Difference between revisions of "Part:BBa K5117038"

Revision as of 14:11, 1 October 2024

PcotYZ-BsRBS-BhBglA-L1-CotY-B0014

This part serves as transcriptional unit composed of:

• promoter PcotYZ of Bacillus subtilis (BBa_K5117021),
• ribosome binding site of Bacillus subtilis (BBa_K5117000),
• bglA gene of Bacillus halodurans encoding a beta-glucosidase (EC 3.2.1.21),
addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA (BBa_K5117026),
• cotY gene of Bacillus subtilis (BBa_K5117022),
• bidirectional terminator B0014 (BBa_B0014).

Sequence and Features

Spore preparation

Spores were prepared by culturing cells in LB medium with chloramphenicol until reaching the exponential growth phase (OD₆₀₀ of 0.4 – 0.6). After washing and resuspension in DSM, the culture was incubated for 24 hours at 37 °C to induce sporulation. The cells were lysed using lysozyme and washed with dH₂O and SDS to remove vegetative cell residues. The spore suspension was adjusted to an OD of 2 for the glucose assay, the pNPG assay, and the pNPAc assay, and to 2.8 for the DNS assay thus allowing to have final OD in reaction was 0.2. For qualitative plate assays an OD of 0.2 was used. Further details are available on the Experimentspage.

First, we prepared spores displaying BhBglA, as this enzyme showed promising results in previous assays involving induced expression (see BBa_K5117017). We aimed to determine whether our glucose assay could effectively measure the glucose concentration resulting from the degradation of 50 mM cellobiose through the immobilized enzymes. The assay was performed according to the protocol described on the Experimentspage, using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit. After a 24-hour incubation period, the glucose assay was carried out, and the absorbance was measured at 560 nm. The results are presented in Fig. 8.

As a control, 50 mM cellobiose, diluted in 1X reaction buffer, was used, and the substrate absorbance was subtracted from the measured values. All three enzymes exhibited comparable absorbance values of approximately 0.2, corresponding to a glucose concentration of 13.8 µM, which, considering the dilution factor, results in 27.6 µM in the reaction (Resultspage, see calibration of glucose for glucose assay). These results suggest that there is no significant difference in glucose production between the three BhBglA linker variants, indicating similar catalytic efficiency for each. The glucose assay appears to be effective, although a relatively high background absorbance though unpurified cellobiose was observed (data not shown), which still allowed for distinguishing enzymatic activity from the control. In the future, we should further investigate cellobiose purification to reduce background signal of cellobiose.

We questioned whether a 24-hour incubation period was beneficial, given the low absorbance observed, which suggested that the enzyme activity might be inhibited by the accumulation of glucose in the reaction medium. Therefore, we decided to discontinue the 24-hour incubation and instead assessed enzyme activity over a shorter time frame of 30 minutes, collecting samples at 10-minute intervals (three samples in total). Additionally, we included spores displaying PpBglB-L3 ( BBa_K5117043) alongside spores displaying BhBglA-L2 ( BBa_K5117039) to compare their activity. The glucose assay was performed according to the protocol (see Experimentspage), and the results are presented in Fig. 9.

W168 control showed no absorbance at all time points, indicating no glucose production and confirming the absence of enzymatic activity in the control spores. BhBglA-L2 ( BBa_K5117039) displayed increasing absorbance values over time, with the highest absorbance at 20 minutes and slightly lower at 30 minutes. This suggests effective enzymatic activity, though the slight decrease could indicate either substrate saturation or variability in the measurements due to the lack of triplicates. PpBglB-L3 ( BBa_K5117043) showed no absorbance, comparable to the W168 control, indicating no enzymatic activity under these conditions. These results suggest that BhBglA-L2 ( BBa_K5117039) effectively degrades cellobiose and produces glucose within the 30-minute incubation period, while PpBglB-L3 ( BBa_K5117043) shows no detectable activity. The peak absorbance of 20 minutes for BhBglA-L2 ( BBa_K5117039) is likely to reflect experimental fluctuations, as triplicates were not performed.

pNPG assay for β-glucosidase activity determination

Based on previous results (see pBS0EX-BhBglA/pBS0EX-BhBglA ( BBa_K5117017) we decided to use p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate to validate the findings from the glucose assay. The assay was performed according to the protocol outlined in the Experiments page. We tested spores displaying (BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040)) as well as (PpBglB-L1 ( BBa_K5117041) and PpBglB-L3 ( BBa_K5117043) in two biological replicates (n = 2), to assess and compare their enzymatic activities using pNPG as a more accessible substrate. The results are shown in Fig. 10.

W168 control shows no absorbance, confirming the absence of enzymatic activity and serving as a baseline for comparison. BhBglA-L1 ( BBa_K5117038) exhibited the highest absorbance (around 2.2), indicating significant enzymatic activity when pNPG was used as a substrate. BhBglA-L2( BBa_K5117039) showed slightly lower activity compared to BhBglA-L1 ( BBa_K5117038), with an absorbance of approx. A₄₀₅= 1.8. BhBglA-L3 ( BBa_K5117040) displayed a high absorbance like BhBglA-L1 ( BBa_K5117038), suggesting comparable activity between these two linkers. Both PpBglB-L1 ( BBa_K5117041) and PpBglB-L3 ( BBa_K5117043) showed no absorbance, indicating no enzymatic activity under the tested conditions.

Overall, these results indicate that BhBglA-L1 ( BBa_K5117038) and BhBglA-L3 ( BBa_K5117040) exhibited the highest activity among the tested variants, while PpBglB-L1 ( BBa_K5117041) and PpBglB-L3 ( BBa_K5117043) showed no significant activity. The use of pNPG as a substrate effectively demonstrated differences in enzyme performance among the linkers and between the two enzymes.

Based on the results of previous assays, immobilized PpBglB on spores was excluded from further testing due to the lack of observable activity. Therefore, we focused on BhBglA which was immobilized on spores fused with three different linkers (BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040)).

Determination of optimal temperature

The optimal temperature for BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039) and BhBglA-L3 ( BBa_K5117040) was determined by evaluating enzyme activity across a temperature range of 40 °C to 90 °C, using pNPG as the substrate to identify the temperature at which each construct displayed maximal catalytic performance, as shown in Fig. 11. The assay was conducted following the standard procedure, with the incubation temperature varied between 40 °C and 90 °C (see Experimentspage).

The W168 control shows no activity at any of the tested temperatures. At 40 °C, all three enzymes BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039) BhBglA-L3 ( BBa_K5117040) exhibit relatively high activity, with BhBglA-L2 ( BBa_K5117039) showing the highest activity, followed by BhBglA-L1 ( BBa_K5117038) and BhBglA-L3 ( BBa_K5117040). The maximum activity for BhBglA-L1 ( BBa_K5117038) and BhBglA-L2 ( BBa_K5117039) is observed at 40 °C, while for L3, the maximum activity is at 50 °C. There is a slight decrease in activity for BhBglA-L1 ( BBa_K5117038) and L2 at 50 °C. Generally, L3 shows higher activity than BhBglA-L1 ( BBa_K5117038) and BhBglA-L2 ( BBa_K5117039) at elevated temperatures. At 60 °C, the activity decreases significantly for all variants, but L3 retains the highest residual activity, followed by BhBglA-L1 ( BBa_K5117038) and BhBglA-L2 ( BBa_K5117039). At higher temperatures (70 °C, 80 °C, and 90 °C), all immobilized enzymes show minimal activity, indicating a sharp decline in enzyme performance, likely due to denaturation. These results suggest that the optimal temperature for BhBglA activity varies between 40 °C and 50 °C depending on the linker, with activity diminishing above 60 °C. BhBglA-L3 appears to retain slightly higher stability at 60 °C compared to the other enzymes that are linked with BhBglA-L1 ( BBa_K5117038) or BhBglA-L2 ( BBa_K5117039).

Thermostability

Additionally, the thermostability of the BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040) was assessed by pre-incubating spore solutions at temperatures ranging from 40 °C to 90 °C for 2 hours, followed by measuring residual enzyme activity with pNPG as shown in Fig. 12. This approach allowed us to evaluate the thermal stability of the immobilized enzymes on spores fused with different linkers by assessing how well each retained activity following heat exposure.

The relative activity of BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039) and BhBglA-L3 ( BBa_K5117040) was analyzed after pre-incubation at various temperatures ranging from 40 °C to 90 °C, followed by a reaction with pNPG for 10 minutes at 50 °C. The measured absorbance values were background corrected and normalized to the corresponding absorbance values obtained with spore solutions stored at room temperature prior to the reaction. After pre-incubation at 40 °C, all three tested immobilized enzymes exhibited some relative activity (25 % to 30 %), with BhBglA-L1 ( BBa_K5117038) and BhBglA-L3 ( BBa_K5117040) showing the highest values. In contrast, no activity was observed for any of enzymes after pre-incubation at temperatures from 50 °C to 90 °C, suggesting that the enzymes underwent heat-induced denaturation or lost their functional capacity at these higher temperatures.

These findings indicate that BhBglA-L1 ( BBa_K5117038) and BhBglA-L3 ( BBa_K5117040) might be more thermostable than BhBglA-L2 ( BBa_K5117039), as they retain higher activity after exposure to 40 °C. However, above 40 °C, all tested enzymes lose their activity, suggesting a temperature limit for functional stability due to heat-induced denaturation. The W168 strain, used as a control, showed no significant activity across all temperatures tested, confirming the specificity of the enzymatic function observed. These results differ from previous literature, which reported an optimal temperature of 45 °C for BhBglA activity, with the enzyme retaining 80% of its activity after incubation at 45 °C for 1 hour (Naz et al., 2010). The discrepancy might be attributed to differences in enzyme immobilization and experimental setups, such as exposure time to heat.

The findings obtained also explain why we observed only minimal glucose production in the glucose assay conducted over 24 hours. The rapid decline in enzyme activity at temperatures above 40 °C likely limited the efficiency of cellobiose degradation, resulting in a low glucose yield. Therefore, for future experiments, the degradation of cellobiose should be carried out at 40 °C to ensure optimal enzyme performance. Additionally, the thermostability assessment should be extended by incubating the spore solution at 40 °C for longer periods prior to the reaction. This approach would provide a more comprehensive evaluation of enzyme stability and suitability for industrial applications at this temperature. By understanding the enzyme's long-term stability at 40 °C, it would be possible to determine whether it can sustain the desired catalytic activity over prolonged industrial processes. It would also be useful to investigate if the enzyme is active at lower temperatures and assess its stability under these conditions. Evaluating the enzyme's activity and stability at temperatures below 40 °C could help determine if the enzyme remains effective in milder conditions.

To strengthen the reliability of these findings, it is essential to perform these experiments in triplicates to confirm the observed trends and ensure reproducibility of the results.

Substrate specificity to prove that spores do not have enzymatic properties

Further, we tested whether the spores interact with p-nitrophenyl acetate (pNPAc), a substrate typically used to determine PETase activity, to rule out any potential intraspecific enzymatic properties of the spores themselves. This was done to ensure that any observed activity was due to the specific β-glucosidase enzymes displayed on the spores and not inherent spore-associated activity, as shown in Fig. 13.

With pNPAc, the absorbance values are low and similar for all constructs, suggesting limited interaction, which confirms that the activity measured is specific to the β-glucosidase enzymes and not due to the inherent properties of the spores. Thus, these results indicate that spores displaying BhBglA (BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040) exhibit specific enzymatic activity towards pNPG, while interaction with pNPAc is minimal, ruling out significant intraspecific enzymatic properties of the spores themselves. This supports the conclusion that the observed activity is due to the expressed β-glucosidase enzymes.

Reusability

Finally, we evaluated the reusability of the spore-displayed enzymes for BhBglA (BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040). The spores were used in five reaction cycles, with a washing step between each cycle. The number of spores in the first cycle was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The washing step involved removing the reaction products by ensuring the spores settled at the bottom of the reaction tube through centrifugation for 5 minutes at 13,000 rpm. The supernatant was discarded, followed by the addition of 1 ml of dH_{2</i>O, another centrifugation step, and subsequent removal of the water. 100 µl of fresh dH2</i>O were added, and the spores were stored until the next usage (20 minutes later). The reaction was conducted with pNPG as the substrate for 15 minutes instead of the usual 10 minutes. After completing the final fifth cycle, the reaction mixture was measured, then incubated for an additional 1 hour, followed by another measurement (indicated as 5* in Fig. 14).}

The normalization was set to the first cycle (100 %) for each enzyme fused to different, and the subsequent cycles were normalized relative to the first cycle for each respective enzyme fused to one linker. The normalization for the control was set to BhBglA-L1 ( BBa_K5117038) to ensure effective comparison. The W168 control showed no activity, confirming the absence of inherent enzymatic function. In the second cycle, a decrease in activity was observed for all linkers, with BhBglA-L1 ( BBa_K5117039) (approx. 40%). Activity continued to decline across subsequent cycles, with BhBglA-L1 ( BBa_K5117040). By the fourth and fifth cycles, enzyme activity was minimal for all linkers, indicating a loss of catalytic performance after repeated use. After the additional 1-hour incubation in the fifth cycle (5*), BhBglA-L1 ( BBa_K5117038) showed an increase in activity (up to 40%), suggesting some enzymatic potential during prolonged incubation. This may indicate slower catalytic degradation of pNPG due to the partial loss of spores.