%0 Journal Article
%T Expression and Purification of Thermostable α-Glucuronidase from Thermotoga maritima<br>极端嗜热菌海栖热袍菌α-葡萄糖醛酸酶的高效表达和重组酶的纯化
%A XUE Ye
%A Min
%A MAO Zhong
%A Gui
%A SHAO Wei
%A Lan
%A <br>薛业敏
%A 毛忠贵
%A 邵蔚蓝
%J 生物工程学报
%D 2004
%I 
%X The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).
%K 海栖热袍菌，α-葡萄糖醛酸酶，高效表达，重组酶纯化，酶学性质
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=D9D01BC909617ED1&yid=D0E58B75BFD8E51C&vid=A04140E723CB732E&iid=E158A972A605785F&sid=D8414BC1307BF1A3&eid=A3F93694B058F76C&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=1&reference_num=16