大肠杆菌基因工程菌诱导表达重组琼胶酶rAga0917的条件优化

Optimization of Recombinant Agarase rAga0917 Expression Conditions in Genetic Engineering Escherichia coli Strain

琼胶酶可催化长链琼脂糖分子内糖苷键水解产生具有生物活性的琼胶寡糖。琼胶酶具有重要的科研和应用价值,从而对琼胶酶的研究也日益增多。为获得基因工程菌株BL21（DE3）ply Ss-p ET28a（＋）-Aga0917重组琼胶酶r Aga0917的高效表达条件,本研究通过单因素实验及正交设计探索了种龄、诱导剂浓度、诱导时机、诱导温度和诱导时间对基因工程菌株表达的r Aga0917酶活力的影响。单因素实验结果显示,种龄6 h、诱导剂终浓度0.05 mmol/L、诱导时机4 h、诱导温度25℃、诱导时间16 h时酶活力最高;正交设计结果显示,r Aga0917的最优表达条件组合为：种龄6 h、诱导时机4 h、诱导剂终浓度0.1 mmol/L、诱导温度16℃。对正交设计结果的直观分析和方差分析结果均表明,四个因素对异源表达目的蛋白琼胶酶酶活力影响的顺序为：诱导时机〉诱导温度〉种龄〉诱导剂终浓度。

Agarases catalyze glycosidic bond within long chain agarose produces agaro-oligosaccharides that shows many biological activities.Agarases play an important role in research and application.Thus,agarase has attracted the attentions of scientists recently.To obtain optimal expression conditions of r Aga0917,the factors of seed age,concentration of isopropyl-β-d-thiogalactoside（IPTG）,induction occasion,induction temperature and induction duration were selected for single factor experiments and orthogonal design.The results of single factor experiment showed that the optimal expression conditions were seed age 6 h,0.05 mmol/L IPTG,induction occasion4 h,25℃ induced for 16 h.According to the results of orthogonal design,the optimized inducing conditions were：seed age 6 h,0.1 mmol/L IPTG,induction occasion 4 h,16℃ induced for 16 h.Both direct calculation and variance analysis of the data from orthogonal design showed that the importance order of the four factors to affect the agarase enzyme activity of r Aga0917 was：induction occasioninduction temperatureseed ageconcentration of IPTG.