Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol
Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyre...
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MDPI AG
2015-02-01
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| Series: | Molecules |
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| Online Access: | http://www.mdpi.com/1420-3049/20/3/3744 |
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| author | Yi-Li Chou Chia-Yun Ko Long-Fang O. Chen Chih-Chung Yen Jei-Fu Shaw |
| author_facet | Yi-Li Chou Chia-Yun Ko Long-Fang O. Chen Chih-Chung Yen Jei-Fu Shaw |
| author_sort | Yi-Li Chou |
| collection | DOAJ |
| description | Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol. |
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| language | English |
| last_indexed | 2024-12-11T17:51:06Z |
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| series | Molecules |
| spelling | doaj.art-a145972bfffc4d69862be10c73bac1d62022-12-22T00:56:13ZengMDPI AGMolecules1420-30492015-02-012033744375710.3390/molecules20033744molecules20033744Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and PhytolYi-Li Chou0Chia-Yun Ko1Long-Fang O. Chen2Chih-Chung Yen3Jei-Fu Shaw4Institute of Biotechnology, National Cheng Kung University, Tainan 70101, TaiwanInstitute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, TaiwanInstitute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, TaiwanDepartment of Biological Science and Technology, I-Shou University, Kaohsiung 82445, TaiwanInstitute of Biotechnology, National Cheng Kung University, Tainan 70101, TaiwanRecombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.http://www.mdpi.com/1420-3049/20/3/3744enzyme purificationenzyme immobilizationBrassica oleracea chlorophyllase 1 (BoCLH1)DIAION®CR11 |
| spellingShingle | Yi-Li Chou Chia-Yun Ko Long-Fang O. Chen Chih-Chung Yen Jei-Fu Shaw Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol Molecules enzyme purification enzyme immobilization Brassica oleracea chlorophyllase 1 (BoCLH1) DIAION®CR11 |
| title | Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol |
| title_full | Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol |
| title_fullStr | Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol |
| title_full_unstemmed | Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol |
| title_short | Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol |
| title_sort | purification and immobilization of the recombinant brassica oleracea chlorophyllase 1 boclh1 on diaion r cr11 as potential biocatalyst for the production of chlorophyllide and phytol |
| topic | enzyme purification enzyme immobilization Brassica oleracea chlorophyllase 1 (BoCLH1) DIAION®CR11 |
| url | http://www.mdpi.com/1420-3049/20/3/3744 |
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