Difference between revisions of "Part:BBa K5117044"

Revision as of 22:44, 1 October 2024

PcotYZ-BsRBS-BhrPET-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)
gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101),

a homologue of the leaf-branch compost cutinase (LCC), codon optimized for Bacillus subtilis (Xi et al. 2021) without any signal peptide,

addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA (BBa_K5117032)

cotY gene of Bacillus subtilis (BBa_K5117022)
bidirectional terminator B0014 (BBa_B0014)

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Immobilization of BhrPET on the spore crust of B. subtilis (spore surface display)

Application: Degradation of PET

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1009
Illegal AgeI site found at 695
Illegal AgeI site found at 740
1000
COMPATIBLE WITH RFC[1000]

Spore preparation

Spores were prepared by culturing cells in LB medium with chloramphenicol until they reached the exponential growth phase (OD₆₀₀ of 0.4–0.6). After washing and resuspension in DSM, the culture was incubated at 37 °C for 24 hours to induce sporulation. The cells were then lysed using lysozyme and washed with dH₂O and SDS to remove vegetative cell residues. The spore suspension was adjusted to an OD₆₀₀ of 2 for the pNPAc assay to achieve a final OD₆₀₀ of 0.2 in the reaction. Further details are available on the Experimentspage.

pNPAc assay for determination of activity

The PETase activity of spores displaying BhrPET (BhrPET-L1 ( BBa_K5117044), BhrPET-L2 ( BBa_K5117045) and BhrPET-L3 ( BBa_K5117046)) was evaluated using an activity assay based on the method described by Xi et al. (2020). To demonstrate the functionality of the immobilized BhrPET, a p--nitrophenyl-acetate (pNPAc) assay was conducted. In this assay, pNPAc is degraded to acetate and p-nitrophenol (pNP), which imparts a yellow color to the solution. Notably, the reaction also occurs in the absence of enzymes, which is referred to as autohydrolysis. To rule out the influence of autohydrolysis on the measured activity values, a negative control without spore solution was included.

The reaction mixture contained phosphate buffer, spores with a final OD₆₀₀ of 0.2, pNPAc at a final concentration of 1 mM, and deionized water, and was incubated at 40 °C for 10 minutes. Detailed protocol can be found on the Experimentspage. Following incubation, the reaction was centrifuged at 13,000 rpm for 1 minute to avoid the spores interfering with the absorbance measurement. After centrifugation, 100 µl of the supernatant was transferred into a 96-well plate for absorbance measurement at 405 nm, performed in triplicates.

The PETase activity assay results for spores displaying BhrPET with different linkers are summarized in Fig. 6. The controls included reactions without spore solution and with W168 spores, which did not display enzymes, serving as negative controls. The absorbance values at 405 nm, with the control subtracted, indicate the enzyme activity. W168 control showed negligible absorbance, confirming the absence of enzymatic activity. Among the variants, BhrPET-L3 exhibited the highest activity, with an absorbance of approximately 0.9, followed closely by BhrPET-L1 at around 0.8. BhrPET-L2 showed a lower activity compared to the other linker variants, with an absorbance of about 0.5. These results suggest that the choice of linkers has a substantial effect on the enzyme's performance, with BhrPET-L3 being the most effective under the tested conditions.

Fig. 6: : Activity assay of BhrPET-displaying spores ((BhrPET-L1 ( BBa_K5117044), BhrPET-L2 ( BBa_K5117045) and BhrPET-L3 ( BBa_K5117046)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.
(see Experimentspage).
Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The assay was performed in one biological replicates (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal.

The initial PETase activity assay showed fluctuations in activity between the constructs, which may have been influenced by the very low yield of spores in the experiment. To address this issue, the spore preparation was repeated twice for subsequent tests to ensure consistent results. The procedure was repeated as previously described. Unfortunately, the yield of BhrPET-L2 spores was too low to perform any experiments. The Fig. 7 displays the mean absorbance values of two replicates, with the control (substrate without spores) subtracted. The results indicate that BhrPET-L1 exhibited the highest enzyme activity, with a maximum absorbance of approximately 2.5, while BhrPET-L3 showed slightly lower activity. Due to the higher performance, all subsequent experiments testing thermostability and optimal temperature were conducted using BhrPET-L1, as its spore yield was also the highest (data not shown).

Additionally, after the spore preparation, the spores appeared impure, which suggests there may have been issues with the sporulation of the PET constructs. This means that the actual enzyme activity might be higher than what was observed.

Fig. 7: : Activity assay of BhrPET-displaying spores ((BhrPET-L1 ( BBa_K5117044), BhrPET-L2 ( BBa_K5117045) and BhrPET-L3 ( BBa_K5117046)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.
(see Experimentspage).
Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The assay was performed in one biological replicate (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal.

Determination of optimal temperature

The optimal temperature for BhrPET-L1 was determined by conducting the enzyme activity assay at varying temperatures ranging from 40 °C to 90 °C for 10 minutes. The results of the temperature-dependent activity assay for BhrPET-L1 are shown in Fig. 8. The graph illustrates the relative absorbance at 405 nm for W168 and BhrPET-L1 spores, with values normalized to the highest observed absorbance (A₄₀₅ = 2.35) for BhrPET-L1 at 50 °C, set as 100 %. The W168 control demonstrated negligible absorbance across all temperatures, confirming the absence of enzymatic activity. BhrPET-L1 showed nearly 90% of its maximum activity at 40 °C and around 80% at 60 °C. However, at 70 °C, enzyme activity sharply declined to approximately 10%, and no activity was detected at 80 °C or 90 °C. These findings suggest that the optimal temperature range for BhrPET-L1 is between 40 °C and 50 °C. Temperatures above 60 °C impair the enzyme's stability and catalytic performance, likely due to the thermal instability of either the enzyme or the substrate.

Fig. 8: : Relative activity of BhrPET-L1 and W168 spores at varying temperatures (40 °C to 90 °C) for determining the optimal temperature for enzyme activity with pNPAc as the substrate (see Experimentspage). The x-axis represents temperature (°C). The y-axis shows relative activity as a percentage, normalized to the highest observed value at 50 °C (100%, A₄₀₅ = 2.35). The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. BhrPET-L1 demonstrated peak activity at 50 °C, with about 90% activity remaining at 40 °C and 80% at 60 °C. A significant reduction in activity was noted at 70 °C, dropping to 10%, and no enzyme activity was detectable at 80 °C or 90 °C. The assay was performed in one biological replicate (N = 1).

Assessment of the thermostability of spore-displayed endoglucanases

Spore solutions were incubated at various temperatures ranging from 40 °C to 90 °C for 2 hours to evaluate the thermostability of the BhrPET-L1 displayed on the spores. After heat treatment, the samples were allowed to cool to room temperature. Subsequently, the residual enzyme activity of BhrPET-L2 was determined using an assay previously described (for 10 minutes at 40 °C). The results of this assessment are presented in Fig. 9. They indicate that BhrPET-L1 retains significant enzymatic activity after incubation at moderate temperatures, demonstrating good thermostability up to 60 °C. The enzyme exhibited peak activity after pretreatment at 40 °C (100%, A₄₀₅ = 2.76). After incubation at 50 °C, approximately 90 % of the enzyme activity was maintained, and around 70 % activity remained at 60 °C. However, a decline in activity was observed at higher temperatures, with residual activity dropping to 15% after pretreatment at 70 °C, 10 % at 80 °C, and only 5 % at 90 °C. These results suggest that BhrPET-L1 maintains its structural stability and enzymatic function up to 60 °C, but loses most of its activity at higher temperatures, indicating reduced thermostability beyond this threshold.

Noteworthy, the obtained results contradict earlier findings from the work of Xi et al (2020), in which almost a linear increase of activity was observed from 30 °C to 90 °C after assessment of the optimal temperature. In addition, with the thermostability experiments performed by Xi et al (2020) it was shown that BhrPET could retain 80 % of its activity when pre-incubated at 80 °C for 2 hours, whereas in our project a significant loss of BhrPET activity was detected after its pre-incubation for 2 hours at 70 °C and higher temperatures. The discrepancies in the results could be influenced by multiple factors. First, Xi et al (2020) applied different assay conditions and used p-nitrophenyl-octanoate and not pNPAc as a substrate. Second, the solutions with spore displaying BhrPET contained some impurities as described above, which could have interfered with the applied assay. Finally, enzyme immobilization on the spore surface could resulted in a negative impact on the protein activity and stability.

Fig. 9: : Relative activity of BhrPET-L1 and W168 spores for determining the thermostability of the enzymes displayed on the spores, which were preincubated at varying temperatures (40 °C to 90 °C) for 2 hours using pNPAc assay (see Experimentspage). Following the pre-incubation, the reaction with pNPAc was conducted for 10 minutes at 50 °C, and the formation of pNP was measured by absorbance at 405 nm. Spores from the W168 strain were used as a control, along with additional control without spore solution. The amount of spores was adjusted to achieve an OD OD₆₀₀ of 0.2 in the reaction mixture. The absorbance from the control without spores was subtracted from the measured values to account for background signal. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures. The assay was performed in one biological replicate (N = 1).

The relative activity of BhrPET-L1 and W168 spores were evaluated after preincubation at various temperatures ranging from 40 °C to 90 °C for 2 hours. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures.

BhrPET-L1 retained substantial activity up to 60 °C, indicating good thermostability under these conditions. However, enzyme activity declined above 60 °C, with minimal activity detected at 70 °C (20 %) and 80 °C (approx. 17%), and almost no activity observed at 90 °C. This indicates that BhrPET-L1 loses both stability and catalytic function at higher temperatures. The W168 control showed negligible activity across all temperatures, confirming that the observed enzymatic activity in BhrPET-L1 is specific to the enzyme displayed on the spores.

BhrPET-L1 demonstrates promising activity within the 40 °C to 50 °C temperature range and maintains good thermostability up to 60 °C, making it suitable for applications within moderate temperature environments. However, the enzyme's stability and activity significantly diminish at temperatures above 60 °C.

References

Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2021). Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and Microbial Technology, 143, 109715. https://doi.org/10.1016/J.ENZMICTEC.2020.109715

Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., & Baratti, J. (2000). Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Applied Microbiology and Biotechnology, 54(2), 173–179. https://doi.org/10.1007/S002530000353

@@ Line 33: / Line 33: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K5117044 SequenceAndFeatures</partinfo>
+===Spore preparation===
+Spores were prepared by culturing cells in LB medium with chloramphenicol until they reached the exponential growth phase (OD<sub>600</sub> of 0.4–0.6). After washing and resuspension in DSM, the culture was incubated at 37 °C for 24 hours to induce sporulation. The cells were then lysed using lysozyme and washed with dH<sub>2</sub>O and SDS to remove vegetative cell residues. The spore suspension was adjusted to an OD<sub>600</sub> of 2 for the pNPAc assay to achieve a final OD<sub>600</sub> of 0.2 in the reaction. Further details are available on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page.
+===pNPAc assay for determination of activity===
+The PETase activity of spores displaying BhrPET (BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>), BhrPET-L2 (<html><a href="https://parts.igem.org/Part:BBa_K5117045"> BBa_K5117045</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) was evaluated using an activity assay based on the method described by <i>Xi et al.</i> (2020). To demonstrate the functionality of the immobilized BhrPET, a <i>p</i>--nitrophenyl-acetate (pNPAc) assay was conducted. In this assay, pNPAc is degraded to acetate and <i>p</i>-nitrophenol (pNP), which imparts a yellow color to the solution. Notably, the reaction also occurs in the absence of enzymes, which is referred to as autohydrolysis. To rule out the influence of autohydrolysis on the measured activity values, a negative control without spore solution was included.
+The reaction mixture contained phosphate buffer, spores with a final OD<sub>600</sub> of 0.2, pNPAc at a final concentration of 1 mM, and deionized water, and was incubated at 40 °C for 10 minutes. Detailed protocol can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page. Following incubation, the reaction was centrifuged at 13,000 rpm for 1 minute to avoid the spores interfering with the absorbance measurement. After centrifugation, 100 µl of the supernatant was transferred into a 96-well plate for absorbance measurement at 405 nm, performed in triplicates.
+The PETase activity assay results for spores displaying BhrPET with different linkers are summarized in Fig. 6. The controls included reactions without spore solution and with W168 spores, which did not display enzymes, serving as negative controls. The absorbance values at 405 nm, with the control subtracted, indicate the enzyme activity. W168 control showed negligible absorbance, confirming the absence of enzymatic activity. Among the variants, BhrPET-L3 exhibited the highest activity, with an absorbance of approximately 0.9, followed closely by BhrPET-L1 at around 0.8. BhrPET-L2 showed a lower activity compared to the other linker variants, with an absorbance of about 0.5. These results suggest that the choice of linkers has a substantial effect on the enzyme's performance, with BhrPET-L3 being the most effective under the tested conditions.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet1.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 6: : Activity assay of BhrPET-displaying spores ((BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>), BhrPET-L2 (<html><a href="https://parts.igem.org/Part:BBa_K5117045"> BBa_K5117045</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.
+(see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page).
+</b> Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD<sub>600</sub> of 0.2 in the reaction mixture. The assay was performed in one biological replicates (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal. </font></center></p>
+The initial PETase activity assay showed fluctuations in activity between the constructs, which may have been influenced by the very low yield of spores in the experiment. To address this issue, the spore preparation was repeated twice for subsequent tests to ensure consistent results. The procedure was repeated as previously described. Unfortunately, the yield of BhrPET-L2 spores was too low to perform any experiments. The Fig. 7 displays the mean absorbance values of two replicates, with the control (substrate without spores) subtracted. The results indicate that BhrPET-L1 exhibited the highest enzyme activity, with a maximum absorbance of approximately 2.5, while BhrPET-L3 showed slightly lower activity. Due to the higher performance, all subsequent experiments testing thermostability and optimal temperature were conducted using BhrPET-L1, as its spore yield was also the highest (data not shown).
+Additionally, after the spore preparation, the spores appeared impure, which suggests there may have been issues with the sporulation of the PET constructs. This means that the actual enzyme activity might be higher than what was observed.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet2.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 7: : Activity assay of BhrPET-displaying spores ((BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>), BhrPET-L2 (<html><a href="https://parts.igem.org/Part:BBa_K5117045"> BBa_K5117045</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.
+(see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page).
+</b> Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD<sub>600</sub> of 0.2 in the reaction mixture. The assay was performed in one biological replicate (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal. </font></center></p>
+===Determination of optimal temperature===
+The optimal temperature for BhrPET-L1 was determined by conducting the enzyme activity assay at varying temperatures ranging from 40 °C to 90 °C for 10 minutes. The results of the temperature-dependent activity assay for BhrPET-L1 are shown in Fig. 8.
+The graph illustrates the relative absorbance at 405 nm for W168 and BhrPET-L1 spores, with values normalized to the highest observed absorbance (A<sub>405</sub> = 2.35) for BhrPET-L1 at 50 °C, set as 100 %. The W168 control demonstrated negligible absorbance across all temperatures, confirming the absence of enzymatic activity. BhrPET-L1 showed nearly 90% of its maximum activity at 40 °C and around 80% at 60 °C. However, at 70 °C, enzyme activity sharply declined to approximately 10%, and no activity was detected at 80 °C or 90 °C. These findings suggest that the optimal temperature range for BhrPET-L1 is between 40 °C and 50 °C. Temperatures above 60 °C impair the enzyme's stability and catalytic performance, likely due to the thermal instability of either the enzyme or the substrate.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet-opt-t.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 8: : Relative activity of BhrPET-L1 and W168 spores at varying temperatures (40 °C to 90 °C) for determining the optimal temperature for enzyme activity with pNPAc as the substrate (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page). </b> The x-axis represents temperature (°C). The y-axis shows relative activity as a percentage, normalized to the highest observed value at 50 °C (100%, A<sub>405</sub> = 2.35). The amount of spores was adjusted to achieve an OD<sub>600</sub> of 0.2 in the reaction mixture. Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. BhrPET-L1 demonstrated peak activity at 50 °C, with about 90% activity remaining at 40 °C and 80% at 60 °C. A significant reduction in activity was noted at 70 °C, dropping to 10%, and no enzyme activity was detectable at 80 °C or 90 °C. The assay was performed in one biological replicate (N = 1). </font></center></p>
+===Assessment of the thermostability of spore-displayed endoglucanases===
+Spore solutions were incubated at various temperatures ranging from 40 °C to 90 °C for 2 hours to evaluate the thermostability of the BhrPET-L1 displayed on the spores. After heat treatment, the samples were allowed to cool to room temperature. Subsequently, the residual enzyme activity of BhrPET-L2 was determined using an assay previously described (for 10 minutes at 40 °C). The results of this assessment are presented in Fig. 9. They indicate that BhrPET-L1 retains significant enzymatic activity after incubation at moderate temperatures, demonstrating good thermostability up to 60 °C. The enzyme exhibited peak activity after pretreatment at 40 °C (100%, A<sub>405</sub> = 2.76). After incubation at 50 °C, approximately 90 % of the enzyme activity was maintained, and around 70 % activity remained at 60 °C. However, a decline in activity was observed at higher temperatures, with residual activity dropping to 15% after pretreatment at 70 °C, 10 % at 80 °C, and only 5 % at 90 °C. These results suggest that BhrPET-L1 maintains its structural stability and enzymatic function up to 60 °C, but loses most of its activity at higher temperatures, indicating reduced thermostability beyond this threshold.
+Noteworthy, the obtained results contradict earlier findings from the work of Xi <i>et al</i> (2020), in which almost a linear increase of activity was observed from 30 °C to 90 °C after assessment of the optimal temperature. In addition, with the thermostability experiments performed by Xi <i>et al</i> (2020) it was shown that BhrPET could retain 80 % of its activity when pre-incubated at 80 °C for 2 hours, whereas in our project a significant loss of BhrPET activity was detected after its pre-incubation for 2 hours at 70 °C and higher temperatures. The discrepancies in the results could be influenced by multiple factors. First, Xi <i>et al</i> (2020) applied different assay conditions and used p-nitrophenyl-octanoate and not pNPAc as a substrate. Second, the solutions with spore displaying BhrPET contained some impurities as described above, which could have interfered with the applied assay. Finally, enzyme immobilization on the spore surface could resulted in a negative impact on the protein activity and stability.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet-thermostability.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 9: : Relative activity of BhrPET-L1 and W168 spores for determining the thermostability of the enzymes displayed on the spores, which were preincubated at varying temperatures (40 °C to 90 °C) for 2 hours using pNPAc assay (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page). </b> Following the pre-incubation, the reaction with pNPAc was conducted for 10 minutes at 50 °C, and the formation of pNP was measured by absorbance at 405 nm. Spores from the W168 strain were used as a control, along with additional control without spore solution. The amount of spores was adjusted to achieve an OD OD<sub>600</sub>  of 0.2 in the reaction mixture. The absorbance from the control without spores was subtracted from the measured values to account for background signal. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures. The assay was performed in one biological replicate (N = 1). </font></center></p>
+The relative activity of BhrPET-L1 and W168 spores were evaluated after preincubation at various temperatures ranging from 40 °C to 90 °C for 2 hours. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures.
+BhrPET-L1 retained substantial activity up to 60 °C, indicating good thermostability under these conditions. However, enzyme activity declined above 60 °C, with minimal activity detected at 70 °C (20 %) and 80 °C (approx. 17%), and almost no activity observed at 90 °C. This indicates that BhrPET-L1 loses both stability and catalytic function at higher temperatures. The W168 control showed negligible activity across all temperatures, confirming that the observed enzymatic activity in BhrPET-L1 is specific to the enzyme displayed on the spores.
+BhrPET-L1 demonstrates promising activity within the 40 °C to 50 °C temperature range and maintains good thermostability up to 60 °C, making it suitable for applications within moderate temperature environments. However, the enzyme's stability and activity significantly diminish at temperatures above 60 °C.
+===References===
+Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2021). Secretory expression in <i> Bacillus subtilis</i> and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and Microbial Technology, 143, 109715. https://doi.org/10.1016/J.ENZMICTEC.2020.109715
+Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., & Baratti, J. (2000). Purification and characterization of a thermostable esterase from the moderate thermophile <i> Bacillus circulans</i>. Applied Microbiology and Biotechnology, 54(2), 173–179. https://doi.org/10.1007/S002530000353