Structure Determination and Development of Purification Process of Taste-modifying protein, Miraculin

调味蛋白 Miraculin 的结构测定及纯化工艺开发

基本信息

批准号：
62880021
负责人：
KURIHARA Yoshie
金额：
$ 8.06万
依托单位：
Yokohama National University. Faculty of Education
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

1. The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. We found that miraculin was extracted with 0.5 M NaCi solution. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel-filtration. The purified miraculin thus obtained gave a single sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure.2. The complete amino acid sequence of miraculin was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content was 24,600. The carbohydrate chains were elucidated to link to Asn-42 and Asn-186.3. The structure of each N-linked oligosaccharides isolated was analyzed. It was found that five oligosaccharides are a series of compounds with xylose-containing common structural core. A variety of oligosaccharide structure are significant for two glycosylation sites, Asn-42 and Asn-186. Two new oligosaccharides with unusual structures containing complex-type were characterized.4. We have developed an enzyme-linked lmmunoabsorbent assay for miraculin. The amounts of miraculin in miracle fruit increased dramatically seven weeks after pollination and reached a maximum at eight weeks. These results suggest that the biosynthesis of miraculin increases dramatically during the final stage of maturation of miracle fruit.

1。改善味觉的蛋白质奇迹蛋白具有将酸味变成甜味的异常特性。我们发现用0.5 M NACI溶液提取奇迹素。通过硫酸铵分馏，CM-隔离离子 - 交换色谱和凝胶滤料从提取的溶液中纯化奇迹素。因此获得的纯化奇迹素在反相高性能液相色谱中给出了一个尖峰，表明它是高度纯的2。奇迹素的完整氨基酸序列是通过自动Edman降解方法确定的。奇迹蛋白是单个多肽，有191个氨基酸残基。基于氨基酸序列和碳水化合物含量的分子量为24,600。阐明了碳水化合物链以与ASN-42和ASN-186.3链接。分析了分离的每种N连接寡糖的结构。发现五种寡糖是一系列含有木糖的公共结构核的化合物。各种寡糖结构对于两个糖基化位点ASN-42和ASN-186具有重要意义。表征了两种具有不寻常结构的新寡糖。4。我们已经开发了一种酶连接的lmmunoabsorbent分析，用于奇迹蛋白。奇迹果实中奇迹素的数量在授粉后七个星期急剧增加，并在八周时达到了最大含量。这些结果表明，奇迹素的生物合成在成熟的奇迹果实的最后阶段急剧增加。