Purification and Characterization of a Novel Redox-Regulated Isoform of Myrosinase (β-Thioglucoside Glucohydrolase) from Lepidium latifolium L.

Myrosinase (ExPASy entry EC 3.2.1.147) is involved in the hydrolysis of glucosinolates to isothiocyanates, nitriles, and thiocyanates that are responsible for various ecological and health benefits. Myrosinase was purified from the leaves of Lepidium latifolium, a high-altitude plant, to homogeneity in a three-step purification process. Purified enzyme exists as dimer in native form (∼160 kDa) with a subunit size of ∼70 kDa. The enzyme exhibited maximum activity at pH 6.0 and 50 °C. With sinigrin as substrate, the enzyme showed <i>K</i>_m and <i>V</i>_max values of 171 ± 23 μM and 0.302 μmol min^–1 mg^–1, respectively. The enzyme was found to be redox-regulated, with an increase in <i>V</i>_max and <i>K</i>_cat in the presence of GSH. Reduced forms of the enzyme were found to be more active. This thiol-regulated kinetic behavior of myrosinase signifies enzyme’s strategy to fine-tune its activity in different redox environments, thus regulating its biological effects

Percentage (%) fatty acid composition of <i>Lepidium latifolium</i> from different locations of Ladakh.

<p>C12:0, lauric acid; C14:0, myristic acid; C15:0, pentadecanoic acid; C16:0, palmitic acid; C16:1 n-7, palmitoleic acid; C18:0, stearic acid; C18:1 n-9, oleic acid; C18:2 n-6, linoleic acid; C18:3 n-3, linolenic acid; SFA, saturated fatty acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Cox: calculated oxidizability value. Each value is a mean of a duplicate analysis performed on different samples.</p

Total crude protein as observed in Kargil (□), Leh (∥∥) and Nyoma (▪) using Bradford reagent.

<p>Means with the different letter represents statistically significant values at <i>p≤0.05</i> using ANOVA.</p

Position wise analysis of total protein, phenols, flavanoids and antioxidant capacity of leaves and roots in <i>Lepidium latifolium</i>.

<p>mg g-1 FW: mg protein (BSA) per gram fresh weight; mg GAE g⁻¹: milligram gallic acid equivalents per gram dry weight of sample; mg QE g⁻¹: milligram quercetin equivalents per gram dry weight of sample; Each value is a mean of all the values obtained for similar position at different locations. * represents statistically significant values among columns at <i>p≤0.05</i></p

Relative proportion of methanolic fraction in different position of leaves and roots of <i>Lepidium latifolium</i> expressed as percentage of total amount of crude extract used for the fractionation (n = 3).

<p>Each value is a mean of a triplicate analysis performed on different samples.</p

Antioxidant capacity in <i>Lepidium latifolium</i> suggested by total phenols, total flavanoids, reducing and chelation power in different leaf positions and roots at three different locations of Ladakh.

<p>mg GAE g⁻¹: milligram gallic acid equivalents per gram dry weight of sample; mg QE g⁻¹: milligram quercetin equivalents per gram dry weight of sample; <i>A₇₀₀</i>: absorbance at 700 nm. Each value is a mean of a triplicate analysis performed on different samples. * represents statistically significant values among rows whereas, different letters represents significant statistical variations at <i>p≤0.05</i> among columns.</p

Elemental analysis in leaves and roots of <i>Lepidium latifolium</i> from three different locations of Ladakh.

<p>%C: percentage carbon; %H: percentage hydrogen; %N: percentage nitrogen; %S: percentage sulfur; N:S: nitrogen to sulfur ratio. Each value is a mean of a duplicate analysis performed on different samples.</p

Locations from where the plants of Lepidium latifolium were collected from Ladakh region of Jammu and Kashmir State, India; Kargil (▪), Leh (•), Nyoma (▴). The figure is for illustrative purposes only.

<p>Locations from where the plants of Lepidium latifolium were collected from Ladakh region of Jammu and Kashmir State, India; Kargil (▪), Leh (•), Nyoma (▴). The figure is for illustrative purposes only.</p

Metabolic content of eight reference glucosinolates in leaves of <i>Lepidium latifolium</i> at three different locations of Kargil, Leh and Nyoma.

<p>* represents statistically significant values at <i>p≤0.05</i>.</p

Antioxidant activity of methanolic plant extracts of <i>Lepidium latifolium</i> at Kargil (□), Leh (∥∥) and Nyoma (▪).

<p>Panel ‘A’ represents superoxide, panel ‘B’ represents hydroxyl and panel ‘C’ represents DPPH radicals. Antioxidant activity was represented as % inhibition of respective free radicals * represents statistically significant values at <i>p≤0.05</i>.</p