Esterase酯水解酵素可對乙醯硫代異丁酸甲酯(methyl DL-β-acetylthioisobutyrate簡稱DL-MATI)進行酵素光學切割產生乙醯硫代異丁酸(D-β-acetylthioisobutyric acid簡稱DAT)。而DAT為合成降血壓藥物Captopril的前趨物。
藉由檢視X-ray繞射定出EST結構與受質之間作用的位置，並由EST和不同受質間作用的相對活性，得知EST對於受質主要有三個辨識位，ester moiety、C-2 site及C-3 site，目前我們分別挑出EST作用於前二辨識位的胺基酸：Phe166、Trp184及Phe203、Ile229做定點突變，且對特定受質作檢測，並經由酵素動力學的計算來觀察突變後酵素對受質的接受度，及突變後對酵素立體選擇水解功能的影響。
此研究論文當中，我們成功的藉由定點突變的方法，取得一酯水解酵素突變株Phe166Ala，可對原本wild-type EST不易水解的受質DL-HEATI進行水解切割，由原本wild-type EST對DL-HEATI的酵素動力學Kcat / Km = 0.8 x 103 mM-1min-1提升至Kcat / Km = 15.3 x 103 mM-1min-1，且仍具有良好的立體選擇性，而此受質為一水溶性較佳的酯類受質，於工業上用途，搭配Phe166Ala的催化作用，便可提升單位時間、反應體積內Captopril的前趨物 - DAT之合成產率。

Esterase is an enzyme that perform stereoselective hydrolysis of methyl DL-β-acetylthioisobutyrate (DL-MATI) to yield D-β-acetylthioisobutyric acid (DAT). The catalytic product ＂DAT＂ is a precursor of an antihypertensive drug--Captopril.
To resolve the crystal structure of EST-substrate complex via X-ray diffraction method and to measure the relative activity of EST with a series of linear ester substrates, we have identified that EST has three potential recognition sites for substrate: ester moiety、C-2 site and C-3 site. Therefore, we selected four target amino acids in EST active site to perform site directed mutagenesis （Phe166, Trp184 specific for ester moiety and Phe203, Ile229 specific for C-2 site）, and investigated the substrate specificity and hydrolytic activity of these four mutants.
In this study, we found an EST mutant (Phe166Ala) has better activity to hydrolyze DL-HEATI than the wild-type EST does. The kinetics parameters of wild-type EST to hydrolyze DL-HEATI was Kcat / Km = 0.8 x 103 mM-1min-1, while the EST mutant (Phe166Ala) was improved to Kcat / Km = 15.3 x 103 mM-1min-1 . Besides, DL-HEATI was 5.3 fold more soluble in water than DL-MATI and DL-HEATI is a better choice than DL-MATI for industrial production of DAT by the enzymatic resolution method. This result suggested that EST mutant ( Phe166Ala ) is a mutant which can kinetically resolve DL-HEATI to yield DAT.

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