Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

Abstract

Chlorophyllase converts chlorophyll and pheophytin into their colorless derivatives (chlorophyllide/ pheophorbide and phytol). This activity can be used in chlorophyll removal from vegetable oils. Chlorophyllase genes from Oscillatoria acuminata (OscChlase) and Citrus aurantium (CitChlase) were isolated, cloned, and expressed in E. coli. Bioinformatics analysis showed that both chlorophyllases shared a conserved GHSXG lipase motif responsible for their catalytic activity. SDS-PAGE and immunoblot assays revealed that both enzymes had a molecular weight of 35 kDa. The purified chlorophyllases were stable at a broad range of temperatures and showed the highest activity at 40 C. OscChlase and CitChlase exhibited the highest activity at pH 6.0 and 7.0, respectively. Enzyme kinetics analysis revealed that OscChlase was able to hydrolyze bacteriochlorophyll-a more efficiently than the recombinant CitChlase (Vmax/Km of 0.38 for OscChlase vs. 0.01 min −1 mg protein for CitChlase). Instead, CitChlase hydrolyzed chlorophyll-b more efficiently than OscChlase. Both enzymes were able to reduce the chlorophyll content of olive (from 623.1 to as low as 87.2 mg per kg oil) and canola oil (from 537.2 to as low as 101.1 mg per kg oil). The ratio of oil to the aqueous reaction media affected chlorophyll hydrolysis (P < 0.05). The lower the oil ratio was (10%), the higher the chlorophyll removal was (75-86%). The efficiency of CitChlase in chlorophyll removal was higher than that of OscChlase at oil ratios of 10 and 20, but lower at 30% ratio (P < 0.05). This is the first report on the application of recombinant OscChlase and CitChlase in chlorophyll removal (up to 86%) from vegetable oils.