Pigment removal from canola oil using chlorophyllase

Abstract

Frost‐damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 µg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water‐soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone‐dried chloroplasts) from 15 to 20‐day‐old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X‐100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg2+ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15µM or 13.5 µg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr.With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (Ki=11 µM or 3.3 µg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity.Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).

Properties of Chlorophyllase from Capsicum annuum L. Fruits

“…Activity was m easured in the tem perature range 3 0 -7 0 °C, and exhibited a maximum at 50 °C. These results were in agreem ent with those found in Olea europaea (Mfnguez-Mosquera et al, 1994) and Canola (Levadoux et al, 1987). D enaturation kinetics were determ ined at four tem peratures (40, 50, 60 and 70 °C).…”

The Role of Pheophorbide a Oxygenase Expression and Activity in the Canola Green Seed Problem

“…Canola is the world's third-most important vegetable oil crop, in significant part due to the low levels of erucic acid and glucosinolates in canola oil (Levadoux et al, 1987;Zhang et al, 2004). However, the chlorophyll (Chl) content is significantly higher than that found in other major vegetable oils and is the biggest quality impediment in the canola oil industry.…”

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

“…The decrease in the catalytic efficiency of enzymes in organic solvent media may be attributed to an increase in the rigidity of the enzyme structure in this environment containing a restricted amount of water as well as to a substrate partitioning effect [4,16]. In addition, because the by-product phytol is hydrophobic, its inhibitory effect on chlorophyllase activity may have increased in the neat organic solvent mixture, affecting hence the catalytic efficiency of the enzyme [3,11,12].…”

“…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].…”