pH Stability Profiles Research Articles

Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2

Poly-3-hydroxybutyrate (P3HB) degradation capabilities of a novel bacterium, Anoxybacillus gonensis G2, were investigated. Both changes on film surfaces of the solution-cast films monitored by scanning electron microscopy (SEM) and weight loss up to 24% after 72 h exposure to A. gonensis G2 cultures indicated secretion of an active esterase responsible for the degradation of P3HB films. Kinetic parameters, V max and K m for the esterase activity of crude enzyme from A. gonensis G2 in the presence of p-nitrophenylbutyrate as substrate were observed as 50 U/L and 0.125 mM, respectively, in 50 mM phosphate buffer, pH 7.5 at 60 °C. The stimulation of the activity by Ca 2+ is an evidence for the requirement of Ca 2+ as a cofactor for the enzyme activity which is a characteristic for lipases/esterases. Inhibition of the esterase activity by metal chelating agents such as ethylenediamine tetraacetate, azide and cyanide has also supported the requirement of a metal ion for the activity. The thermal and pH stability profiles for the enzyme showed that the thermophilic bacterium A. gonensis G2 secretes an extracellular thermoalkalophilic PHB depolymerase active at 60 °C, and stable at this temperature for 120 min at pH 7.5 and for 24 h at pH 7.5–9.5 range at 4 °C by retaining over 75% of its initial activities.

Diphenolases from two cultivars of cherry laurel ( Laurocerasus officinalis Roem.) fruits at an early stage of maturation

Diphenolases from two cherry laurel cultivars ( Laurocerasus officinalis Roem. ‘Globigemmis’ and ‘Oxygemmis’) were highly active against 3-(3,4-dihydroxyphenyl)propionic acid (DHPPA) at acidic pH values with temperature optima of 50 and 40 °C, respectively. Although the pH-stability profiles showed that both enzymes were fully stable at pH 7.0, their stabilities decreased significantly at alkaline pH values. Thermal-stabilities of the cherry laurel diphenolases indicated that enzymes from the two cultivars share similar thermodynamic properties and heat-sensitivities as a result of heat-inactivation. In addition, ascorbate and metabisulfite, at 1 mM final concentrations, almost completely inhibited the oxidation of DHPPA by the enzymes, indicating the sensitivities of the cherry laurel diphenolases from the two cultivars towards general Polyphenol oxidases inhibitors. It can be concluded that the crude enzymes prepared from the cherry laurel fruits of the two cultivars, at an early stage of development, possess diphenolase activities sharing similar behaviours.

High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZαA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPα-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4 L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000 U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000 U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter λP L, yielding 600 U/g or 54,000 U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.

Characterization of the interactions between polymethacrylate‐based aqueous polymeric dispersions and aluminum lakes

Instability in film coating formulations can arise from interactions between aluminum lake pigments and aqueous polymeric dispersions. The purpose of this study was to characterize the interactions between three polymethacrylate‐based aqueous polymeric dispersions (Eudragit® RS 30 D, Eudragit® L 30 D‐55, and Eudragit® NE 30 D) and aluminum lakes. Particle size measurements, pH stability profiles, zeta potential measurements, and microscopy were used to study mixed dispersions of the polymeric latices and the lakes. Interactions leading to dispersion instability were related to the surface charge of the components in the formulation. Interactions between the ionic polymers and the lakes arose from instability of the lakes outside a certain pH range resulting in the release of electrolytes, which led to aggregation of the polymeric particles. Interactions between the nonionic polymer and the lakes were related to the polymer modifying the surface charge of the lakes, resulting in aggregation of the pigment particles. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1937–1947, 2001

Titration calculations of foot-and-mouth disease virus capsids and their stabilities as a function of pH

Foot-and-mouth disease virus (FMDV), a non-enveloped picornavirus, is sensitive to acidic conditions. At pH values below 7 the icosahedral virus capsid, formed from 60 copies of a protomer containing four polypeptides (VP1 to 4), dissociates into 12 pentamers, releasing the viral RNA. Evidence suggests that this acid lability may assist FMDV cell entry via an endosomal pathway. Calculations of titration curves and pH-stability profiles are presented for three different strains of FMDV, O1BFS, A1061 and A22 Iraq, and compared with experimental data for complete virions and empty capsids (which lack RNA). The finite difference Poisson-Boltzmann method was used for the calculation of electrostatic free energies with the solvent treated as a dielectric continuum. The inter-pentamer interface in the virus is formed by two protomers related by 2-fold icosahedral symmetry. As a simple model for inter-pentamer interactions, a dimer and two separate protomers were compared. The association free energy was computed by integrating the difference between the titration curves of the two species. The calculations reproduced the observed decrease in capsid stability at acidic pH but not the difference in pH sensitivities of the two type A viruses. It is shown that only residues within 15 Å of the interface play a significant role in determining acid lability. For the experimentally studied pH range (5 to 7.6), histidine residues were found to dominate the pH-dependence of the stability. Two histidine residues in VP3, H142 and H145, are shown to have the greatest effect by virtue of their interactions with many polar residues across the inter-pentamer interface; the interaction of H142 with an α-helix in the opposite pentamer contributes only a small proportion of the destabilization energy.

Purification and Characterization of Cynomolgus Monkey Tryptase

Cynomolgus monkey tryptase was purified to homogeneity from lung tissue. Reducing SDS-PAGE analysis of the monkey enzyme produced a doublet at 30–32 kDa, which reacted with antibodies against human lung tryptase on a Western blot. N-terminal sequence analysis of the monkey enzyme yielded a sequence that was identical to human tryptase out to 15 residues. Gel filtration chromatography either in the presence or absence of heparin indicated that the monkey enzyme had a molecular mass of approximately 250 kDa and 140 kDa, respectively, consistent with the formation of a tetramer that can bind heparin. Other similarities between human and monkey tryptase included the ability to degrade vasoactive intestinal peptide and a resistance to inhibition by biological serine protease inhibitors. However, the two enzymes displayed markedly different pH stability profiles. Monkey tryptase was unstable at pH values over 7.0, even in the presence of heparin, displaying a half-life of 10.9 min at pH 8.0. In addition, the stabilizing effect of heparin was pH dependent, being most prevalent at lower pH values. Therefore, the biological activity of monkey tryptase may be controlled by both pH and the availability of heparin.

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7.

Effect of immobilization on the physical and kinetic properties of soluble and insoluble trypsin–albumin polymers

Bovine trypsin was crosslinked to human serum albumin (HSA) with glutaraldehyde to form soluble and insoluble copolymers. The physical and kinetic properties of trypsin and trypsin-HSA polymers were compared. Trypsin was heat labile, retaining only 24% of its enzymic activity after heating for 5 min at 60 degrees C. In contrast, under the same condition both the soluble and insoluble trypsin-HSA polymers showed enhanced resistance to heat in-activation, retaining 81 and 100% of their original activities, respectively. The trypsin-HSA polymers also showed shifts in pH optima, an increase in activation energy, and a broadening of their pH stability profiles.

Rat liver aldehyde dehydrogenase. II. Isolation and characterization of four inducible isozymes.

The purification and properties of 4 inducible cytosolic rat liver aldehyde dehydrogenase isozymes are described. Based on their behavior during purification and their properties, the activities can be grouped into 2 classes. The isozyme inducible in normal liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the tumor-specific isozyme found in hepatocellular carcinomas have apparent molecular weights of 110,000, prefer NADP+ as coenzyme, and preferentially oxidize benzaldehyde-like aromatic aldehydes, but not phenylacetaldehyde. They also have identical pH profiles and responses to effectors. These isozymes differ slightly in isoelectric point and thermal stability. The normal liver phenobarbital-inducible isozyme and the isozyme appearing during the promotion phase of hepatocarcinogenesis appear to be identical. Both have apparent molecular weights of 165,000, are NAD-specific and prefer aliphatic aldehydes. They can oxidize phenylacetaldehyde, but not benzaldehyde-like aromatic aldehydes. They also have identical pH and thermal stability profiles and responses to effectors. While the 4 inducible isozymes share identical subunit molecular weights (54,000) with the normal liver millimolar Km aldehyde dehydrogenases, they are distinctly different enzymatic species. The interrelationships of the various normal liver and inducible rat liver aldehyde dehydrogenases are discussed.

Growth and trophic factors.

Sedimentation velocity analyses of the isolated subunits of mouse 7S nerve growth factor (NGF) and their binary complexes have been performed. The 2.5S (beta) form of NGF binds 2 alpha subunits independent of the presence of gamma subunits. Two gamma subunits are also complexed by 2.5S NGF; however, this interaction is dependent on the presence of a carboxyl terminal arginine residue on the beta polypeptide chain. The partial loss of this residue in 2.5S NGF preparations results in a ternary complex(es) with a reduced sedimentation coefficient(s). The two binary complexes formed (alpha 2-beta and beta-gamma 2) show markedly different pH stability profiles. The alpha and gamma subunits do not form a demonstrable complex, but apparently can interact in the ternary complex to render it more stable than the sum of the alpha-beta and beta-gamma interactions.

EFFECT OF SLOW INTRAPARTICLE DIFFUSION ON pH-STABILITY PROFILES OF IMMOBILIZED ENZYME

Stabilities of immobilized enzymes at varying pH under different levels of intraparticle diffusion limitation have been analyzed theoretically and numerically. Unlike pH-activity profiles, which are broadened at high levels of diffusion limitation, pH-stability profiles are narrowest at intermediate levels of limitation. When diffusion limitation is cither absent or very severe, pH-stability profiles are identical in shape, those under severe limitation having half-lives exactly double those free of limitation at all pHs.

Renaturation of [formula omitted] green-fluorescent protein

Renaturation of Aequorea green-fluorescent protein (A-GFP) was achieved for the first time following denaturation in guanidine-HCl or acid. Denaturation was accompanied by the concerted loss of visible fluorescence, alteration of absorption characteristics, and large negative deflection of CD signal in the far UV. Dialysis of a guanidine-denatured sample at pH 8 resulted in 64% renaturation (return to native absorption) and neutralization of an acid-denatured sample restored 90% of the native absorption. Renatured GFP is highly fluorescent and indistinguishable from native GFP with respect to the shape of excitation and emission spectra. Both native and denatured proteins exhibit resistance to trypsin hydrolysis and have identically broad pH and heat stability profiles, all of which suggest full renaturation.

Association of cyclic nucleotide phosphodiesterase of buffalo spermatozoa with sperm chromatin

Buffalo sperm heads contain more than 50% of the total cyclic AMP-phosphodiesterase activity (EC 3.1.4.17) present in spermatozoa. Its distribution in sperm heads revealed no activity in acrosome and other membrane structures present in the head. All the cyclic AMP-phosphodiesterase activity was found firmly bound to sperm chromatin which could not be solubilized. In addition to cyclic AMP, cyclic GMP was also hydrolysed by chromatin preparation. The rate of hydrolysis was 2.5-times more rapid with cyclic AMP than with cyclic GMP at their optimum pH of 7.5 and 8.0, respectively. The pH and heat stability profiles, inhibition studies and the effect of divalent metal ions indicated that the two activities are not associated witht the same protein. Mixed substrate analysis showed two sites at which the hydrolysis of cyclic AMP and cyclic GMP is catalysed. Chromatin cyclic nucleotide phosphodiesterases exhibited kinetics typical of one enzyme species both for cyclic AMP ( K m = 100 μM; V = 100 nmol/min per mg protein) and cyclic GMP ( K m = 23 μM; V = 0.4 nmol/min per mg protein). Each cyclic nucleotide was found to be a competitive inhibitor of the hydrolysis of the other with a K i value of 30.18 μM for cyclic AMP hydrolysis and 256 μM for cyclic GMP hydrolysis. Hill coefficients of 1.0 obtained in the presence of cyclic AMP for cyclic GMP hydrolysis and vice-versa indicated no allosteric interactions. It is suggested that chromatin cyclic nucleotide phosphodiesterase may have a role post fertilization in cell growth and differentiation with no role in sperm motility which is regulated by similar enzymes present in sperm flagella.

Preliminary crystallographic data for an extracellular proteolytic enzyme from a strain of Arthrobacter

The chapter discusses endopeptidases and focuses on their three main types: (1) acid proteases, (2) diisopropylphosphofluoridate (DFP)-sensitive alkaline proteases, and (3) metal chelator-sensitive neutral proteases. The acid proteases that include the Aspergillus saitoi acid protease, Rhizopus chinensis acid protease, and Paecilomyces varioti acid protease are characterized by low pH activity and stability profiles, insensitivity to metal chelating agents, phosphorylating compounds, such as DFP and thiol poisons. These enzymes show limited esterolytic activity and are reminiscent of the animal pepsins and rennins. The most widely distributed group of proteolytic enzymes of both microbial and animal origin is that of the the serine proteases, generally identified by their sensitivity to organophosphorus reagents, such as DFP and isopropylmethylphosphofluoridate. In addition to the three broad classes of endopeptidases, a number of different kinds of proteolytic enzymes are produced by bacteria and molds. Another unique group of proteases, which in some cases shares properties with one of the other groups, is the bacterial cell wall lysing proteases. These enzymes by virtue of the specific bonds cleaved have uniquely difierent characteristics from the other enzymes.