Immobilization of Nuclease p1 on Chitosan Micro-spheres

L. E. Shi, Z. X. Tang, Y. Yi, J. S. Chen, W. Y. Xiong and G. Q. Ying

Abstract
This paper presents the preparation of chitosan micro-spheres by the cross-linking method. Effects of various factors on enzyme activity were investigated, such as glutaraldehyde concentration, cross-linking time, enzyme amount, pH, and immobilized time. The immobilized conditions were optimized by orthogonal experiments. Characterizations of immobilized nuclease p1 were also evaluated. Through orthogonal optimization, optimal immobilization conditions were as follows: glutaraldehyde mass fraction w = 0.20 %, enzyme amount 6.0 mL (enzyme activity 2160 U), immobilized time 4.0 h and pH 5.8. Optimal pH of immobilized enzyme was 5.8. Optimal temperature of immobilized enzyme was 80 °C. Thermal, operational and storage stabilities of the enzyme were improved after it was immobilized on chitosan micro-spheres. Michaelis constant Km of immobilized enzyme was 96.58 mg mL–1 by Lineweaver-Burk plot at 70 °C.

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Keywords
Chitosan micro-spheres, nuclease p1, immobilization, characterizations