Flavobacterium meningosepticum之β-葡萄糖甘酵素的反應機制研究

Abstract

本研究對自黃質菌（Flavobacterium meningosepticum）中選殖出基因並在E. coli.表現之β-糖甘酵素進行性質與動力學研究。 由E. coli.細胞粗提取液經硫酸銨鹽沉澱，再以陽離子交換樹脂管柱HiTrap SP於 pH6.9和pH6.6下進行兩次管柱層析分離，可獲得純度達95%以上之酵素，其分子量為 78KDa 。在較高酵素濃度時，不需添加其它物質，酵溶液可維持30天活性穩定（磷酸鹽濃度 20mM , PH6.9, 4℃）。在可測量的範圍內，反應最佳之酸鹼值為4.5 。葡萄糖對此酵素有產物抑制效果，其Ki 為4.6mM。此酵素對受質糖基部份的選擇甚嚴，對非糖基部份的選擇性則較寬鬆許多。其對p-nitrophenyl β-D-glucopyranoside的 Km 值為0.815mM （PH6.8），與野生種酵素（Km=0.65mM, pH7.0）性質相差不大。 由氫核磁共振圖譜觀察酵素分解受質PNPG時，產物為保留形態 （retention configuration）的β-form葡萄糖。將酵素對不同受質所得之 kcat值以 Bronsted equation 分析所得圖形為向下彎曲的兩相圖。顯示其為兩步驟（two-step）之催化反應，可能包含glucosylation與 deglucosylation兩步驟。當受質為含有較佳離去基者 （pKa<7, β1g=0），其反應速率決定步驟為deglucosylation step；當受質為含有較差離去基者 （pKa<7, β1g= -0.8）時，其速率決定步驟應為glucosylation step ，而反應達過渡狀態時，推測glycosidic C-O鍵已幾乎完全斷裂。

A β-Glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in E. coli. The cloned enzyme was purified to homogeneity by following ammonium sulfate fractionation and a Hitrap SP column applying at pH6.9 and pH6.6, consecutively. The concentrated enzyme (0.4mg/ml) was stable at 4 ℃ (pH 6.9, 20mM phosphate buffer) up to 30 days. The optiomal pH is at pH0.5. Glucose presents a productinhibition with Ki=4.6 mM. The enzyme displayed a restrict specificity on the glycon portion of substrate. Km value of p-nitrophenyl β-D-glucopyranoside (PNPG) for the cloned enzyme is 0.815 mM, which is similar to that of wild type (0.65mM). By NMR spectroscopy study, the enzyme catalyzes hydrolysis of p-nitrophenyl β-D-glucopyranoside(PNPG) with the retention of anomeric configuration. A preliminary study of Bronsted relationship showed a concaveg-downward plot. These provide a strong indication for a two-step mechanism: formation (step 1)and breakdown (step 2)of glucosylenzyme intermediate. The reaction rate-limiting-step (r. l. s.) of good substrates (pKa of leaving group<7) is the breakdown of glucosy-enzyme intermediate whereas the r.l. s. of poor substrates (pKa of leaving group>7) is the formation of the intermediate. The Bronsted constant for poor substrates is β1g= -0.8. Suggest that an extensive amount of charge be developed on the oxygen atom of the leaving phenols at eh transition-state.