College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Int J Mol Sci. 2022 Jun 13;23(12):6588. doi: 10.3390/ijms23126588.
Type I L-asparaginase from Z-1 (BlAase) was efficiently produced and secreted in RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in . First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.
来自 Z-1（BlAase）的 I 型 L-天冬酰胺酶在 RIK 1285 中高效表达和分泌，但产量低，不适合工业用途。因此，本研究采用组合方法提高 BlAase 在. 中的合成。首先，选择了十五个单强启动子替代原始启动子 P43，其中 PyvyD 实现了最高的 BlAase 活性（436.28 U/mL）。其次，使用四个启动子（PyvyD、P43、PaprE 和 PspoVG）构建双启动子系统，相对较高的 BlAase 表达水平以提高 BlAase 产量，启动子 PaprE-PyvyD 的引擎达到 502.11 U/mL。通过修饰启动子 PaprE-PyvyD 的关键部分，还提高了 BlAase 的活性（568.59 U/mL）。第三，当替换启动子 PyvyD 的核糖体结合位点（RBS）序列时，BlAase 活性达到 790.1 U/mL，比原始启动子 P43 菌株高 2.27 倍。在 10 L 发酵罐中培养 36 h 后，BlAase 的表达水平达到 2163.09 U/mL，比使用启动子 P43 的初始菌株高 6.2 倍。此外，还评估了 BlAase 在薯片丙烯酰胺迁移中的应用潜力。结果表明，结合烫漂和 BlAase 处理，可去除炸薯片 89.50%的丙烯酰胺。这些发现表明，转录和翻译技术的组合是提高重组蛋白产量的有效策略，BlAase 可以成为控制食品加工中丙烯酰胺的候选酶。
