In Vitro Stability of Phytase from Recombinant Bacteria E. Coli BL21 (DE3) EAS1-AMP

The objective of the research was to inquire the Km, Vm, activity, intracellular phytase stability exposed to pH variation, temperature variation and protease (pepsin and pancreas) in vitro. The phytase was produced from recombinant bacteria E. coli BL21(DE3) EAS1-AMP using 1.5 mM IPTG as inducer. Intracellular enzyme was extracted via freeze shock and centrifugation. Pure enzyme was acquired through NI-NTA agarose column. The enzyme was then tested for Km, Vm, phytase activity and stability against pH, temperature and protease. Treatment levels for stability against protease were P0: without protease, P1: addition of pepsin, P2: addition of pepsin and pancreas, and the data were statistically analyzed using analysis of variance of one-way Completely Randomized Design. Crude intracellular phytase had Vm 6.39 υM/sec, Km 34.82 υM, and 277 units activity. Intracellular phytas was stable at pH 4–6 and 0–550 C. Protease level influenced the activity of intracellular phytase (P<0.05). Intracellular phytase was stable against pepsin but not pancreas. Keywords: Km, Vm, activity, intracellular phytase, pH, temperature, protease

β-Catenin is a pH sensor with decreased stability at higher intracellular pH.

β-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R-β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation

Stability of casein micelles cross-linked with genipin: a physicochemical study as a function of pH

Chemical or enzymatic cross-linking of casein micelles (CMs) increases their stability against dissociating agents. In this paper, a comparative study of stability between native CMs and CMs cross-linked with genipin (CMs-GP) as a function of pH is described. Stability to temperature and ethanol were investigated in the pH range 2.0-7.0. The size and the charge ($\zeta$-potential) of the particles were determined by dynamic light scattering. Native CMs precipitated below pH 5.5, CMs-GP precipitated from pH 3.5 to 4.5, whereas no precipitation was observed at pH 2.0-3.0 or pH 4.5-7.0. The isoelectric point of CMs-GP was determined to be pH 3.7. Highest stability against heat and ethanol was observed for CMs-GP at pH 2, where visible coagulation was determined only after 800 s at 140 $^\circ$C or 87.5% (v/v) of ethanol. These results confirmed the hypothesis that cross-linking by GP increased the stability of CMs.Comment: 5 pages, 2 figures, International Dairy Journal, 201

Hydrogen-bonded multilayer thin films and capsules based on poly(2-n-propyl-2-oxazoline) and tannic acid : investigation on intermolecular forces, stability, and permeability

In recent years, hydrogen-bonded multilayer thin films and capsules based on neutral and nontoxic building blocks have been receiving interest for the design of stimuli-responsive drug delivery systems and for the preparation of thin-film coatings. Capsule systems made of tannic acid (TA), a natural polyphenol, as a hydrogen bonding donor and poly(2-n-propyl-2-oxazoline) (PnPropOx), a polymer with lower critical solution temperature around 25 degrees C, as a hydrogen bonding acceptor are advantageous over other conventional hydrogen-bonded systems because of their high stability in physiological pH range, biocomparibility, good renal clearance, stealth behavior, and stimuli responsiveness for temperature and pH. In this work, investigations on the interactive forces in TA/PnPropOx capsule formation, film thickness, stability, and permeability are reported. The multilayer thin films were assembled on quartz substrates, and the layer-by-layer film growth was investigated by UV-vis spectroscopy, atomic force microscopy, and profilometry. Hollow capsules were fabricated by sequential coating of TA and PnPropOx onto CaCO3 colloidal particles, followed by template dissolution with a 0.2 M ethylenediaminetetraacetic acid solution. The obtained capsules and multilayer thin films were found to be stable over a wide pH range of 2-9. It is found that both hydrogen bonding and hydrophobic interactions are responsible for the enhanced stability of the capsules at higher pH range. Swelling followed by dissolution of the capsules was observed at a pH value lower than 2, while the capsules undergo shrinking at a pH value higher than 8 and finally transform into a particle-like morphology before dissolution. The TA/PnPropOx capsules reported here could be used as a temperature-responsive drug delivery system in controlled drug delivery applications

The oxalato complexes of titanium(IV)—I : Mononuclear Ti(OH)2(C2O4)22− in solution

Molecular weight, electrodialysis and anion exchange measurements between pH 1 and 4 showed that the titanyl oxalate anion is present in solution as mononuclear Ti(OH)2(C2O4)22 units. pH studies of solutions of (NH4)2TiO(C2O4)2·H2O in 0.5 M NaClO4 medium and computer evaluation and simulation by LETAGROP and HALTAFALL showed that the behaviour of such solutions can be simulated by applying stability constants of log β1 = 7.90 ± 0.02 and log β2 = 13.24 ± 0.07, when using hydrolysis constants given by Nazarenko et al.\ud \ud In an attempt to determine the stability constant β2 spectrophotometrically using an exchange method with pyrocatechol, a mixed ligand complex was found with a Ti:pyrocatechol:oxalate ratio of 1 : 1 : 1

Co-populated Conformational Ensembles of β(2)-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

Ordered assembly of monomeric human β(2)-microglobulin (β(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 °C), wild-type β(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, β(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of β(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of β(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid

Effect of Water Content on the Thermal Inactivation Kinetics of Horseradish Peroxidase Freeze-Dried from Alkaline pH

The thermal inactivation of horseradish peroxidase freeze-dried from solutions of different pH (8, 10 and 11.5, measured at 25 C) and equilibrated to different water contents was studied in the temperature range from 110 to 150 C. The water contents studied (0.0, 1.4, 16.2 and 25.6 g water per 100 g of dry enzyme) corresponded to water activities of 0.0, 0.11, 0.76 and 0.88 at 4 C. The kinetics were well described by a double exponential model. The enzyme was generally more stable the lower the pH of the original solution, and for all pH values, the maximum stability was obtained at 1.4 g water/100 g dry enzyme. Values of z were generally independent of water content and of the pH of the original solution, and in the range of 15–25 °C, usually found in neutral conditions, with the exception of the enzyme freeze dried from pH 11.5 and equilibrated with phosphorus pentoxide, where a z-value of the stable fraction close to 10 C was found

Functionalisation of pea protein by tryptic hydrolysis – Characterisation of interfacial and functional properties

With regard to applications in dispersed systems (i.e. emulsions), improving the poor solubility of pea protein in the pH-range applicable to foods (pH 3 to pH 7) is a prerequisite. To achieve this, a pea protein concentrate was produced on a lab scale using alkaline extraction and subsequent enzymatic hydrolysis to degrees of 2 and 4%. Solubility was improved and interfacial properties were influenced. All samples led to the formation of emulsions but displayed a tendency towards wider oil-droplet size distributions at pH close to the isoelectric point. Using microscopy, this increase could be attributed to the formation of aggregates, which in turn can be ascribed to lack of repulsion caused by the low absolute values of ζ-potentials. The same lack of repulsion led to stronger and more elastic interfacial films at pH 4 and 5 than at pH 7. Moreover, film strength increased significantly with increasing degree of hydrolysis. Dilatational experiments imply that hydrolysis enhances in-plane structural rearrangements. Thus, it is concluded that tryptic hydrolysis has the potential to improve the overall stability of emulsions.BMBF, 01EA1408C, Kompetenzcluster Ernährungsforschung: NutriAct - Ernährungsintervention für gesundes Altern, Technische Universität Berli