Difference between revisions of "Part:BBa K5193003"

Revision as of 06:02, 29 September 2024

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.

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

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.

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.

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).

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.

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.

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.

Additionally, we sent our lip4 post sample to WeiPu Shanghai for detailed GCMS analysis. 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 percentages, 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.

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 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.

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. 12a) . 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. 12b).

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