以酵素工程法開發黏質沙雷氏菌BCRC10948之新型具有高催化效率短鏈脫氫酶還原酶

Engineer a novel short-chain dehydrogenase/reductase that exhibits enhanced catalytic efficiency and exquisite substrate specificity from Serratia marcescens BCRC 10948

鄒宇(Tsou, Yu)

指導教授 ： 王雯靜

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2015年)

摘要

(R)-Phenylephrine [(R)-PE] 是一種α1腎上腺素受體興奮劑，相較於其他類腎上腺素藥物其副作用較低，目前被廣泛用於舒緩鼻充血和延長麻醉時效。利用化學合成方式生產(R)-PE時，非藥用(S)-PE幾乎占整體產量的一半，且生產過程需用到觸媒並在高溫高壓下進行，不僅成本高且對環境不友善。近期中興大學許文輝教授及楊明德教授實驗室從Serratia marcescens BCRC 109485中分離出一株具有可以將1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) 轉換成(R)-PE的short-chain dehydrogenase/reductase (SDR)，稱之為SmSDR。從SmSDR的轉換結果中發現鏡像異構物選擇性[(R)-PE)]高達99%，但整體產率及轉換率不佳，無法達到工業化應用的標準。此篇研究即是與中興大學合作，希望利用晶體繞射學技術解出SmSDR的蛋白質結構，之後再更進一步分析其結構特性、催化機制以及反應物專一性，並依據這些資訊來設計定點突變，即是利用蛋白質工程的方式改良SmSDR的催化效率使之有潛力應用在藥物生產工業上。首先我們成功的培養出SmSDR晶體並解出具有1.47Å高解析度的蛋白質結構，接著用電腦模擬的方式建立出蛋白質與反應物的作用關係，依據這些資訊來設計突變點，最後得到具有不改變鏡像選擇性且酵素活性提高的雙點突變株SmSDR-F98YF202L，且在生物轉化過程中提高了(R)-PE)的轉換率，證明了有效的突變位點的確可以使SmSDR的催化效力增加，而我們的最終目標為將SmSDR之產率及轉換率改良到足以應用在工業化生產製程上。

關鍵字

苯腎上腺素 ； 短鏈脫氫酶/還原酶 ； 酵素工程 ； 生物催化法 ； 黏質沙雷氏菌

並列摘要

(R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist widely used as a nasal decongestant and a cardiac agent without major side effects opposing to other adrenergic drugs such as ephedrine. In addition, the current mass-production procedure usually consists of (S) chiral form (50%). In an end to increase the specificity, a bio-catalytic transformation procedure using a novel short-chain dehydrogenase/reductase (SDR) from Serratia marcescens BCRC 10948 (Peng, G. J. et al.) to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into an enantioseletive (R)-PE (more than 99%) has been attempted. However, this method performs relatively low conversion yield and productivity. In this study, we aim to determine the crystallographic structure of SmSDR as a structural basis to engineer high-activity SmSDR variants. Here, we report the 1.47 Å atomic-resolution apo-form structure. A liganded complex was built using Discovery Studio. Several mutants were predicted and characterized based on a structure-guided approach. A double mutant SmSDR-F98YF202L was found to display the highest activity. Furthermore, this mutant demonstrated a much higher conversion yield and productivity in the whole-cell assay, suggesting a valuable engineered variant for pharmaceutical applications.

並列關鍵字

Phenylephrine ； Short-chain dehydrogenase/reductase ； Enzyme engineering ； Biocatalysis ； Serratia marcescens

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