High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiae and its purification and immobilization by one-step method
Jian-Ju He, Xinxin Liu, Ying Li, Zhe Wang, Hongling Shi, Yunchao Kan, Lunguang Yao, Cun‐Duo Tang
Abstract
Nicotinamide riboside kinase (NRK) plays an important role in the synthesis of β -nicotinamide nucleotide (NMN). NMN is a key intermediate of NAD + synthesis, and it actually contribute to the well-being of our health. In this study, gene mining technology was used to clone nicotinamide nucleoside kinase gene fragments from S. cerevisiae , and the Sc NRK1 was achieved a high level of soluble expression in E. coli BL21. Then, the reScNRK1 was immobilized by metal affinity label to optimize the enzyme performance. The results showed that the enzyme activity in the fermentation broth was 14.75 IU/mL, and the specific enzyme activity after purification was 2252.59 IU/mg. After immobilization, the optimum temperature of the immobilized enzyme was increased by 10°C compared with the free enzyme, and the temperature stability was improved with little change in pH. Moreover, the activity of the immobilized enzyme remained above 80% after four cycles of immobilized reScNRK1, which makes the enzyme more advantageous in the enzymatic synthesis of NMN.