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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  2019, Vol. 39 Issue (7): 71-78    DOI: 10.13523/j.cb.20190710
技术与方法     
氨基载体共价结合固定化海洋假丝酵母脂肪酶 *
朱衡1,2,林海蛟1,2,张继福3,张云1,孙爱君1,胡云峰1,**()
1 中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室 广州 510301
2 中国科学院大学 北京 100049
3 广东省中医院 广州 510120
Covalent Immobilization of Marine Candida Rugosa Lipase Using Amino Carrier
Heng ZHU1,2,Hai-jiao LIN1,2,Ji-fu ZHANG3,Yun ZHANG1,Ai-jun SUN1,Yun-feng HU1,**()
1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301,China
2 University of Chinese Academy of Sciences,Beijing 100049,China
3 Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120,China
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摘要：

共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5.0、0.1%戊二醛、0.25g载体、交联0.5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83.01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。
关键词： 氨基载体;  脂肪酶;  戊二醛;  共价交联;  固定化酶    
Abstract:

Covalent binding is one important immobilization method of industrial enzyme, which uses stable covalent bonds to immobilize industrial enzymes, creates multi-point covalent connections between carrier and enzyme, and prepares immobilized enzymes with good stability and possesses practical application value. Marine Candida rugosa lipase was immobilized using amino carrier through covalent binding method and relative inexpensive glutaraldehyde was used as the crosslinking reagent. By using single factor and orthogonal experiment, the optimal immobilization conditions were determined as follows:25℃, pH5.0,0.1% glutaraldehyde,0.25g carrier, crosslinked 0.5h,immobilization time 1h, enzyme loading 800U, with the final obtained enzyme activity being 83.01U/g. Compared with free enzyme, the optimal pH of immobilized lipase was shifted to alkaline direction, the optimal reaction temperature was increased by 10℃, the thermal stability and acid-alkali stability of immobilized enzyme were better than that of free enzyme, and the reusability and storage stability were better than that of free enzyme. Meanwhile, crosslinking agent was found to be an important factor in the preparation of immobilized lipase. Thus it is of great significance to explore new crosslinking agent to improve immobilization effect. A good foundation for the immobilization technique and industrial application of marine candida rugosa lipase were laid.
Key words: Amino carrier    Lipase    Glutaraldehyde    Covalent crosslinking    Immobilized enzyme
收稿日期: 2018-12-29 出版日期: 2019-08-05
ZTFLH:  Q814.2  
基金资助: * 广东省海洋渔业科技攻关与研发方向项目(A201701C12);中国科学院战略性先导科技专项(XDA11030404);中国科学院“科学”号高端用户项目(KEXUE2018G05);广东省自然科学基金资助项目(2018A030313151)
通讯作者: 胡云峰     E-mail: yunfeng.hu@scsio.ac.cn
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朱衡
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引用本文:

朱衡,林海蛟,张继福,张云,孙爱君,胡云峰. 氨基载体共价结合固定化海洋假丝酵母脂肪酶 *[J]. 中国生物工程杂志, 2019, 39(7): 71-78.

Heng ZHU,Hai-jiao LIN,Ji-fu ZHANG,Yun ZHANG,Ai-jun SUN,Yun-feng HU. Covalent Immobilization of Marine Candida Rugosa Lipase Using Amino Carrier. China Biotechnology, 2019, 39(7): 71-78.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190710    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I7/71

图1  脂肪酶固定化条件的优化
图2  固定化酶与游离酶酶学性质表征
序号 载体量(g) 固定化时间(h) 温度(℃) pH 酶活(U/g) 相对酶活(%)
1 0.25 1 25 5 83.010 752 9 100
2 0.25 2 30 6 59.704 299 32 71.923 57
3 0.25 3 35 7 35.725 805 37 43.037 56
4 0.5 1 30 7 48.413 977 89 58.322 54
5 0.5 2 35 5 38.360 214 51 46.211 14
6 0.5 3 25 6 51.881 718 44 62.5
7 0.75 1 35 6 58.602 152 16 70.595 86
8 0.75 2 25 7 45.000 000 69 54.209 85
9 0.75 3 30 5 47.338 710 9 57.027 2
K1 178.440 9 190.026 9 179.892 5 170.188 2
K2 138.655 9 143.064 5 155.457 168.709 7
K3 150.940 9 134.946 2 132.688 2 129.139 8
R 27.499 99 55.080 65 47.204 3 41.048 39
表1  脂肪酶固定化条件优化正交试验结果与分析
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