Isolation, Purification, Immobilization and Characterization of Chitosan Lipoxygene
Hanan Khaled Farahat
- 2015
- p. ill. 21 cm.
Supervisor: Mona Bakr
Thesis (M.A.)—Nile University, Egypt, 2015 .
"Includes bibliographical references"
Contents: 1.2 Structure of Enzyme 1.3 Role of iron 1.4 Mechanism of Action 1.5 Roles of substrates 1.6 LOX inhibition 1.6.1 Mechanisms of LOX inhibition 1.7 LOX in human 1.7.1 LOX and Cancer 1.7.1.i Procarcinogenic effect 1.7.1.ii Anticarcinogenic effect 1.7.1.iiiAntagonistic effects of LOX products 1.7.2 Inflammation 1.7.3 Skin Cancer 1.8 LOX from Avocado 1.9 Avocado LOX pathway Chapter II 2. Literature Review 2.1 Nanobiotechnology 2.2 Definition 2.3 Bionanotechnology 2.4 Enzymes in Biotechnology 2.5 Enzyme properties 2.6 Enzyme instability 2.7 Enzyme immobilization 2.8 Different techniques used for immobilization 2.8.1 Adsorption 2.8.2 Covalent bonding 2.8.3 Affinity immobilization 2.8.4 Entrapment 2.9 Materials used for immobilization 2.9.1Natural Polymers as supports 2.9.1.i Alginate 2.9.1.ii Chitosan and Chitin 2.9.1.iiii Collagen 2.9.1.iv Carrageena 2.9.1.v Gelatin 2.9.1.vi Cellulose 2.9.1.vii Strach 2.9.1.viii Pectin 2.9.1.ix Sepharose 2.9.2 Synthetic Polymers as supports 2.9.3 Inorganic materials as supports 2.9.3.i Zeolites 2.9.3.ii Ceramics 2.9.3.iii Celite 2.9.3.iv Silica 2.9.3.v Glass 2.9.3.vi Activated Carbon 2.9.3.vii Charcoal 2.10 Applications and scope 2.10.1 Applications of immobilized enzymes 2.10.1.i Applications in Biosensors 2.10.1.ii Applications in medical application 2.10.1.iii Applications in Antibiotic production 2.10.1.iv Applications in food industry 2.10.1.v Applications in biodiesel production 2.10.1.vi Applications in bioremediation 2.10.2 LOX in baking industry 2.11 Plant pigment 2.12 Flour bleaching activities 2.13 Chitin and chitosan 2.13.1 Chitin processing 2.13.2 Properties of chitin and chitosan 2.13.2.i Chemical properties of chitosan 2.13.2.ii Biological properties of chitosan 2.13.3 Molecular weight of chitosan 2.13.4 Toxicity test of chitosan Chapter III 3.1 Materials 3.2 Characterization Technology 3.3 Methodology 3.3.1 LOX enzyme extraction from Avocado 3.3.2 Protein content determination 3.3.2.i Lowry method 3.3.2.ii Procedure of Lowry method 3.3.3 Activity with various substrates 3.3.4 Michaelis Menten Kinetic parameters 3.3.5 Temperature optimum 3.3.6 pH optimum 3.3.7.i Chitosan NP preparation 3.3.7.ii Immobilization of LOX on Chitosan NP 3.3.8 Stain removal and Basic degree of whitness 3.3.8.i Test procedure 3.3.8.ii Reflectance measurement 3.3.9 FTIR test Chapter IV 4. Results and Discussion 4.1 Enzyme Extraction 4.2 Protein content determination 4.3 Activity with various substrates 4.4 LOX enzyme kinetic parameters 4.5 Temperature effect on avocado LOX activity 4.6 pH effect on avocado LOX activity 4.7 Chitosan nanoparticles preparation 4.8 LOX enzyme immobilization on chitosan nanoparticles 4.9 Cleanability test 4.9.1 Soil removal 4.9.2 Evaluation 4.9.2.i Stain removal 4.9.2.ii Basic degree of whitness 4.10 FTIR Spectroscopy
Abstract: Lipoxygenases (LOX) are a class of oxidoreductases (non-hem, ferrum-free, transferring oxygen and hydrogen) and they are wide-spread in plants and animals. Lipoxygenases enzymatic activity has a significant detrimental effect on the organoleptic and nutritional values of minimally processed fruits and vegetables. These group of enzymes catalyze the oxidation of polyunsaturated fatty acids (PUFA) stimulating the commencement and propagation of the free radical chain reaction. Natural forms of the enzyme's substrate are polyunsaturated fatty acids, appearing in plants (linoleic (LA), linolenic (LN) and arachidonic acid (ANA), methyl/ethyl esters of linoleic acid). The aim of the study is to isolate and purify lipoxygenase from avocado, to determine enzyme activity, kinetics of reaction and determine the kinetic parameters. Also determine the optimum temperature and pH at which the enzyme works best. To increase the chemical and thermal stability of the enzyme, we immobilized the enzyme on the surface of chitosan nanoparticles. We tested the activity of the enzyme as a bleaching agent in detergent industry. Our results indicate that the activity of the enzyme increased by 30% upon immobilization.