PS-TEPA、PS-g-PEG_（600）树脂合成及其固载α-淀粉酶研究
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摘要
本论文以聚苯乙烯树脂球为对象,通过氯乙酰化后,分别接枝四乙烯五胺或聚乙二醇链,合成两种新型树脂:PS-TEPA树脂、PS-g-PEG600树脂,用于α-淀粉酶的固定化。系统研究了树脂的接枝条件,固定化酶特性,采用荧光光谱分析方法考察了四乙烯五胺与α-淀粉酶、聚乙二醇600与α-淀粉酶相互作用。主要研究内容如下:
1.单因素扫描实验表明:以二氯甲烷作分散剂,分散8 h,在合成温度为85℃,反应时间为12 h,投料比为32 g四乙烯五胺/g树脂条件下,从氯乙酰化聚苯乙烯树脂合成PS-TEPA型树脂的增重率和氮元素含量最大,分别为75%和8%;用1,4-二氧六环作分散剂,分散4 h,在合成温度为35℃,合成时间为6 h,投料比为2.875 g聚乙二醇钠/g树脂条件下,从氯乙酰化聚苯乙烯树脂合成PS-g-PEG600型树脂的增重率和氧元素含量最大,分别为37%和13%。
2.固定化时间8 h,温度为30℃,pH值为8.0是PS-TEPA树脂对α-淀粉酶的最佳固载条件,酶活吸附率和蛋白吸附率最高可分别达到83%和72%;固定化时间10 h,温度为40℃,pH值为7.0是PS-g-PEG600树脂对α-淀粉酶的最佳固载条件,酶活吸附率和蛋白吸附率最高可分别达到60%和56%;在一定的浓度范围内,添加戊二醛对固定化酶的活性没有影响。
3.固定化酶与自由酶的催化动力学性能研究结果表明:酶在固定化后的米氏常数Km值下降,对底物的亲和力上升;两种固定化酶的最大反应初速度vmax均比自由酶有所提高;酶在固定后的最适反应温度从40℃分别提高到50℃(PS-TEPA固定化酶)和60℃(PS-g-PEG600固定化酶),最适pH值范围从pH 5~8变宽为pH 7~8(PS-TEPA固定化酶)和pH 7~10(PS-g-PEG600固定化酶),增强了酶适应pH值波动的能力;固定化酶的抗洗脱和批式操作性能较好;酶的失活半衰期t1/2在固定化后显著延长,在70℃下,t1/2从1 h(自由酶)延长到4.5 h(PS-TEPA)和7.5 h(PS-g-PEG600)。
4.应用荧光光谱、同步荧光光谱和紫外吸收光谱法分别研究了TEPA和PEG600与α-淀粉酶相互作用的机理。PEG600对α-淀粉酶有荧光敏化作用,斯卡查德(Scatchard)方程离解常数随温度的升高而增大;TEPA对α-淀粉酶有荧光淬灭作用,由Stern-Volmer方程和Lineweaver-Burk双倒数函数方程计算得到的淬灭类型为动、静态淬灭的结合,温度越高,静态淬灭越明显;TEPA与α-淀粉酶之间存在非辐射能量转移作用,平均结合位点数为1.31;同步荧光光谱证明添加PEG600会对酪氨酸残基的微环境产生很大的影响,添加TEPA只改变了色氨酸残基的微环境。
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.
1.单因素扫描实验表明:以二氯甲烷作分散剂,分散8 h,在合成温度为85℃,反应时间为12 h,投料比为32 g四乙烯五胺/g树脂条件下,从氯乙酰化聚苯乙烯树脂合成PS-TEPA型树脂的增重率和氮元素含量最大,分别为75%和8%;用1,4-二氧六环作分散剂,分散4 h,在合成温度为35℃,合成时间为6 h,投料比为2.875 g聚乙二醇钠/g树脂条件下,从氯乙酰化聚苯乙烯树脂合成PS-g-PEG600型树脂的增重率和氧元素含量最大,分别为37%和13%。
2.固定化时间8 h,温度为30℃,pH值为8.0是PS-TEPA树脂对α-淀粉酶的最佳固载条件,酶活吸附率和蛋白吸附率最高可分别达到83%和72%;固定化时间10 h,温度为40℃,pH值为7.0是PS-g-PEG600树脂对α-淀粉酶的最佳固载条件,酶活吸附率和蛋白吸附率最高可分别达到60%和56%;在一定的浓度范围内,添加戊二醛对固定化酶的活性没有影响。
3.固定化酶与自由酶的催化动力学性能研究结果表明:酶在固定化后的米氏常数Km值下降,对底物的亲和力上升;两种固定化酶的最大反应初速度vmax均比自由酶有所提高;酶在固定后的最适反应温度从40℃分别提高到50℃(PS-TEPA固定化酶)和60℃(PS-g-PEG600固定化酶),最适pH值范围从pH 5~8变宽为pH 7~8(PS-TEPA固定化酶)和pH 7~10(PS-g-PEG600固定化酶),增强了酶适应pH值波动的能力;固定化酶的抗洗脱和批式操作性能较好;酶的失活半衰期t1/2在固定化后显著延长,在70℃下,t1/2从1 h(自由酶)延长到4.5 h(PS-TEPA)和7.5 h(PS-g-PEG600)。
4.应用荧光光谱、同步荧光光谱和紫外吸收光谱法分别研究了TEPA和PEG600与α-淀粉酶相互作用的机理。PEG600对α-淀粉酶有荧光敏化作用,斯卡查德(Scatchard)方程离解常数随温度的升高而增大;TEPA对α-淀粉酶有荧光淬灭作用,由Stern-Volmer方程和Lineweaver-Burk双倒数函数方程计算得到的淬灭类型为动、静态淬灭的结合,温度越高,静态淬灭越明显;TEPA与α-淀粉酶之间存在非辐射能量转移作用,平均结合位点数为1.31;同步荧光光谱证明添加PEG600会对酪氨酸残基的微环境产生很大的影响,添加TEPA只改变了色氨酸残基的微环境。
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.
引文
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