利用基因轉殖水稻生產醫藥工業用酵素

Rice ( Oryza sativa L. )is one of the most important and most widely cultivated crops in Taiwan.However, because the cost of cultivation is increasing every year, and the upcoming strong competition of imported rice from other countries after joining the WTO, the economic value of growing rice is downsizing.In addition to reduce the production cost, to use the rice plants as bioreactors to produce pharmaceutical proteins becomes a new way for increasing economic values of rice and may solve the problems we are facing.The general purpose of this proposal is to transfer genes of several pharmaceutical enzymes, such as L-hydantoinase, L-aminoacylase and transglutaminase into rice using genetic engineering techniques, and expect to increase the additional value of rice.Hydantoinase is involved in pyrimidine degradative process.Hydantoinases are divided into D-, L-and D, L-forms according to substrate enanthioselectivity.Generally, hydantoinases are low substrate specificity and catalyze 5-substituted hydantoins and dihydropyrimidine.L-hydantoin derivatives cleavage enzymes include L-carboxymethylhydantoinase, L-carboxyethylhydantoinase and ATP dependent N-methylhydantoinase.A novel substrate dependent hydantoinase was found from Arthrobacter aurescens DSM 3745.L-hydantoinases are divergent and mostly with unknown physiologic roles.Transglutaminaseactivity is found to be widely distributed in various cells and body fluids of mammals.They are thought to play important roles in cellular processes involving blood clotting, wound healing, coagulating of semen, neuronal differentation, and stabilization of the cell cytoskeletal network.In this study, we expect 1 )to obtain transgenic rice plants expressing hydantoinase and other pharmaceutical enzymes; 2 )to screen for thermostable L-hydantoinase-producing bacteria, clone the thermostable L-hydantoinase gene and have it overexpressed in Escherichia coli and rice plants; 3 )to screen for thermostable L-aminoacylase bacteria, clone the thermostable L-aminoacylase gene and have it overexpressed in Escherichia coli and rice plants; 4 )to screen for transglutaminase gene and have it expressed in rice plants.After performing this project, we expect to obtain several kinds of transgenic rice plants each contains different type of high-value protein gene.These transgenic rice plants will serve as bioreactors to produce high-value-protein to increase the economic value of rice and thus open up a new direction of our rice production in the future.水稻是本省重要的農作物之一, 其栽培面積十分廣大.但因栽培成本不斷提高, 使得種植水稻之經濟效益相對降低.加上目前政府正積極運作加入世界貿易組織, 唯市場開放後, 水稻的經濟價值即將面臨重大的入關壓力.因此, 除了研究降低生產成本之外, 提高水稻生產之附加價值, 利用水稻當作生物反應器來生產高經濟價值蛋白質或人類用藥, 成為另一條提升水稻價值之有效途徑.本計畫擬利用基因工程技術將醫藥工業用酵素, 如DHT、NCA、麩胺酸醯基轉移酵素等基因轉殖入水稻中, 生產高經濟價值之重組蛋白質進而提高水稻之附加價值.Hydantoinase為pyrimidine降解過程中的一個酵素, 依其對於基質的光學選擇性, 可分為D型、L型及所謂D, L型hydantoinase.Hydantoinase對基質的專一性低, 可水解不同的5-substituted hydantoins及dihydropyrimidine.目前發現的L型分解hydantoin衍生物的酵素有L-carboxymethylhydantoinase、L-carboxyethylhydantoinase及ATP依賴性N-methylhydantoinase等.在Arthrobacter aurescens DSM 3745則發現一種新奇的hydantoinase其受質有不同的光學選擇性.這些L型hydantoinases相當歧異, 且大多不清楚其生理功能.麩胺酸醯基轉移酵素廣泛的存在於哺乳類動物體內, 它是一種醯基轉移酵素, 可以催化由蛋白質上麩胺酸側鏈所提供的γ-carboxyamide基進行醯基轉移反應, 而造成蛋白質產生分子間或分子內的交叉架構.麩胺酸醯基轉移酵素的活性可在細胞組織及體液中被偵測到, 它同時也被認為和許多細胞的生理功能有關.本計畫擬完成下列工作項目: 1 )獲得帶有醫藥工業用酵素基因之轉殖水稻; 2 )選殖耐熱性L-hydantoinase基因, 轉移基因入水稻中; 3 )選殖耐熱性L-aminoacylase基因, 轉移基因入水稻中; 4 )選殖transglutaminase基因, 轉移基因入水稻中.執行本計畫後, 預期育成含有多種高價值蛋白之轉基因水稻, 做為生產這些高價值蛋白之工廠, 以開創稻米產業之新方向.本計畫若能成功, 其預期之經濟效益將相當大, 不僅可降低加入WTO後稻米市場開放之衝擊, 確保台灣水稻之永續經營, 並可使水資源有效再利用, 影響國計民生甚鉅