Fortified continous catalytic properties of immobilized nuclease P₁ with surface activated HA amino resin

The effect of HA amino resin (immobilized enzyme widely-used carrier) geometry and surface activated process on the immobilized nuclease P₁'s properties was studied. In addition, the dynamic analysis and continuous catalytic properties were also investigated. FESEM, BET and FTIR characterization were utilized to verify that HA amino resin had a great deal of enzymes available hole and the scope of the main hole was 4-30 nm in the immobilized process. Compared with free nuclease P₁, the acid resistance and heat resistance of the immobilized enzymes were improved. The study on Michael's Mention kinetics indicated that the substrate affinity and maximum reaction rate of immobilized enzymes decreased; the reusability of after crosslinking group was significantly enhanced compared to physical adsorption and chemical crosslinking groups. The optimized conditions of immobilization were as follows: the enzyme to carrier ratio of 3:20 (mass ratio), enzyme concentration of 0.8 g·L^-1 and 10 h immobilized time at pH 6.0. Under these conditions, the immobilized enzyme activity was about 10013 U·g^-1. Furthermore, the operating conditions of column flow reactor were designed and optimized. The continuous running time of the reactor was up to 120 h at 30 g·L^-1 products nucleotide concentration (hydrolysis rate of 60%), when the reaction temperature was 65℃ with substrate flow rate of 0.75 ml·min^-1. This work would be beneficial to the application of nuclease P₁ in nucleotide continuous industrial production.