Part:BBa_K5193002

exoglucanase and endoglucanase

The following are Team PuiChing_Macau 2024 contributions.

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, BBa_K2929003 from Thermotoga maritima) to hydrolyse the cello-oligosaccharides and cellobiose inside the plant cell walls into glucose monomers [1]. Moreover, we have cex from Cellulomonas fimi, P6: BBa_K118022 and cex with cenA from Cellulomonas fimi, P7: BBa_K118022_BBa_K118023, composite part BBa_K5193002, a combination of thermostable exoglucanase [3] and thermostable endoglucanase [4]. By inserting the two genes into the MCS blocks (with two T7 promoters), the engineered bacteria can therefore produce two types of enzyme in huge amounts simultaneously, providing multiple catalytic domains and enhancing efficiency [5]. We used the two enzymes to break down cellulose in plant cell walls [1], therefore releasing a greater amount of essential oil.

To investigate the plant cell wall degradation efficiency from our engineered bacteria, we use the DNS (3,5-dinitrosalicylic acid) method. This method allows us to access the amount of reducing sugars liberated during hydrolysis [2].

After adding CMC (Carboxymethyl cellulose solution) bacteria 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 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.

Figure 2. The optical density absorbance at 540 nm of solutions with bacterial culture containing 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.

The graph shows that the OD value of P7 exhibits the most significance, which means that it has the most impact, among the three, on the hydrolysis of the plant’s cell wall. Meanwhile, P1 stood at the second place, and P6 had the lowest OD value. All plasmids had a higher absorbency in comparison to our PET11a control.

DNS over time

In order to investigate our enzymes’ capacity in different incubation durations, we have conducted an overtime DNS assay. We prepared nine test tubes containing the same solution for different tests. First, we tested the OD value of the solution without any incubation. We then incubated the rest of the prepared solutions for 5, 10, 15, 30, 45, 60, 90, 120, and 150 minutes respectively. The results are shown in Figure 3.

Figure 3. The optical density absorbance of bacterial culture solutions containing cellulase after being incubated in different time durations.

As shown in the graph, as the incubation time increases, the absorbance of all solutions rises until 120 minutes, at which β-glucosidase (P1) met its peak. Thus we chose to incubate the solution for 120 for any further experiments.

Protein Detection Using Coomassie Blue and Western Blot

To validate the expression of our desired protein, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis (using FLAG tag antibody to trap our proteins).

Prior to performing the experiments, we added 0.5M IPTG for induction over 6 hours and 16 hours for the demonstration of the result. As seen in Figure 4 and 5, the results for all three cellulases across both induction times are similar at around 51 to 52 kDa. We, therefore, chose to utilize the 6 hour induction time for further experiments.

Figure 4. Images of SDS-PAGE, flag-tag antibody, dyed with coomassie blue, 6 hours IPTG induction of all proteins.

Figure 5. Western blot with flag-tag antibody of bglA (P1), cex (P6) and cex_cenA (P7) after 6 and 16 hours of incubation.

Yield test

To further validate our test results, we had done a yield test. Before the distillation process, we soaked 100g of dried lavender with our enzymes for different 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. We then soaked it at 50°C for 10 minutes and extracted the oil using distillation. The results are shown in figure 6. 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. Results are shown in Figure 6.

Figure 6. Comparison of the essential oil yield of samples treated with enzyme extracts between reacting at room temperature and at 50°C. 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.

It is steadily evident that in room temperature, 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.

Moreover, after the reaction had occurred in 50C, 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 at 50°C and at room temperature. 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.

GCMS results

We first incubated flowers (raw ingredient) with cellulase crude enzyme at 50C for 10 minutes, allowing the reaction to take place. We sent out the final oil product to Metware China and WeiPu Shanghai for Gas Chromatography–Mass Spectrometry (GC-MS, equipment: Agilent 8890-7000D) analysis.

The total ion current (TIC) chromatogram delineates 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 all cellulase treated essential oil (P1, P7, P1+P7 and P3+P7) is higher (Fig. 8, 10, 12 and 14). We also found out that the abundance of the compounds in cellulase is higher than that of our positive control, essential oil with PET11a (Fig. 7, 9, 11 and 13). Moreover, essential oil treated with P1+P7 exhibits the highest abundance in increasing linalool and linalyl acetate concentration among the four cellulase enzymes, which means that essential oil treating with P1+P7 enzyme extracts will, comparatively, be more effective in increasing the two compound’s concentration.

Figure 7, 8, 9, 10, 11, 12, 13, 14. The TIC graph of different cellulase enzyme extracts, namely bglA(P1, blue), cex_cenA(P7, pink), P1+P7(red) and P3+P7(green), versus water and PET11a (black). The two conspicuous peaks are linalool and linalyl acetate, at 10.9 and 13.7 min RT correspondingly.

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. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030
3. Saxena H, Hsu B, de Asis M, Zierke M, Sim L, Withers SG, Wakarchuk W. Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484. Biochem Cell Biol. 2018 Feb;96(1):68-76. doi: 10.1139/bcb-2017-0150. Epub 2017 Oct 5. PMID: 28982013. https://pubmed.ncbi.nlm.nih.gov/28982013/
4. Chen, Y.-P., Hwang, I.-E., Lin, C.-J., Wang, H.-J., & Tseng, C.-P. (2012). Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli. Journal of Applied Microbiology, 112(3), 455–463. doi:10.1111/j.1365-2672.2012.05232.x
5. Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PMID: 27702471.