Zeolite inorganic supports for BSA immobilization: comparative study of several zeolite crystals and composite membranes

Zeolites due to their low toxicity and high compatibility are considered new biomaterials for medical applications. The surface adsorption behaviour of zeolite crystals and composite membranes was discussed in this research. The zeolite materials were synthesized by hydrothermal syntheses using different reaction gels to modulate the Br?nsted acidity of the microporous structures. Spectrophotometric analyses were used to evaluate protein adsorption on these surfaces. This study revealed that zeolite chemical composition and structure influenced the kinetics of protein adsorption. Zeolite Y surface adsorbed greater amount of BSA than the other structures. The percentage of adsorption increases with temperature and depends on the pH of the solution, being highest at the pI of the protein. The influence of the membrane configuration on the protein adsorption was studied using different zeolite structures and crystallization types. It seems that the observed differences could depend on the type of hydrothermal crystallization inside the inorganic support.     

Tyrosyl Radical in the W164Y Mutant of P. eryngii Versatile Peroxidase: an EPR and DFT/PCM Study

Versatile peroxidases (VP) constitute a new class of high redox potential fungal enzymes that are able to degrade lignin and large substrate molecules. These enzymes catalyze the oxidation of substrates at an exposed tryptophan radical formed by a long-range electron transfer mechanism to heme following the activation by H₂O₂. In a previous paper, it was demonstrated using electron paramagnetic resonance (EPR) and electron-nuclear double resonance experiments on wild-type VP that Trp164 was the radical site and that it was in a hydrogen-bonded neutral form. In this paper, the W164Y variant is analyzed and it is shown that also the variant is able to form the so-called Compound I (VPI) in the form of protein radical, although in different yields with respect to the wild type. The X-band EPR experiments in combination with density functional theory/polarizable continuum model calculations show that the W164Y mutant is able to form a neutral radical on Tyr164 residue, after activation by H₂O₂, but in contrast to Trp164, tyrosine is not expected to be hydrogen bonded.     

Tyrosine or Tryptophan? Modifying a Metalloradical Catalytic Site by Removal of the Cys–Tyr Cross-Link in the Galactose 6-Oxidase Homologue GlxA

The concerted redox action of a metal ion and an organic cofactor is a unique way to maximize the catalytic power of an enzyme. An example of such synergy is the fungal galactose 6-oxidase, which has inspired the creation of biomimetic copper oxidation catalysts. Galactose 6-oxidase and its bacterial homologue, GlxA, possess a metalloradical catalytic site that contains a free radical on a covalently linked Cys–Tyr and a copper atom. Such a catalytic site enables for the two-electron oxidation of alcohols to aldehydes. When the ability to form the Cys–Tyr in GlxA is disrupted, a radical can still be formed. Surprisingly, the radical species is not the Tyr residue but rather a copper second-coordination sphere Trp residue. This is demonstrated through the introduction of a new algorithm for Trp-radical EPR spectra simulation. Our findings suggest a new mechanism of free-radical transfer between aromatic residues and that the Cys–Tyr cross-link prevents radical migration away from the catalytic site.     

The Use of PAMAM Dendrimers as a Platform for Laccase Immobilization: Kinetic Characterization of the Enzyme

The kinetic behavior of the enzyme laccase in solution and immobilized onto carbon platforms using poly(amido amine) (PAMAM) dendrimers has been investigated. The results with the immobilized enzymes have demonstrated that almost ten times more enzyme on the carbon support is required for satisfactory kinetic rates to be achieved. Furthermore, the study as a function of the substrate concentration revealed that the kinetic behavior of the enzyme in solution fits the Michaelis?CMenten model. However, when the enzyme is immobilized onto the carbon surface, the catalyzed reaction follows a particular kinetic behavior with apparent positive cooperativity. The highest activity with laccase (in solution or immobilized) is achieved around pH?4.5, and the substrate conversion rate clearly diminishes with rising pH. The optimum temperature lies around 60?°C. The enzyme displays good catalytic activity in a wide range of pH and temperature values. The stability tests evidenced that there is no appreciable reduction in the enzymatic activity after immobilization within the first 30?days. Taking into account both the kinetic and stability tests, one can infer that the use of PAMAM dendrimers seems to be a very attractive approach for the immobilization of enzymes, as well as a feasible and useful methodology for the anchoring of enzymes with potential application in many biotechnological areas.     

Casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis

We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.     

Computational study on the origin of the stereoselectivity for the photochemical denitrogenation of diazabicycloheptene

The origin of the inversion stereoselectivity of housane formation via photochemical nitrogen extrusion of diazabicycloheptene (DBH) has been investigated using reaction path computations and multireference second-order perturbation theory within a CASPT2//CASSCF scheme. We show that the primary photoproduct of the reaction is an exo-axial conformer of the diazenyl diradical ((1) DZ) which displays a cyclopenta-1,3-diyl moiety with a Cs-like structure. (1) DZ is selectively generated via decay at a linear-axial conical intersection, and it is located in a shallow region of the ground state potential energy surface that provides access to five different reaction pathways. Reaction path analysis (including probing with classical trajectories) indicates that production of inverted housane can only occur via impulsive population of an axial-to-equatorial pathway, and it is thus inconsistent with thermal equilibration of the primary (1) DZ conformer. Similarly, according to the same analysis, the decrease of inversion stereoselectivity and even the retention (stereochemical memory effect) observed for suitably substituted DBHs are explained by dynamics effects where the axial-to-equatorial impulsive motion is restrained by the inertia and/or steric hindrance of the substituents. These results shade light on the poorly understood mechanisms that allow a photochemical reaction, in which a large amount of energy is deposited in the reactant by photon absorption, to show a high degree of stereoselectivity.     

Preface

Journal of Solid State Electrochemistry -