Zhang Yue, Dong Weiliang, Lv Ziyao, Liu Jiawei, Zhang Wenmin, Zhou Jie, Xin Fengxue, Ma Jiangfeng, Jiang Min
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing, 211800, People's Republic of China.
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, 211800, People's Republic of China.
Mol Biotechnol. 2018 Sep;60(9):681-689. doi: 10.1007/s12033-018-0103-6.
Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10 cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.
利用枯草芽孢杆菌的聚γ-谷氨酸合成酶A蛋白（PgsA）作为锚定基质，成功地将枯草芽孢杆菌168的漆酶CotA展示在大肠杆菌细胞膜上。进一步分析表明，融合蛋白PgsA/CotA能有效地转运到大肠杆菌细胞表面，酶活性为65 U/10个细胞。与野生型CotA相比，表面展示的CotA具有更好的酶学性质，包括更高的热稳定性（70℃孵育5小时后活性高于90%，90℃孵育5小时后活性接近40%）和更强的抑制剂耐受性（分别与200和400 mM NaCl孵育时活性约为80%和65%）。此外，全细胞系统对蒽醌染料酸性蓝62、三苯甲烷染料孔雀石绿和偶氮染料甲基橙具有较高的酶活性，孵育5小时后脱色率分别为91%、45%和75%。
