Part:BBa_K5193003

lip4 (Candida rugosa)

This is a type of lipase used to esterify alcohol and acid into ester. In order to enhance the scent of our essential oil, we aimed to increase the amount of ester by using lipase (lip4) to catalyze acid and alcohol into ester [1]. See figure 1 for the mechanism of lipase-catalyzed esterification.

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) 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 of all proteins, induced with IPTG for 6h.
Figure 3. Western Blot induced with IPTG for 6h 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.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
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• 21
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  Illegal BglII site found at 815
  
• 23
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  Illegal NgoMIV site found at 841
  
• 1000
  COMPATIBLE WITH RFC[1000]