Işık Perçin

Mannose‐specific lectin isolation from Canavalia ensiformis seeds by PHEMA‐based cryogel

Mannose‐specific lectin Concanavalin A (Con A) was purified from Canavalia ensiformis seeds. For this purpose, mannose attached poly(hydroxyethyl methacrylate) (PHEMA) cryogel was prepared by cryopolymerization. Mannose was used as the affinity ligand and was covalently attached onto the PHEMA cryogel via carbodiimide activation. The PHEMA cryogel containing 23.3 mmol mannose/g polymer were used in the binding studies. Con A binding with the mannose attached PHEMA cryogel from Con A aqueous solution was 5.2 mg/g at pH 7. Maximum binding capacity for Con A from C. ensiformis seed extract was 39 mg/g. Con A was eluted with 0.3 M galactose, and the purity of Con A was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was observed that the mannose attached PHEMA cryogel can be used without significant decrease in Con A binding capacity after six binding‐elution cycles.

Macroporous PHEMA-based cryogel discs for bilirubin removal

Abstract A novel N-methacryloyl-L-tryptophan methyl ester (MATrp) containing poly (hydroxyethyl methacrylate) cryogel (PHEMATrp) disc was prepared for removal of bilirubin (BR) out of human plasma. PHEMATrp cryogel disc was produced by bulk polymerization, with high gelation yield up to 92% and characterized by swelling tests, scanning electron microscopy (SEM), elemental analysis, Brunauer– Emmett–Teller (BET) analysis, contact angle measurements and surface energy calculations. BR adsorption studies were performed in a batch system, and the maximum BR adsorption capacity was found as 22.2 mg/g cryogel disc.

Purification of urease from jack bean (Canavalia ensiformis) with copper (II) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) cryogels†

Jack bean (Canavalia ensiformis) is the source of interesting proteins that contribute to modern biochemistry, and urease is the primary of these proteins. Owing to its role and occurrence in nature, urease has become a part of extensive studies. In this study, jack bean urease (JBU) was purified by immobilized metal affinity chromatography using Cu2+ chelated poly(hydroxyethyl methacrylate‐N‐methacryloyl‐(l)‐histidine methyl ester) [PHEMAH‐Cu2+]–based cryogels. PHEMAH‐Cu2+ cryogel was synthesized and characterized for swelling degree, morphology (by SEM), N‐methacryloyl‐(l)‐histidine methyl ester and Cu2+ incorporation (by elemental analysis and atomic absorption spectrophotometry). The binding of JBU to PHEMAH‐Cu2+ cryogel was optimized by examining the effect of pH, flow rate and JBU concentration on binding. The maximal binding of JBU was 23.2 mg/dry gram of adsorbent. The maximal binding of JBU extracted from jack bean meal was 67.8 mg/dry gram of adsorbent. The elution of JBU from cryogel column was accomplished by 1.0 M NaCl in 20 mM phosphate buffer (pH 8.0). Molecular weight and purity of JBU from jack bean meal was estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. It was observed that JBU could be repeatedly bound and eluted from (PHEMAH)‐Cu2+ cryogel with less than 10% loss in column capacity. Copyright

Metal-immobilized magnetic nanoparticles for cytochrome C purification from rat liver.

Cu2+‐immobilized magnetic poly(hydroxyethylmethacrylate‐N‐methacryloyl‐(l)‐histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant‐free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu2+ was measured using atomic absorption spectrophotometry. N‐Methacryloyl‐(l)‐histidinemethylester and Cu2+ content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu2+‐immobilized mPHEMAH nanoparticles was 1180 m2/g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu2+‐immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu2+‐immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu2+‐immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu2+‐immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS‐PAGE.

Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for prostate specific antigen binding

The aim of this study was to prepare Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads for prostate specific antigen (PSA) binding and to study binding capacities of the beads using lectin-glycoprotein interactions. Firstly, iron oxide nanoparticles were synthesized by co-precipitation method and then, beads were synthesized by dispersion polymerization in the presence of iron oxide nanoparticles. Con A molecules were both covalently immobilized onto the beads directly and through the spacer arm (1,6-diaminohexane-HDMA). The total PSA and free PSA binding onto the mPGMA-HDMA-Con A beads were higher than that of the mPGMA-Con A beads. Maximum PSA binding capacity was observed as 91.2 ng/g. Approximately 45% of the bound PSA was eluted by using 0.1 M mannose as elution agent. The mPGMA-HDMA-Con A beads could be reused without a remarkable decrease in the binding capacities after 5 binding-desorption cycles. Serum fractions were analyzed using SDS-PAGE. The mPGMA-HDMA-Con A beads could be useful for the detection of PSA and suggested as a model system for other glycoprotein biomarkers.

Tentacle-type immobilized metal affinity cryogel for invertase purification from Saccharomyces cerevisiae

Abstract The aim of this study is to investigate the usability of cryogel columns for the purification of invertase from Saccharomyces cerevisiae. Poly(2-hydroxyethyl methacrylate) monolithic columns were produced via cryogelation. Ester groups of the poly(2-hydroxyethyl methacrylate) structure were then converted to imine groups by the reaction with poly(ethylene imine) in the presence of NaHCO3. Transition metal ions, Cu(II), Co(II), and Ni(II), were chelated on the PEI-modified cryogel columns. Purification of invertase from natural source namely S. cerevisiae was also studied, and the purification fold values were obtained as 41.350, 44.714, and 30.302 for Cu(II)-chelated, Co(II)-chelated, and Ni(II)-chelated PHEMA/PEI columns, respectively.

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs

ABSTRACT In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe3+) cryogel discs were prepared. The PHEMAGA/Fe3+ cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe3+ cryogel discs had large pores ranging from 10 to 100 µm with a swelling degree of 9.36 g H2O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe3+ cryogel discs were investigated. Maximum catalase adsorption capacity (62.6 mg/g) was obtained at pH 7.0, 25°C, and 3 mg/ml initial catalase concentration. The PHEMAGA/Fe3+ cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe3+ cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.

Composite cryogels for lysozyme purification

Beads‐embedded novel composite cryogel was synthesized to purify lysozyme (Lyz) from chicken egg white. The poly(hydroxyethyl methacrylate‐N‐methacryloyl‐L‐phenylalanine) (PHEMAPA) beads of smaller than 5 µm size were synthesized by suspension polymerization and then embedded into a poly(hydroxyethyl methacrylate) (PHEMA)‐based cryogel column. The PHEMAPA bead‐embedded cryogel (BEC) column was characterized by swelling tests, scanning electron microscopy (SEM), surface area measurements by the Brunauer–Emmett–Teller (BET) method, elemental analysis, and flow dynamics. The specific surface area of the PHEMAPA BEC was found as 41.2 m2/g using BET measurements. Lyz‐binding experiments were performed using aqueous solutions in different conditions such as initial Lyz concentration, pH, flow rate, temperature, and NaCl concentration of an aqueous medium. The PHEMAPA BEC column could be used after 10 adsorption–desorption studies without any significant loss in adsorption capacity of Lyz. The PHEMAPA BEC column was used to purify Lyz from chicken egg white, and gel electrophoresis was used to estimate the purity of Lyz. The chromatographic application of the PHEMAPA BEC column was also performed using fast protein liquid chromatography.

Gelatin-loaded p(HEMA-GMA) cryogel for high-capacity immobilization of horseradish peroxidase

Abstract Poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [p(HEMA-GMA)] cryogel discs were prepared under sub-zero temperatures. Gelatin was attached covalently on the p(HEMA-GMA) cryogel discs and reversible immobilization of horseradish peroxidase (HRP) was performed. The p(HEMA-GMA) cryogel discs were characterized by swelling tests, scanning electron microscopy, and surface area measurements. HRP immobilization capacity of p(HEMA-GMA)/gelatin cryogel discs was 24.8 mg/g. Removal of phenol from aqueous solutions was performed using HRP immobilized p(HEMA-GMA)/gelatin cryogel. It was observed that within 2 h of contact time, the percentage of phenol removal reaches up to 91% in the presence of H2O2.

Microcontact imprinted plasmonic nanosensors: Powerful tools in the detection of salmonella paratyphi

Identification of pathogenic microorganisms by traditional methods is slow and cumbersome. Therefore, the focus today is on developing new and quicker analytical methods. In this study, a Surface Plasmon Resonance (SPR) sensor with a microcontact imprinted sensor chip was developed for detecting Salmonella paratyphi. For this purpose, the stamps of the target microorganism were prepared and then, microcontact S. paratyphi-imprinted SPR chips were prepared with the functional monomer N-methacryloyl-L-histidine methyl ester (MAH). Characterization studies of the SPR chips were carried out with ellipsometry and scanning electron microscopy (SEM). The real-time Salmonella paratyphi detection was performed within the range of 2.5 × 106–15 × 106 CFU/mL. Selectivity of the prepared sensors was examined by using competing bacterial strains such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The imprinting efficiency of the prepared sensor system was determined by evaluating the responses of the SPR chips prepared with both molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Real sample experiments were performed with apple juice. The recognition of Salmonella paratyphi was achieved using these SPR sensor with a detection limit of 1.4 × 106 CFU/mL. In conclusion, SPR sensor has the potential to serve as an excellent candidate for monitoring Salmonella paratyphi in food supplies or contaminated water and clearly makes it possible to develop rapid and appropriate control strategies.