Abstract
Sulforaphane and other natural isothiocyanates released from the respective plant glucosinolates by the plant enzyme myrosinase (β-thioglucoside glucohydrolase) show extensive anticancer and antimicrobial effects. In this study, myrosinase from garden cress (Lepidium sativum) seeds was purified to electrophoretic homogeneity by a fast and easy strategy consisting of fractionation by isoelectric precipitation with ammonium sulphate (AS) and affinity chromatography using sulforaphane (SFN) attached to cellulose resin. The overall purification of enzyme with respect to crude extract was 169-fold and recovery of 37%. Under non-reducing conditions, two protein bands exhibiting myrosinase activity with masses of about 114 and 122 kDa, respectively, and a 58 kDa protein band with no activity were detected by SDS-PAGE and zymography on polyacrylamide gel. MALDI-Tof/Tof of tryptic fragments obtained from the respective protein bands detected sequence motifs homologous to the regions responsible for glycoside-substrate binding and similarities to members of the enzyme subfamilies β-glucosidases and myrosinases GH. The enzyme hydrolyzed both the natural (sinigrin, sinalbin, glucoraphanin) and the synthetic (p-nitrophenol-β-D-glucopyranoside (pNPG)) substrates. The highest catalytic activity of purified enzyme was achieved against sinigrin. The KM and Vmax values of the enzyme for sinigrin were found to be 0.57 mM, and 1.3 mM/s, respectively. The enzyme was strongly activated by 30 μM ascorbic acid. The optimum temperature and pH for enzyme was 50 °C and pH 6.0, respectively. The purified enzyme could be stored at 4 °C and slightly acidic pH for at least 45 days without a significant decrease in specific activity.
Highlights
Myrosinases (β-thioglucoside glucohydrolase, EC 3.2.1.147) are a family of enzymes with β-glucosidase activity that is involved in the cleavage of S-glycosyl and O-glycosyl bonds [1] and release toxic volatile compounds such as nitriles, epitionitriles, thiocyanates, isothiocyanates and others from various plant glucosinolates [2]
The results described above show that myrosinase from the seeds of L. sativum is able to split both types of β-glycosides, S- and O, at different levels, but the presence of the glucose moiety, and the β-configuration of the glycosidic linkage, formed between the
The main objective of this study was to develop an effective method for the purification of myrosinase from L. sativum
Summary
Myrosinases (β-thioglucoside glucohydrolase, EC 3.2.1.147) are a family of enzymes with β-glucosidase activity that is involved in the cleavage of S-glycosyl and O-glycosyl bonds [1] and release toxic volatile compounds such as nitriles, epitionitriles, thiocyanates, isothiocyanates and others from various plant glucosinolates [2]. Myrosinase and glucosinolates are physically separated from each other and are stored either in different cell compartments [3] or different plant tissues [4] to avoid an undesirable hydrolysis. Myrosinase and glucosinolates are a prominent part of the defense mechanism that is activated by the mechanical damage of plants caused by herbivores or insects [5]. Glucosinolates and their hydrolytic products are involved in allelopathic interactions among plants, which are applied in the agriculture industry and ecological farming for crop protection [6,7].
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