Characterization of a family I-liked alkaline-resistant inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus furiosus for rapid determination of sugar nucleotidyltransferase at high temperature

详细信息    查看全文

• 作者：Xufan Yan ; Qing Dong ; Minhui Zheng ; Ziwen Yang
• 关键词：Alkaline-resistant ; Family-I inorganic pyrophosphatase ; Glucose-1-phosphate uridylyltransferase ; Hyperthermophilic enzyme ; Pyrococcus furiosus
• 刊名：Journal of Molecular Catalysis B: Enzymatic
• 出版年：30 December, 2013
• 年：2013
• 卷：98
• 期：Complete
• 页码：15-20
• 全文大小：915 K

文摘

A gene-encoded inorganic pyrophosphatase (AE009950.1: AAL80381.1) from hyperthermophilic archaeon Pyrococcus furiosus (PfPPase) was cloned and expressed in Escherichia coli BL21 (DE3). PfPPase showed 95% identity with family I PPases from Pyrococcus horikoshii OT3. The recombinant PfPPase had a molar mass of 22 kDa and was activated by Mg²⁺. The optimum temperature and pH of PfPPase were 95 掳C and 9.5, respectively. PfPPase showed the half-lives of heat inactivation about 6.85 脳 10³, 2.76 脳 10³ and 0.85 脳 10³ min at 85, 95 and 100 掳C in the presence of Mg²⁺, respectively; 3.94 脳 10³, 1.90 脳 10³ and 0.49 脳 10³ min at 85, 95 and 100 掳C in the absence of Mg²⁺, respectively. Inorganic pyrophosphate was the most specific substrate for PfPPase. The K_m and the maximum velocity (V_max) of the enzyme were 173 渭M and 55.9 渭mol min^鈭? mg^鈭?, respectively, at pH 9.5 and 95 掳C. A simple and efficient colourimetric coupled enzyme assay to determine the activity of thermophilic glucose-1-phosphate uridylyltransferase from P. furiosus (PfG1PUTase) with PfPPase at high temperatures was also developed.