环氧树脂固定化的<i>Bacillus</i> sp. DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用 <sup>*</sup>

doi:10.13523/j.cb.1911045

中国生物工程杂志

2020, Vol. 40

Issue (4): 49-58 DOI: 10.13523/j.cb.1911045

技术与方法

环氧树脂固定化的Bacillus sp. DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用 ^*

董璐^1,²,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东省海洋药物重点实验室广州 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 Extracellaluar Proteases of Bacillus sp. DL-2 Using Epoxy Resin to Asymmetrically Hydrolyze (±)-1-Phenylethyl Acetate

DONG Lu^1,²,ZHANG Ji-fu⁴,ZHANG Yun¹,HU Yun-feng^1,^3,^**(

)

1 Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medical, 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：

环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp. DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:pH 8.0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e.e.p=97.5%的(R)-1-苯乙醇(产率为45.0%)和e.e.s=99.2%的(S)-乙酸苏合香酯(产率为83.9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e.e.p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。

关键词： 环氧树脂ES-103B; 海洋微生物; 固定化酶; 不对称水解; (R)-1-苯乙醇; (S)-乙酸苏合香酯

Abstract:

Epoxy group, a very active group, can react with biomolecules such as enzymes, proteins, nucleic acid and form covalent bonds, which is beneficial for the immobilization of biomolecules. Enzymes immobilized on carriers via covalent binding exhibit better stability and reusability than free enzymes. The extracellular proteases of marine bacterium Bacillus sp. DL-2 were immobilized by covalent bonding onto epoxy resin ES-103B. After the the optimization of immobilization conditions, pH 8.0 extracellular proteases solution, 25 g/L epoxy resin ES-103B, and 45 ℃ for 8 h were determined as the optimal conditions for immobilized proteases to asymmetrically hydrolyze (±)-1-phenylethyl acetate, which generated (R)-1-phenylethanol with the e.e.p being 97.5% and the yield being 45.0%, and generated (S)-1-phenylethyl acetate with the e.e.s being 99.2% and the yield being 83.9%, respectively. The the immobilized enzyme could be reused to resolve (±)-1-phenylethyl acetate for eight times and the e.e.p still retained over 90%. Additionally, the immobilized extracellular proteases showed good storage stability at 4℃.

Key words: Epoxy resin ES-103B Marine bacterium Immobilized enzyme Asymmetric hydrolysis (R)-1-phenylethanol (S)-1-phenylethyl acetate

收稿日期: 2019-11-28 出版日期: 2020-05-18

ZTFLH:

Q502

基金资助: * 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);广东省自然科学基金项目(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)

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

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引用本文:

董璐,张继福,张云,胡云峰. 环氧树脂固定化的Bacillus sp. DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(4): 49-58.

DONG Lu,ZHANG Ji-fu,ZHANG Yun,HU Yun-feng. Immobilization of Extracellaluar Proteases of Bacillus sp. DL-2 Using Epoxy Resin to Asymmetrically Hydrolyze (±)-1-Phenylethyl Acetate. China Biotechnology, 2020, 40(4): 49-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1911045 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I4/49

图1 使用环氧树脂固定化的Bacillus sp. DL-2胞外蛋白酶拆分(±)-乙酸苏合香酯

表1 九种环氧树脂固定化的Bacillus sp. DL-2胞外蛋白酶拆分(±)-乙酸苏合香酯的比较

图2 胞外蛋白酶固定化条件的优化

图3 ES-103B固定化的胞外蛋白酶不对称水解(±)-乙酸苏合香酯制备的(R)-1-苯乙醇和(S)-乙酸苏合香酯的气相图谱

图4 固定化的胞外蛋白酶的重复利用性和储存稳定性

表2 ES-103B固定化的Bacillus sp. DL-2胞外蛋白酶与硅藻土固定化的此酶拆分(±)-乙酸苏合香酯的比较

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