Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (4): 576-581.doi: 10.3969/j.issn.2095-4344.2372

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Immobilization of lactase on magnetic chitosan microspheres and its effect on enzymatic properties

Li Li1, Ma Li2   

  1. 1Chengdu Agricultural College, Chengdu 611130, Sichuan Province, China; 2Xihua University, Chengdu 610039, Sichuan Province, China
  • Received:2019-12-31 Revised:2020-01-07 Accepted:2020-03-11 Online:2021-02-08 Published:2020-11-21
  • About author:Li Li, Master, Lecturer, Senior engineer, Chengdu Agricultural College, Chengdu 611130, Sichuan Province, China

Abstract: BACKGROUND: The free enzyme has the problems of poor stability and inability to be reused during application. Using magnetic polymer microspheres as the carrier of the binding enzyme to prepare immobilized enzyme can maintain the natural activity of the enzyme, and can be reused, but also provide convenient conditions for automatic production management.
OBJECTIVE: Magnetic chitosan microspheres as carrier of binding enzyme were used to prepare the immobilized lactase, which is easy to recycle, can be reused, and has high enzyme activity and stability.
METHODS: By inputting a certain amount of magnetic chitosan microspheres into the phosphoric acid buffer for swelling for 2 hours, the swelling magnetic chitosan microspheres were collected with a magnet and added to a certain concentration of lactase phosphate buffer. They were shocked in a constant temperature shaker for 1 hour and preserved in refrigerator at 4 °C. The microspheres were precipitated with a magnet to pour out the supernatant. After full washing with buffer solution, the immobilized lactase was obtained. The properties of the magnetic microspheres (the amount of glutaraldehyde used in the preparation of microspheres was 2, 4, 6, 8, 10, 12, 14 mL), the amount of enzymes (0.5, 1, 1.5, 2 g/L) added, the pH (6.4, 6.8, 7.0, 7.2) of the buffer, and the immobilization time (1, 2, 5, 10, 15, 20 hours) were tested to determine the optimal immobilization conditions.
RESULTS AND CONCLUSION: (1) The optimum conditions for immobilized lactose with magnetic chitosan microspheres were as follows: magnetic chitosan microspheres prepared with 10 mL of glutaraldehyde were selected as the immobilized carrier of lactase. The amount of enzyme added was 0.3 g/L, pH 7.0 and the immobilization time was 5 hours. (2) Compared with the free enzyme, immobilized lactase showed a wider range of reaction temperature and pH value. (3) The ability of immobilized enzyme binding substrate was enhanced. (4) After repeated use of the immobilized enzyme five times, the enzyme activity remained 65%. (5) The storage stability of lactase was also improved after immobilization.

Key words: material, magnetic microsphere, polymer microsphere, biomaterials, chitosan, immobilized enzyme, magnetic response,  , lactase

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