帶有金屬紫質之Coprinus cinereus 過氧化氫酵素之動力學及光化學特性研究

Abstract

Coprinus cinereus過氧化氫酵素 ( CIP ) 是一種在真菌中含有血基質(鐵原紫質IX)的酵素，在提供過氧化氫的環境下，可進行酚類化合物氧化的催化反應。藉由大腸桿菌大量表現無糖基化的重組CIP，可探討金屬原紫質IX (metallo-protoporphyrinIX)在重組CIP(rCIP)催化活性中的功能，以及其在生物太陽能電池上的發展潛力。以包含體的形式表現的重組CIP，會經過復性以及凝膠過濾管柱純化。純化後的重組CIP具有高純度並且對過氧化氫以及2,2’-azino-bis ( 3-ethylbenzothiazoline- 6-sulfonate ) ( ABTS )具有高比活性 ( 大約20000 U/mg )。從酵素動力學的結果顯示，重組CIP在催化ABTS和H2O2的能力上與重組HRP相比具有較高的催化活性。置換不同金屬原紫質IX之重組CIP在30℃、pH 5的條件測定其動力學參數，置換鐵原紫質、鈷原紫質、鎳原子、質銅原紫質或是鋅原紫質為中心的重組CIP對於ABTS或是H2O2，皆得到類似的Km值，而Vmax的高低則是照rCIP(FePPIX) > rCIP(NiPPIX) >rCIP(CoPPIX) > rCIP(CuPPIX) > rCIP(ZnPPIX)的順序遞減。相較於重組CIP (鐵原紫質IX)的過氧化氫酵素活性，置換鈷原紫質IX、鎳原紫質 IX、銅原紫質IX及鋅原紫質IX為中心的重組CIP分別具有17% 、53%、1% 以及 0.4% 的過氧化氫酵素活性。對重組CIP置換不同金屬原紫質IX後的紫外光-可見光以及螢光光譜研究其光學特性，在時間-解析螢光光譜中的皮秒級螢光衰減發現，置換鋅原紫質IX為中心的重組CIP相較於溶於THF的單體鋅原紫質IX具有較長的S1激發態螢光生命期。

Corprinus cinereus peroxidase (CIP) is a fungal heme (Fe-protoporphyrin-IX)-containing enzyme catalyzing the oxidation of phenolic compounds in the presence of H2O2. To explore the function of metallo-protoporphyrin-IX (metal-PPIX) in the catalytic activity of CIP as well as its potential in the development of bio-solar cell the nonglycosylated recombinant CIP (rCIP) was overexpressed in E. coli BL21 (DE3). As an inclusion body form, the rCIP was then renatured and purified on a gel filtratioin column. The purified rCIP exhibited high purity and high specific activity of about 20000 U/mg protein toward H2O2 and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). Enzyme Knetic study showed that rCIP exhibits higher catalytic activity toward H2O2 and ABTS than that of rHRP. The kinetics of rCIP reconstituted with various metal-PPIXs was determined at 30°C and pH 5. The Km of Fe-PPIX, Co-PPIX, Ni-PPIX, Cu-PPIX or Zn-PPIX-reconstituted rCIP for ABTS or H2O2 were similar, but the Vmax of reconstituted rCIPs were decreased in an order of rCIP(FePPIX) > rCIP(NiPPIX) > rCIP(CoPPIX) > rCIP(CuPPIX) > rCIP(ZnPPIX). The peroxidase activity of rCIP reconstituted with CoPPIX, NiPPIX, CuPPIX and ZnPPIX were about 17%, 53%, 1% and 0.4% of the activity of rCIP(FePPIX) respectively, The metal-PPIX-reconstituted rCIPs were then characterized their UV-Visible and fluorescence spectra. In time-resolve fluorescence sepectrometric study, fluorescence decay of ZnPPIX-reconsituted rCIP had been observed that rCIP(ZnPPIX) exhibit a longer fluorescence lifetime of S1 excited state than that of ZnPPIX in THF.