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 graph 1 for the mechanism of lipase-catalyzed esterification.

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 graph 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]

GCMS results

We first incubate flowers (raw ingredient) with lip4 crude enzyme at room temperature for half an hour, allowing the reaction to take place. Additionally, we tried incubating our essential oil extract (distilled) with enzyme extract of lip4 for 2.5 hours in a 37C 200 rpm shaker. For both groups, we centrifuged the mixture at 4956 x g for 10 minutes before taking the upper oil layer (desired product) to a new tube. The final esterified oil product went through Gas Chromatography–Mass Spectrometry (GC-MS, equipment: Agilent 8890-7000D) to validate the change of chemical composition in essential oil with and without post-treatment. We selected the top 10 Log2 fold changes components to compare.

Figure 2. 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 3-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 3. Lip4 enzyme crude extract added to the freshly produced essential oil compared to pET11a (AFTER steam distillation).

The content of 3-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 might be more efficient to add our lip4 extract to the flowers before distillation to enhance ester content.

Figure 4. The TIC graph of lavender oil treated with lip4 after extraction.

The total ion current (TIC) chromatogram depicts the relative abundance of detected compounds at different retention times. By assigning peaks to different compounds with retention time, we can identify the amount of different compounds in the lavender oil sample. Linalool and linalyl acetate are the two major components of lavender essential oil, with 39.529% and 22.2% relative percentage, respectively.

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 5 and 6. 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 caused 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 7, at the 12th hour, the relative OD600 (optical density at 600 nm) of bacterial culture added lip4 post lavender oil, at 2 ug per ml, is much lower than most samples. In short, lip4 post treatment improves the bacterial inhibition ability of lavender oil.

Figure 7. 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. 8a) . When compared to blank (nothing added) with diluted bacterial culture, only adding 1 ug per ml oil can significantly reduce the area covered by bacteria (leaving colonies). Similarly, in the original bacterial concentration, both 4 ug and 2 ug 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. 8b).

Figure 8 a and b. (a) The plate with bacteria (diluted 10^5 times) and that with lip4 post-treated lavender oil (4 ug and 2 ug 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 1ug PET11a control treated lavender oil, and 1ug 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 815
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 841
  
• 1000
  COMPATIBLE WITH RFC[1000]