Molecular Dissection and Application of an Enzyme-Inhibitor System with Special Reference to a Proteinase That Specifically Decomposes a Hardly Soluble Protein in Food Seeds.

酶抑制剂系统的分子解剖和应用，特别是专门分解食品种子中难溶蛋白质的蛋白酶。

• 批准号: 10660118
• 负责人: ABE Keiko
• 金额: $ 2.5万
• 依托单位: The University of Tokyo
• 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-10660118/
• 关键词: wheat grains; cysteine proteinases; gliadain; cystatins; flour processing

A recombinant enzyme was produced from gliadain we found as a new cysteine proteinase in wheat seeds. It actually decomposed, though in vitro, gliadin as a major wheat protein and was found to be involved in the proteolysis of the storage protein during germination. Next, we cloned wheat cystatins by screening with oryzacystatin cDNA as a probe. Three cDNA clones encoding cystatins were thus obtained and it was disclosed that all these cystatins belong to the so-called "phytocystatin" family. In the mean time, we produced a recombinant species of gliadain by fusion with glutathione S-transferase (GST). The resultant GST-progliadain elicited a proteolytic activity under a weakly acidic condition, which was strongly inhibited by a commercially available preparation of egg cystatin as well as by oryzacystatin.The activity was also inhibited to a similar extent by two wheat cystatins we obtained recombinantly. All these results suggest the possibility that a system of the proteolysis with wheat cysteine proteinases and its regulation with wheat cystatins can be industrially applied to food processing for flour quality improvement.

从小麦种子中发现的一种新的半胱氨酸蛋白酶--麦醇溶蛋白酶中重组得到一种酶。它实际上分解，虽然在体外，麦醇溶蛋白作为一个主要的小麦蛋白质，并被发现参与蛋白质水解的储存蛋白在发芽过程中。其次，我们以oryzacystatin cDNA为探针，通过筛选克隆了小麦半胱氨酸蛋白酶抑制剂。由此获得了编码半胱氨酸蛋白酶抑制剂的三个cDNA克隆，并且公开了所有这些半胱氨酸蛋白酶抑制剂都属于所谓的“植物半胱氨酸蛋白酶抑制剂”家族。同时，我们通过与谷胱甘肽S-转移酶（GST）融合，制备了重组麦醇溶蛋白。在弱酸性条件下，GST-前麦醇溶蛋白具有蛋白水解活性，该活性可被市售的卵半胱氨酸蛋白酶抑制剂和谷半胱氨酸蛋白酶抑制剂所强烈抑制，也可被我们重组获得的两种小麦半胱氨酸蛋白酶抑制剂所抑制。所有这些结果表明，小麦半胱氨酸蛋白酶的蛋白质水解系统及其小麦半胱氨酸蛋白酶抑制剂的调节系统可以工业化应用于食品加工以改善面粉品质。

项目成果

J.Funaki,: "Purification and characterization of a neutral protease that contributes to the unique flavor and texture of tofu-misozuke." J.Food Biochem.21. 191-202 (1997)

J.Funaki：“中性蛋白酶的纯化和表征，有助于形成豆腐味噌的独特风味和质地。”

Y. Kusakabe: "Identification of two α-subunit species of GTP-binding proteins, Gαl5 and Gαq, expressed in rat taste buds."Biochim. Biophys. Acta. 1403. 265-272 (1998)

Y. Kusakabe：“大鼠味蕾中表达的两种 α-亚基种类 Gα15 和 Gαq”。Biochim。1403。265-272（1998）。

N. Kudo: "Crystallization and preliminary X-ray diffraction studies of a rice cysteine proteinase inhibitor, oryzacystatin-I"J. Biochem.. 123. 568-570 (1998)

N. Kudo：“水稻半胱氨酸蛋白酶抑制剂 oryzacystatin-I 的结晶和初步 X 射线衍射研究”J。

M. Asano-Miyoshi: "Identification of taste tissue-specific cDNA clones from a subtraction cDNA library of rat circumvallate and foliate papillae"J. Biochem.. 124. 927-933 (1998)

M. Asano-Miyoshi：“从大鼠外周和叶状乳头的消减 cDNA 文库中鉴定味觉组织特异性 cDNA 克隆”J.

T. Misaka: "A gustatory cyclic nucleotide-gated channel. CNG gust is expressed in the retina"NeduroReport. 10. 743-746 (1999)

T. Misaka：“味觉环核苷酸门控通道。CNG 味觉在视网膜中表达”NeduroReport。