Mustafa Teke

Immobilization of pancreatic lipase on chitin and chitosan.

Abstract In this study, porcine pancreatic lipase (EC 3.1.1.3) was immobilized on chitin and chitosan by adsorption and subsequent crosslinking with glutaraldehyde, which was added before (conjugation) or after (crosslinking) washing unbound proteins. Conjugation proved to be the better method for both supports. The properties of free and immobilized enzymes were also investigated and compared. The results showed that the pH optimum was shifted from 8.5 to 9.0 for both the immobilized enzymes. Also, the optimum temperature was shifted from 30 to 40°C for chitin‐enzyme and to 45°C for chitosan‐enzyme conjugates. The immobilization efficiency is low, but the immobilized enzymes have good reusability and stability (storage and operational). Besides these properties, the immobilized lipases were also suitable for catalyzing esterification reactions of fatty acids and fatty alcohols, both with a medium chain length. According to our results, esterification activities of immobilized lipases were two‐ and four‐fold higher for chitosan‐ and chitin‐enzyme, than for the free enzyme, respectively. The immobilization procedure shows a great potential for commercial applications of the immobilized lipase, a relatively low cost commercial enzyme.

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.

A comparative study for lipase immobilization onto alginate based composite electrospun nanofibers with effective and enhanced stability

In this study, lipase was successfully immobilized on polyvinyl alcohol/alginate and polyethylene oxide/alginate nanofibers that were prepared by electrospinning. Results showed that nanofibers (especially polyvinyl alcohol/alginate) enhanced the stability properties of lipase. When the free lipase lost its all activity after 40-60min at high temperatures, both lipase immobilized nanofibers kept almost 65-70% activity at the same time. The lipase immobilized poly vinyl alcohol/alginate and polyethylene oxide/alginate nanofibers protected approximately all of their activities until pH 9. Lipase immobilized polyvinyl alcohol/alginate and polyethylene oxide/alginate nanofibers maintained 60% of their activities after 14 and 7 reuses, respectively. The morphology of nanofibers was characterized by Scanning Electron Microscope, Fourier Transform Infrared Spectroscopy and Thermal Gravimetric Analyzer. As a result, this nanofiber production method, electrospinning, is simple, versatile and economical for preparing appropriate carrier to immobilize the enzymes.

A bio-imprinted urease biosensor : Improved thermal and operational stabilities

Despite the increasing number of applications of biosensors in many fields, the construction of a steady biosensor remains still challenging. The high stability of molecularly bio-imprinted enzymes for its substrate can make them ideal alternatives as recognition elements for sensors. Urease (urea aminohydrolase, EC 3.5.1.5), which catalysis the hydrolysis of urea to ammonia and carbon dioxide, has been used in immobilized form in artificial kidney for blood detoxification. According to one report approximately half a million patients worldwide are being supported by haemodialysis. In this study, the enzyme of urease was first complexed by using a substrate analogue, thiourea, in aqueous medium and then this enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, urease noncomplexed with thiourea was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability, pH stability and thermal stability, and also, linear ranges of two biosensors were compared with each other.

Immobilization of bovine catalase onto magnetic nanoparticles.

The scope of this study is to achieve carrier-bound immobilization of catalase onto magnetic particles (Fe3O4 and Fe2O3NiO2 · H2O) to specify the optimum conditions of immobilization. Removal of H2O2 and the properties of immobilized sets were also investigated. To that end, adsorption and then cross-linking methods onto magnetic particles were performed. The optimum immobilization conditions were found for catalase: immobilization time (15 min for Fe3O4; 10 min for Fe2O3NiO2 · H2O), the initial enzyme concentration (1 mg/mL), amount of magnetic particles (25 mg), and glutaraldehyde concentration (3%). The activity reaction conditions (optimum temperature, optimum pH, pH stability, thermal stability, operational stability, and reusability) were characterized. Also kinetic parameters were calculated by Lineweaver–Burk plots. The optimum pH values were found to be 7.0, 7.0, and 8.0 for free enzyme, Fe3O4-immobilized catalases, and Fe2O3NiO2 · H2O-immobilized catalases, respectively. All immobilized catalase systems displayed the optimum temperature between 25 and 35°C. Reusability studies showed that Fe3O4-immobilized catalase can be used 11 times with 50% loss in original activity, while Fe2O3NiO2 · H2O-immobilized catalase lost 67% of activity after the same number of uses. Furthermore, immobilized catalase systems exhibited improved thermal and pH stability. The results transparently indicate that it is possible to have binding between enzyme and magnetic nanoparticles.

Immobilization of Phospholipase A2 on Porous Glass and Its Application for Lowering Serum Cholesterol Concentration

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn‐2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50°C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.

TiO2 beads and TiO2-chitosan beads for urease immobilization

The aim of the present study is to synthesize TiO2 beads for urease immobilization. Two different strategies were used to immobilize the urease on TiO2 beads. In the first method (A), urease enzyme was immobilized onto TiO2 beads by adsorption and then crosslinking. In the second method (B), TiO2 beads were coated with chitosan-urease mixture. To determine optimum conditions of immobilization, different parameters were investigated. The parameters of optimization were initial enzyme concentration (0.5; 1; 1.5; 2mg/ml), alginate concentration (1; 2; 3%), glutaraldehyde concentration (1; 2; 3% v/v) and chitosan concentration (2; 3; 4 mg/ml). The optimum enzyme concentrations were determined as 1.5mg/ml for A and 1.0mg/ml for B. The other optimum conditions were found 2.0% (w/v) for alginate concentration (both A and B); 3.0mg/ml for chitosan concentration (B) and 2.0% (v/v) for glutaraldehyde concentration (A). The optimum temperature (20-60°C), optimum pH (3.0-10.0), kinetic parameters, thermal stability (4-70°C), pH stability (4.0-9.0), operational stability (0-230 min) and reusability (20 times) were investigated for characterization. The optimum temperatures were 30°C (A), 40°C (B) and 35°C (soluble). The temperature profiles of the immobilized ureases were spread over a large area. The optimum pH values for the soluble urease and immobilized urease prepared by using methods (A) and (B) were found to be 7.5, 7.0, 7.0, respectively. The thermal stabilities of immobilized enzyme sets were studied and they maintained 50% activity at 65°C. However, at this temperature free urease protected only 15% activity.

A bio-imprinted ascorbate oxidase biosensor

Interest in bio-imprinting techniques has increased because it allows some stability characteristics of enzymes to be improved. In this study, we developed a simple way to improve the thermal and pH stabilities of ascorbate oxidase biosensor. The membrane of a Clark oxygen electrode was coated by a bioactive layer containing ascorbate oxidase and gelatin cross-linked by glutaraldehyde. Citrate was used to imprint the ascorbate oxidase molecularly. The optimum temperature and pH of both unmodified and citrate modified biosensors were investigated, by comparing their resulting stability. Also, calibration graphs and operational stabilities were compared with each other. The results showed that this simple way should be used to improve the stabilities of a biosensor.

Two Biosensors for Phenolic Compounds Based on Mushroom (Agaricus bisporus) Homogenate: Comparison in Terms of Some Important Parameters of the Biosensors

Abstract Interest in molecular imprinted polymer techniques has increased because they allows for the improvement of some stability characteristics of enzymes. The high stability of molecularly imprinted enzymes for a substrate can make them ideal alternatives as recognition elements for sensors. A bioimprinted mushroom tissue homogenate biosensor was constructed in a very simple way. For this purpose, sulfite was used. The enzyme, polyphenol oxidase, was first complexed by using a competitive inhibitor, sulfite, in aqueous medium and then the enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, polyphenol oxidase uncomplexed with sulfite was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability and thermal, pH, and operational stability; also, the linear ranges of the two biosensors were compared with each other.

Development of a new biosensor for determination of catalase activity.

Catalase is one of the major antioxidant enzymes that catalyzes the hydrolysis of H2O2. The aim of this study was to suggest a new method for the assay of catalase activity. For this purpose, an amperometric biosensor based on glucose oxidase for determination of catalase activity was developed. Immobilization of glucose oxidase was made by a cross-linking method with glutaraldehyde on a Clark-type electrode (dissolved oxygen probe). Optimization and characterization properties of the biosensor were studied and determination of catalase activity in defined conditions was investigated in artificial serum solution. The results were compared with a reference method.