Master Degree / Yüksek Lisans Tezleri

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Access Right: Open AccessSubject: search.filters.subject.Immobilized enzymes--Biotechnology

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Now showing 1 - 4 of 4
  • Master Thesis
    Immobilization of Lipase From Candida Rugosa on Hydrophobic and Hydrophilic Supports
    (Izmir Institute of Technology, 2001) Öztürk, Banu; Harsa, Hayriye Şebnem; Harsa, Hayriye Şebnem; 03.08. Department of Food Engineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
    The aim of this study is to find the optimum conditions for the immobilization of Candida rugosa lipase and also to select the most suitable support maximizing the immobilized enzyme yield under these optimum conditions.Prior to the immobilization studies, optimum working conditions of soluble Candida rugosa lipase were investigated. Candida rugosa lipase expressed maximum activity in pH 6.5 phosphate buffer with 1.0 M NaCl solution at 30 0C. Indeed, lipase was fully active between 4 and 37 0C. Under these conditions, activity of free lipase was 12.2 U/mg enzyme.Immobilization studies were carried out under constant temperature and stirring rate. Immobilization of lipase on hydrophobic support, octyl-sepharose, was very rapid and the equilibrium was reached in 10 minutes. Immobilized enzyme ratio was maximized by the use of pH 7.0 phosphate buffer with 1.0 M NaCl at 37 0C. Also, optimum solid-liquid ratio was found to be 0.1 when 1 ml support was added to 9 ml of enzyme solution. Effect of enzyme loading on lipase immobilization on octyl-sepharose was investigated at two different temperatures. At 20 0C and in the specified range of enzyme concentration (0.25-75 mg/ml), the experimental data fitted well to the linear isotherms with a C value of 138.2. Also, at 4 0C, results were similar and the experimental data could be expressed with the linear isotherm with a C value of 209.1. Under these conditions, activity of immobilized enzyme was 410 U/ml hydrated support.In the case of immobilization on hydrophilic support, chitosan, the reaction was much slower and the amount of the immobilized enzyme was maximized at the end of 24th hour. Optimum conditions of immobilization were determined as pH 6.5 and 20 0C. In this case, addition of salt decreased the immobilization and, therefore, low ionic strengths favored immobilization. Again, optimum solid-liquid ratio was found to be around 0.1 when the 1 ml support was added to 9 ml. of enzyme solution. The effect of enzyme loading was investigated at 20 0C and in the specified range of concentration, data fitted well to the Langmuir isotherm where the constants were calculated to be qm.200 U adsorbed enzyme/ml hydrated support and K. 72.5 U/ml solution.The storage stability of chitosan immobilized lipase was investigated at 4 0C under two different conditions. When immobilized lipase was kept dry for 25 days, 50 % of the initial activity remained. However, if the same lipase was kept in phosphate buffer of pH 6.5, activity loss was only 10 % after a period of 2 months. Complementary work was also carried out to find the differences in the behavior of free and chitosan immobilized lipase against different substrates. The results showed that the presence of 33 % olive oil and 45.5 % corn oil in substrate yielded the maximum activity for free and chitosan immobilized lipases, respectively.
  • Master Thesis
    Multivariate Statistical Optimization of Enzyme Immobilization Onto Solid Matrix Using Central Composite Design
    (İzmir Institute of Technology, 2013) Arpakcı, Tuğba; Özdemir, Durmuş; Özdemir, Durmuş; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of Technology
    In recent years, scientist have been used alternative technology in order to increase enzyme stability and also reduce the cost of production of enzyme. Immobilization methods have attracted the attention of scientists due to its advantages in comparison with soluble enzyme or other methods. Immobilization process can be affected by many factors for this reason it is important to optimize the effective factors in order to enhance success of this process. In preliminary studies, Bradford protein assay was used for determination of protein concentration. In order to increase sensitivity and accuracy of this assay, Bradford protein assay was combined with a multivariate calibration methods. Genetic Inverse Least Squares (GILS) and Partial Least Squares (PLS) were used for multivariate calibration. Calibration model was constructed for various concentration of Bovine Serum Albumin (BSA). Standard Error of Calibration (SEC) and Standard Error of Prediction (SEP) were calculated and results of multivariate calibration method were compared with univariate calibration methods and each other. In this study, the bovine serum albumin immobilization studies were carried out. The bovine serum albumin was immobilized on chitosan nanoparticles and effective factors such as chitosan concentration, immobilization time, pH and temperature were optimized by using central composite design (CCD). Central composite design is used to investigate interaction between these parameters and to find the optimum values of effective factors.
  • Master Thesis
    Immobilization of Thermophilic Recombinant Esterase Enzyme by Microencapsulation in Alginate-chitosan/Caci2 Polyelectrolyte Beads
    (Izmir Institute of Technology, 2011) Tercan, Cisem; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of Technology
    In recent years, enzyme immobilization has gained importance for design of artificial organs, drug delivery systems, and several biosensors. Polysaccharide based natural biopolymers used in enzyme or cell immobilization represent a major class of biomaterials which includes agarose, alginate, dextran, and chitosan. Especially, chitosan has used many biomedical applications, including tissue engineering, because of its biodegradability and biocompatibility, non-toxicity and degradation in the body. In this research, Recombinant esterase enzyme was purified from Thermophilic Bacillus sp. That was isolated from Balçova (Agamemnon) Geothermal region in İzmir by using one-step affinity purification chromatography. In the second step, purified enzyme encapsulated in alginate-chitosan/CaCl2 polyelectrolyte beads that were prepared by adding dropwise a protein-containing sodium alginate mixture into a chitosan-CaCl2 crosslinker solution. And then the polyelectrolyte beads were stabilized in at the same crosslinker solution 30 minutes more. In the third step, the effect of different conditions were tested such as temperature and pH, bead diameter, reuse of beads. Also the effects of inhibition of CaCl2, ZnCl2, MgCl2, CuSO4, MgSO4, Sodium dodecyl sulfate (SDS) and Triton X-100 onto the immobilized and free enzyme activity were studied. In the last step, analysis of surface morphologies of polyelectrolyte beads were determined and examined by means of Scanning Electron Microscope.
  • Master Thesis
    Immobilization of Thermophilic Recombinant Esterase Enzyme by Entrapment in Coated Ca-Alginate Beads
    (Izmir Institute of Technology, 2009) Gülay, Seçkin; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of Technology
    Recently, industrial enzymes produced by micro-organisms are being utilized widely, especially from thermophilic ones due to their ability to withstand intense heat. Esterase enzymes from thermophilic micro-organisms are special interest in a variety of biotechnological applications because of their many useful properties. Due to potential use as biocatalysts in variety of biotechnological applications, esterase enzyme isolated from Balçova (Agamemnon) geothermal site were aimed to be immobilized via a costeffective protocol, in order to be re-used over very long periods of time. Previously, the gene encoding thermophilic esterase from the thermal environmental samples, isolated from Balçova (Agamemnon) geothermal site, was cloned and respective protein was expressed in Escherichia coli in our group. In this study using that recombinant esterase enzyme, expression, purification and immobilization studies were carried out. The esterase enzyme was immobilized in the Ca-alginate beads which were coated with silica and the effects of the temperature and pH on the immobilized enzyme was determined and diameter of the beads, reuse and surface of the beads were analyzed. Immobilization yield for coated beads was determined as 71.27% and compared with non-coated ones which were 45.80%. Analysis of surface morphologies of beads was compared with Scanning Electron Microscope.