Synthesis Of PS-TEPA And PS-g-PEG₆₀₀ For The Immobilization Of α-amylase

In this study, chloroacetylated polystyrene microspheres were grafted with TEPA and PEG600 respectively. The obtained PS-TEPA and PS-g-PEG600 resin were applied in the immobilization ofα-amylase. The conditions for grafting, the characters of immobilized enzyme and the fluorescence spectrum of the interaction betweenα-amylase and TEPA (or PEG600) were systematically investigated.1. After dispersing in methylene dichloride for 8 h, the optimal conditions for the synthesis of PS-TEPA from chloroacetylated polystyrene were 85℃, 12 h, the feed rate of TEPA to resin was 32:1. The weight gain rate was 75% and the content of nitrogen was 8%. The optimal conditions for the synthesis of PS-g-PEG600 from chloroacetylated polystyrene were 35℃, 6 h, when dispersing in 1,4-dioxane for 4 h, the feed rate of PEG600 to resin was 2.875:1. The weight gain rate was 37% and the content of nitrogen was 13%.2. The suitable conditions of the immobilization ofα-amylase on PS-TEPA were 30℃, pH 8.0 and 8 h of reaction time. The maximal adsorption rate ofα-amylase on PS-TEPA was 83% (enzymatic activity) or 72% (protein content). The suitable conditions of the immobilization ofα-amylase on PS-g-PEG600 were 40℃, pH 7.0 and 10 h of reaction time. The maximal adsorption rate ofα-amylase on PS-g-PEG600 was 60% (enzymatic activity) or 56% (protein content). The addition of glutaraldehyde had no harmful effect on the activity of immobilized enzymes.3. Compared to the free enzyme, the immobilized enzymes exhibited a higher affinity to the substrates, the values of Km were decreased, the values of vmax were increased. The optimum reaction temperatures were raised from 40℃(free enzyme) to 50℃(PS-TEPA) and 60℃(PS-g-PEG600) respectively. The optimum reaction pH ranges were widened from pH 7~8 (free enzyme) to pH 7~10 (PS-TEPA) and pH 5~8 (PS-g-PEG600) separately. The desorption rate ofα-amylase from the carriers were relatively lower. The half life times ofα-amylase t1/2 were prolonged after the immobilization, from 1 h (free enzyme) to 4.5 h (PS-TEPA) and 7.5 h (PS-g-PEG600) at 70℃.4. The interactions ofα-amylase with TEPA and PEG600 were investigated by fluorescence, synchronous fluorescence and UV absorption spectrometry. PEG600 sensitized the fluorescence ofα-amylase. The dissociation constant solving Scatchard equation was increased with the increased temperature. TEPA had a quenching effect on the fluorescence ofα-amylase. The quenching type was a complex of dynamic and static quenching according to the fitting results of Stern-Volmer and Lineweaver-Burk equation. The effect of non-radiation energy translocation existed between TEPA andα-amylase. The average number of binding site was 1.31. The synchronous fluorescence spectrum shew that the addition of TEPA variated the micro-environment of tryptophan residue and PEG600 had significant influence on the micro-environment of tyrosine residue.