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.
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.
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan. qbowchou@gmail.com.|Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan. qbowchou@gmail.com.
authors.forename: Yi-Li
authors.initials: YL
authors.lastname: Chou
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan. g874222@gate.sinica.edu.tw.
authors.forename: Chia-Yun
authors.initials: CY
authors.lastname: Ko
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan. ochenlf@gate.sinica.edu.tw.
authors.forename: Long-Fang O
authors.initials: LF
authors.lastname: Chen
authors.affiliation: Department of Biological Science and Technology, I-Shou University, Kaohsiung 82445, Taiwan. rover4rover4@isu.edu.tw.
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Yen
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan. shawjf@isu.edu.tw.|Department of Biological Science and Technology, I-Shou University, Kaohsiung 82445, Taiwan. shawjf@isu.edu.tw.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan. shawjf@isu.edu.tw.
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D001937:Brassica / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D002734:Chlorophyll / Q000378:metabolism*; D000077194:Chlorophyll A; D002735:Chlorophyllides / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010836:Phytol / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014753:Vinyl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D002735:Chlorophyllides; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011137:Polystyrenes; D011994:Recombinant Proteins; D014753:Vinyl Compounds; D002734:Chlorophyll; D010836:Phytol; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase; C004985:divinyl benzene; D000077194:Chlorophyll A