Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology.
Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology.
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DOI：
10.1016/s0168-1656(97)01693-3
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发表时间：
1997-05
影响因子：
4.1
通讯作者：
J. Ahn;Young Phil Lee;J. Rhee
中科院分区：
文献类型：
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作者：
J. Ahn;Young Phil Lee;J. Rhee
采用响应面法对荧光假单胞菌脂肪酶在大肠杆菌中以包涵体形式表达的复性条件进行了优化。杆菌选择蛋白质浓度、pH值和盐酸胍浓度3个交互作用因素作为响应面变量。在4 ° C和−15°C的低温下，蛋白质浓度在选定范围内（50-340 μg ml−1）不影响复性产率，但在25°C下是一个关键因素，复性产率随蛋白质浓度增加而显著降低。在所有的实验条件下，pH和盐酸钆是显着的因素。但最适pH值随温度和添加剂的变化趋势不明显，仅在中性pH值下最适。甘油使最适GdnHCl浓度向高侧移动，精氨酸向低侧移动。因此，可以得出结论，甘油和精氨酸剥夺和补充GdnHCl，分别。在这项研究中，响应面使得有可能成功地调查在体外复性的最佳条件，并阐明复性因子之间的相互作用与最少数量的实验。在响应面法确定的最佳条件下，复性率可达90%左右，比传统方法提高了30%。
Response Surface Methodology (RSM) was used to investigate optimal refolding conditions of Pseudomonas fluorescens lipase which was expressed as inclusion body in E. coli. Three interacting factors, protein concentration, pH and guanidine hydrochloride (GdnHCl) concentration were selected as the variables of RSM. The protein concentration did not affect the refolding yield within the selected range (50–340 μg ml−1) at low temperatures of 4 and −15°C, but it was a critical factor at 25°C and refolding yield significantly decreased with increasing protein concentration. The pH and GdnHCl were significant factors in all experimental conditions. But there was no trends of optimal pH depending on temperature and additives, just showing the optimum at neutral pH. Glycerol shifted optimal GdnHCl concentration to higher side, while arginine to lower side. Therefore, it was concluded that glycerol and arginine deprives and supplements GdnHCl, respectively. In this study, RSM made it possible to investigate successfully the optimal conditions of in vitro refolding and to elucidate interactions between refolding factors with a minimum number of experiments. Under the optimal condition determined by RSM, approximately 90% refolding yield was obtained and it was a 30% increase over the conventional method.