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Cellulase   Pectinase   Lipase  

Cellulase

BBa_K2929003 bglA, BBa_K118022 cex, BBa_K5193002 cex_cenA


To increase the yield of our essential oils, we aim to break down plant cell walls to allow more substances to be extracted. Thus we used β-glucosidase (P1, bglA, BBa_K2929003 from Thermotoga maritima) to hydrolyse the cello-oligosaccharides and cellobiose inside the plant cell walls into glucose monomers [1]. Moreover, we have cex from Cellulomonas fimi, P6: BBa_K118022 and cex with cenA from Cellulomonas fimi, P7: BBa_K118022_BBa_K118023, composite part BBa_K5193002, a combination of thermostable exoglucanase [3] and thermostable endoglucanase [4]. By inserting the two genes into the MCS blocks (with two T7 promoters), the engineered bacteria can therefore produce two types of enzyme in huge amounts simultaneously, providing multiple catalytic domains and enhancing efficiency [5]. We used the two enzymes to break down cellulose in plant cell walls [1], therefore releasing a greater amount of essential oil.

To investigate the plant cell wall degradation efficiency from our engineered bacteria, we use the DNS (3,5-dinitrosalicylic acid) method. This method allows us to access the amount of reducing sugars liberated during hydrolysis [2].

After adding CMC (Carboxymethyl cellulose solution) bacteria culture, we first incubated the solution for 2 hours at room temperature, 50°C, and 90°C in order to let the reaction take place. However, we found out that the most significant impact on the result is when the incubation takes place at 50°C. (See Fig 1.) Therefore we chose to incubate the solution for 2 hours in 50°C. After adding some 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.

Figure 1. The measured optical density absorbance after incubating at different temperatures. TBS is buffer (control) and PET11a is bacteria with empty vector (control). P1 is bglA, P6 is cex, P7 is cex _cenA.

Figure 2. The optical density absorbance at 540 nm of solutions with bacterial culture containing cellulose after 2 hours of incubation at 50°C. TBS is a buffer (control) and PET11a is a bacteria with an empty vector (control). P1 is bglA, P6 is cex, P7 is cex_cenA.

The graph shows that the OD value of P7 exhibits the most significance, which means that it has the most impact, among the three, on the hydrolysis of the plant’s cell wall. Meanwhile, P1 stood at the second place, and P6 had the lowest OD value. All plasmids had a higher absorbency in comparison to our PET11a control.


DNS over time

In order to investigate our enzymes’ capacity in different incubation durations, we have conducted an overtime DNS assay. We prepared nine test tubes containing the same solution for different tests. 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 3.


Figure 3. The optical density absorbance of bacterial culture solutions containing cellulase after being incubated in different time durations.

As shown in the graph, as the incubation time increases, the absorbance of all solutions rises until 120 minutes, at which β-glucosidase (P1) met its peak. Thus we chose to incubate the solution for 120 for any further experiments.


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 (using FLAG tag antibody to trap our proteins).

Prior to performing the experiments, we added 0.5M IPTG for induction over 6 hours and 16 hours for the demonstration of the result. As seen in Figure 4 and 5, the results for all three cellulases across both induction times are similar at around 51 to 52 kDa. We, therefore, chose to utilize the 6 hour induction time for further experiments.

Figure 4. Images of SDS-PAGE, flag-tag antibody, dyed with coomassie blue, 6 hours IPTG induction of all proteins.
Figure 5. Western blot with flag-tag antibody of bglA (P1), cex (P6) and cex_cenA (P7) after 6 and 16 hours of incubation.

Yield test

To further validate our test results, we had done a yield test. Before the distillation process, we soaked 100g of dried lavender with our enzymes for different durations and temperatures. We first soaked the plant at room temperature for 30 minutes and measured the volume of lavender oil that is being extracted. We then soaked it at 50°C for 10 minutes and extracted the oil using distillation. The results are shown in figure 6. Moreover, to test our enzymes’ ability to improve the yield, we combined our enzymes into two groups, namely β-glucosidase (P1) with cex_cenA (P7) and therm_pelA (P3) with P7. Results are shown in Figure 6.

Figure 6. Comparison of the essential oil yield of samples treated with enzyme extracts between reacting at room temperature and at 50°C. PET11a is a bacteria with an empty vector (control). P1 is bglA, P3 is therm_pelA, P5 is pelA, P6 is cex, P7 is cex _cenA.

It is steadily evident that in room temperature, the yield of the combination of P3 and cex_cenA (P7) is presented to be the most significant, while the combination of P1 and P7 is comparatively lower. P1, P6 and P7 are seen to have a lower yield, with P7 being the highest by having slightly beyond 1.5 mL and P6 being the lowest, having slightly over 1 mL.

Moreover, after the reaction had occurred in 50C, the total volume of essential oil being extracted after reacting with the combination of P3 and P7 is shown to have the highest impact with more than 1.8 mL of essential oil, followed by the combination of P1 and P7, having 1.8 mL. In short, the combination of two enzyme extracts, P3 and P7 as well as P1 and P7, demonstrates significant improvement of oil yield. On the contrary, the volume of essential oil measured after reacting with P1, P6 and P7 respectively, is seen to have a lower yield. With P6 having the lowest yield of slightly lower than 1.2 mL; and P7 with around 1.7 mL, yielding the highest among the three plasmids.

In order to choose the best reacting temperature, we also compared the yield between reacting at 50°C and at room temperature. As shown, all of the data demonstrated that the yield of extraction after being reacted at 50°C is higher than that at room temperature.

GCMS results

We first incubated flowers (raw ingredient) with cellulase 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. At Retention Time RT = 10.90340476 min, we identified the peak of linalool; at RT = 13.70656667, we found the peak of linalyl acetate. Compared with the abundance of linalool and linalyl acetate in the negative control group, essential oil with water, we found that the abundance of these two compounds in all cellulase treated essential oil (P1, P7, P1+P7 and P3+P7) is higher (Fig. 8, 10, 12 and 14). We also found out that the abundance of the compounds in cellulase is higher than that of our positive control, essential oil with PET11a (Fig. 7, 9, 11 and 13). Moreover, essential oil treated with P1+P7 exhibits the highest abundance in increasing linalool and linalyl acetate concentration among the four cellulase enzymes, which means that essential oil treating with P1+P7 enzyme extracts will, comparatively, be more effective in increasing the two compound’s concentration.
Figure 7, 8, 9, 10, 11, 12, 13, 14. The TIC graph of different cellulase enzyme extracts, namely bglA(P1, blue), cex_cenA(P7, pink), P1+P7(red) and P3+P7(green), 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. Ramani G, Meera B, Vanitha C, Rajendhran J, Gunasekaran P. Molecular cloning and expression of thermostable glucose-tolerant β-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization. J Ind Microbiol Biotechnol. 2015 Apr;42(4):553-65. doi: 10.1007/s10295-014-1549-6. Epub 2015 Jan 28. PMID: 25626525.
  2. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030
  3. Saxena H, Hsu B, de Asis M, Zierke M, Sim L, Withers SG, Wakarchuk W. Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484. Biochem Cell Biol. 2018 Feb;96(1):68-76. doi: 10.1139/bcb-2017-0150. Epub 2017 Oct 5. PMID: 28982013. https://pubmed.ncbi.nlm.nih.gov/28982013/
  4. Chen, Y.-P., Hwang, I.-E., Lin, C.-J., Wang, H.-J., & Tseng, C.-P. (2012). Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli. Journal of Applied Microbiology, 112(3), 455–463. doi:10.1111/j.1365-2672.2012.05232.x
  5. Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PMID: 27702471.

Pectinase

BBa_K5193000 pelA_therm and BBa_K5193001 pelA

p3 - NSP4-themostable pelA

p5 - NSP4-pelA


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

Lipase

lip4 BBa_K5193003

Esters are responsible for many of the pleasant and characteristic scents found in essential oils. They contribute to the complex aroma profiles that make essential oils appealing for use in perfumery, aromatherapy, and other applications. Second, esters often have calming and soothing effects. This makes essential oils containing esters valuable in promoting relaxation and reducing stress. For example, lavender essential oil, which contains esters like linalyl acetate, is known for its calming properties.Third, esters can have certain therapeutic properties. Some esters have anti-inflammatory, analgesic (pain-relieving), or antibacterial effects. This makes them potentially useful in natural medicine and skincare products. Finally, esters can enhance the stability and longevity of essential oils.

In order to enhance the quality of our essential oil, we aimed to increase the amount of ester by using lipase (lip4) (BBa_K5193003) to catalyze acid and alcohol into ester [1]. See Figure 1 for the mechanism of lipase-catalyzed esterification.

Figure 1. Lipase-catalyzed synthesis of ester through direct esterification, alcoholysis or acidolysis. Source: Kuo, C.-H. et. al. 2020. [2]

Protein Detection with Coomassie Blue and Western Blot

To confirm whether our engineered bacteria successfully produced our desired protein, lip4, we used FLAG tag antibody to trap our proteins. We used Coomassie Blue and Western Blot to detect the protein. In figure 2 and 3, we can see that the size of lip4 is approximately 59 kDa.

Figure 2. Protein FLAG tag antibody binding experiment dyed with coomassie blue, induced with IPTG for 6 hours. Figure 3. Western Blot with flag tag antibody, induced with IPTG for 6 and 16h respectively.

GCMS results

To investigate the impact of our lipase, we sent our samples to Metware China for GMCS to assess the component difference of essential oil with or without Lipase treatment. In these, we allow 30mins Lipase crude enzyme / pET11a (crude enzyme) control incubation with dry lavender petals at room temperature, allowing the reaction to take place.

The total ion current (TIC) chromatogram depicts the relative abundance of detected compounds at different retention times. At Retention Time RT = 10.90340476 min, we identified the peak of linalool; at RT = 13.70656667, we found the peak of linalyl acetate. Compared with the abundance of linalool and linalyl acetate in the negative control group, essential oil with water, we found that the abundance of these two compounds in lip4 pretreated oil is higher (Fig. 4). We also found out that the abundance of the compounds in lip4 is higher than that of lip4 post, showing that adding lip4 enzyme extract before distillation may be more effective in increasing linalool and linalyl acetate concentration than after distillation (Fig. 5).

Figure 4 and 5. The TIC graph of lip4, green, versus water, black, and lip4, green, versus lip4 post, red. The two conspicuous peaks are linalool and linalyl acetate, at 10.9 and 13.7 min RT correspondingly.

We validated the change of chemical composition in the essential oils with and without post-treatment. We selected the top 10 Log2 fold changes components to compare.

Figure 6. The component difference analysis at two fold change of essential oil compared to pET11a (added Lip4 enzyme crude BEFORE steam distillation).

As can be seen from the graph, the content of 2-methyl, 3-phenylpropyl ester increased the most with a positive 3.97 Log2 fold change when compared to essential oil added with pET11a control crude enzyme.

Figure 7. Lip4 enzyme crude extract added to the freshly produced essential oil compared to pET11a (AFTER steam distillation).

In addition, we would like to investigate if adding lipase to essential oil would have additional quality improvement. Through GCMS experiment, we revealed that the content of 2-methyl, 3-phenylpropyl ester increased the most, with a positive 3.65 Log2 fold change when compared to the pET11a group. We can therefore conclude that it is more efficient to add our lip4 extract to the flowers and essential oil product to enhance ester content.


Flavor analysis

From the detected component of the oil, we analyzed the flavor differences of lip4 pre and post treated oil with water treated oil (control). Our radar chart with top ten annotated flavors of the differentiated substance shows that lip4 incubation enhanced the sweet flavor of the oil at 20 substances, whereas lip4 post treatment enhanced the green flavor the most at 21 substances.

Figure 8 and 9. The first graph: lip4 vs water treated lavender oil flavor differential analysis. The second graph: lip4 post vs water treated lavender oil flavor differential analysis.

Antibacterial effect

Ka Hong Wong from the University of Macau taught and guided our students to conduct experiments on the antibacterial effect of our lavender essential oil. Lavender essential oil has been proved to have antimicrobial properties, as essential oil alters the strain’s sensitivity to antibiotics by altering the permeability of the outer membrane of bacteria [3]. We added different concentrations of our pretreated essential oil to the bacterial culture and spread them on agar plates. Oil with lipase pretreatment demonstrates a significant antibacterial effect. As can be seen from figure 11, at the 12th hour, the relative OD600 (optical density at 600 nm) of bacterial culture added lip4 post lavender oil, at 2 μg per ml, is much lower than most samples. In short, lip4 post treatment improves the bacterial inhibition ability of lavender oil.

Figure 10. The relative OD600 value of bacterial culture on the plate with lavender oil treated with different enzymes. Control is the essential oil treated with PET11a empty vector enzyme extract.

We also spread bacterial culture on agar plates (no antibiotic added). Our lavender oil with lip4 postreatment shows strong inhibition to the bacteria (See Fig. 11a) . When compared to blank (nothing added) with diluted bacterial culture, only adding 1 μg per ml oil can significantly reduce the area covered by bacteria (leaving colonies). Similarly, in the original bacterial concentration, both 4 μg and 2 μg per ml of lavender oil show notable antibacterial ability. In addition, when compared to blank and PET11a control treated essential oil, lip4 post significantly reduced the number of colonies (See Fig. 11b).

Figure 11 a and b. (a) The plate with bacteria (diluted 10^5 times) and that with lip4 post-treated lavender oil (4 μg and 2 μg per ml added to original conc. and 1 ug per ml added to diluted culture). (b) The plate with bacteria culture (diluted 10^5 times), with 1 μg per ml PET11a control treated lavender oil, and 1 μg per ml lip4 post-treated lavender oil.

References:

  1. Tang SJ, Sun KH, Sun GH, Chang TY, Lee GC. Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli. Protein Expr Purif. 2000 Nov;20(2):308-13. doi: 10.1006/prep.2000.1304. PMID: 11049754.
  2. Kuo, C.-H.; Huang, C.-Y.; Lee, C.-L.; Kuo, W.-C.; Hsieh, S.-L.; Shieh, C.-J. Synthesis of DHA/EPA Ethyl Esters via Lipase-Catalyzed Acidolysis Using Novozym® 435: A Kinetic Study. Catalysts 2020, 10, 565. https://doi.org/10.3390/catal10050565
  3. Wińska K, Mączka W, Łyczko J, Grabarczyk M, Czubaszek A, Szumny A. Essential Oils as Antimicrobial Agents-Myth or Real Alternative? Molecules. 2019 Jun 5;24(11):2130. doi: 10.3390/molecules24112130. PMID: 31195752; PMCID: PMC6612361.