Abstract: Four amino acid residues( F60,A62,T102 and Y135) were selected and five single and multiple mutants of Nuc2 were constructed. After in vitro expression and purification,enzyme activity,thermostability and secondary structure of these nucleases were determined. The results indicated that the enzyme activity of T102 M and F60 A /A62 L / T102 M / Y135 V had significant differences with wild- type( WT) after variance analysis( p < 0.05),and the nuclease activity of F60 A / A62 L / T102 M / Y135 V was totally disabled. The thermostability of T102 M and F60 A / A62 L was much weaker than that of WT. Besides,the α- helical content of T102 M and F60 V / A62 L in their secondary structure was much lower than that of WT,and it was showed from the CD spectra that the secondary structure of F60 A / A62 L / T102 M / Y135 V was in a state of random coil. Therefore,T102 is a pivotal site to maintain the nuclease activity,thermostability and secondary structure of Nuc2.And the interaction among F60,A62,T102 and Y135 is also significant to maintain its stability of secondary structure.