大腸桿菌表現Coprinus cinereus過氧化氫酵素之特性研究

Abstract

Coprinus cinereus peroxidase (CIP) 為含血基質的過氧化氫酵素，可在過氧化氫存在下將酚類化合物及其衍生物氧化。CIP於大腸桿菌中表現，其生物物理以及生化特性將被進一步的分析。大腸桿菌表現無醣基化的重組CIP，其為無活性的包涵體，接著於4 ℃，1.8 M urea，10 mM CaCl2 和5 μM hemin條件下再摺疊達七小時。具有活性的CIP經過純化其產量為15 %，且經由2,2’-azino-bis (3-ethylbenzothiazoline -6-sulfonate) (ABTS) 的測定，其比活性為23544 U/mg。重組CIP最適的催化條件為pH 5以及 35 ℃。而重組CIP的熱穩定性可經由活性分析、UV-Vis光譜分析、掃描式熱差分儀 (differential scanning calorimetry, DSC)、和旋光光譜儀 (circular dichroism, CD) 等方法來測定。EDTA可以增加失活的速率，而鈣離子有可逆的影響。在DSC實驗顯示熔點 (Tm) 為 48 ℃，去摺疊的熱力學參數為ΔHcal = 348.3 KJ/mol、ΔS = 1.09 KJ/mol。經CD光譜研究證實，溫度高於55 ℃會造成重組CIP構形改變。CIP比HRP有較高的催化效率，而其高催化特性在未來的研究是極具潛力的。

Coprinus cinereus peroxidase (CIP) is a heme-containing enzyme that oxidizes a wide variety of phenolic compounds and derivatives in the presence of H2O2. To further characterize the biophysical and biochemical properties of CIP was overexpressed in Escherichia coli BL21 (DE3). The nonglycosylated recombinant CIP was expressed as an inactive inclusion body and subsequent by renatured under 4 ℃ in the presence of 1.8 M urea, 10 mM CaCl2 and 5 μM hemin for 7 h. The yield of active recombinant CIP was about 15 % with the specific activity of 23544 U/mg by using 2,2’-azino-bis (3-ethylbenzothiazoline -6-sulfonate) (ABTS) as substrate. The highest activity could be observed at around pH 5 and 35 ℃. The thermostability of the recombinant CIP were studied by activity assay, UV-Vis spectrophotometer analysis, differential scanning calorimetry (DSC), and circular dichroism (CD). EDTA increased the rate of inactivation, which could be reversed by Ca2+ ion. DSC experiments show the transition temperature (Tm) for recombinant CIP is 48 ℃. The enthalpy (ΔH) and entropy (ΔS) of the recombinant CIP in thermal denaturation were calculated as ΔHcal = 348.3 KJ/mol, ΔS = 1.09 KJ/mol/K. Circular dichroism studies prove that temperatures higher than 55 ℃ induced a change in the conformation of the recombinant CIP. CIP exhibits higher catalytic activity than that of HRP, suggesting a potential for future application.