木腐黴菌之阿拉伯呋喃糖苷酶的過量表現與催化功能研究

Abstract

本研究旨在探討木腐黴菌 (Trichoderma koningii G-39) 阿拉伯呋喃糖苷酶ABF基因在酵母菌Pichia pastoris表現及其催化功能的研究。經 由胺基酸序列比對之後，該酵素屬於family 54 的水解酶 (EC 3.2.1.55)。 基因重組後的野生株酵素及突變酵素均可由胞外粗提液經0 ~ 80% 硫酸銨鹽沉澱，以HiTrap SP陽離子交換樹脂管柱於20 mM，pH 4.5 NaOAc含120 mM NaCl緩衝液下獲得純度95% 的酵素，其單體分子量約50 kDa。阿拉伯呋喃糖苷酵素在25℃下，於pH 2.0 ~ pH 8.3之間有極佳的pH值穩定度。而酵素對PNPAF (p-nitrophenyl-α-L-arabinofuranoside)及2,4-DNXP (2,4-dinitrophenyl-β-D-xylopyranoside) 之pH profile皆呈現鐘型曲線趨勢，且有轉醣之特性，因此推測酵素可能以雙取代構型保留 (double displacement & retention of anomeric configuration) 反應。 該酵素屬於雙功能的催化水解酶，其催化五環受質PNPAF的反應速率是催化六環受質PNPA (p-nitrophenyl-α-L-arabinopyranoside) 的500倍、PNXP (p-nitrophenyl-β-D-xylopyranoside) 的400倍、2,4-DNXP的6倍，顯然對五環受質具有較強之專一性。為了探討五環受質和六環受質與酵素的鍵結情形，利用MAF (methyl-α-L-arabinoside) 當抑制劑，分別與PNPAF和2,4-DNXP反應，推測得知五環受質和六環受質與酵素的鍵結作用位置是在不同的地方。

The gene from Trichoderma koingii G-39 encoding a bifunctional enzyme with both α-L-arabinofuranosidase (ABF) and β-xylosidase activities was successfully expressed in Pichia pastoris. This enzyme was further recognized as a α-L-arabinofuranosidase (EC 3.2.1.55) and belongs to the family 54 glycohydrolase. The recombination enzyme can be efficiently purified to >95 % homogeneity by SP (cationic) column. The physical and chemical property of the recombination enzyme is similar to that of the native enzyme. Based on the reversible inhibition study, we conclude that the enzyme possesses two different catalytic domains toward p-nitrophenyl-α-L- arabinofuranoside (PNPAF) and 2,4-dinitrophenyl-β-D-xylopyranoside (2,4-DNXP) with the kcat / Km values of 75512, 5370 S-1M-1, respectively. The transglycosylation activity and the pH-dependent hydrolysis (a bell-shaped curve) on both PNPAF and 2,4-DNXP reveal that the enzyme may catalyze the hydrolysis of PNPAF & 2,4-DNXP with the retention of anomeric configuration. Since no significant activity decreases on E271Q, D302N, E305Q, and E389Q. E271, D302, E305 and E389 are unlikely to be the catalytic essential group for both enzymatic functions.