Our team’s goal was to create a new method for chlorophyll removal from green seed canola oil. After months of hard work and experimentation, our team was able to demonstrate the following:

1. Our water soluble chlorophyll-binding protein, 6GIX, can be produced and purified
2. 6GIX can be emulsified and can be used to remove chlorophyll from canola oil

Using an IPTG-inducible system, our team was able to successfully produce and purify 6GIX in E.coli BL21 (DE3). An SDS-PAGE gel with whole cell lysate and purified elutions of 6GIX without a signal peptide showed strong bands of the correct size (21kDa) in the whole cell lysate and elution fraction 2. No bands were seen in the corresponding empty vector control lanes.

Figure 1. SDS-PAGE gel showing whole cell lysate and elutions after purification of no signal peptide 6GIX and empty vector control. Arrow denotes correct band length for 6GIX protein.

To make sure that the bands seen on the SDS-PAGE are of the correct protein, we ran a western blot using Anti-His MAb (from mouse) primary antibody and Anti-mouse IgG conjugated with HRP secondary antibody. This western blot confirmed the identity of our protein at the correct band length.

Figure 2. Western blot showing whole cell lysate and elutions after purification of no signal peptide 6GIX and empty vector control. Arrow denotes correct band length for 6GIX protein.

Using our phase diagram models, our team identified five candidates for optimal emulsion conditions, all intended to produce a Winsor 1 emulsion.

Figure 3: Absorbance at 670nm (correlated to chlorophyll concentration) of oil after emulsification at increasing volume fractions of surfactant

Of these five conditions, the emulsion system with a surfactant volume of 10% (C9 emulsion) was shown to be the most effective in capturing chlorophyll. This is ideal in industrial conditions as a larger pure oil phase allows for a higher yield of oil. It also minimizes downstream processing steps, making it more economically efficient.

Using C9 emulsion conditions, an emulsion test was performed comparing the effectiveness of BSA, 6GIX, and a buffer control.

Figure 4: Emulsions tested in lab. BSA and 6GIX were emulsified at the same concentration.

The above figure shows that the aqueous microemulsion resulting from the emulsification of 6GIX is significantly more green than the BSA or buffer controls. This shows that 6GIX functions in emulsion to succesfully bind and remove chlorophyll from green seed canola oil.