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

• Published in: Molecules (Basel, Switzerland) Switzerland), 2015-01, Vol.20 (3), p.3744-3757
• Main Authors: Chou, Yi-Li, Ko, Chia-Yun, Chen, Long-Fang O, Yen, Chih-Chung, Shaw, Jei-Fu
• Format: Article
• Language: English
• Subjects: Brassica oleracea; Escherichia coli
• ISSN: 1420-3049
• DOI: 10.3390/molecules20033744

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 registered 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 plus or minus 0.43 mg/g gel) and enzyme activity (46.3 plus or minus 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 degree C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION registered 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.