Conclusion 51 



STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM POLYPHENOL 



OXIDASES 52 



Introduction 52 



Materials and Methods 53 



Extraction of Mushroom, Potato, Lobster, and Shrimp PPO ... 53 



Purification of Mushroom, Potato, Lobster, and Shrimp PPO . . 54 



Protein Quantitation and Enzyme Purity Determination 54 



Enzyme Activity Assay 55 



Anti -lobster PPO Antibody Production and Purification .... 55 



Molecular Weight Determination of Anti-lobster PPO Antibody . 56 

 Antibody Titer Determination by Enzyme-linked Immunosorbent 



Assay (ELISA) 57 



Analysis of Antigenic Properties of PPO 58 



Immunoblotting 58 



Spectropolarimetric Analysis of PPO 59 



Results and Discussion 60 



Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis 



(SDS-PAGE) Profile of PPO 60 



Antibody Production and Molecular Weight Determination. ... 63 



Antibody Titer Determination 63 



Immunological Characteristics of PPO 70 



Spectropolarimetric Analysis of PPO 75 



Conclusion 89 



INHIBITION MECHANISM OF KOJIC ACID ON SOME PLANT AND CRUSTACEAN 



POLYPHENOL OXIDASES 90 



Introduction 90 



Materials and Methods 91 



Extraction and Purification of Potato PPO 92 



DEAE-cellulose Chromatography 93 



Gel Filtration 93 



Extraction and Purification of Apple PPO 94 



Hydroxyl apatite Chromatography 94 



Extraction and Purification of Grass Prawn PPO 95 



Extraction and Purification of Lobster PPO 96 



Extraction and Purification of white shrimp PPO 97 



Protein Quantitation and Enzyme Purity Determination 98 



Enzyme Activity Assay 98 



Effect of Kojic Acid on Enzyme Activity 99 



Enzyme Kinetics Study 100 



Effect of Pre-incubation Temperature on PPO Inhibition by 



Kojic Acid 102 



Effect of Kojic Acid on the Hydroxyl ation Capability of PPO . 103 



Effect of Kojic Acid on Oxygen Uptake by PPO Reaction .... 103 



Effect of Kojic Acid on Reduction of Cupric Copper 104 



Effect of Kojic Acid on Quinone Products 104 



Enzymatic Activities of Kojic Acid-treated PPO 105 



Statistical Analysis 106 



iv 



