Functional analysis of rice aspartic proteinase, oryzasin and application to milk clotting

大米天冬氨酸蛋白酶、米扎星的功能分析及其在凝乳中的应用

基本信息

批准号：
10680160
负责人：
ASAKURA Tomiko
金额：
$ 1.6万
依托单位：
Atomi Junior College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680160/
关键词：
aspartic proteinase rice soybean アスパラギン酸プロテアーゼ

项目摘要

A number of proteinases occur in food plant seeds. These enzymes play a crucial role in the metabolism of seeds by processing newly synthesized proteins into mature forms during ripening and by decomposing storage proteins into amino acids during germination. However, details of plant proteinases are not so well known as those of animal proteinases. We purified aspartic proteinase (AP) from rice and named oryzasin. Oryzasin was inhibited by pepstatin which is the specific inhibitor for APs. Oryzasin hydrolyzed glutelin but did not affect to prolamin. This enzyme clotted a skim milk solution and formed gel like soft cheese. It yielded the same k-casein digest pattern as those of chymosin and pepsin producing. Plant APs, structurally different from animal and microbial APs, has a large insertion sequence that accounts for one fourth of the molecular size, though the significance of this interesting insertion for any plant AP remains to be interpreted. A good example can be provided by oryzasins 1. We have constructed a system for expression of oryzasin 1 to investigate the effect of the deletion on the enzymatic activity, we found that the mutant was activated as well as the wild oryzasin 1 under an optimally acidic pH condition, with the conclusion that the presence of this insertion is not necessary for the AP activity. We carried out experiments of finding out a new AP that may occur in soybean as well. These were found to share 60-80% similarity to one another, each conserving the insertion which that characterizes to APs of plant origin. A histochemical study using an anti-oryzasin 1 antibody showed that oryzasin 1 was expressed in aleurone layer and endosperms.

食品植物种子中发生了许多蛋白酶。这些酶通过在成熟期间将新合成的蛋白质加工成成熟的形式，并在发芽期间将储存蛋白分解为氨基酸，在种子的代谢中起着至关重要的作用。然而，植物蛋白酶的细节并不像动物蛋白酶那样众所周知。我们从水稻中纯化了天冬氨酸蛋白酶（AP），并命名为Oryzasin。 pepstatin抑制or Zasin，pepstatin是AP的特异性抑制剂。 Oryzasin水解谷蛋白，但不影响丙酰蛋白。这种酶凝结了脱脂牛奶溶液，并像软奶酪一样形成凝胶。它产生的K-casein摘要模式与胰岛素和胃蛋白酶产生的模式相同。与动物和微生物AP结构不同的植物AP具有较大的插入序列，该序列占分子大小的四分之一，尽管这种有趣的插入对于任何植物AP的重要性仍有待解释。 Oryzasins 1可以提供一个很好的例子。我们已经构建了一个表达Oryzasin 1的系统来研究缺失对酶活性的影响，我们发现在最佳酸性pH条件下激活了突变体以及野生的Oryzasin 1，并且结论该插入物的存在是不需要用于插入的插入量。我们进行了实验，以发现大豆可能发生的新AP。发现这些彼此共享60-80％的相似性，每一个都保留了特征于植物起源AP的插入。使用抗氧化酶1抗体的组织化学研究表明，牛鼠素1在核龙层和胚乳中表达。