Studies on the immobilization of glucuronidase (part 1)

beta-Glucuronidase (EC 3.2.1.31) was immobilized on various organic and inorganic carriers by different methods. Optimum coupling conditions have been worked out. The immobilization were characterized and compared to each other. Parameters resulting in most stable preparations with high activities are discussed.     

Poly[(N-isopropylacrylamide-co-acrylamide-co-(hydroxyethylmethacrylate))] thermoresponsive microspheres: An accurate method based on solute exclusion technique to determine the volume phase transition temperature

Poly[(N-isopropylacrylamide-co-acrylamide-co-(hydroxyethylmethacrylate))] [poly(NIPAAm-co-AAm-co-HEMA)] copolymer was synthesized as a new thermoresponsive material possessing a lower critical solution temperature (LCST) around 37 °C in phosphate buffer, pH 7.4, at a solution concentration of 1%, w/v. The influence of polymer concentration on LCST was determined by cloud point measurements and by microcalorimetric analysis. The copolymer was transformed in hydrogel microspheres by suspension reticulation of OH groups with glutaraldehyde. The volume phase transition temperature (VPTT) of microspheres was determined by a new approach, which involves measurement of the increase in concentration of a blue dextran (BD) solution at different temperatures in the presence of dry microspheres. The minimum BD concentration that gives reliable and reproducible results was determined to be 1 mg/ml. However, the higher is the concentration of BD in solution the smaller is the error. Contrary to solution of the linear polymer which displays a sharp phase transition temperature, the dependence of water regain of the hydrogel with temperature lasts from 4 °C to 50 °C.     

壳聚糖固定化总状毛霉MR137-3蛋白酶的性质

以壳聚糖为载体,戊二醛作交联剂,将总状毛霉MR137—3蛋白酶固定在壳聚糖上。研究了戊二醛浓度,给酶量,处理时间对MR137—3蛋白酶固定化的影响。同时对固定化酶与游离酶的热稳定性、最适pH、最适温度以及脲、有机溶剂、金属离子的影响等理化性质进行了探讨。     

A new process for preparing dialdehyde by catalytic oxidation of cyclic olefins with aqueous hydrogen peroxide

A. novel peroxo-niobophosphate was synthesized for the first time and used as a catalyst in the oxidation reaction of cyclic olefins with aqueous hydrogen peroxide to prepare dialdehy-des. The catalyst was characterized by elemental analysis, thermographic analyses, IR, UV/vis, 31P NMR and XPS spectra as [ π-C5H5N(CH2 )13 CH3 ]2 [ Nb4O6 (O2 )2 (PO4 )2 ] ·6H2O (PTNP). It showed high selectivity to glutaraldehyde in the catalytic oxidation of cyclopentene with aqueous hydrogen peroxide in ethanol.     

W-SBA-15的原位合成及其在环戊烯氧化反应中的催化性能研究

将WO_3物种原位引入介孔分子筛SBA-15的骨架内，首次得到具有选择氧化活性 中心的W-SBA-15分子筛，并用XRD，BET，SEM及TEM等手段进行了表征。该新型催化 剂在环戊烯催化氧化合成戊二醛的反应中表现出优异的催化性能，戊二醛的选择发 生性高达84%，收率亦达到79%，超过所有的负载型含钨催化剂，具有极好的应用前 景。     

Selection of carrier for immobilization of fructosyltransferase from Aureobasidium pullulans

The study deals with the development of an immobilized biocatalyst for the production of fructooligosaccharides. Several commercial anion-exchange resins and polymethacrylate carriers were tested for the immobilization either by the direct attachment of fructosyltransferase or by the attachment accompanied by simultaneous glutaraldehyde crosslinking in the pH range from 5.7 to 7.1. On the basis of the efficiency of immobilization and the storage stability, Amberlite IRA 900 and Dowex Marathon MSA were selected as the best carriers for fructosyltransferase immobilization by direct attachment at pH 5.7. Regular paper An erratum to this article is available at .     

Melibiase in Immobilized Cells of Watermelon

Cells of suspension culture Citrullus vulgaris cv. “Samara” were permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest melibiase activity was at pH 5.4 and 60°C. The hydrolysis of substrate was linear for 3.5 h, reaching 65–70% conversion of the substrate. The cells, characterized by high enzyme activity and stability in long-term storage, showed convenient physico-mechanical properties (physical protection from shear forces and easy separation of product from biocatalysts).__________Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 53–55, January–February, 2005.     

NMR structural study of hydrogels based on partially deacetylated hyaluronan

Hydrogels have been prepared using two partially deacetylated hyaluronan (deHA) samples having a molar ratio of free amino groups to total initial N-acetamido groups equal to 23 and 33%, respectively. Taking advantage of such primary amino functionalities, polymeric networks could be easily obtained in aqueous media by means of two different crosslinking procedures, namely: a) Ugi multicomponent reactions and, more simply, b) glutaraldehyde reticulation. In this paper, we wish to report on the synthesis of deHA based hydrogels and, in particular, on their structural elucidation as obtained by NMR studies.

Structure of HA (R = COCH₃) and de-HA (R = H) repeat units.     

Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

Development of enzyme-based bar code-style lateral-flow assay for hydrogen peroxide determination

A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG-horseradish peroxidase conjugate (Gt anti-M IgG-HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3′,5,5′-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H₂O₂) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H₂O₂ in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H₂O₂-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 μM and 100 μM.