糖祿錠(acarbose)是α-glucosidase的抑制劑，廣泛用於第二型糖尿病的治療，acarbose主要結構含有acarviosine與maltose。ATase酵素(Acarviosyl transferase)不受acarbose抑制，可將acarviosine切下，並以α-(1,4) 鍵結轉至另個糖類分子上，是唯一具有此功能的轉糖酶。因此針對此催化特性，本研究之目的包括：(i)利用ATase提高糖祿錠在發酵液的產率。(ii)探討ATase之催化機制。(iii)表現基因重組ATase以提供X-ray結晶學研究。經由利用ATase與放射線菌發酵液作用，可轉換acarviosine至glucose與salicin上，唯使用maltose做為接受者時，acarbose產量無明顯增加。我們再以大腸桿菌表現融合蛋白ZZ-ATase，探討ZZ domain對於ATase功能之影響。除去ZZ domain的ATase對受質對acarbose之動力學常數為Km= 0.85 mM與Vmax為0.21 mM/hr，與ZZ-ATase酵素對acarbose之動力學常數Km=0.86 mM，Vmax=0.24 mM/hr比較，顯示ZZ domain對ATase酵素活性影響並不大。
進一步為解出ATase與其受質(acarbose)之複合體結構，以定點突變方法生產出突變株ATase (E435Q)，以降低其催化活性。實驗結果顯示E435Q之催化活性與ATase比較，只剩約4%之活性。解開ATase (E435Q)與Acarbose之複合體結構是探討ATase催化機制之重要步驟。

sp. fermentation broth to increase the yield of acarbose, (ii) to study the catalytic mechanism of ATase, and (iii) to express recombinant ATase for X-ray crystallography study.
In our study, ATase transfer the acarviosine group from the acarbose analogues to glucose or salicin but not maltose. We have expressed the fusion protein ZZ-ATase in E.coli BL21(DE3) strain. The ZZ domain is added to facilitate the purification and thrombin cleavage site was also engineered into the linker between ZZ and ATase to remove the ZZ domain after purification. Comparison of the kinetics parameters of ZZATase (Km=0.86 mM and Vmax=0.24 mM/hr) and ATase (Km=0.85 and Vmax=0.21 mM/hr) showed the ZZ domain didn’t interfere the catalytic activity of ATase. We designed a mutant of ATase, named E435Q, to reduce its catalytic efficiency but it will not affect its affinity with acarbose. Enzymatic analysis of the E435Q mutant revealed that the catalytic activity of the mutant was decreased to about 4% of the ATase. The mutant will be used for co-crystallization study.

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