Chlorophyllase biocatalysis in an aqueous/miscible organic solvent medium containing canola oil

Abstract

Chlorophyllase catalyzes the bioconversion of chlorophyll into chlorophyllide by replacing the phytol group with a hydrogen atom. There is an increased interest in the biotechnological application of chlorophyllase for the removal of green pigments from edible oil and its potential as an alternative to the use of the conventional bleaching technique. Partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity in an aqueous/miscible organic solvent system containing refinedbleached-deodorized (RBD) canola oil, using chlorophyll and pheophytin as substrate models. The results indicated that chlorophyllase biocatalysis could be successfully carried out in an aqueous/miscible organic system containing RBD canola oil. The presence of 20% RBD canola oil decreased the hydrolytic activity of chlorophyllase by 2.2 and 6.7 times, using chlorophyll and pheophytin as substrates, respectively. In addition, acetone acted as an activator of chlorophyllase activity at low concentrations and an inhibitor at higher ones. The optimal reaction conditions for chlorophyllase biocatalysis in the aqueous/miscible organic system were determined to consist of 20% RBD oil and 10% acetone at a 200 rpm agitation speed and at a temperature and substrate concentration of 35°C and 12.6 µM for chlorophyll, and 30°C and 9.3 µM for pheophytin.Papers no. J10850 in JAOCS 81, 927-932 (October 2004).

Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media

“…3a) show that the maximal activity of chlorophyllase from P. tricornutum was obtained at reaction temperature of 35°C. Similar optimal temperature was also reported for the same enzyme in the aqueous/miscible organic solvent [5] as well as in the ternary micellar system [6,13]. This optimal temperature of 35°C falls within the range of 30 to 40°C reported in literature for chlorophyllase from other plant [7].…”

“…3b), an activation energy (E a ) of 105.0 kJ/mol was obtained for the chlorophyllase-catalyzed hydrolysis of chlorophyll in the neat organic solvent mixture. This E a is higher than that obtained for the same reaction (66.9 kJ/mol), but in the aqueous/miscible organic solvent containing 10% acetone [5]. The increase in E a in the neat organic solvent mixture may be due to the use of a suspended solid enzyme.…”

“…However, the decrease in chlorophyllase activity, obtained at temperature above 35°C (Fig. 3a), was much lower than that reported for the same enzyme in the aqueous/ miscible organic solvent mixture [5] and the ternary micellar system [6]. This limited decrease in chlorophyllase activity in the neat organic solvent mixture may be due to a better enzyme stability as compared to that in other reaction media.…”

“…The use of chlorophyllase in the removal of green pigments from edible oils could be of great interest [3]. The development of a biotechnological process involving the use of chlorophyllase for the bioconversion of the hydrophobic chlorophylls and their derivatives into their hydrophilic chromophore moieties was reported previously by our group [4,5].…”