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.

许多蛋白酶存在于食用植物种子中。这些酶在种子的新陈代谢中发挥着至关重要的作用，在成熟过程中将新合成的蛋白质加工成成熟形式，并在发芽过程中将储存蛋白质分解成氨基酸。然而，植物蛋白酶的细节并不像动物蛋白酶那样广为人知。我们从大米中纯化了天冬氨酸蛋白酶（AP），并将其命名为 oryzasin。 Oryzasin 被胃酶抑素抑制，胃酶抑素是 AP 的特异性抑制剂。米扎辛水解谷蛋白但不影响谷醇溶蛋白。这种酶使脱脂牛奶溶液凝结并形成像软奶酪一样的凝胶。它产生与凝乳酶和胃蛋白酶产生相同的 k-酪蛋白消化模式。植物 AP 在结构上不同于动物和微生物 AP，具有占分子大小四分之一的大插入序列，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。 oryzasin 1 就是一个很好的例子。我们构建了 oryzasin 1 的表达系统来研究缺失对酶活性的影响，我们发现突变体在最佳酸性条件下与野生 oryzasin 1 一样被激活。 pH 条件，结论是该插入的存在对于 AP 活性不是必需的。我们进行了实验，寻找大豆中也可能出现的新AP。发现它们彼此之间有 60-80% 的相似性，每个都保留了植物来源 AP 所特有的插入。使用抗 oryzasin 1 抗体进行的组织化学研究表明，oryzasin 1 在糊粉层和胚乳中表达。