Difference between revisions of "Part:BBa K2929003"

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) to hydrolyse the cello-oligosaccharides and cellobiose inside the plant cell wall into glucose monomers.[1]

The activity of cellulase was measured by the DNS (3,5-dinitrosalicylic acid) method through the amount of reducing sugars liberated during hydrolysis.[2]

After adding CMC (Carboxymethyl cellulose solution) into our cell 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>

     <img src="" style="width: 600px;">

   <figcation>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.</figcation>
   

     <figure>

         <img src="" style="width: 600px;">

       <figcation>Figure 2. The optical density absorbance at 540 nm of 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.</figcation>
       <figcation>As seen in the graph, the OD value of P7 had the most significant amount of. Whereas the P1 stood in second place. And the P6 has the lowest OD value. But all promoters had a higher absorbency in comparison to our PET11a control.</figcation>
 

Yield test<h4>

To further validate our test results, we had done a yield test. 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 plant at room temperature for 30 minutes and measured the volume of lavender oil that is being extracted, the results are shown in figure 1. We then soaked it at 50°C for 10 minutes and extracted the oil using distillation. The results are shown in figure 2. 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. The results are shown in both Figure 1 and Figure 2.

 </p>
 <figure>

<img src="" style="width: 600px;">

   <figcation>Figure 1. The volume of essential oil being extracted after reacting with our enzymes in room temperature for 30 minutes. 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.</figcation>
   

It is steadily evident that 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.

   

   <figure>

<img src="" style="width: 600px;">

     <figcation>Figure 2. The volume of essential oil being extracted after reacting with our enzymes in 50°C for 30 minutes. 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.</figcation>
     

As seen in the chart, 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 in 50°C and in room temperature. The result is shown in figure 3.

   

   <figure>

<img src="" style="width: 600px;">

     <figcation>Figure 3. Comparison of the yield between reacting in room temperature and in 50°C. PET11a is bacteria with empty vector (control). 
       P1 is bglA, P3 is therm_pelA, P5 is pelA, P6 is cex, P7 is cex _cenA.</figcation>
   

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.

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. Islam F, Roy N. Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. Islam and Roy BMC Res Notes (2018) 11:445 https://doi.org/10.1186/s13104-018-3558-4

Usage and Biology

The Thermatoga maritima Beta-glucosidase A enzyme expresses well under the T7 promoter in a pET28a, even without IPTG induction, due to a leaky promoter. The protein is mostly soluble based on small scale expression tests and runs at ~55 kDa on an SDS-Page gel.

Figure 1. Soluble expression for the wild-type protein, the pET28a vector was known to have a leaky promoter so the bands at ~55kDa (expected mass 52kDa) indicate expression, as they are absent in lanes used for mOrange and Antibody expression (lanes 2-10).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 998
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 927
  Illegal SapI site found at 379