyzes the added synigrin. Figure 4: Kohlrabi matrix samples with addition of sinigrin potassium salt after GC/MS analysis (**a**) control (H\({}_{2}\)O); (**b**) iron(II) sulfate (FeSO\({}_{4}\)); (**c**) iron(III) sulfate (Fe\({}_{2}\)(SO\({}_{4}\))\({}_{3}\)); (**d**) saturated solution of calcium chloride (CaCl\({}_{2}\)); (**e**) silver nitrate (AgNO\({}_{3}\)); (**f**) copper sulfate (CuSO\({}_{4}\)); (**g**) anhydrous sodium sulfate salt (Na\({}_{2}\)SO\({}_{4}\)). Figure 3: Volatile products from the interaction between glucosinolate (sinigrin or glucotropaeolin) and an enzymatic reaction blocking agent (without enzyme presence); 2-phenylacetonitrile (_m/z_ = 117.2); but-3-enemitrile (_m/z_ = 41.3); (a, b, c—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). The presence of 2-phenylacetonitrile was not recorded in the sequence with the addition of synigrin, while it was detected in the samples with the addition of glucotropaeolin. The highest amount of 2-phenylacetonitrile was found for copper(II) ions, half less when iron(II) ions were used, and negligible amounts were found in the calcium chloride and iron(III) solutions. Isothiocyanatomethylbenzene was also found only in the samples to which glucotropaeolin was added. However, the presence of this isothiocyanate was not confirmed in the samples with the addition of synigrin. The only peak for isothicyanatomethylbenzene was in the control sample (H\({}_{2}\)O). The profile of 4-methylsulfonylbutanenitrile and 5-methylsulfanylpentanenitrile average peak area depending on various environmental conditions for samples with the addition of synigrin and glucotropaeoline was similar. The higher content of 4-methylsulfanylbutanenitrile than the control was observed in both samples with the addition of sinigrin and glucotropaeolin in aqueous solutions of copper(II) sulfate, and iron(II) salts. In aqueous solution of silver salt 4-methylsulfanylbutanenitrile was not found. Concerning the control sample, an increased amount of this compound is observed in the presence of copper ions, while in the medium of silver and iron(III) ions, the presence of this nitrile was not observed.