chlorophyll-a and divinyl-benzene

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.

Topics: Brassica; Carboxylic Ester Hydrolases; Catalysis; Chelating Agents; Chlorophyll; Chlorophyll A; Chlorophyllides; Cross-Linking Reagents; Enzymes, Immobilized; Escherichia coli; Half-Life; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Phytol; Polystyrenes; Recombinant Proteins; Vinyl Compounds

A simple, reliable, and gentle saponification method for the quantitative determination of carotenoids in green vegetables was developed. The method involves an extraction procedure with acetone and the selective removal of the chlorophylls and esterified fatty acids from the organic phase using a strongly basic resin (Ambersep 900 OH). Extracts from common green vegetables (beans, broccoli, green bell pepper, chive, lettuce, parsley, peas, and spinach) were analyzed by high-performance liquid chromatography (HPLC) for their content of major carotenoids before and after action of Ambersep 900 OH. The mean recovery percentages for most carotenoids [(all-E)-violaxanthin, (all-E)-lutein epoxide, (all-E)-lutein, neolutein A, and (all-E)-beta-carotene] after saponification of the vegetable extracts with Ambersep 900 OH were close to 100% (99-104%), while the mean recovery percentages of (9'Z)-neoxanthin increased to 119% and that of (all-E)-neoxanthin and neolutein B decreased to 90% and 72%, respectively.

Topics: Carotenoids; Chlorophyll; Chromatography, High Pressure Liquid; Esters; Fatty Acids; Styrenes; Vegetables; Vinyl Compounds