木腐菌β-木糖甘酵素反應機制研究

Abstract

木糖甘酵素 (β-xylosidase) 能將木寡糖 (xylooligosaccharide) 或木雙糖 (xylobiose) 水解成木糖單糖 (xylose)，是木聚糖 (xylan) 水解系統中，一個很重要的酵素。本實驗以1 % 木聚糖加0.1 % 木糖單糖為唯一碳源，可自Trichoderma koningii G-39這個菌種中，大量誘導出木糖□酵素II，經一簡單的純化步驟可得純度達95 % 以上的酵素，並對此酵素的性質與反應機構做一深入的研究。 木糖甘酵素II是一個單分子 (monomric) 的醣蛋白質(glycoprotein) ，分子量為104 kDa，在37℃時，於pH 2.5 ~ pH 7.4之間穩定性佳，最適化之pH值在3.7 ~ 4.5之間，而最佳催化溫度為50℃左右，溫度高於60℃以上穩定性不佳。 金屬離子､EDTA對酵素活性沒有影響，xylose對此酵素有產物抑制作用，其Ki值為4.3±0.2 mM，另δ-xylonolactone亦有抑制效果，其Ki值為3.5±0.2 mM，兩者為競爭性抑制劑。 為探討β-木糖□酵素之特異性及詳細的反應機制﹐我們合成一系列含有不同離去基之受質。由所測得之Km 與kcat得知β-木糖□酵素對含有不同離去基之受質特異性並不大。 以氫核磁共振光譜觀察酵素分解受質DNXP時，產物為保留形態 (retention configuration) 的β-木糖單糖，顯示其應為兩步驟 (two-step)的催化反應: 第一步為xylosylation (形成xylosyl-enzyme中間體) ，第二步進行水解反應，形成dexylosylation中間體而得產物。以受質離去基之pKa對log kcat 及log kcat /Km 作圖可得Bronsted plot 。由Bronsted plot結果顯示，pKa對log kcat呈單一線性關係 (βlg = 0) 其斜率為零，表示反應之速率決定步驟與離去基之強弱無關，因此dexylosylation step應為催化反應之速率決定步驟 (rate-limiting step)。 二級同位素效應 (secondary deuterium, kinetic isotope effect) 之實驗得知，毫無明顯之同位素效應﹐由此可見此催化反應進行類似SN2之機制﹐其過渡狀態結構應近似xylocosyl-enzyme中間體。另此酵素有極佳之transxylosylation的活性，故極具有機合成上應用之潛力。

A xylosidase II (Xyl II) was induced and purified from the culture filtrate of Trichoderma koningii G-39, grown on 1% oat spelt xylan and 0.1% xylose as carbon sources. Purification steps involved ethanol precipitation and cation-exchanged chromatography. Xyl II is a monomeric glycoprotein with an estimated Mr value of 104 kDa and pI = 4.6. It is stable in pH 2.5 ~ 7.4 at 37 oC and posses its pH and temperature optima in the range of 3.7 ~ 4.5 and 55 ~ 60 oC, respectively. By NMR spectroscopy study, the enzyme catalyzes hydrolysis of p-nitrophenyl D-xylopyranoside with the retention of anomeric configuration. Indicate that a two-step mechanism, formation and breakdown of the xylosyl-enzyme intermediate, is involved. Measurement of kcat and Km values for a series of arylxylosides allowed constructing a Bronsted plot. The flat shape (i.e. lg = 0) of the plot suggests that the rate-limiting step of enzymatic hydrolysis of all tested aryl-xylosides be the breakdown of xylosyl-enzyme intermediate. Secondary deuterium kinetic isotope effects (kH/kD) measured for 2,4-dinitrophenyl-D-xylopyranoside is 1.02. Suggest that the transition-state for breakdown of the xylosyl-enzyme intermediate be SN2-like. Xylose and xylonolactone were competitive inhibitors with Ki values of 4.3 and 3.5 mM, respectively. In addition, Xyl II showed a promising transxylosylation activity, which is potentially useful in xylosides synthesis.