Heterologous expression and characterization of Penicillium citrinum nuclease P1 in Aspergillus niger and its application in the production of nucleotides

Abstract

Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg2+, Zn2+ and Cu2+, and inhibited by addition of Ca2+, Fe2+, Mn2+, Ni2+, Co2+, Mg2+, K+ and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5‘- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.