Crude enzyme immobilization-based cell-free system for efficient N-acetylneuraminic acid biosynthesis aided by N-terminal coding sequence screening
Peng Wen, Xueqin Lv, Long Liu, Jianghua Li, Guocheng Du, Yanfeng Liu
Systems Microbiology and Biomanufacturing ›› 2023, Vol. 3 ›› Issue (3) : 479-488.
Crude enzyme immobilization-based cell-free system for efficient N-acetylneuraminic acid biosynthesis aided by N-terminal coding sequence screening
N-acetylneuraminic acid (NeuAc) is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests, crucian eggs, caviar and human breast milk. The extraction process is complicated, resulting in the disadvantages of low NeuAc content and low recovery rate. In this study, a crude enzyme immobilization-based cell-free system (CEICFS) was developed for efficient NeuAc biosynthesis. First, N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid aldolase (NanA) were obtained by high-throughput screening. And these sequences resulted in up to 1.5-fold (1.2-fold) increase in AGE (NanA) enzyme levels. And then, a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino resin. Subsequently, NeuAc production from GlcNAc using CEICFS in one reactor was carried out, resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/h. Further, the enzyme activity was still higher than 75% after five repeated uses. The functional properties of CEICFS were studied and compared to those of the free enzyme, immobilization can extend the application of enzyme to some harsh environments, such as low temperature and acidic environment. Therefore, CEICFS with excellent heat resistance, storage stability and reusability exhibit great potential for industrial application.
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