Wen Qingshi, Zhang Jie, Miao Rongxin, Zhang Bingyun, Yan Ziyi, Ying Hanjie, Wang Junzhi
Jiangsu Industrial Technology Research Institute, Nanjing Institute of White-Biotech Co.Ltd, Nanjing, China.
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, China.
Bioprocess Biosyst Eng. 2023 Apr;46(4):499-505. doi: 10.1007/s00449-022-02827-4. Epub 2023 Feb 17.
The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200 mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6 mM, and the productivity was 12.6 mM L h, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.
目前工业生产三磷酸胞苷（CTP）的生物催化方法存在反应速率损失的问题。这是由醋酸盐浓度逐渐增加引起的，醋酸盐会抑制酶的活性并降低最终产量。这项工作通过对CTP生产系统中的一种酶——CMP激酶（CMPK）进行计算机辅助设计，为解决这一问题提供了一种可能的解决方案，以提高其在高醋酸盐浓度溶液中的稳定性。受天然嗜盐酶特性的启发，将碱性和中性表面残基替换为酸性氨基酸。这种蛋白质设计策略有效地提高了CMPK在工作条件下（醋酸盐浓度超过1200 mM）的活性。耐盐CMPK应用于CTP的补料分批生产。最大滴度为201.4±1.6 mM，生产率为12.6 mM L h，分别比使用野生型CMPK的工艺提高了26.4%和27.8%。
