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.

Experiment details: New Parts

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. 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. (from top to bottom) TIC graph of lavender oil treated with lip4 (after extraction), lip4, and water.

The total ion current (TIC) chromatogram shows 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. Generally, the TIC pattern of lip4 post-treatment, lip4, and water are similar, with conspicuous peaks at 11 and 13.7 minutes.

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]. Oil with lipase pretreatment demonstrates a significant antibacterial effect. As can be seen from figure 5 a and b (violet bar), the OD600 (optical density at 600 nm) of culture at 1 ug/mL of lip4 lavender oil is much lower than that of lavender oil (incubated with water). Overall, both lavender oil samples demonstrate excellent antibacterial effects at concentrations higher than 2 ug/mL.

Figure 5a, 5b. (a) The OD600 value of bacterial culture with lavender oil treated with water at different concentrations across 12 hours. (b) The OD600 value of bacterial culture with lavender oil treated with lip4.

We also spread bacterial culture on agar plates (no antibiotic added). Our lavender oil shows strong inhibition to the bacteria. When compared to blank (added nothing) with diluted bacterial culture, only adding 1 ug can significantly reduce the area covered by bacteria (leaving colonies). Similarly, in original concentration, both 4 ug and 2 ug of lavender oil show notable antibacterial ability.

Figure 6. The plate with bacteria (diluted 10^5 times) and that with lip4 treated lavender oil (4 ug and 2 ug added to original conc. and 1 ug added to diluted culture).

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]