Sun Xiaoyue, Shen Wei, Gao Yanyun, Cai Menghao, Zhou Mian, Zhang Yuanxing
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Protein Expr Purif. 2019 Jan;153:97-104. doi: 10.1016/j.pep.2018.09.002. Epub 2018 Sep 8.
Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation. By domain and disorder examination, we found that the protein was completely functional by expressing the C terminal fragment alone. For the final strain we constructed (HMS-ompA-CF), the extracellular enzyme activity reached 375 U/ml in shake flask and 1789 U/ml in fed batch cultivation (5 L bioreactor). And the final protein yield reached 0.58 g/L in fed batch cultivation. We determined that the optimal pH and temperature for the shortened alginate lyase were 7.0 and 39 °C, respectively.
海藻酸盐裂解酶消化是将海藻酸盐降解为寡糖的有效方法，这些寡糖在化学、制药和食品工业等各个领域都很有用。在这里，我们确定了来源于弧菌属QY102的海藻酸盐裂解酶的序列，并在大肠杆菌中建立了其异源表达。我们通过用大肠杆菌特异性信号肽ompA替换原始分泌序列来提高其分泌效率。我们在摇瓶培养中成功纯化了全长蛋白，然而，在补料分批培养过程中发生了降解。通过结构域和无序检查，我们发现单独表达C末端片段时该蛋白具有完全功能。对于我们构建的最终菌株（HMS-ompA-CF），摇瓶中的胞外酶活性达到375 U/ml，补料分批培养（5 L生物反应器）中达到1789 U/ml。补料分批培养中的最终蛋白产量达到0.58 g/L。我们确定缩短的海藻酸盐裂解酶的最佳pH和温度分别为7.0和39°C。
