Hayrettin Tümtürk

Stability of α-amylase immobilized on poly(methyl methacrylate-acrylic acid) microspheres

Poly(methyl methacrylate-acrylic acid) microspheres were prepared and the acid groups were activated by using either carbodiimide (CDI) or thionyl chloride (SOCl2). alpha-Amylase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. The relative activities were found to be 80.4 and 67.5% for carbodiimide and thionyl chloride bound enzymes, respectively. Maximum activities were obtained at lower pHs and higher temperatures upon immobilization compared to free enzyme. No change in Vmax and approximately 12-fold increase in K(m) were observed for immobilized enzymes. The enzyme activities, after storage for 1 month, were found to be 24.5 and 52.5% of the initial activity values for CDI and SOCl2 activated matrices, respectively. On the other hand the free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca2+ ions demonstrated higher stability, such as SOCl2 immobilized enzyme retained 83.7% and CDI immobilized enzyme retained 90.3% of the original activity of the enzyme. The immobilized enzymes that were used 20 times in 3 days in repeated batch experiments demonstrated that, in the absence of Ca2+ ions about 75% and in the presence of Ca2+ ions greater than 90% of the original enzyme activity was retained.

Covalent immobilization of α-amylase onto poly(2-hydroxyethyl methacrylate) and poly(styrene-2-hydroxyethyl methacrylate) microspheres and the effect of Ca2+ ions on the enzyme activity.

Abstract α-Amylase (1,4-α- d -glucan-glucanohydrolase; EC 3.2.1.1, Type VI-B from porcine pancreas, extra pure 29 units mg−1) was covalently immobilized on poly (2-hydroxyethyl methacrylate), p(HEMA), and poly (styrene- 2- hydroxyethyl methacrylate), p(St-HEMA) microspheres, which were activated by using epichlorohydrin (ECH). The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. For the assays carried out at 25°C and pH 6.9, the relative activities were found to be 61.7 and 67.0% for ECH-activated P(HEMA) and P(St-HEMA) bound enzymes, respectively. The maximum activities were obtained at lower pH values and higher temperatures upon immobilization compared to free enzyme. Kinetic parameters were calculated as 2.51, 22.4 and 6.62 g dm−3 for Km and 1.67×10−3, 1.63×10−3 and 1.35×10−3 g dm−3 min−1 for Vmax in the case of free, P(HEMA) and P(St-HEMA) bound enzymes, respectively. Enzyme activity was found to be ca. 38.9% for ECH-activated P(HEMA) bound enzyme after storage for 1 month. On the other hand, free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments that were carried out in the presence of Ca2+ ions demonstrated higher stability. The enzymes immobilized in the presence of Ca2+ ions retained 90.7 and 80.0% of their original activities even after 30 days for ECH-activated P(HEMA) and P(St-HEMA) systems, respectively. In repeated batch experiments, 20 uses in 3 days, in the absence of Ca2+ ions, retention of 79% of the original enzyme activities was observed for ECH-activated P(HEMA) immobilized enzymes. On addition of Ca2+ ions to the assay medium, 90.0 and 80.0% of retention was observed for ECH-activated P(HEMA) and P(St-HEMA) systems, respectively.

Immobilization of invertase attached to a granular dimer acid-co-alkyl polyamine

Abstract Invertase was immobilized onto the granular dimer acid-co-alkyl polyamine after activation with carbodiimide. The Km value for immobilized enzyme (53.6 mM) was much greater than that of the free enzyme (20.6 mM). Vmax values were 6.44×10−5 mol dm−3 min−1 and 5.45×10−5 mol dm−3 min−1 for free and bound, respectively. The optimal pH values for free and covalently bonded enzymes were 4.56 and 5.50, respectively. The optimum temperature for both free and covalent invertase was 55°C. The enzyme activities, after storage for 1 month, were found to be 21.0 and 99.0% of the initial activity values for free and covalently bonded, respectively. The immobilized enzyme that was used 50 times in 5 days in repeated batch experiments showed 100% of its original activity.

The effect of gel composition on the adsorption of invertase on poly(acrylamide/maleic acid) hydrogels

Abstract The effects of external stimuli such as pH of solution, temperature, substrate concentration of solution and storage stability on the invertase adsorption capacity of poly(acrylamide/maleic acid) [P(AAm/MA)] hydrogels, synthesized by gamma irradiation of ternary mixtures of AAm/MA/Water, were investigated. The adsorption capacities of the hydrogels were found to increase from 4.0 to 13.3 mg invertase/g dry gel with increasing amount of MA in the gel system, while P(AAm) gel adsorbed only 3.1 mg invertase/g dry gel. Kinetic parameters were calculated as 20.6 mM for K m and 6.44×10 −5 mol/dm 3 min for V max for free enzyme and in the range of 23.6–57.7 mM for K m and 8.62×10 −5 –1.05×10 −4 mol/dm 3 min for V max , depending on the amount of MA in the hydrogel. Enzyme activities were found to increase from 50.0 to 74.0% with increasing amount of MA in the gel system and retained their activities for one month storage. The enzyme activities, after storage at 4°C for one month, were found to be 21.0 and 50.0–74.0% of the initial activity values for free and adsorbed enzyme, respectively. The optimal pH values for free and adsorbed enzymes were determined as 4.56 and 4.56–5.00. The optimum temperature for free and adsorbed enzymes was 55°C. Adsorption studies show that, not only gel composition but also the stimuli, temperature and pH of the solution, play important roles on the invertase adsorption capacity of poly(AAm/MA) hydrogels.

Immobilization of β-galactosidase on Novel Polymers Having Schiff Bases

Abstract: We have developed a strategy to immobilize β-galactosidase as a model enzyme by using polymeric supports having Schiff bases, which were prepared from (aminomethyl)polystyrene and 2-phenlyindole-3-carboxaldehyde by condensation. β-galactosidase was immobilized onto the new polymer supports via covalent bonds. The influence of temperature, pH, reusability, and storage capacity on the free and immobilized β-galactosidase was investigated. Our results indicate that the (aminomethyl)polystyrene with Schiff bases is most suitable for the immobilization of β-galactosidase. These kinds of new supports can be used for the immobilization of β-galactosidase due to their strong storage capacity and reusability.

Covalent Immobilization of a α‐Amylase onto Poly(methyl methacrylate‐2‐hydroxyethyl methacrylate) Microspheres and the Effect of Ca2+ Ions on the Enzyme Activity

α-Amylase was covalently immobilized onto poly(methyl methacrylate-2-hydroxyethyl methacrylate) microspheres, which were activated by using either epichlorohydrin (ECH) or cyanuric chloride (C3N3Cl3). The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. For the assays carried out at 25 °C and pH 6.9, the relative activities were found to be 73.0% and 90.8% for epichlorohydrin and cyanuric chloride bound enzymes, respectively. Upon immobilization, the maximum activities were obtained at lower pH values and higher temperatures as compared with the free enzyme. Kinetic parameters were calculated as 2.51 g/L, 28.54 g/L and 15.50 g/L for Km and 1.67 × 10−3 gL−1 min−1 2.89 × 10−4 gL−1 min−1 and 1.89 × 10−3 gL−1 min−1 for Vmax for free, epichlorohydrin and cyanuric chloride bound enzymes, respectively. Enzyme activities were found to be ca. 32.7% for ECH and 41.1% for C3N3Cl3 activated matrices after storage for one month. On the other hand the free enzyme lost its activity completely within 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca2+ ions demonstrated higher stability in the presence of these ions. The enzymes immobilized in the presence of Ca2+ ions retained 90.6% and 90.8% of the original activities even after 30 days in the case of ECH and C3N3Cl3 activations, respectively. In repeated batch experiments, i.e., 20 uses of the enzyme in 3 days; in the absence of Ca2+ ions retentions of 79.2% and 77.1% of the original enzyme activities were observed for ECH and C3N3Cl3 immobilized enzymes, respectively, whereas, in the case of addition of Ca2+ ions to the assay medium, these values were enhanced to 95.3% and 92.2%.

The effect of external stimuli on the invertase adsorption capacity of poly(N‐vinyl‐2‐pyrrolidone‐co‐itaconic acid) hydrogels

Polyelectrolyte Poly(N-vinyl-2-pyrrolidone-co-itaconic acid) hydrogels (P(VP/IA)) with varying compositions were prepared from ternary VP/IA/water mixtures. The effect of external stimuli such as pH of the solution, temperature, substrate concentration of solution, and storage stability on the invertase adsorption capacity of P(VP/IA) hydrogels was investigated. The adsorption capacity of the hydrogels was found to increase from 4.4 to 18.4 mg invertase/g dry gel with increasing amount of IA in the gel system, while P(VP) gel adsorbed only 3.1 mg invertase/g dry gel. Kinetic parameters were calculated as Michaelis-Menten constant K m = 20.6 mmol L -1 and maximum velocity V max = 6.44 × 10 -5 mol dm -3 min -1 for free enzyme and in the range of K m = 26.4-41.1 mmol L -1 and V max = 6.35.10 -5 -6.66.10 -5 mol dm -3 min -1 . depending on the amount of IA in the hydrogel. Enzyme activities were found to increase from 59.0% to 72.0% with increasing amount of IA in the gel system and retained their activities for one month storage. The enzyme activities, after storage for one month at 4 °C, were found to be 21.0% and 59.0-74.0% of the initial activity values for free and adsorbed enzyme, respectively. The optimal pH values for free and adsorbed enzymes were determined as 4.56 and 5.00, respectively. The optimum temperature for free and adsorbed enzymes was 55°C. Adsorption studies have shown that not only the gel composition but also the stimuli, temperature and pH of the solution play an important role on the invertase adsorption capacity of poly(VP/IA) hydrogels.

Acetylcholinesterase immobilized onto PEI-coated silica nanoparticles

Polyethyleneimine (PEI) coated-silica nanoparticles were prepared by the Stöber method. The formation and the structure of the nanoparticles were characterized by ATR-FT-IR spectroscopy and transmission electron microscopy (TEM). TEM images of the silica and PEI-coated nanoparticles revealed that they were well dispersed and that there was no agglomeration. The acetylcholineesterase enzyme was immobilized onto these nanoparticles. The effects of pH and temperature on the storage stability of the free and immobilized enzyme were investigated. The optimum pHs for free and immobilized enzymes were determined as 7.0 and 8.0, respectively. The optimum temperatures for free and immobilized enzymes were found to be 30.0 and 35.0°C, respectively. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) were investigated for the free and immobilized enzyme. The storage stability of acetylcholinesterase was increased when immobilized onto the novel PEI-coated silica nanoparticles. The reuse numbers of immobilized enzyme were also studied. These hybrid nanoparticles are desirable as carriers for biomedical applications.

Monomer-bound ion pairs in the stereoregular polymerization of propylene oxide by the Pruitt-Baggett catalyst

Abstract The Pruitt-Baggett adduct (PBA) (MW = 438 g mol−1) that formed from the reaction of FeCl3 with propylene oxide (PO) was hydrolysed in diethyl ether (ether) solutions at different r = H 2 O Fe molar ratios. The hydrolysates ( PBH r ), which were insoluble in ether, could be converted into catalytically active form ( PBC r ) by a thermal treatment. PBC r (r was soluble in etheric solvents and in pyridine. These solutions, in contrast to PBA and PBH r , were electrical conductors. Profound differences in the electrical conductivity of PBC r was observed by varying the hydrolysis ratio r. The observed conductivity decayed and finally vanished with a kinetically second-order process. This is explained by the free ions, formed from PBC 0.67 (MW = 4400 g mol−1), being bound together with an ether (or PO) molecule to yield ether- (or PO-) bound ion pairs (PBCB). PBC 0.67 and the electrical conductivity of its freshly prepared solutions could be retrieved by ‘driving off’ (by drying under vacuum at 40°C) the ‘binding molecule’ (ether or PO) from PBCB. In contrast to this system, the electrical conductivity of solutions of PBC 0.20 (which shows inferior catalytic activity) did not change with ageing.

Covalent immobilization of invertase on chemically activated poly(2-hydroxyethyl methacrylate) microbeads

Properties of invertase immobilized on poly(2-hydroxyethyl methacrylate) microbeads activated by epichlorohydrin or cyanuric chloride were studied. After 20 repeated uses for 3 days, the activity of the immobilized enzyme was 92–93%.