磁性壳聚糖复合微球固定化果胶酶及反应动力学研究
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摘要
果胶酶是分解植物主要成分之一果胶质的酶类,主要应用于果汁果酒行业。但是由于液态酶用量大,成本较高,加上精制困难,保存期短,难以回收,影响了其在食品工业中的应用。固定化酶可以在一定程度上弥补这一缺陷,同时还可减少外源物质的污染。但果胶物质在植物细胞壁中并不单独存在,它通常与纤维素互相结合,果胶酶没有纤维素酶的协同作用,不能破解纤维素构成的致密保护结构,因此对果胶质的水解便会削弱。目前对果胶酶的固定化虽然已有所研究,但以磁性载体对果胶酶的固定化研究较少,对果胶酶和纤维素酶的共固定化更鲜见报道。本文以自制的磁性壳聚糖复合微球作为载体对果胶酶进行了固定化;并对其反应动力学进行了研究,为固定化酶催化反应器的设计及操作条件的确定提供良好的理论依据,为固定化酶在工业化生产中的实际应用提供一个可靠的技术参考,具有重大的现实意义。同时根据果胶酶和纤维素酶的协同效应,应用联合固定化技术对双酶的共固定化进行了初步探索,为这两种酶开辟了新的应用领域。其主要研究结果如下:
1、试验首先通过化学共沉淀法合成了纳米级Fe3O4粒子,并将其作为磁核利用乳化交联法制备出磁性壳聚糖复合微球,利用TEM、SEM、FT-IR、激光粒度仪及紫外分光光度计等分别对微球的粒径、形貌、结构、粒度分布和磁响应性进行了表征。结果表明所制得的磁性壳聚糖微球的粒径在50nm,分布较窄,且呈规则的球形,红外光谱测定了微球的特征官能团结构,且表明已包覆了Fe3O4粒子,分光光度法表明磁性微球具有很强的磁响应性,是一种固定化酶的优良载体。
2、通过酶促反应动力学试验,得到pH、温度、产物浓度、底物浓度对酶催化反应速度均有较大的影响,且产生了产物抑制和高浓度底物抑制作用。并求出了固定化果胶酶的米氏常数K' m=2.67 ,游离酶的米氏常数K m=2.25,即固定化酶对底物的亲和力减小,这可能与固定化载体壳聚糖的空间障碍和扩散限制影响有关;同时求取产物抑制常数和底物抑制常数分别为K is=0.234和K iP=0.386,并得到了产物抑制和底物抑制下的速率方程。
3、通过研究分配效应、外扩散限制及内扩散限制对固定化酶反应系统的影响,给出了各因素影响下的动力学模型。
4、利用吸附法对果胶酶和纤维素酶进行共固定化试验,优化了共固定化的条件,即戊二醛浓度为5%、两酶量比例为3:2、吸附时间为6h。并通过对游离态的和共固定化后的两酶的酶学性质对比得到:共固定化后两酶的最适温度升高;最适pH值降低,且范围拓宽;热稳定性均有所提高;果胶酶半衰期为45天,纤维素酶为32天。
Pectinase is enzymes of decompose pectin of the plant. It is mainly applicated in dustry of fruit juice and wine. But because of the liquid enzyme's large dosage, high cost, difficult refine, short shelf life, the difficulty in recovery, influence its application in food industry. Then immobilized enzyme may make up the defects in certain degree. At the same time, it can also reduce the pollution of exogenous substance on fruit products. But pectin in the plant cell wall doesn't exist solely. It generally exist combinateing with cellulose. Pectinase can't decode compact protective structure of cellulose without the synergism of cellulase, so can't hydrolyze well pectin.
At present, immobilization of pectinase has been researched. But study on immobilization of pectinase by magnetic carrier is less. Studies on the Co-immobilization of pectinase and cellulase have rarely been reported. In this article, the pectinase was immobilized on the magnetic chitosan microspheres, and studied kinetics of reaction on immobilized pectinase. It provided a theoretical basis for the design of immobilized enzyme reactor and determination of the operating conditions. Meanwhile, according to the synergistic effect of pectinase and cellulose, preliminary exploration on immobilized bi-enzyme by applying technology of co-immobilization, opened up new fields of application for the two kinds of enzymes.
The main results were as follows:
1. First nanometer Fe3O4 particle was synthesized by coprecipitation, then magnetic chitosan microspheres were prepared by the suspension cross-linking technique. Finally, the size, morphology, structure, size distribution and magnetic response properties were studied by means of TEM, SEM, FT-IR, MASTERSIZE. The results indicate: the size of magnetic chitosan microspheres were about 50 nano-sizes, distribution was narrow, and the morphology was all sphericity. FT-IR results show the structure of magnetic microspheres and the Fe3O4 particle was coated completely by the chitosan. The spectrophotometry indicate:magnetic microspheres have strong magnetic response properties. It is a kind of superior carrier of immobilization of enzyme.
2. Through the experiment of kinetics of enzyme-catalyzed reaction, results showed that pH, temperature, substrate concentration, product concentration had strong effects to the rate of the enzyme catalysis reaction, and the product inhibition and high concentration substrate inhibition were produced. Km of immobilized pectinase was 2.67, and liquid pectinase was 2.25. So the affinity immobilized pectinase to substrate was reduced. This may be the result of diffusion limitation and steric hindrance of carrier. Inhibition constant of product and substrate were 0.234 and 0.386 respectively, accordingly gained reaction rate equation.
3. By the study on allocation effect, outer diffusional limitation, inner diffusional limitation, allocation effect on immobilized enzyme reaction system, kinetics model and were given respectively.
4. By the test of co-immobilization of pectinase and cellulase with adsorption method, the optimum conditions of the reaction were as follows: glutaraldehyde concentration 5%, the volume of two enzymes ratio 3:2, adsorption time 6h. Through the comparison of free pectinase and cellulose with co-immobilized two enzymes, the result of experiment on the property of enzyme were as follows: their optimal temperature is rise, optimal pH is low, but range wide, thermal stability were all enhanced, half life time of pectinase and cellulose were 45d and 32d respectively. The results had industrial application value.
1、试验首先通过化学共沉淀法合成了纳米级Fe3O4粒子,并将其作为磁核利用乳化交联法制备出磁性壳聚糖复合微球,利用TEM、SEM、FT-IR、激光粒度仪及紫外分光光度计等分别对微球的粒径、形貌、结构、粒度分布和磁响应性进行了表征。结果表明所制得的磁性壳聚糖微球的粒径在50nm,分布较窄,且呈规则的球形,红外光谱测定了微球的特征官能团结构,且表明已包覆了Fe3O4粒子,分光光度法表明磁性微球具有很强的磁响应性,是一种固定化酶的优良载体。
2、通过酶促反应动力学试验,得到pH、温度、产物浓度、底物浓度对酶催化反应速度均有较大的影响,且产生了产物抑制和高浓度底物抑制作用。并求出了固定化果胶酶的米氏常数K' m=2.67 ,游离酶的米氏常数K m=2.25,即固定化酶对底物的亲和力减小,这可能与固定化载体壳聚糖的空间障碍和扩散限制影响有关;同时求取产物抑制常数和底物抑制常数分别为K is=0.234和K iP=0.386,并得到了产物抑制和底物抑制下的速率方程。
3、通过研究分配效应、外扩散限制及内扩散限制对固定化酶反应系统的影响,给出了各因素影响下的动力学模型。
4、利用吸附法对果胶酶和纤维素酶进行共固定化试验,优化了共固定化的条件,即戊二醛浓度为5%、两酶量比例为3:2、吸附时间为6h。并通过对游离态的和共固定化后的两酶的酶学性质对比得到:共固定化后两酶的最适温度升高;最适pH值降低,且范围拓宽;热稳定性均有所提高;果胶酶半衰期为45天,纤维素酶为32天。
Pectinase is enzymes of decompose pectin of the plant. It is mainly applicated in dustry of fruit juice and wine. But because of the liquid enzyme's large dosage, high cost, difficult refine, short shelf life, the difficulty in recovery, influence its application in food industry. Then immobilized enzyme may make up the defects in certain degree. At the same time, it can also reduce the pollution of exogenous substance on fruit products. But pectin in the plant cell wall doesn't exist solely. It generally exist combinateing with cellulose. Pectinase can't decode compact protective structure of cellulose without the synergism of cellulase, so can't hydrolyze well pectin.
At present, immobilization of pectinase has been researched. But study on immobilization of pectinase by magnetic carrier is less. Studies on the Co-immobilization of pectinase and cellulase have rarely been reported. In this article, the pectinase was immobilized on the magnetic chitosan microspheres, and studied kinetics of reaction on immobilized pectinase. It provided a theoretical basis for the design of immobilized enzyme reactor and determination of the operating conditions. Meanwhile, according to the synergistic effect of pectinase and cellulose, preliminary exploration on immobilized bi-enzyme by applying technology of co-immobilization, opened up new fields of application for the two kinds of enzymes.
The main results were as follows:
1. First nanometer Fe3O4 particle was synthesized by coprecipitation, then magnetic chitosan microspheres were prepared by the suspension cross-linking technique. Finally, the size, morphology, structure, size distribution and magnetic response properties were studied by means of TEM, SEM, FT-IR, MASTERSIZE. The results indicate: the size of magnetic chitosan microspheres were about 50 nano-sizes, distribution was narrow, and the morphology was all sphericity. FT-IR results show the structure of magnetic microspheres and the Fe3O4 particle was coated completely by the chitosan. The spectrophotometry indicate:magnetic microspheres have strong magnetic response properties. It is a kind of superior carrier of immobilization of enzyme.
2. Through the experiment of kinetics of enzyme-catalyzed reaction, results showed that pH, temperature, substrate concentration, product concentration had strong effects to the rate of the enzyme catalysis reaction, and the product inhibition and high concentration substrate inhibition were produced. Km of immobilized pectinase was 2.67, and liquid pectinase was 2.25. So the affinity immobilized pectinase to substrate was reduced. This may be the result of diffusion limitation and steric hindrance of carrier. Inhibition constant of product and substrate were 0.234 and 0.386 respectively, accordingly gained reaction rate equation.
3. By the study on allocation effect, outer diffusional limitation, inner diffusional limitation, allocation effect on immobilized enzyme reaction system, kinetics model and were given respectively.
4. By the test of co-immobilization of pectinase and cellulase with adsorption method, the optimum conditions of the reaction were as follows: glutaraldehyde concentration 5%, the volume of two enzymes ratio 3:2, adsorption time 6h. Through the comparison of free pectinase and cellulose with co-immobilized two enzymes, the result of experiment on the property of enzyme were as follows: their optimal temperature is rise, optimal pH is low, but range wide, thermal stability were all enhanced, half life time of pectinase and cellulose were 45d and 32d respectively. The results had industrial application value.
引文
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