Juglans regia and Ribes nigrum as potential nutraceuticals: Source of thermostable superoxide dismutase enzyme.

In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%-40% and 40%-80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%-40%) exhibited purification level of 3.09- (W) and 3.22- (M) fold with specific activity 79.32 Umg-1 (W) and 125.23 Umg-1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33 mM (W) and 2.86 mM (M), whereas the activation energy (Ea ) calculated as 24.52 KJ mol-1 (W) and 26.25 KJ mol-1 (M). Na+ , Mn2+ , and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7 mM (W) and 0.9 mM (M), suggesting the more prominent binding affinity and effectiveness. PRACTICAL APPLICATIONS: Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day-to-day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal-containing enzymes that act as a main line of defense against oxidative stress. Antioxidant-based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.

Polyphenol metabolism in differently colored cultivars of red currant (Ribes rubrum L.) through fruit ripening.

MAIN CONCLUSION: Rare red currants colors caused by low anthocyanin content in the pink and a lack of anthocyanins in the white cultivar correlated with low ANS gene expression, enzyme activity, and increased sugar/acid ratios. Changes in the contents of polyphenols, sugars, and organic acids in berries of the three differently colored Ribes rubrum L. cultivars ('Jonkheer van Tets', 'Pink Champagne', and 'Zitavia') were determined by LC-MS and HPLC at 4 sampling times during the last month of fruit ripening. The activities of the main flavonoid enzymes, chalcone synthase/chalcone isomerase (CHS/CHI), flavanone 3-hydroxylase (FHT), and dihydroflavonol 4-reductase (DFR), and the expression of anthocyanidin synthase (ANS) were additionally measured. Despite many attempts, activities of flavonol synthase and glycosyltransferase did not show reliable results, the reason of which they could not be demonstrated in this study. The pink fruited cultivar 'Pink Champagne' showed generally lower enzyme activity than the red cultivar 'Jonkheer van Tets'. The white cultivar 'Zitavia' showed very low CHS/CHI activity and ANS expression and no FHT and DFR activities were detected. The DFR of R. rubrum L. clearly preferred dihydromyricetin as substrate although no 3',4',5'-hydroxylated anthocyanins were present. The anthocyanin content of the red cultivar slightly increased during the last three weeks of ripening and reached a maximum of 890 mg kg-1 FW. Contrary to this, the pink cultivar showed low accumulation of anthocyanins; however, in the last three weeks of ripening, their content increased from 14 to 105 mg kg-1 FW. Simultaneously, the content of polyphenols slightly decreased in all 3 cultivars, while the sugar/acid ratio increased. The white cultivar had no anthocyanins, but the sugar/acid ratios were the highest. In the white and pink cultivars, reduction/lack of anthocyanins was mainly compensated by increased relative concentrations of hydroxycinnamic acids and flavonols.

The role of C-terminal amino acid residues of a Δ6-fatty acid desaturase from blackcurrant.

Δ6-fatty acid desaturase is an important enzyme in the catalytic synthesis of polyunsaturated fatty acids. Using domain swapping and a site-directed mutagenesis strategy, we found that the region of the C-terminal 67 amino acid residues of Δ6-fatty acid desaturase RnD6C from blackcurrant was essential for its catalytic activity and that seven different residues between RnD6C and RnD8A in that region were involved in the desaturase activity. Compared with RnD6C, the activity of the following mutations, V394A, K395I, F411L, S436P, VK3945AI and IS4356VP, was significantly decreased, whereas the activity of I417T was significantly increased. The amino acids N, T and Y in the last four residues also play a certain role in the desaturase activity.

Identification and functional analysis of the genes encoding Delta6-desaturase from Ribes nigrum.

Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Phosphoenolpyruvate carboxykinase and its potential role in the catabolism of organic acids in the flesh of soft fruit during ripening.

Previous studies of grapes and tomatoes have shown that the abundance of phosphoenolpyruvate carboxykinase (PEPCK) increases in their flesh at the start of ripening, and that this coincides with a decrease in its citrate and/or malate content. Thus, PEPCK might function in the catabolism of organic acid anions during the ripening of these fruits. In the present study, the abundance of PEPCK was determined in the flesh of blueberries, raspberries, red currants, and strawberries at different stages of their development. In addition, changes in the amounts of citrate, malate, soluble sugars, isocitrate lyase, NADP-malic enzyme, phosphoenolpyruvate carboxylase, and pyruvate, orthophosphate dikinase in the flesh were determined. PEPCK was not detected in strawberry flesh, in which there was no dissimilation of malate or citrate. In the flesh of the other fruits, the abundance of PEPCK increased during ripening to an amount that was similar to that in grapes and tomatoes. In the flesh of blueberries and red currants, PEPCK was most abundant when there was dissimilation of malate. In the flesh of raspberries, PEPCK was most abundant when there was dissimilation of malate and citrate. These results are consistent with PEPCK playing a role in the dissimilation of citrate and/or malate in the flesh of these fruits during ripening. However, PEPCK was also present in the flesh of blueberries, raspberries, and red currants when there was no dissimilation of malate or citrate, and this raises the possibility that PEPCK might have additional functions. Dissection of blueberries provided evidence that both PEPCK and phosphoenolpyruvate carboxylase were present in the same cells, and possible functions for this are discussed.