Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme beta-galactosidase (beta-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, beta-Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin beta-D-galactopyranoside (RGB) when used as the substrate for beta-Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the beta-Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.
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PMID:Photolithographic fabrication of poly(ethylene glycol) microstructures for hydrogel-based microreactors and spatially addressed microarrays. 1809 67

We report a method to load proteins into polymer-based sustained-release systems without exposing them to water-oil or water-air interfaces, factors known to denature proteins. By dispersing a dextan solution containing a protein into a PEG solution containing small amount of alginate, a stable aqueous-aqueous "emulsion" was formed. The poly-anionic alginate generated a diffuse double layer around each dextran droplet to prevent them from contacting with each other and fusing to a block phase. Proteins distributed in the stabilized dextran droplets by preferential partition favoring dextran. Freeze-drying this emulsion resulted in protein-loaded dextran particles, 1-2 microm in diameter and 1.6 g/cm(3) in density. The particles were harvested by washing the lyophilized powder using organic solvents to remove the PEG continuous phase. An activity assay of encapsulated beta-galactosidase indicated that protein activity was preserved during the particle-forming process including the step of sonicating the particles in organic solvents. The dextran particles also improved release profile and integrity of proteins when encapsulated in degradable polymer sustained-release systems. The aqueous-aqueous emulsion offers a convenient way to prepare solvent-resistant protein-polysaccharide particles that can easily be incorporated in a variety of polymer-based pharmaceutical dosage forms and medical devices such as microspheres, scaffolds and drug-eluting stents for sustained-release protein delivery.
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PMID:Preparing polymer-based sustained-release systems without exposing proteins to water-oil or water-air interfaces and cross-linking reagents. 1841 40

A method suitable for large-scale isolation of beta-galactosidase from a suspension of disintegrated E. coli cells has been developed. In an aqueous two-phase system consisting of PEG 6000 and potassium phosphate, all cell debris and the major part of the proteins and nucleic acids were partitioned to the denser salt phase. Seventy-five percent of the beta-galactosidase was recovered in the lighter PEG phase, giving a purification ratio of about 12.
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PMID:A Process for large-scale isolation of beta-galactosidase from E. coli in an aqueous two-phase system. 1855 82

We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to beta-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient K(p) for these proteins was related to measured interfacial potential differences Deltaphi using the simple thermodynamic model, In K(p) = In K(o) + (F/RT)Z(p) deltaphi. The protein net charge Z(p) was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Z(p) deltaphi was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The beta-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The beta-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. (c) 1994 John Wiley & Sons, Inc.
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PMID:Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning. 1861 79

This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two-phase extration. The genetic modifications consisted of the fusion of charged peptide tails to beta-galactosidase and charge-change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl-dextran (DEAE-dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the beta-galactosidase, which carries a net negative charge, partitioned to the DEAE-dextran-rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein-rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient (K(p)) of up to 37-fold. However, these changes could not be solely attributed to charge-based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate-containing phase, and displayed four- to sevenfold shifts in K(p) as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of beta-galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2- to 15-fold shifts in K(p). (c) 1995 John Wiley & Sons, Inc.
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PMID:Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Charge directed partitioning. 1862 63

This study investigates a method of preparing hazard-resistant protein-loaded polysaccharide glassy microparticles using freezing-induced phase separation method without exposure to water/oil, water/air interface and cross-linking reagents. Model protein (such as bovine serum albumin, myoglobin and beta-galactosidase (beta-Gal)) was dissolved in water together with dextran and polyethylene glycol (PEG), followed by a freezing process to form a temperature-stabilized aqueous-aqueous emulsion wherein dextran separated out as the dispersed phase with protein partitioned in preferentially. The frozen sample was freeze-dried and washed with dichloromethane (DCM) to remove the PEG continuous phase, after which protein-loaded polysaccharide particles, 1-4 microm in diameter, were harvested. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) patterns showed that the particles were in glassy state. These glassy polysaccharide microparticles can well protect the delicate structure of proteins and preserve their bioactivities under deleterious environment interacting with organic solvents, vortex and centrifugation processes that often involve during the formulation processes leading to polymer-based sustained-release systems. Therefore, this freezing-induced phase separation method is a mild and effective way to encapsulate protein into hazard-resistant polysaccharide glassy particles, which ensure its stability in subsequent formulating processes that leads to polymer-based sustained-release system.
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PMID:Preparation of polysaccharide glassy microparticles with stabilization of proteins. 1883 46

We report on a hybridization assay using DNA microarrays for the quantification of amplification products of the uidA gene of E. coli. Using the stopped-PCR strategy, the amplified target DNA was strongly dependent on the applied gene copies. The quantification was carried out by a flow-through chemiluminescence microarray readout system. The DNA microarrays were based on a poly(ethylene glycol)-modified glass substrate. The probes on the surface were 18 or 25 nucleotides long and the quantified PCR product was 60 nucleotides. The amplification was stopped after 25 cycles; at this point amplification was in the middle of the logarithmical phase, and the spread between different DNA starting concentrations reached the maximum. A conjugate of streptavidin and horseradish peroxidase (HRP) bound to the biotinylated strands on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The detection limit for the gene uidA (beta-galactosidase) of E. coli was 1.1 x 10(5) copies/mL. This system allowed for a sensitive detection and quantification of E. coli in a concentration range from 10(6) to 10(9) copies/mL.
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PMID:Quantification of E. coli DNA on a flow-through chemiluminescence microarray readout system after PCR amplification. 1943 Jan 51

An extracellular beta-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5 A, alpha = 109.1, beta = 97.3, gamma = 114.5 degrees . The crystals diffracted to 1.8 A resolution using a rotating-anode generator and to 1.2 A resolution using a synchrotron source. On the basis of the Matthews coefficient (V(M) = 3.16 A(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme.
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PMID:Crystallization and preliminary diffraction analysis of a beta-galactosidase from Trichoderma reesei. 1965 34

The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.
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PMID:Characterization of galactosyl derivatives obtained by transgalactosylation of lactose and different polyols using immobilized beta-galactosidase from Aspergillus oryzae. 1989 2

While formulating proteins into solid particles prior to microsphere preparation is regarded as an effective way to stabilize such macromolecules, the protein particles may still contact the aqueous continuous phase and be re-dissolved. Dissolved proteins may not only leak into the aqueous continuous phase (resulting in reduced loading efficiency), but also contact water-oil (the hydrophobic polymer solution) interfaces, factors known to denature proteins. To avoid dissolution of solidified protein particles, we developed a microencapsulation procedure involving a hydrophilic "oil" (hO) continuous phase to which the hydrophobic solution of the controlled-release polymer was dispersed. The hydrophilic "oil" phase was a glycerol-based liquid mixed with ethylene glycol and polyvinyl alcohol solution to adjust viscosity and surface tension. This non-water hydrophilic continuous phase is immiscible with the hydrophobic polymer solution yet unable to dissolve pre-formulated protein particles. After the embryonic microspheres loaded with the protein particles were formed in this hydrophilic "oil" phase, the formulation was transferred into a cold ethanol bath where the microspheres were immediately hardened due to extracting the organic solution by ethanol. This method was examined by microencapsulating bovine serum albumin (BSA) and beta-galactosidase (beta-gal) into polylactide-co-glycolide (PLGA) microspheres for encapsulation efficiency, release kinetics and bioactivity preservation. As measured using size exclusion chromatography (SEC-HPLC), up to 90% added BSA was encapsulated in microspheres, and the release kinetics of the protein was adjusted by selecting surfactants used in microencapsulation emulsification. The assay of enzymatic activity of beta-galactosidase in hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) indicated that over 90% of the protein recovered from the microspheres was active.
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PMID:Preparation of protein-loaded sustained-release microspheres via 'solid-in-oil-in-hydrophilic oil-in-ethanol (S/O/hO/E)' emulsification. 2048 70


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