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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2020, Vol. 40 Issue (4): 49-58    DOI: 10.13523/j.cb.1911045
    
Immobilization of Extracellaluar Proteases of Bacillus sp. DL-2 Using Epoxy Resin to Asymmetrically Hydrolyze (±)-1-Phenylethyl Acetate
DONG Lu1,2,ZHANG Ji-fu4,ZHANG Yun1,HU Yun-feng1,3,**()
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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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     
Received: 28 November 2019      Published: 18 May 2020
ZTFLH:  Q502  
Corresponding Authors: Yun-feng HU     E-mail: yunfeng.hu@scsio.ac.cn
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Cite this article:

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.

URL:

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

Fig.1 Asymmetric hydrolysis of (±)-1-phenylethyl acetate by immobilized extracellaluar proteases of Bacillus sp. DL-2 derived from epoxy resins
环氧树脂类型 ES-1 ES-103B ES-107 EP200 LX-
1000EP		 LX-
1000EPC		 LX-
1000EPF		 LX-
EP120		 MC-
300EP
第一次 e.e.p (%) 93.3 93.9 74.4 68.2 87.2 79.7 93.2 67.0 85.5
C (%) 5.1 6.7 2.2 1.3 2.3 1.3 3.4 1.1 1.7
第二次 e.e.p (%) 47.9 84.3 —— —— —— —— 67.1 —— ——
C (%) 0.4 2.4 —— —— —— —— 2.4 —— ——
Table 1 Comparation of the asymmetric hydrolysis of (±)-1-phenylethyl acetate by immobilized extracellaluar proteases of Bacillus sp. DL-2 derived from 9 different epoxy resins
Fig.2 The optimization of immobilization conditions of extracellaluar proteases (a) Effect of temperature on the activity of immobilized enzyme (b) Effect of immobilization time on the activity of immobilized enzyme (c) Effect of carrier concentration on the activity of immobilized enzyme (d) Effect of buffer pH on the activity of immobilized enzyme
Fig.3 The gas chromatogram of (R)-1-phenylethanol and (S)-1-phenylethyl acetate prepared through asymmetric hydrolysis of (±)-1-phenylethyl acetate using the extracellaluar proteases immobilized by ES-103B (a) standards of (±)-1-phenylethyl acetate and (±)-1-phenylethanol (b) (R)-1-phenylethanol prepared after enzymatic kinetic resolution (c) (S)-1-phenylethyl acetate prepared after enzymatic kinetic resolution
Fig.4 The reusability and storage stability of immobilized extracellaluar proteases (a) Reusability in the hydrolysis of (±)-1-phenylethyl acetate catalyzed by immobilized extracellular proteases for the preparation of (R)-1-phenylethanol (b) Reusability in the hydrolysis of (±)-1-phenylethyl acetate catalyzed by immobilized extracellular proteases for the preparation of (S)-1-phenylethyl acetate (c) Storage stability in the hydrolysis of (±)-1-phenylethyl acetate catalyzed by immobilized extracellular proteases for the preparation of (R)-1-phenylethanol (d) Storage stability in the hydrolysis of (±)-1-phenylethyl acetate catalyzed by immobilized extracellular proteases for the preparation of (S)-1-phenylethyl acetate
固定化载体 (R)-1-苯乙醇 (S)-乙酸苏合香酯
e.e.p
(%)		 C
(%)		 YR
(%)		 酶浓度
(mg/ml)		 底物浓度
(mmol/L)		 e.e.p > 90%
的重复使用次数		 e.e.s
(%)		 C
(%)		 YR
(%)		 酶浓度
(mg/ml)		 底物浓度
(mmol/L)		 e.e.s > 90%的
重复使用次数
环氧树脂ES-103B > 97 23 45 320 5 8 > 99 58 84 320 5 1
硅藻土 97 18 41 360 5 2 > 99 64 71 440 2.5 1
Table 2 Comparation of extracellaluar proteases of Bacillus sp. DL-2 immobilized by ES-103B and by kieselguhr in the asymmetric hydrolysis of (±)-1-phenylethyl acetate
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