Xue Ye-Min, Mao Zhong-Gui, Shao Wei-Lan
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):554-60.
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).
海栖热袍菌中发现的木聚糖分解酶具有极高的热稳定性，在工业应用中具有相当大的潜力。然而，编码这些酶的基因表达水平非常低。克隆了海栖热袍菌ATCC 43589的α-葡萄糖醛酸酶基因aguA，并在几种不同载体的大肠杆菌菌株中进行表达。α-葡萄糖醛酸酶在携带质粒pET-28a(+)的大肠杆菌BL21-CodonPlus(DE3)-RIL中过量表达，占细胞内可溶性部分总蛋白的约20%。结果证明了以下假设：精氨酸（AGA/AGG）和异亮氨酸（AUA）的稀有密码子会影响嗜热细菌海栖热袍菌的aguA基因在大肠杆菌中的表达。纯化过程包括热处理和固定化金属亲和色谱两步，从1升诱导培养物中获得了超过13.5毫克的纯酶。纯化后的酶在SDS聚丙烯酰胺凝胶电泳上显示为单一条带，纯化倍数为5.1倍，产率为55.1%。重组α-葡萄糖醛酸酶的最佳活性在pH 6.0和85℃下，在85℃孵育1小时后，该酶保留了70%的活性。通过IPTG研究了重组菌株BL21-CodonPlus(DE3)-RIL/pET-28a-aguA表达的诱导条件，包括诱导时间和持续时间。结果表明，在指数生长期（OD600为0.7 - 0.8）诱导后5小时，热稳定α-葡萄糖醛酸酶的活性达到最大值。
