Sulphoraphane Affinity-Based Chromatography for the Purification of Myrosinase from Lepidium sativum Seeds

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.

The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba

“…The catalytic ability of crude Myr preparation to decompose GRN and form SFN was confirmed by an RP-HPLC analysis. We observed almost a 100% conversion of GRN to SFN under the used experimental conditions (Figure 3) at a reaction rate comparable to that measured with a purified enzyme [63,89,90]. Based on this observation, it can be concluded that the isolated GRN and the crude Myr preparation represent a suitable GLS-Myr system for the generation of SFN in biological samples under in situ conditions.…”

“…The essential task was to find out if an appropriate amount of free SFN is generated during the enzymatic cleavage of GRN, which could exhibit any biological effects. We subjected the isolated GRN to enzyme hydrolysis with a crude Myr preparation obtained from swollen garden cress seeds by isoelectric precipitation with ammonium sulfate [63]. Although Myr preparation is not an electrophoretically homogeneous enzyme, it has a few advantages against purified enzymes [63], such as easy and fast preparation with a high yield and better storage stability at 4 • C (Figure S5, Supplementary Material).…”

“…We subjected the isolated GRN to enzyme hydrolysis with a crude Myr preparation obtained from swollen garden cress seeds by isoelectric precipitation with ammonium sulfate [63]. Although Myr preparation is not an electrophoretically homogeneous enzyme, it has a few advantages against purified enzymes [63], such as easy and fast preparation with a high yield and better storage stability at 4 • C (Figure S5, Supplementary Material). The catalytic ability of crude Myr preparation to decompose GRN and form SFN was confirmed by an RP-HPLC analysis.…”

“…To release the aglycone from GRN, we used a crude preparation enriched with Myr from garden cress seeds obtained by precipitation of the seed homogenate with ammonium sulfate [63]. This preparation showed an activity of 0.2 ± 0.02 µmol/(min × mg).…”

“…This preparation showed an activity of 0.2 ± 0.02 µmol/(min × mg). Myr can be further purified practically to electrophoretic homogeneity by affinity chromatography with immobilized SFN [63]. However, for the effective release of SFN from GRN, such a highly pure preparation is not necessary, and the crude preparation obtained by precipitation with ammonium sulfate was sufficient.…”