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1.
Gulay Bayramoglu Begum Altintas M. Yakup Arica 《Applied microbiology and biotechnology》2013,97(3):1149-1159
Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase. 相似文献
2.
Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads 总被引:1,自引:0,他引:1
A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract. 相似文献
3.
M. Yakup Arica Hande Soydogan Gulay Bayramoglu 《Bioprocess and biosystems engineering》2010,33(2):227-236
Poly(2-hydroxyethyl methacrylate/ethylenglycol dimethacrylate) beads were grafted with poly(glycidylmethacrylate) via surface
initiated atom transfer radical polymerization. Epoxy groups of the grafted polymer were modified in to sulfone groups. Sulfonated
beads were characterized by swelling studies, FT-IR, SEM and elemental analysis, and were used for reversible immobilization
of lipase. Under given experimental conditions, the beads had an adsorption capacity of 44.7 mg protein/g beads. The adsorbed
lipase on beads retained up to 67.4% of its initial activity. The immobilized lipase exhibited improved thermal and storage
stabilities over those of the free enzyme. The immobilized lipase could desorb 1.0 M NaCl solution at pH 8.0, and the sulfonated
beads can be repeatedly charged with fresh enzyme after inactivation upon use. 相似文献
4.
Caragounis A Du T Filiz G Laughton KM Volitakis I Sharples RA Cherny RA Masters CL Drew SC Hill AF Li QX Crouch PJ Barnham KJ White AR 《The Biochemical journal》2007,407(3):435-450
Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor. 相似文献
5.
Fibrous poly(styrene-d-glycidylmethacrylate) (P(S-GMA)) brushes were grafted on poly(styrene-divinylbenzene) (P(S-DVB)) beads using surface initiated-atom transfer radical polymerization (SI-ATRP). Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The multi-modal ligand attached beads were used for reversible immobilization of catalase. The influences of pH, ionic strength and initial catalase concentration on the immobilization capacities of the P(S-DVB)-g-P(S-GMA)-TEDETA beads have been investigated. Catalase adsorption capacity of P(S-DVB-g-P(S-GMA)-TEDETA beads was found to be 40.8 ± 1.7 mg/g beads at pH 6.5 (with an initial catalase concentration 1.0 mg/mL). The Km value for immobilized catalase on the P(S-DVB-g-P(S-GMA)-TEDETA beads (0.43 ± 0.02 mM) was found about 1.7-fold higher than that of free enzyme (0.25 ± 0.03 mM). Optimum operational temperature and pH was increased upon immobilization. The same support was repeatedly used five times for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. 相似文献
6.
Adlard PA Bica L White AR Nurjono M Filiz G Crouch PJ Donnelly PS Cappai R Finkelstein DI Bush AI 《PloS one》2011,6(3):e17669
We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function. 相似文献
7.
In this work, a new methodology is developed for selection of affinity ligands towards the enzyme “trypsin” using quartz crystals
microbalance (QCM) technique. To achieve this goal, the surface amination of gold plated QCM crystals was achieved in 13.56
MHz plasma polymerization system by using ethylenediamine. Three different ligands (i.e., 4-aminobenzamidine, 4-aminobenzoic
acid, and phenylalanine) were immobilized on the aminated QCM crystals surface via glutaraldhyde coupling. All three ligand
immobilized QCM crystals were characterized and compared under different experimental conditions. It was observed that the
benzamidine ligand showed higher affinity to trypsin with a dissociation constant on the order of 1.76 × 10−9 M, which is within the range of 10−4–10−8 M for affinity ligands. Thus, its selectivity was suitable for purification of trypsin from biological fluids. 相似文献
8.
Chow C Gauci CG Vural G Jenkins DJ Heath DD Rosenzvit MC Harandi MF Lightowlers MW 《Experimental parasitology》2008,119(4):499-505
Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype. 相似文献
9.
Gulay Bayramoglu Ihsan Gursel Yagmur Tunali M. Yakup Arica 《Bioresource technology》2009,100(10):2685-2691
The removal of phenol (Ph) and 2-chlorophenol (2-CPh) from aqueous solution by native and heat inactivated fungus Funaliatrogii pellets were investigated. The effects of contact time, solid/liquid ratio, optimum pH and temperature on the phenols removal capacity by the pellets were established. The removal efficiency of phenols increased significantly with increasing biomass dose. The optimum pH was detected to be 8.0. The second-order equations are described and evaluated on the basis of a comparative estimation of the corresponding coefficients. The phenol removal equilibrium isotherm was modeled by the Langmuir equations. The enthalpy change values were obtained between −7.62 and −10.64 kJ/mol. This indicated that the uptake of phenols either on native or heat inactivated fungal pellets was based on a physical adsorption process. 相似文献
10.
Gulay Mann Simon Diffey Brian Cullis Fermin Azanza David Martin Alison Kelly Lynne McIntyre Adele Schmidt Wujun Ma Zena Nath Ibrahim Kutty P. Emmett Leyne Lynette Rampling Ken J. Quail Matthew K. Morell 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,118(8):1519-1537
While the genetic control of wheat processing characteristics such as dough rheology is well understood, limited information
is available concerning the genetic control of baking parameters, particularly sponge and dough (S&D) baking. In this study,
a quantitative trait loci (QTL) analysis was performed using a population of doubled haploid lines derived from a cross between
Australian cultivars Kukri × Janz grown at sites across different Australian wheat production zones (Queensland in 2001 and
2002 and Southern and Northern New South Wales in 2003) in order to examine the genetic control of protein content, protein
expression, dough rheology and sponge and dough baking performance. The study highlighted the inconsistent genetic control
of protein content across the test sites, with only two loci (3A and 7A) showing QTL at three of the five sites. Dough rheology
QTL were highly consistent across the 5 sites, with major effects associated with the Glu-B1 and Glu-D1 loci. The Glu-D1 5 + 10 allele had consistent effects on S&D properties across sites; however, there was no evidence for a positive effect
of the high dough strength Glu-B1-al allele at Glu-B1. A second locus on 5D had positive effects on S&D baking at three of five sites. This study demonstrated that dough rheology
measurements were poor predictors of S&D quality. In the absence of robust predictive tests, high heritability values for
S&D demonstrate that direct selection is the current best option for achieving genetic gain in this product category.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献