Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non-covalent immobilization to poly(vinylimidazole)/clay hydrogel.

The characterization of the hydrogel was performed using Fourier-transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7-derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K m, V m, and k cat of immobilized enzyme were 4.4, 1.7, and 7.5-fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG≠ (activation free energy) and ΔG≠ E-T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG≠ ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2-fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS≠ value implied that the immobilized form of the enzyme was more ordered than its free form.

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.

Water miscible mono alcohols' effect on the proteolytic performance of Bacillus clausii serine alkaline protease.

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the Vm, kcat and kcat/Km values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the Km value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (KI) were in the range of 1.32-3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG(≠) and ΔG(≠)E-T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG(≠)ES value of the enzyme remained almost the same. The constant Km and ΔG(≠)ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The kcat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG(≠) and ΔG(≠)E-T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

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

In this study chick pea ß-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 ß-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 ß-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 ß-galactosidase from chick pea, and has a big potential use for industrial applications.

Three-phase partitioning as a rapid and easy method for the purification and recovery of catalase from sweet potato tubers (Solanum tuberosum).

Three-phase partitioning (TPP) was used to purify and recover catalase from potato crude extract. The method consists of ammonium sulfate saturation, t-butanol addition, and adjustment of pH, respectively. The best catalase recovery (262 %) and 14.1-fold purification were seen in the interfacial phase in the presence of 40 % (w/v) ammonium sulfate saturation with 1.0:1.0 crude extract/t-butanol ratio (v/v) at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the enzyme showed comparatively purification and protein molecular weight was nearly found to be 56 kDa. This study shows that TPP is a simple, economical, and quick method for the recovering of catalase and can be used for the purification process.