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

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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.