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Molecules 2015, 20(3), 3744-3757; doi:10.3390/molecules20033744

Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol

1
Institute of Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan
2
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan
3
Department of Biological Science and Technology, I-Shou University, Kaohsiung 82445, Taiwan
4
Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan
*
Authors to whom correspondence should be addressed.
Academic Editor: Roberto Fernandez-Lafuente
Received: 4 December 2014 / Revised: 16 February 2015 / Accepted: 17 February 2015 / Published: 24 February 2015
(This article belongs to the Section Molecular Diversity)
View Full-Text   |   Download PDF [947 KB, uploaded 24 February 2015]   |  

Abstract

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. View Full-Text
Keywords: enzyme purification; enzyme immobilization; Brassica oleracea chlorophyllase 1 (BoCLH1); DIAION®CR11 enzyme purification; enzyme immobilization; Brassica oleracea chlorophyllase 1 (BoCLH1); DIAION®CR11
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MDPI and ACS Style

Chou, Y.-L.; Ko, C.-Y.; Chen, L.-F.O.; Yen, C.-C.; Shaw, J.-F. 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 2015, 20, 3744-3757.

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