In this study, we established a new methodology for preparing 5'-nucleotidase (5'-NT) with the aim of enhancing our understanding of its enzyme activity and laying a basis for regulating the content of umami-enhancing nucleotides in pork. 5'-NT was prepared with Sephadex gel filtration and reverse-phase high-performance liquid chromatography, and its enzymatic properties and catalytic activity were evaluated. The results show that the molecular weight of the prepared 5'-NT was 57 kDa, the optimal catalytic temperature was 40 °C, and the optimal pH was 8. Zn2+, and sucrose showed inhibitory effects on the activity of 5'-NT, while K+, Na+, Ca2+, Mg2+, glucose, fructose, and trehalose promoted the activity of the studied compound. The prepared 5'-NT exhibited higher catalytic activity and selectivity against IMP compared with its commercial counterpart, while its catalytic activity against XMP was not significant (p > 0.05). In brief, we established a new methodology for preparing 5'-NT, enhancing our understanding of its enzyme activity and providing a solid basis for regulating the content of umami-enhancing nucleotides in pork through the control of endogenous 5'-NT activity.
Keywords: 5′−nucleotidase; isolation and purification; pork; umami.