Akgeyik, Aytul Uzun; Akyildiz, Esma; Karaoglu, Hakan; Beris, Fatih Saban

β-glucosidases belong to the class of glycoside hydrolases (GHs) and can mediate the cleavage of β-1-4 glycosidic bonds in alkyl or aryl-β-D-glucosides, amino, cyanogenic glucosides, and oligo-or disaccharides. The gene encoding β-glucosidase of Anoxybacillus flavithermus DSM2641T was subcloned and expressed in E. coli BL21 Star(DE3) cells with pET100 Directional TOPO Expression Kit. The recombinant AFBGL was purified by a Ni-NTA affinity chromatography. The enzyme was 53 kDa and had 5.13 × 105 µmol/min/mg protein of Vmax and 422.3 µM of Km towards p-nitrophenyl β-D-glucoside as a main substrate. The optimum temperature and pH of the purified enzyme were 65°C and 6.8, respectively. We also determined kinetic parameters of AFBGL against the other artificial substrats, pNPC, pNPX, and oNPG. The enzyme had the Vmax and Km for 5.13 × 105 µmol/min/mg protein and 0.42 mM for pNPG, 0.007 µmol/min/mg protein and 0.19 mM for pNPC, 0.01 µmol/min/mg protein and 11.97 mM for pNPX, 0.06 µmol/min/mg protein and 0.134 mM for oNPG, respectively. AFBGL's substrate affinity was found to be more directed towards oNPG. Although 0.5 mM Mg2+ increases the enzyme's activity 1.6-fold, higher concentrations of Mg2+ and the presence of K+, Fe2+, Mn2+, Zn2+, Cu2+, and Al3+ decreases its activity. AFBGL retained 50% of the initial activity towards pNPG for 25 min and the activity was not detected after 225 min. According to amino acid motifs, AFBGL belongs to family 1 of glycosylhydrolase and 4/7 super family. So, AFBGL is a member of GH1, as the amino acid sequence contained a GH1 specific domain.

Kural, Birgül Vanizor; Küçük, Nurçin; Yücesan, Fulya Balaban; Örem, Asim

Of Brassicaceous plants, kale (Brassica oleraceae L. var. acephala DC) contains polyphenols, flavonoids, isoflavones and glucosinolates and so has antioxidant and anticarcinogenic properties. Antioxidants inhibit negative effects of free radicals and may, therefore, protect tissues against oxidative damage. Oxidation of lipoproteins is a key event in the development of atherosclerosis. In the current study, the levels of total phenolic and flavonoid contents and total antioxidant capacity of methanolic and aqueous extracts of kale leaves were determined. In addition, the susceptibility of isolated lipoproteins - very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to the Cu 2+-induced oxidation with various concentrations of metanolic and aqueous extracts was evaluated as t-lag values. Although aqueous extract had higher total antioxidant capacity, methanolic extract had higher total phenolic and flavonoid content (P<0.05). On the other hand, both extracts inhibited lipid peroxidation in both isolated VLDL and LDL. Inhibitory effect of extracts or increasing t-lag values, mainly in methanolic extract was found to be related to increasing the concentration of extracts. It was concluded that because of high antioxidant capacity and phenolic content, kale showed a protective effect on the oxidation of lipoproteins. Therefore, it may be speculated that kale consumption may play an important protective role in the cardiovascular and other related diseases resulting from imbalance of oxidant and antioxidant status.