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

食用植物种子中含有大量的蛋白酶。这些酶在种子的新陈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的形式，并在萌发过程中将贮藏蛋白质分解成氨基酸。然而，植物蛋白水解酶的细节并不像动物蛋白水解酶那样广为人知。我们从大米中纯化了天冬氨酸蛋白酶(AP)，并将其命名为oryzasin。谷胱甘肽可被AP的特异性抑制剂胃抑素抑制。谷氨酸对谷蛋白有水解性，但对醇溶蛋白无影响。这种酶凝结了脱脂牛奶溶液，形成了像软奶酪一样的凝胶。它产生了与凝乳酶和胃酶产生相同的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任何植物AP的意义仍有待解释。我们构建了oryzasin 1的表达系统，考察了缺失对酶活性的影响，发现突变体和野生oryzasin 1在最适的酸性条件下都被激活，从而得出结论：该插入不是AP活性所必需的。我们开展了寻找大豆中可能存在的一种新的AP的实验。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的AP的特征插入。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。

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.