Trying to optimize our production of essential oils and increasing their yield, we used a new plasmid named pelA (P5, BBa_K5193001), which is a bio-enzyme that is effective in the production of essential oils.

In addition, we also wanted to strengthen cell wall decomposition during the distillation process, so we added a temperature stabilizing element. Therefore, we have another plasmid, thermostable pelA (P3, therm_pelA, BBa_K5193000).

Temperature

1.1 Design

We conducted a series of tests to investigate the activity of enzymes at different incubation temperatures. Sixteen test tubes were prepared, each containing the same solution for different tests.

1.2 Build

We have prepared two different enzymes, namely thermostable pelA (P3, therm_pelA, BBa_K5193000) and pelA (P5, BBa_K5193001) to investigate our enzymes’ activity, which therm_pelA is a thermostable enzyme. We incubated them at different temperatures hoping to find out whether those thermostable enzymes are able to work in high temperatures.

1.3 Test

To test our enzyme’s thermostability, we designed a test. Prior to any further experiments, we tested a solution without any incubation, then we incubated another solution at room temperature, 50°C, and 90°C respectively in order to find out the result.

Initially, we added the enzyme extracts (50μL) and the substrate (50μL) to each of the four test tubes and labeled them correspondingly.

For the one without incubation, we added the DNS reagent (200μL) directly into the tube after adding our enzyme extract, while the others were incubated at room temperature, 50°C and 90°C for 2.5 hours respectively in order to react. After incubating the solutions, DNS reagent (200μL) was added for another incubation at 92°C for 10 minutes.

Finally, the solutions were then transferred into 96-well plates to measure optical density (absorbance at 540 nm) with a plate reader. The results are shown in Figure 1.

1.4 Learn

After conducting the tests, it is evident that incubation under 50°C exhibited the most significant result, displaying the highest OD value compared to all other conditions. This indicates that incubation at 50°C is the most suitable temperature, regardless of whether it is for cellulase or pectinase.

Based on these findings, we have decided to use 50°C as the incubation temperature for any other subsequent tests.

Bacterial culture vs enzyme extract

2.1 Design

In our preliminary tests, we also tested the effect of the enzyme in bacterial culture. We hypothesized that thermostable enzyme P3 will demonstrate a better enzymatic activity at 50°C.

2.2 Build

To confirm that our hypothesis was correct, we set up a test merely testing for the bacterial culture to see whether the thermostable enzyme P3(thermostable pelA, therm_pelA, BBa_K5193000) would have a higher value of optical density absorbance than non-thermo enzyme P5 (pelA, BBa_K5193001), which means the amount of reduced sugars liberated during hydrolysis on pectinase.

2.3 Test

Initially, we centrifuged 1mL of the cell culture, which was stored in a -80 fridge or induced for 6 hours with IPTG. After taking away 945 μL of the supernatant and resuspending, we added 50μL of the pectin and 50μL of bacterial culture into the test tubes and incubated the mixture at 50°C incubation for 2 hours, we then added 1:1 DNS reagent (DNS: reacting mixture) to the solutions and incubated the final solution at 92°C for 10 minutes.

2.4 Learn

This showed that in the form of bacterial culture, thermostable pelA (P3, therm_pelA) exhibited a relatively lower ability to undergo hydrolysis at 50°C compared to that of pelA (P5), a non-thermostable bacterial culture.

3.1 Design

As our preliminary result using bacterial culture directly in DNS assay contrasts with our hypothesis that thermostable enzyme P3 should be able to hydrolyze more pectin at 50°C than P5, we decided to test whether a sonified and concentrated enzyme extract will support our hypothesis. Additionally, we tried to find the best method to use these enzymes for our later yield experiment in a steam distillation setting.

3.2 Build

In order to prove that the thermo_pelA will still function in 50°C incubation temperature, we conducted a test comparing whether concentrated enzymes or bacteria would have a greater ability to reduce sugars liberated during hydrolysis on pectinase.

3.3 Test

We first centrifuged for ten minutes, followed by using a sonication to break the cell wall, and a millipore protein filter to concentrate our enzyme. This concentrated enzyme extract can be stored in a 4C fridge for later usage (within 7 days). We added this solution with 1% pectin in 1:1 ratio. 50μL of the pectin and 50μL of enzyme or bacterial culture were added into the test tubes. We then incubated the mixture at 50°C incubation for 2.5 hours (enzymes), added DNS reagent 1:1 (DNS: reacting mixture) and incubated this final solution at 92°C for 10 minutes.

3.4 Learn

As shown in Fig 1&2, it’s clear that the optical density absorbance of thermostable pelA enzyme extracts contains a greater value than that of pelA. Thus we chose to do further experiments with enzymes for any pectinase tests.