Immobilization of High Temperature Resistant Enzymes Based on Magnetic Chitosan Composites

doi:10.13523/j.cb.2209059

China Biotechnology

2023, Vol. 43

Issue (2/3): 83-94 DOI: 10.13523/j.cb.2209059

Immobilization of High Temperature Resistant Enzymes Based on Magnetic Chitosan Composites

NIE Ming-fu^1,²,LI You-ran^1,²,SHI Gui-yang^1,^2,^**(

)

1 National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China
2 School of Biotechnology, Jiangnan University, Wuxi 214122, China

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Abstract

Biocatalyst enzymes are sensitive to the environment, and high temperature has a significant effect on enzyme inactivation among various physical, chemical and biological factors. The construction of heat-resistant specific matrix and the immobilization of the enzyme can not only improve the stability of the enzyme at high temperature, but also realize the reuse of the catalyst, which greatly improves the industrial application value of the enzyme. In this study, chitosan hydrogels with excellent mechanical properties and high temperature resistance were prepared by means of dissolution and hydrogen bond rearrangement of alkaline solution under freezing conditions, and the immobilized high temperature resistant enzyme was further prepared by chemical cross-linking. The optimal conditions for the fixation of high temperature resistant enzyme were determined by single factor experiment: the mass fraction of chitosan was 5%, the mass fraction of sodium alginate was 0.5%, the mass concentration of ZnSO₄ solution was 20 g/L, and the volume fraction of EGDE was 0.8%. Compared with the free enzyme, the immobilized enzyme showed improved acid-base resistance and thermal stability, optimum temperature increase of 5℃ (from 90℃ to 95℃), and also maintained high activity over a wide range of pH and temperature. At the temperature of 80℃, the relative enzyme activity remained above 75% after 8 repeated reactions. Thermogravimetric analysis (TGA) was used to study the state and thermal stability of water in the hydrogels. The immobilized enzyme preparation method established in this study is helpful to solve the problem that industrial enzymes are easy to be inactivated at high temperature and difficult to be used continuously.

Key words： Chitosan High temperature resistance Immobilized enzyme Hydrogel Fe₃O₄

Received: 21 September 2022 Published: 31 March 2023

ZTFLH:

Q819

Corresponding Authors: **Gui-yang SHI E-mail: gyshi@jiangnan.edu.cn

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Cite this article:

NIE Ming-fu, LI You-ran, SHI Gui-yang. Immobilization of High Temperature Resistant Enzymes Based on Magnetic Chitosan Composites. China Biotechnology, 2023, 43(2/3): 83-94.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2209059 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I2/3/83

Fig.1 Determination of enzyme activity of chitosan fixation enzyme under original conditions (a)The preparation process of chitosan composite (b)The enzyme activity of immobilized enzyme under the condition of chitosan matrix

Fig.2 Effects of different concentrations of chitosan on the activity and mechanical strength of immobilized enzymes (a) The change of relative enzyme activity with the mass fraction of chitosan (b)The activity changes of chitosan immobilized enzymes with different mass fractions under different reaction times (c) The change of pressure with the mass fraction of chitosan

Fig.3 Effects of different mass fractions of sodium alginate on the activity and mechanical strength of immobilized enzymes (a)The change of relative enzyme activity with the mass fraction of sodium alginate (b)The activity changes of immobilized alginate containing different mass fractions under different reaction times (c) The change of pressure with the mass fraction of sodium alginate

Fig.4 Effects of different metal ions on the activity and mechanical strength of immobilized enzymes (a)The change of relative enzyme activity with different types of coagulants (b)The activity changes of immobilized enzymes in different coagulants under different reaction times (c)The change of pressure with the type of coagulant

Fig.5 Effect of metal ion concentration on immobilized enzyme activity and mechanical strength (a)The change of relative enzyme activity with the concentration of coagulants (b)The activity changes of immobilized enzymes reacting with different mass concentrations of coagulants under different reaction times (c)The change of pressure with the concentration of solidified liquid

Table 1 Effects of different ZnSO₄ solution concentrations on enzyme loading

Fig.6 Effect of concentration of EGDE solution on immobilized enzyme activity and mechanical strength (a) The change of relative enzyme activity with different volume concentrations of crosslinkers (b)The activity changes of immobilized enzymes reacting with different volume concentrations of crosslinker under different reaction times (c) The change of pressure with the volume concentration of different crosslinkers

Fig.7 Heat resistance and infrared analysis of immobilized enzyme

Fig.8 SEM photos of different materials (a) Sodium alginate (b) Pure chitosan gel (c) Immobilized enzyme prepared under optimal conditions (d) The trailing phenomenon caused by adding high concentration of sodium alginate

Fig.9 Comparison of enzymatic properties of immobilized and free enzymes (a)The effect of pH on the activities of immobilized and free enzymes (b) The effect of temperature on the activities of immobilized and free enzymes (c) Stands for comparison of thermal stability between immobilized and free enzymes


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