环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶<sup>*</sup>

doi:10.13523/j.cb.1912027

中国生物工程杂志

2020, Vol. 40

Issue (5): 57-68 DOI: 10.13523/j.cb.1912027

技术与方法

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶^*

朱衡^1,^2,³,张继福⁴,张云¹,胡云峰^1,^3,^**(

)

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 南方海洋科学与工程广东省实验室(广州) 广州 511458
⁴ 广东省中医院广州 510120

Immobilization of Marine Candida Lipase Using Novel Epoxy Cross-linker and Amino Carrier

ZHU Heng^1,^2,³,ZHANG Ji-fu⁴,ZHANG Yun¹,HU Yun-feng^1,^3,^**(

)

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 Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou),Guangzhou 511458,China
⁴ Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,China

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摘要：

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。

关键词： 聚乙二醇缩水甘油醚; 氨基载体; 脂肪酶; 固定化; 酶学性质

Abstract:

After immobilization, the stability and environmental tolerance of the free enzyme were optimized, which can improve the utilization rate of enzymes and reduce the production costs in diverse fields such as food, medicine, chemical industry, environment and leather, and thus possesses great application potentials. The utilization of novel crosslinking agents in immobilized enzyme process has greatly advanced the study of immobilized enzymes.The optimized crosslinking and immobilization conditions were obtained by using the new crosslinking agent polyethylene glycol diglycidyl ether (PEGDGE), which was immobilized with the amino carrier LX-1000HA, and by using single factor and orthogonal experiment optimization: crosslinking temperature 30℃, crosslinking time 2h, crosslinking agent concentration 0.75%, pH 7.0, enzyme amount 800U, carrier volume 0.5g, immobilized time 2h, immobilized temperature 45℃. Using the optimized immobilization process, the immobilized enzyme LX-1000HA-PEGDGE-CRL was prepared to achieve 160.81U/g which was about twice of the activities of immobilized enzyme LX-1000HA-GA-CRL(obtained by LX-1000HA and glutaraldehyde crosslinked lipase) and LX-1000EA-PEGDGE-CRL(obtained by short-chain amino carrier LX-1000EA and PEGDGE crosslinked lipase). The optimal reaction temperature of immobilized enzyme LX-1000HA-PEGDGE-CRL was found to be 15℃ higher than that of free enzyme; the immobilized enzyme could still remain 70% of its original activity after incubation at 70℃ for 3h; the immobilized enzyme could remain about 65% of its original activity after recycling for 6 times; the immobilized enzyme also behaved good tolerance to acid and alkali condition and good storage stability, the immobilized enzyme could remain about 70% of the initial enzyme activity after storage at 4℃ for 30 days. After the comparation of immobilized enzyme LX-1000HA-PEGDGE-CRL with free enzyme, immobilized enzyme LX-1000HA-GA-CRL and immobilized enzyme LX-1000EA-PEGDGE-CRL, the immobilized enzyme LX-1000HA-PEGDGE-CRL behaved better application effect in thermo-tolerance and reusability.

Key words: Polyethylene glycol diglycidyl ether Amino-carrier Lipase Immobilization Enzymatic property

收稿日期: 2019-12-18 出版日期: 2020-06-02

ZTFLH:

Q814.2

基金资助: * 广东省自然科学基金(2018A030313151);广东省海洋渔业科技攻关与研发方向项目(A201701C12);中国科学院“科学”号高端用户项目(KEXUE2018G05);中国科学院战略性先导科技专项(XDA11030404);南方海洋科学与工程广东省实验室(广州)人才团队引进重大专项(GML2019ZD0406)

通讯作者: 胡云峰 E-mail: yunfeng.hu@scsio.ac.cn

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引用本文:

朱衡,张继福,张云,胡云峰. 环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶^*[J]. 中国生物工程杂志, 2020, 40(5): 57-68.

ZHU Heng,ZHANG Ji-fu,ZHANG Yun,HU Yun-feng. Immobilization of Marine Candida Lipase Using Novel Epoxy Cross-linker and Amino Carrier. China Biotechnology, 2020, 40(5): 57-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1912027 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I5/57

图1 LX-1000HA固定化LIPASE过程示意图

表1 正交试验因素水平表

图2 单因素试验确定交联及固定化条件

表2 脂肪酶固定化条件优化正交试验结果与分析

表3 计算过程

图3 固定化酶和游离酶的酶学性质比较

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