Myrosinases (thioglucosidases) catalyze the hydrolysis of a class of compounds called glucosinolates, of which the aglycones show various biological functions. It is often necessary to minimize the loss of myrosinase activity during thermal processing of cruciferous vegetables. Myrosinase was isolated from a popular spice, white mustard (Sinapis alba), and its thermal inactivation kinetics was investigated. The enzyme was extracted from white mustard seeds and purified by a sequential processes of ammonium sulfate fractionation, Concanavalin A-Sepharose column chromatography, and gel permeation chromatography. At least three isozymes were revealed by Concanavalin A-Sepharose column chromatography. The purity of the major myrosinase was examined by native polyacrylamide gel electrophoresis and on-gel activity staining with methyl red. The molecular weight of the major enzyme was estimated to be 171 kDa. When the consecutive step model was used for the thermal inactivation of the major myrosinase, its inactivation energy was 44.388 kJ/mol for the early stage of destruction and 32.019 kJ/mol for the late stage of destruction. When the distinct two enzymes model was used, the inactivation energy was 77.772 kJ/mol for the labile enzyme and 95.145 kJ/mol for the stable enzyme. The thermal inactivation energies lie within energy range causing nutrient destruction on heating.

Many insects are able to feed on crucifers despite the presence of a potent activated defense system known as the mustard oil bomb. In damaged tissue, mustard oil glucosides (glucosinolates) are hydrolyzed by the enzyme myrosinase to form toxic mustard oils (isothiocyanates). Here, we analyzed how the the cabbage stem flea beetle Psylliodes chrysocephala, a key pest of oilseed rape, copes with this chemical defense. First, we found that P. chrysocephala prevents the activation of ingested glucosinolates by two different strategies, a) by sequestering glucosinolates and b) by converting glucosinolates to desulfo-glucosinolates. Our next aim was to identify the sulfatase enzyme(s) responsible for the detoxification of glucosinolates in P. chrysocephala. Nine arylsulfatase-like genes were identified in the transcriptome of P. chrysocephala, and five of them showed glucosinolate sulfatase activity upon heterologous expression in Sf9 cells. By using RNAi, we confirmed that PcGSS1 and PcGSS2 are active towards benzenic and indolic glucosinolates in P. chrysocephala adults in vivo. However, in feeding experiments, the proportion of sequestered and desulfated glucosinolates ranged from 26 to 35% which suggests that these strategies alone are likely not sufficient to overcome the chemical plant defense. Indeed, P. chrysocephala additionally conjugates isothiocyanates to glutathione and metabolizes them via the conserved mercapturic acid pathway. In summary, the cabbage stem flea beetle avoids isothiocyanate formation by specialized strategies (sequestration and desulfation), but also relies on a conserved detoxification pathway to prevent toxicity of isothiocyanates.

돌산갓의 독특한 향미와 잠재된 항균성을 김치의 맛과 저장성 향상에 이용하기 위한 기초자료로서 갓의 myrosinase를 분리 정제하여 그 특성을 밝히고, 갓김치 숙성 중 myrosinase 활성도 변화를 측정하였다. 갓의 myrosinase를 DEAE Sephadex, chromatofocusing 및 Con A Sepharose column chromatography에 의해 정제한 결과 비활성은 7107배 증가하였고 수율은 18.8%였다. 정제된 효소의 최적 pH는 5.9였으며, 등전점은 4.6, 분자량은 약 129 kD, Km은 0.206 mM, Vmax는 2.039 μM·min-1·mg protein-1로 나타났다. 또한 myrosinase의 activator인 ascorbic acid는 0.6 mM에서 최대 효소활성을 보이다가 그 이후는 점차 효소활성의 감소를 보여 2.0 mM 이상의 농도에서는 효소활성을 거의 완전히 상실시켰다. 갓김치의 저장 중 myrosinase 활성 변화를 측정한 결과 김치 제조 직후에 약 70 nmol/min/mg protein이던 것이 20℃에서 3일 이상 저장으로 급격히 그 활성을 잃어 4일 후에는 50% 이상의 활성을 손실하고 10일 후에는 거의 활성이 없었다.