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

许多蛋白酶存在于食用植物的种子中。这些酶在种子的代谢中起着至关重要的作用，在成熟过程中将新合成的蛋白质加工成成熟的形式，并在萌发过程中将储存的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为人所知。我们从水稻中纯化了天冬氨酸蛋白酶（AP），命名为oryzasin。Oryzasin被胃抑素抑制，胃抑素是APs的特异性抑制剂。稻谷蛋白对谷蛋白有水解作用，但对蛋白无影响。这种酶使脱脂牛奶溶液凝结，形成像软奶酪一样的凝胶。它产生了与产生凝乳酶和胃蛋白酶相同的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，它们有一个大的插入序列，占分子大小的四分之一，尽管这个有趣的插入对任何植物AP的意义仍有待解释。oryzasins 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，我们发现突变体和野生的oryzasin 1在最佳酸性条件下被激活，结论是这个插入的存在对AP活性不是必需的。我们还进行了在大豆中发现一种新的AP的实验。结果发现，它们之间有60-80%的相似性，每个都保留了植物源ap的插入特征。利用抗oryzasin 1抗体进行组织化学研究表明，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.