海藻糖合成酶的固定化研究
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摘要
海藻糖在自然界中广泛存在于低等植物、细菌、真菌、酵母、昆虫及无脊椎动物中，在许多情况下，该糖是昆虫血糖的主要成分。海藻糖是一种非特异性的保护剂，似乎对所有的生物分子都具有一定的保护功能。它可以保护生物膜、蛋白质、脂质体及敏感细胞壁免受干旱、冷冻、渗透压的变化等造成的伤害，由于这些独特的生物学功能，海藻糖在工业上可用于不稳定食品、化妆品和医药的保存。
本课题目的在于寻找一条可行的海藻糖生产途径—固定化酶法。具体的步骤包括：1、从土壤中分离获得菌株GX—0012，产生一种海藻糖合成酶，能转化麦芽糖合成海藻糖，进一步研究表明该酶是一种胞内酶。2、将菌株GX—0012进行大量的培养，富集发酵菌丝体，经超声破碎、浸提、离心后获得粗酶液。3、粗酶液经硫酸铵分级沉淀后，收集相应的活性部分，用葡聚糖凝胶进一步纯化，通过电泳来测定海藻糖合成酶的纯度和分子量。4、将粗酶液用壳聚糖凝胶固定化，制得固定化酶，固定化酶的活性分析是通过测定固定化酶与麦芽糖反应后混合物中的海藻糖含量来实现的。
SDS聚丙烯酰胺凝胶电泳测定结果显示，该海藻糖合成酶有两条亚基带，测定它们的分子量分别为55000Da和50000Da。
研究表明制备固定化酶最佳的条件是：戊二醛的浓度为0.5％；交联的PH值为8.0；交联的温度为15℃；交联的最佳时间为12小时；液态酶与壳聚糖凝胶的质量比为1∶1，按此条件制备的固定化酶反应的活性最高，生成的海藻糖量最多，海藻糖的最高含量能达到40％左右。另外，固定化酶转化麦芽糖为海藻糖的最佳反应时间为18小时，这时可以获得最高含量的海藻糖。
另外通过高效阴离子色谱仪、高效液相色谱仪、旋光仪、傅立叶红外光谱仪、液质联用仪几种测试手段，最终可以确认：海藻糖合成酶能够将麦芽糖转化为D—(+)—海藻糖，即自然界中存在最多的(α，α)型海藻糖。
Trehalose exists in nature widely, including lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. In most cases trehalose is the major sugar in the blood of insects. Trehalose is an specific protectant, which seems to protect all biological molecules. It can protect biological membranes, proteins, liposomes, and sensitive cytoderms etc. from damage due to drought, freezing and osmotic pressure changes. Because of its distinctive biological functions, trehalose may possibly have various industrial applications such as a preservative material for unstable food, cosmetics and medicines.
On the basis of them my studies is carrying on in order to find one efficient way of actual industrious production-immobilized enzyme method. Concrete step include: 1. separate a kind of bacterium GX?0012 from soil and produce trehalose synthase enzyme and transform maltose to trehalose, following study find that trehalose synthase enzyme is a kind of enzyme in the cell. 2. substantive cultivate bacterium GX?0012 and enrich culture mycelium and the cells harvested were disrupted by ultrasonic, followed by leaching and centrifugation to obtain crude enzyme solution. 3. The crude enzyme was fractionated with ammonium sulfate and the active fractions were collected, dialyzed, concentrated and successively purified by column chromatography packing Sephadex G-100. The purity and molecular weight of the enzyme were determined by SDS polyacrylamide gel electrophoresis. 4. Coarse enzyme solution were immobilized by polysaccharide gel and produced immobilized enzyme. Activity analysis of immobilized enzyme were rea
lized by trehalose content in reaction mixture of immobilized enzyme and
maltose.
SDS polyacrylamide gel electrophoresis determined result showed that trehalose synthase enzyme appeared to be a dimmer. Their molecular weight was 55000 Da and 5000 Da respectively.
The study result showed that optimal condition of producing immobilized enzyme: the concentration of aldehyde is 0.5%; the pH value of unite is 8.0; the temperature of unite is 15癈; the optimal time of unite is 12 hour; the ratio of quality for liquid stated enzyme and polysaccharide gel is 1:1; in the condition of these immobilized enzyme reaction activity is the highest and producing trehalose quantity is the most, the most content of trehalose can arrive to around 40%. Otherwise, the optimal reaction time is 18 hour for immobilized enzyme changing maltose to trehalose and we can acquire the highest content of trehalose. Otherwise by HPAEC,HPLC etc determined means, at last we can affirmed that immobilized trehalose synthase enzyme can transform maltose to D- (+) -trehalose and D- (+) -trehalose is the most (a , a )-trehalose in the nature.
本课题目的在于寻找一条可行的海藻糖生产途径—固定化酶法。具体的步骤包括：1、从土壤中分离获得菌株GX—0012，产生一种海藻糖合成酶，能转化麦芽糖合成海藻糖，进一步研究表明该酶是一种胞内酶。2、将菌株GX—0012进行大量的培养，富集发酵菌丝体，经超声破碎、浸提、离心后获得粗酶液。3、粗酶液经硫酸铵分级沉淀后，收集相应的活性部分，用葡聚糖凝胶进一步纯化，通过电泳来测定海藻糖合成酶的纯度和分子量。4、将粗酶液用壳聚糖凝胶固定化，制得固定化酶，固定化酶的活性分析是通过测定固定化酶与麦芽糖反应后混合物中的海藻糖含量来实现的。
SDS聚丙烯酰胺凝胶电泳测定结果显示，该海藻糖合成酶有两条亚基带，测定它们的分子量分别为55000Da和50000Da。
研究表明制备固定化酶最佳的条件是：戊二醛的浓度为0.5％；交联的PH值为8.0；交联的温度为15℃；交联的最佳时间为12小时；液态酶与壳聚糖凝胶的质量比为1∶1，按此条件制备的固定化酶反应的活性最高，生成的海藻糖量最多，海藻糖的最高含量能达到40％左右。另外，固定化酶转化麦芽糖为海藻糖的最佳反应时间为18小时，这时可以获得最高含量的海藻糖。
另外通过高效阴离子色谱仪、高效液相色谱仪、旋光仪、傅立叶红外光谱仪、液质联用仪几种测试手段，最终可以确认：海藻糖合成酶能够将麦芽糖转化为D—(+)—海藻糖，即自然界中存在最多的(α，α)型海藻糖。
Trehalose exists in nature widely, including lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. In most cases trehalose is the major sugar in the blood of insects. Trehalose is an specific protectant, which seems to protect all biological molecules. It can protect biological membranes, proteins, liposomes, and sensitive cytoderms etc. from damage due to drought, freezing and osmotic pressure changes. Because of its distinctive biological functions, trehalose may possibly have various industrial applications such as a preservative material for unstable food, cosmetics and medicines.
On the basis of them my studies is carrying on in order to find one efficient way of actual industrious production-immobilized enzyme method. Concrete step include: 1. separate a kind of bacterium GX?0012 from soil and produce trehalose synthase enzyme and transform maltose to trehalose, following study find that trehalose synthase enzyme is a kind of enzyme in the cell. 2. substantive cultivate bacterium GX?0012 and enrich culture mycelium and the cells harvested were disrupted by ultrasonic, followed by leaching and centrifugation to obtain crude enzyme solution. 3. The crude enzyme was fractionated with ammonium sulfate and the active fractions were collected, dialyzed, concentrated and successively purified by column chromatography packing Sephadex G-100. The purity and molecular weight of the enzyme were determined by SDS polyacrylamide gel electrophoresis. 4. Coarse enzyme solution were immobilized by polysaccharide gel and produced immobilized enzyme. Activity analysis of immobilized enzyme were rea
lized by trehalose content in reaction mixture of immobilized enzyme and
maltose.
SDS polyacrylamide gel electrophoresis determined result showed that trehalose synthase enzyme appeared to be a dimmer. Their molecular weight was 55000 Da and 5000 Da respectively.
The study result showed that optimal condition of producing immobilized enzyme: the concentration of aldehyde is 0.5%; the pH value of unite is 8.0; the temperature of unite is 15癈; the optimal time of unite is 12 hour; the ratio of quality for liquid stated enzyme and polysaccharide gel is 1:1; in the condition of these immobilized enzyme reaction activity is the highest and producing trehalose quantity is the most, the most content of trehalose can arrive to around 40%. Otherwise, the optimal reaction time is 18 hour for immobilized enzyme changing maltose to trehalose and we can acquire the highest content of trehalose. Otherwise by HPAEC,HPLC etc determined means, at last we can affirmed that immobilized trehalose synthase enzyme can transform maltose to D- (+) -trehalose and D- (+) -trehalose is the most (a , a )-trehalose in the nature.
引文
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