Studies On The Preparation And Properties Of Immobilized Alcoholoxidase

Immobilized enzyme technique is an important component of modern bioengineering and biotechnology. The application of immobilized enzymes have gone deep into the fields of industry, medicine, analytical and separation techniques, chemical synthesis, and so oa In this thesis, the methods of immobilization of enzymes and changes in properties of immobilized enzymes have been reviewedTwo kinds of immobilized enzymes were prepared by using alcoholoxidases obtained from Candida boidirii and Pichia pastoris, respectively, as model and using chitoson and cellulose as carriers. The properties of the immobilized enzymes are investigatedAlcoholoxidase from Candida boidirri was immobilized on glutaraldehyde activated chitosan made from shrimp chitin. Enzyme activity for immobilized alcoholoxidase was 770 U/g chitosan (wet weight), which represents approximately 60% activity when compared with alcoholoxidase in solution. The optimum pH and temperature for enzyme activity changes upon immobilization . Optimum pH becomes more acidic, while the optimum temperature increases by 10 癈. Also, immobilized enzyme can function over a wider temperature range. The bound enzyme showed excellent stability to repeated use and retained approximately 86% of its initial activity after 8 cycles of use.We determined the kinetics parameters of alcoholoxidase solutions and immobilized alcoholoxidases, including Michaelis constant, maximum reaction velocity, and inhibition constant Km and Vmax studies indicated that immobilized enzyme displayed greater transport resistance than the soluble enzyme. The immobilized alcoholoxidase showed inhibition at high substrate concentration.Alcoholoxidase from Pichia pastoris was immobilized on aminized cellulose, using glutaraldehyde as crosslinking agent The optimum temperature of the immobilized enzyme can reach 35癈. After incubated at 50癈 for Ih, its residual activity is higher than 75%. The bound enzyme showed excellent stability to repeated use and retained approximately 220% ofits initial activity after 8 cycles of use. Being immobilized on cellulose, Alcoholoxidase from Pichia pastoris has some degree of increase in thermal stability and durability for thermal treatment and repeated use.The yellow substance produced by the reaction of ammoniurn salts, acetyl acetone with aldehyde group can absorb UV-Vis light and emit fluorescence. All factors which influence the reaction speed of the yellow product were investigated by UV-Vis and Fluorescence Spectrometry. Under ambient temperature, the yellow substance product of the reaction of formaldehyde with 4mol/L ammoniurn citrate and 0.04mol/L acetyl acetone can be quickly developed in 24 seconds. The linear correlation of formaldehyde measured by fluorescence is 0.4- 1.0 u g/ml. The method has been applied to measure formaldehyde concentrations in tap water with satisfactory results.