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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。这些酶在种子的代谢中起着至关重要的作用，它们在成熟过程中将新合成的蛋白质加工成成熟形式，并在萌发过程中将贮藏蛋白质分解成氨基酸。然而，植物蛋白酶的详细情况不如动物蛋白酶那样为人所熟知。我们从水稻中纯化了天冬氨酸蛋白酶（AP），并命名为oryzasin。谷氨酰胺酶抑制剂胃蛋白酶抑制剂是AP的特异性抑制剂。谷氨酰胺水解谷蛋白，但不影响醇溶蛋白。这种酶使脱脂牛奶溶液凝结，并形成类似软奶酪的凝胶。它产生的k-酪蛋白消化模式与凝乳酶和胃蛋白酶产生的相同。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的意义仍有待解释。一个很好的例子是oryzasins 1。我们构建了oryzasin 1的表达系统，研究了缺失对酶活性的影响，我们发现突变体在最适酸性pH条件下与野生型oryzasin 1一样被激活，结论是该插入的存在对于AP活性不是必需的。我们进行了寻找一种可能也存在于大豆中的新AP的实验。这些被发现彼此共享60-80%的相似性，每一个都保存了植物来源的AP的特征插入。利用抗oryzasin 1抗体的组织化学研究表明，oryzasin 1在糊粉层和胚乳中表达。