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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (2/3): 83-94    DOI: 10.13523/j.cb.2209059
技术与方法     
基于磁性壳聚糖复合材料对耐高温酶的固定化研究*
聂铭甫1,2,李由然1,2,石贵阳1,2,**()
1 粮食发酵与食品生物制造国家工程研究中心 江南大学 无锡 214122
2 江南大学生物工程学院 无锡 214122
Immobilization of High Temperature Resistant Enzymes Based on Magnetic Chitosan Composites
NIE Ming-fu1,2,LI You-ran1,2,SHI Gui-yang1,2,**()
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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摘要：

生物催化剂酶对环境较为敏感,在各种物理、化学和生物因素中,高温对酶的失活效应尤为显著。构建耐热的特异性基质,对酶进行固定化,既有利于提高酶在高温条件下的稳定性,又可实现催化剂的重复使用,极大提升了酶的工业应用价值。以壳聚糖和海藻酸钠为原料,利用碱性溶液冷冻条件下的溶解及氢键重排的方法,制备出力学性质优良且耐高温的壳聚糖复合水凝胶,进一步采用化学交联法制备固定化耐高温酶。通过单因素实验确定最优的固定耐高温酶的条件:壳聚糖质量分数5%,海藻酸钠质量分数 0.5%,ZnSO4溶液质量浓度20 g/L,交联剂乙二醇二缩水甘油醚(EGDE)体积分数0.8%。与游离酶相比,固定化酶的抗酸碱能力和热稳定性提高,最适温度升高5℃(从90℃升高到95℃),并在较宽pH和温度范围内也能保持较高酶活。在80℃下,重复反应8次以后,相对酶活仍能保持在75%以上。利用热重分析(TGA)研究了此类水凝胶中水的状态和热稳定性。建立的固定化酶制备方法,有助于解决工业酶高温情况下易失活,且难以连续使用的问题。
关键词： 壳聚糖;  耐高温;  固定化酶;  水凝胶;  四氧化三铁    
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
收稿日期: 2022-09-21 出版日期: 2023-03-31
ZTFLH:  Q819  
基金资助: *国家重点研发计划(2020YFA0907704);国家自然科学基金(32172174)
通讯作者: **石贵阳     E-mail: gyshi@jiangnan.edu.cn
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引用本文:

聂铭甫, 李由然, 石贵阳. 基于磁性壳聚糖复合材料对耐高温酶的固定化研究*[J]. 中国生物工程杂志, 2023, 43(2/3): 83-94.

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.

链接本文:

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

图1  壳聚糖基质条件下固定化酶的制备
图2  壳聚糖质量浓度对固定化酶活性和力学强度的影响
图3  海藻酸钠添加量对固定化酶活性和力学强度的影响
图4  不同金属离子对固定化酶活和力学强度的影响
图5  ZnSO4溶液浓度对固定化酶活和力学强度的影响
ZnSO4浓度/(g/L) 载酶量/(mg/g)
0 10.67
5 9.14
10 9.26
20 12.88
40 10.54
表1  不同ZnSO4溶液浓度对载酶量的影响
图6  EGDE溶液的浓度对固定化酶活和力学强度的影响
图7  固定化酶的热重及红外分析
图8  不同材料的SEM照片
图9  固定化酶和游离酶的酶学性质比较
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