Team:ULaval/Proof Of Concept

Proof of Concept
The final goal of this project is to produce a dextranase that can be directly added into the ropy maple syrup and degrade dextrans. For this to work, the dextranase must be stable in the conditions of the maple syrup, as its pH, and decrease the viscosity of the ropy maple syrup. The dextranase is expressed and purified before performing enzymatic assays to determine the conditions in which its activity and stability are optimal. Those tests also gave information about how the dextranase behaves in different conditions (pH & temperature). With those promising enzymology Results, we moved forward to the proof of concept: a small scale treatment directly in ropy maple syrup. Then, to add more quantitative data to support this proof of concept, we also did rheological assays to quantify the changes in the treated ropy syrup’s viscosity.

Small Scale Treatement

The first experiment was done to make sure that our dextranase is working in the conditions of the ropy maple syrup. A reaction composed of 50μL of purified G. algens’ dextranase in 500μL(36μM) of ropy maple syrup at room temperature reacted on an agitating plate at 200 rpm for 5 min. In parallel, a control was done by adding 50μL of water in the ropy maple syrup to verify if the dilution factor had an important effect on viscosity.

In the industry, ropy maple syrup is identified by the formation of a 10 cm filament when a spatula is dipped in the barrel of syrup and then elevated until the filament breaks. We used the same kind of protocol to test the ropy condition of the maple syrup. Since we are doing the experiment on a way smaller scale, we used a toothpick and evaluated the viscosity with a filament of approximately 2 cm.

We observe the efficiency of our enzymatic treatment in the video below. Indeed, the 2 cm filament is what we saw on average with our negative control (on the left). The second sample of 500μL of the same ropy maple syrup was treated for 5 min with our enzyme. We observed no more filament when the toothpick test was performed with the treated sample. This result proves that the dextranase degrades dextran in ropy maple syrup making our proof of concept.

Ropy maple syrup + water
Ropy maple syrup + dextranase

Rheological assays

Even though the small sample test is quite impressive, we wanted to add quantitative data to strengthen the proof of concept. We used a rheometer to assess the viscosity of the maple syrup. This device applies shear stress on the sample and evaluates its behaviour on a time scale set by the user. We used a similar concentration of dextranase(35.5 μM) for those assays as for the one in the small scale test. Also, a negative control was performed with the enzyme buffer.

At 20 °C (top), the ropy syrup with buffer and dextranase have respectively a viscosity of 0.25 Pa s and 0.22 Pa s. The ropy syrup alone increases in viscosity with increasing fluctuations during the sampling time. At 10 °C (bottom), the ropy syrup with buffer and dextranase have respectively a viscosity of 0.47 Pa s and 0.42 Pa s.