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An Enzymatic Process for the Synthesis of L-Homophenylalanine
L-homophenylalanine is one of the most important intermediates for the synthesis of angiotensin-converting enzyme inhibitor (ACEI) type drugs for the treatment of hypertension and congestive heart failure. The chriality of homophenylalanine used for the synthesis of ACEI type pharmceticals is critical for the efficacy. L-homophenylalanine is currently produced by either chemical resolution or the asymmetric hydrogenation, which are in general expensive and usually result in the emission of toxic wastes. To this end, biotransformation involving the employment of lipase or alcalase followed by kinetic resolution has been development. However, these processes suffer from the disadvantages of low conversion, leaving 50 % of D-HPA ester un-reacted. An improved process employing whole cells co-expressing N-acylamino acid racemase and L-aminoacylase for the synthesis of L-homophenylalanine from N-carbamoyl-D,L-homophenylalanine has been reported. Although higher conversion can be achieved with the whole cell process, it may suffer from some disadvantages such as low stability and high mass transfer resistance. In light of this, we propose in this study to develop a novel process employing immobilized N-acylamino acid racemase and L-aminoacylase. Specifically, the work involves in this study will include the expression and purification of N-acylamino acid racemase and L-aminoacylase, the immobilization of the said enzymes, the characterization of the immobilized enzymes, the development of batch and continuous enzymatic processes for L-homophenylalanine production, medium engineering for improved substrate solubility, the development of novel matrix for enzyme immobilization and the process of L-homophenylalanine recovery.
L-同苯丙胺酸(L-homophenylalanine)為血管收縮素轉化酶抑制劑(Angiotensin-converting enzyme inhibitor , ACEI)類高血壓用藥之重要原料之一。由於HPA之對掌性對ACEI之活性極具重要性，因此建立L-同苯丙胺酸之生產製程具有相當高之重要性。目前L-同苯丙胺酸之生產係以化學分割法 (chemical resolution)或不對稱氫化法 (asymmetric hydrogenation)達成的。然而，這兩個化學法均有成本過高且過程複雜與高污染之缺點。為了克服這些問題，利用lipase或alcalase配合動態分割(kinetic resolution)的生物轉換法（biotransformation）乃應運而生。但由於這些酵素製程只能將外銷旋原料中的L-HPA ester轉化為產物，其轉換率、e.e. 值及產率均偏低，因此仍有其改善空間。以N-carbamoyl-D,L-homophenylalanine為原料使用共表現N-acylamino acid racemase與L-aminoacylase之大腸桿菌全細胞之生物轉換製程已經見於文獻，本計畫擬針對全細胞生物轉換製程之缺點，建立一較佳之固定化酵素製程。本計畫第一年度之主要工作項目為建立該二酵素之發酵生產/純化流程、原料N-carbamoyl-L-HPA與N-carbamoyl-D-HPA之製備與純化、以商用基材進行酵素固定化與固地化酵素之定性，批次製程之探討等。第二計畫年度之主要工作項目為依第一年度之結果嘗試新固地化基材之開發、探討共固定化之最適條件、建立連續式固定化酵素製程、進行反應介質工程提昇反應物溶解度、建立產物分離純化程序等。完成本計畫在學術、工業應用、人才培育等方面均將具有相當之貢獻。
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