Part:BBa_K5193001

pelA (Pectrobacterium astrospecticum)

This is a pectinase from Pectrobacterium astrospecticum with no thermostable ability. In the part design, we added a NSP4 tag before the sequence to enhance secretion of the protein. We used it to hydrolyze pectin.

As EO extraction is often completed with distillation at high temperatures, specific enzymes were selected for their thermostable capabilities. Beside utilizing cellulases to break down cell walls and increase the yield of essential oils (EOs). We also applied a similar method with NSP4-themostable pelA (from Thermotoga maritima BBa_K5193000) [1] and NSP4-pelA (from Pectobacterium astrospecticum BBa_K5193001) to hydrolyse pectin, a component of the middle lamella and the primary cell wall.

The activity of pectinase was measured by the DNS (3,5-dinitrosalicylic acid) method through the amount of reducing sugars produced during hydrolysis of the polysaccharide. [2]

After adding 1% pectin solution to our bacteria culture, we first incubated the solution for 2 hours at room temperature, 50°C and 90°C in order to allow for a complete reaction between the pectinase and its substrate. However, we found that similar to cellulase, the most significant impact on the absorbance reading is when the incubation takes place at 50°C. (See Fig 1.) Therefore we chose to incubate the solution for 2 hours at 50°C subsequently. After adding DNS reagent to the solution, we incubated the solution again for another 10 minutes at 50°C to stop the reaction. We then added our solution into a 96-well transparent plate for OD measurement at 540 nm. The results are shown below. (See Fig 2.)

Figure 1. The measured optical density absorbance after incubating at different temperatures for 2 hours. TBS buffer (control); PET11a empty vector (control); P3 is therm_pelA; P5 is pelA.

Figure 2. The optical density absorbance at 540 nm of pectinase after 2 hours of incubation at 50°C (bacteria culture). TBS buffer (control); PET11a empty vector (control).

As seen in Fig. 2, the OD value of P5 is the highest, whereas P3 is second. But both enzymes yield a higher absorbency in the well in comparison to our PET11a control and TBS buffer control.

Figure 3. The optical density absorbance at 540 nm of pectinase after 2 hours of incubation at 50°C (crude enzyme). TBS buffer (control); PET11a empty vector (control).

We further conducted the same test with the crude enzyme extract of pectinase. P3 yielded the highest absorbance, with both enzymes being higher than both controls.

Protein Detection Using Coomassie Blue and Western Blot

To validate the expression of our desired protein, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis (with flag tag antibody).

For P3 and P5, we performed the same experiment as for the cellulases. A similar tendency is observed as above with both 6 hours and 16 hours of IPTG induction for P3 and P5 showing similar results as well, with P5 at 104 kDA and P3 at 43 kDa.

Figure 4. Protein FLAG tag antibody binding experiment dyed with coomassie blue, induced with IPTG for 6 hours.

Figure 5. Western blot with flag-tag antibody, induced with IPTG for 6 and 16h respectively.

DNS over time

In order to investigate our enzymes’ capability in different incubation durations, we conducted a test by preparing nine test tubes containing the same solution and incubating them for various periods of time. First, we tested the OD value of the solution without any incubation. We then incubated the rest of the prepared solutions for 5, 10, 15, 30, 45, 60, 90, 120 and 150 minutes respectively. The results are shown in Figure 6.

Figure 6. The absorbance of the solutions after incubation across 0 to 150 minutes.

As shown in Fig 6, as the incubation time increases, the absorbance of all solutions has a general increasing trend.

Yield test

To further validate our test results, we completed a yield test with the use of our enzymes. Before the distillation process, we soaked the same amount of dried lavender with our enzymes for different time durations and temperatures. We first soaked the flowers at room temperature for 30 minutes in a TBS buffer and our enzymes, after that, we measured the volume of lavender oil that was extracted by distillation, the results are shown in Figure 5. We further tested the thermostability of our enzymes in a yield test by soaking the plants at 50°C in the same solution for 10 minutes and extracted EO using distillation. The results are shown in Figure 5. Moreover, to test our enzymes’ ability to improve yield, we combined our enzymes into groups, such as P3 with P7. The results are shown below.

Figure 7. Comparison of the yield between reacting in room temperature and in 50°C. PET11a empty vector (control). P1 is bglA; P3 is therm_pelA; P5 is pelA; P6 is cex; P7 is cex _cenA.

For P5, the amount of oil extracted with the incubation in room temperature is similar to that of P3 and the PET11a control. However, P3 has a notable higher yield with incubation at 50°C than P5.

In short, the combination of two enzyme extracts, P3 and P7 demonstrates visible improvement of EO yield. In contrast, the individual tests of P3 and P7 had shown a much lower volume of EO.

In order to choose the best reacting temperature, we also compared the yield between reacting in 50°C and in room temperature. As shown, most of the data demonstrated that the yield of extraction after being reacted at 50°C is higher than that of room temperature, with the exception of water (negative control).

References:

1. Berensmeier, S., Singh, S.A., Meens, J. et al. Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase. Appl Microbiol Biotechnol 64, 560–567 (2004). https://doi.org/10.1007/s00253-003-1446-9
2. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030