Part:BBa_K5193000

thermostable pelA (thermotoga maritima)

This is a thermostable pectinase (pelA) and we used it with NSP4 tag to facilitate excretion of the protein. This enzyme can hydrolyze pectin in high temperature (optimal 50-60C).

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.

It is evident that in room temperature, the combination of P3 and P7 yields the most amount of EO compared to all of the enzymes and the combination of P1+P7. At 50°C, the same trend is observed across the enzymes and enzyme groups, with the total volume of EO extracted with the combination of P3 and P7 having shown to have the highest yield at more than 1.8 mL.

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 (control).

GCMS results

We first incubated flowers (raw ingredient) with pectinase crude enzyme at 50C for 10 minutes, allowing the reaction to take place. We sent out the final oil product to Metware China and WeiPu Shanghai for Gas Chromatography–Mass Spectrometry (GC-MS, equipment: Agilent 8890-7000D) analysis.

The total ion current (TIC) chromatogram delineates the relative abundance of detected compounds at different retention times (RT). At RT = 10.90340476 min, we identified the peak of linalool, a naturally occurring terpene alcohol found in many flowers; at RT = 13.70656667, we found the peak of linalyl acetate, a principal component of the EOs from lavender. Compared with the abundance of linalool and linalyl acetate in the negative control group, EOs extracted with only steam distillation, we found that the abundance of these two compounds in all pectinase treated essential oil (P3, pelA_therm and P3+P7) is higher (Fig.8 and 9). We also found out that the abundance of the compounds in pectinase is higher than that of our positive control, EOs extracted with PET11a (Fig. 8 and 10). Moreover, essential oil treated with P3 exhibits the highest abundance in increasing linalool and linalyl acetate concentration among the two pectinase enzymes, which means that essential oil treating with P3 enzyme extracts will, comparatively, be more effective in increasing the two compound’s concentration.

Figure 8,9,10,11. The TIC graph of different pectinase EO extracts, namely pelA_therm (P3, purple), pelA_therm+cenA (P7, pink), versus water and PET11a (black). The two conspicuous peaks are linalool and linalyl acetate, at 10.9 and 13.7 min RT correspondingly.

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