醯亞胺水解酵素催化p-D,L-hydroxyphenyl hydantoin 水解的探討

Abstract

醯亞胺水解酵素(imidase)是工業上生產D-4-hydroxyphenyl glycine(DHPG)的一個重要酵素.而DHPG是生產抗生素,例如: Cefoperazone, Cefpiramide, Amoxicillin和Cefadroxil等的重要中間產 物. 目前生產DHPG的問題在於是否可以得到一適當的酵素, 用來分離不具 光學活性的D,L-4-hydroxyphenyl hydantoin, 進而得到具光學活性的產 物. 我們可以用醯亞胺水解酵素(imidase, D-hydantoinase, or dihydropyrimidinase)來達成此一目的. 而醯亞胺水解酵素一般的來源是 Agrobacterium sp.,Pseudomonas striata, 牛肝和鼠肝. 在台灣由豬肝 中純化醯亞胺水解酵素, 相信可提高豬肝的附加價值, 使台灣養豬戶不必 擔心因市場價格下降而滯消的問題. 在本論文中, 我們證實從臺灣豬肝 分離出醯亞胺水解酵素可以水解p-D,L-hydroxyphenyl hydantoin, 同時 具有旋光選擇性可以用來生產D-4-hydroxyphenyl glycine(DHPG). Imidase is an industrially important enzyme leading to the productionof D-4-hydroxyphenylglycine(DHPG). DHPG in turn is the key intermediate for the manufacture of important antibiotics such as cefoperazone, cefpiramide, amoxicillin, aspoxicillin and cefadroxil. To be able to produce DHPG, one needs an enzyme that can selectively hydrolyze D-4- hydroxyphenyl hydantoin from its racemic mixure. This enzyme is imidase, also known as D-hydantoinase, dihydropyrimidinase, dihydropyrimidine hydrase, and dihydropyrimidine amidohydrolase. In the past, imidase have been purified from Agrobacterium sp., Pseudomonas striata, beef liversand rat livers. While Taiwan is an major pig exporting country, we trust successful industrial applications of the imidase from pig livers will provide a tremendous value to our agriculture sector. In this thesis, a methodology of purifying imidase from pig livers hasbeen developed. It has been shown that, indeed, the pig liver imidase can selectivelycatalyze p-D,L-hydrophenyl hydantoin which in turn leads to the production of D- hydrophenylglycine(DHPG). The optimum reaction condition for the pig liverimidase appears to be at pH 8.5 and 60 C. The obervation is well in line withthe results shown in the literature. This research has provided important technical insights towards successful bioreactor design and scale-up for theproduction of a wide spectrum of antibiotics.