Birnur Akkaya

Production and immobilization of a novel thermoalkalophilic extracellular amylase from bacilli isolate

A Thermoalkalophilic amylase was produced from an environmental bacterial isolate. The enzyme was then immobilized through its amino groups onto the epoxy rings of magnetic poly glycidyl methacrylate [m-poly (GMA)] beads. The free enzyme was active within a large pH range, between 7 and 12 and displayed the optimum activity at 95°C and pH 10. The immobilization appeared to increase the stability of the enzyme as its bound form showed optimum activity at 105°C and pH 11.0. Kinetic studies demonstrated that immobilized enzyme had higher K(m) and lower V(max) values. The activity of the free and bound enzyme was determined, at 37°C and pH 10.0 and pH 11.0, respectively, in the presence of various organic solvents and detergents (5%, v/v). Results obtained indicated that detergents, sodium dodecyl sulfate (SDS) and TritonX-100, caused six fold increase and that various organic solvents also increased the activity of the amylase.

IgG purification by bentonite-acrylamide-histidine microcomposite.

In this work, a new microcomposite composed of bentonite, acrylamide and histidine, as a pseudospecific ligand, was synthesized by bulk polymerization. The aim of this study was to improve IgG adsorption capacity of bentonite by incorporating histidine. The surface areas of the bentonite and bentonite-acrylamide-histidine microcomposites were 33.4 and 1.42 m(2)/g, respectively. The amount of histidine was found to be 50 μmol/g bentonite via elemental analysis. Adsorption capacity was at the value of 100mg/g from aqueous solution while adsorption capacity was 108 mg/g from human plasma with a purity of 90%. IgG biomolecules were able to be adsorbed and desorbed five times by using the same microcomposites without significant loss in their adsorption capacity.

Concanavalin a Immobilized Monosize and Magnetic Poly(glycidyl Methacrylate) Beads for Use in Yeast Invertase Adsorption

Concanavalin A (Con A) immobilized monosize and magnetic poly(glycidyl methacrylate)[m-poly(GMA)] beads were investigated for specific adsorption of yeast invertase from aqueous solutions. m-Poly(GMA) beads (1.6 μ m in diameter) were prepared by dispersion polymerization in the presence of Fe3O4 nanopowder. The epoxy groups of m-poly(GMA) beads were opened by base catalyses. Then, Con A was immobilized by covalent binding onto the beads. Con A immobilization amount was 12.5 mg/g. The invertase adsorption capacity of the m-poly(GMA)/Con A beads was 111 mg/g. The maximum invertase adsorption on the m-poly(GMA)/Con A beads was observed at pH 5.5. The optimum activity for both free and adsorbed invertase was observed at 50°C. Vmax values were determined as 330 U/mg and 292 U/mg enzyme, for free and adsorbed invertase, respectively. KM values were found to be the same for free and adsorbed invertase (20 mM). Adsorption of invertase via Con A improved the pH stability of invertase. Thermal stability also increased with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments.

Production, purification, and characterization of metalloprotease from Candida kefyr 41 PSB

A thermostable metalloprotease, produced from an environmental strain of Candida kefyr 41 PSB, was purified 16 fold with a 60% yield by cold ethanol precipitation and affinity chromatography (bentonite-acrylamide-cysteine microcomposite). The purified enzyme appeared as a single protein band at 43kDa. Its optimum pH and temperature points were found to be 7.0 and 105°C, respectively. Km and Vmax values of the enzyme were determined to be 3.5mg/mL and 4.4μmolmL-1min-1, 1.65mg/mL and 6.1μmolmL-1min-1, using casein and gelatine as the substrates, respectively. The activity was inhibited by using ethylenediamine tetraacetic acid (EDTA), indicating that the enzyme was a metalloprotease. Stability of the enzyme was investigated by using thermodynamic and kinetic parameters. The thermal inactivation profile of the enzyme conformed to the first order kinetics. The half life of the enzyme at 95, 105, 115, 125 and 135°C was 1310, 610, 220, 150, and 86min, respectively.

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H2O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.

Cross-Linked Bentonite-Acrylamide-histidine-Based Metal-Chelate Affinity Microcomposites for Lysozyme Separation From Egg White

Among different adsorbents, a new support for immobilized metal affinity chromatography was used for lysozyme separation from egg white. For this purpose, acrylamide, bisacrylamide, bentonite, and L-histidine in the presence of an initiator (azobisisobutyronitrile, AIBN) were mixed and bentonite-acrylamide-histidine microcomposites were prepared by bulk polymerization. Characterization studies were performed by SEM, FT-IR, XRD, and BET. Specific surface area of the bentonite and BABH microcomposite was found to be 33.4 and 1.42 m2/g, respectively. Cu2+ ions (20 µmol/g) were chelated on these bentonite-acrylamide-histidine (50 µmol/g L-histidine incorporation) microcomposites. The lysozyme adsorption capacity of the Cu2+ ions chelated microcomposites increased the lysozyme adsorption up to 100 mg/g while the lysozyme adsorption capacity of the bentonite-acrylamide-histidine was 24 mg/g. More than 89% of the adsorbed lysozyme was desorbed in 1 h in the desorption medium containing 1.0 M NaCl. Bentonite-acrylamide-histidine-Cu(II) was used for the purification of lysozyme from chicken egg white. The purity of lysozyme was estimated by SDS-PAGE. Specific activity of the purified lysozyme was found as 50,000 U/mg using Micrococcus lysodeikticus as substrate. The new metal-chelate affinity microcomposites were suitable for repeated use for five cycles without a noticeable loss of capacity.

Characterization of thermostable β-amylase isozymes from Lactobacillus fermentum

A strain of Lactobacillus fermentum producing two isozymes of a 20kDa β-amylase was isolated from the faecal sample of a newborn. The starin was identified by sequencing its 16S rRNA gene. The two β-amylase isozymes were resolved and visualized by two dimensional protein gel electrophoresis (2-D gel electrophoresis). Some of the physical and biochemical properties of the enzymes were characterized. The β-amylase displayed two optimum pH s, 5.0 and 10.0 and two optimum temperatures, 45°C and 37°C, respectively. The isozymes hydrolyzed different substrates: glycogen at pH 5.0, and corn starch at pH 10.0. The activity did not require Ca2+, though the activity at pH 10.0 was enhanced in the presence of 5.0mM and 10.0mM CaCl2, 110% and 130%, respectively.

New low-cost composite adsorbent synthesis and characterization

Abstract In this research, a novel composite, poly(acrylamide-expanded perlite) [P(AAm-EP)], was synthesized and characterized. The chemical synthesis was achieved by using free-radical polymerization and a number of structural characterization methods, including Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Brunauer–Emmett–Teller (BET)-porosity, and swelling tests. Free-radical polymerization of acrylamide (AAm) over expended perlite (EP) was successfully performed. The effect of reaction variables, such as dosage of the initiator, total concentration of the reactants, reactant ratio, mixing time, temperature, and reaction time, were investigated in detail. Expended perlite was cross-linked with acrylamide to enhance its chemical resistance. P(AAm-EP) composite has a specific surface area of 31.7 m2 g−1.

Bentonite-acrylamide-histidine-Cu(II) Microcomposite for Cytochrome c Adsorption

Bentonite-acrylamide-histidine with an average particle diameter of 200-1000 μm was produced by bulk polymerization of bentonite, acrylamide, bisacrylamide, AIBN and histidine. Bentonite-acrylamide-histidine-Cu(II) microcomposite was prepared by loading copper ions. The microcomposite was characterized by scanning electron microscopy (SEM), specific surface area and elemental analysis. Cytochrome c adsorption studies were performed, for the first time, by using the new metal chelated affinity support (bentonite-acrylamide-histidine-Cu(II)) microcomposite in batchwise experiments.