在大肠杆菌中表达的枯草芽孢杆菌尿酸氧化酶的克隆、纯化及部分特性分析
Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.
作者信息
Pfrimer Pollyana, de Moraes Lidia Maria Pepe, Galdino Alexsandro Sobreira, Salles Loise Pedrosa, Reis Viviane Castelo Branco, De Marco Janice Lisboa, Prates Maura Vianna, Bloch Carlos, Torres Fernando Araripe Gonçalves
机构信息
Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900 Brasília (DF), Brazil.
出版信息
J Biomed Biotechnol. 2010;2010:674908. doi: 10.1155/2010/674908. Epub 2010 Feb 4.
Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.
尿酸氧化酶（EC 1.7.3.3）是一种参与嘌呤代谢的酶，可用于治疗痛风，并作为检测尿酸的诊断试剂。为了出于生物技术目的大量生产这种酶，编码枯草芽孢杆菌尿酸氧化酶的基因被克隆并在大肠杆菌中进行异源表达。在大肠杆菌中的时间进程诱导显示出一种表观分子量约为60 kDa的诱导蛋白。使用镍-氮三乙酸（Ni-NTA）柱通过单步程序纯化可溶性重组酶。与粗提物相比，该酶纯化了2.1倍，产率为56%。基质辅助激光解吸电离飞行时间（MALDI-TOF）分析显示出一个质量为58675 Da的离子，这与重组蛋白的预期质量相符。纯化后的酶的最适pH和温度分别为8.0和37℃，在-20℃和4℃孵育72小时后仍保留其90%的活性。
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