摘要
α 葡萄糖醛酸酶作为木聚糖降解的限速酶之一 ,在木聚糖类半纤维素的生物转化中起着重要的作用。海栖热袍菌Thermotogamaritima是一个嗜极端高温的厌氧细菌 ,其产生的极耐热性酶类具有非常可观的工业应用前景。但热袍菌属Thermotoga的基因在大肠杆菌中的表达一般较困难。研究了T .maritima中的极耐热性α 葡萄糖醛酸酶基因在大肠杆菌不同菌株中的表达水平及纯化技术。结果表明 ,稀有密码子AGA、AGG和AUA限制了该基因在大肠杆菌中的表达 ,在大肠杆菌BL2 1 CodonPlus(DE3) RIL可得到高效表达 ,重组蛋白表达量达 2 0 % ,比酶活比野生菌株提高 5倍 ;重组蛋白经热处理和金属Ni2 + 的亲和层析提纯后 ,达到了电泳纯 ,提纯倍数为 5 1倍 ,收率为5 5 1 %。对重组菌诱导表达条件的研究表明 ,营养丰富的TB培养基有助于重组菌的生长 ,重组菌生长至OD6 0 0 为0 7～ 0 8时添加IPTG诱导
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 α glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The α 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 α glucuronidase was found at pH 6 0 and 85℃,the enzyme retained 70% of its activity after 1 h of incubation at 85℃. 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 α glucuronidase reach the maximum in 5 hour after inducted at the exponential phase ( OD 600 of 0 7～0 8).
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第4期554-560,共7页
Chinese Journal of Biotechnology
基金
国家轻工总局 2 11专项基金资助~~
关键词
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
Thermotoga maritima , α glucuronidase, gene overexpression, recombinant enzyme purification, characterization
