Yonca Avcı Duman

Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique.

In this study chick pea β-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 10.1 purification fold with 133% recovery of β-galactosidase. SDS-PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50°C. Enzyme was further characterized by the Arrhenius activation energy and Michael-Menten kinetic constants. Activation energy (Ea) was calculated by using Arrhenius equation and determined to be 15.52kcalmol(-1). Km value of purified enzyme was estimated for the o-nitrophenyl β-d galactopyranoside (ONPG) substrate as 1.09mM, while its maximum velocity, Vm was 0.90U/mL/min at 37°C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of β-galactosidase from chick pea, and has a big potential use for industrial applications.

Aqueous two-phase (PEG4000/Na2SO4) extraction and characterization of an acid invertase from potato tuber (Solanum tuberosum).

Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development, and stress responses. To study their isolation and purification from potato, an attractive system useful for the separation of biological molecules, an aqueous two-phase system, was used. The influence of various system parameters such as type of phase-forming salts, polyethylene glycol (PEG) molecular mass, salt, and polymer concentration was investigated to obtain the highest recovery of enzyme. The PEG4000 (12.5%, w/w)/Na2SO4(15%, w/w) system was found to be ideal for partitioning invertase into the bottom salt-rich phase. The addition of 3% MnSO4 (w/w) at pH 5.0 increased the purity by 5.11-fold with the recovered activity of 197%. The Km and Vmax on sucrose were 3.95 mM and 0.143 U mL−1 min−1, respectively. Our data confirmed that the PEG4000/Na2SO4 aqueous two-phase system combined with the presence of MnSO4 offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics of temperature and pH stability for potato invertase prepared from an ATPS make the enzyme a good candidate for its potential use in many research and industrial applications.

Analysis of plant growth and biochemical parameters in Amsonia orientalis after in vitro salt stress

This study aimed to better understand the limited natural distribution of the endangered ornamental plant Amsonia orientalis Decne. by focusing on salt stress, a common limiting factor of plant growth. Plants were subjected to in vitro salt stress at concentrations between 25 and 150 mM. In general, shoot and root lengths, root number, and total protein, chlorophyll a and carotenoid content were negatively influenced at NaCl concentrations above 25 mM. Hydrogen peroxide, malondialdehyde and proline content all gradually increased with increasing salt concentration. Activity levels of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were all at their maximum in plants cultured in medium containing 50 mM NaCl. Compared to control cultures, an overall upward trend in POD activity was observed with increasing salt concentration, while the activity levels of SOD and CAT increased at lower concentrations but were limited at elevated concentrations of NaCl. These results suggest that A. orientalis prefers soils with no or very low salt but can tolerate NaCl up to a concentration of 50 mM.

Purification of peroxidase from Amsonia orientalis by three-phase partitioning and its biochemical characterization

ABSTRACT The present work describes the purification and characterization of peroxidase from the medicinal plant, Amsonia orientalis, for the first time. The activity recovery for peroxidase was 162% with 12.5-fold purification. Optimal purification parameters were 20% (w/v) (NH4)2SO4 saturation at pH 6.0 and 25°C with 1.0:1.0 (v/v) ratio of crude extract to t-butanol ratio for 30 min. The molecular mass of the enzyme was found to be ca. 59 kDa. Peroxidase showed Km values of 1.88 and 2.0 mM for pyrogallol and hydrogen peroxide, respectively. FeSO4, CuSO4, HgCl2, MnSO4 and MgSO4 did not inhibit the enzyme activity.

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

ABSTRACT Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.