Thermoanaerobacter thermohydrosulfuricus lipase (TTL)

Thermoanaerobacter thermohydrosulfuricus lipase (TTL) is a thermophilic lipase that has been studied in the potential degradation of polyethylene terephthalate (PET). This protein expresses well in E.coli and sufficient quantity can be purified by IMAC test is ability to cleave ester linkages.

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To characterize TTL, enzyme assays are performed with TTL-wt (wild type) and TTL-Nle (Norleucine) at different temperature to obtain Michaelis Menten parameters such as kcat, KM, and specificity constant. To prepare TTL-Nle, selective pressure incorporation (SPI) was performed. Methionine-auxotrophic cells were grown in minimal media with limiting 30 µM Methionine at 37℃ for 10 hours until depletion of Methionine. Then, 1 mM Norleucine was added to the culture. Enzymes are first purified using IMAC and then size exclusion chromatography. Before starting assay, PBS buffer is heat up to the desired temperature. To prepare reaction mixture, pure enzyme is mixed with different concentrations of substrate (para-nitrophenylbutyrate, pNOB, and para-nitrophenyloctanoate, NPO) and PBS in a quartz cuvette. The cuvette is inverted three times quickly for proper mixing and then, placed in a spectrophotometer to monitor absorbance at 410 nm for 1 to 3 minutes. The slope within the first 3 seconds is taken for initial rate.

Figure 1. Michaelis-Menten Esterase Activity Assay of Thermoanaerobacter thermohydrosulfuricus lipase with 4-nitrophenyl butyrate (pNOB). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365-0.11 µM. The rate of change of absorbance was monitored at 410 nm for 1 min. A) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 25 B) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 60 ℃ C) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25 ℃ D) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue.

Michaelis Menten parameters for TTL-wt and TTL-Nle when para-nitrophenylbutyrate (pNOB) is used

TTL-wt
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 2.4x10^-2± 6.0x10^-3 3.2 ± 0.26 1.3x10² ± 3.6x10¹

25 2.0x10^-1 ± 8.3x10^-2 3.3 ± 0.49 1.9x10¹ ± 1.0x10¹

TTL-Nle
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 5.9x10^-2± 1.8x10^-2 6.9 ± 0.72 1.2x10² ± 3.8x10¹

25 4.2x10^-2 ± 2.4x10^-2 1.3 ± 0.27 3.1x10¹ ± 1.9x10¹

Figure 2. Michaelis-Menten Esterase Activity Assay of Thermoanaerobacter thermohydrosulfuricus lipase with 4-nitrophenyl octanoate (NPO). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365-0.11 µM. The rate of change of absorbance was monitored at 410 nm for 1 min. A) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 25 ℃ B) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 60 ℃. C) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25℃. D) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 60℃

Michaelis Menten parameters for TTL-wt and TTL-Nle when para-nitrophenyloctanoate (NPO) is used

TTL-wt
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 1.7x10^-3± 1.5x10^-3 6.3 ± 0.50 3.6x10³ ± 3.3x10³

25 1.7x10^-3 ± 2.7x10^-3 2.0 ± 0.36 1.2x10³ ± 1.1x10³

TTL-Nle
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 2.0x10^-4± 1.9x10^-4 5.3 ± 0.48 2.7x10⁴ ± 2.5x10⁴

25 3.6x10^-3 ± 5.2x10^-3 2.0 ± 0.41 5.6x10² ± 8.2x10²

The enzyme affinity of (Km) wild-type TTL (TTL-wt) to the pNOB substrate was observed to be lower at 60°C. The Km value at 25°C of 2.0x10^-1µM. decreased to 2.4x10^-2µM at 60℃. This indicates that at 25℃ more of the substrate must be present to saturate the enzyme thus having a lower affinity for the substrate. However, the catalytic activity of wild-type TTL did not change in response to temperature as they are both at approximately 3.2 to 3.3 s^-1. The wild-type catalytic of NPO increased at 60℃ in comparison to 25℃. As a result, the specificity constant increases with temperature when pNOB and NPO are used as substrates. Both observations of TTL catalytic efficiency towards NPO and pNOB are consistent with TLL having maximal activity at thermophilic temperatures (Haernvall et al. 2022). The trends observed in the TTL-wt were also observed in the TTL with norleucine introduced (TTL-Nle), apart from the catalytic activity with the pNOB substrate. The catalytic activity of Nle TTL to pNOB increased from 1.3 s^-1 at 25℃ to 6.9 s^-1 at 60℃.

The results from the NPO assays suggest that the incorporation of norleucine greatly improved the catalytic of TTL. When comparing the results of the 60°C assays the enzyme affinity decreased from 1.7x10^-3µM for the wild type to 2.0x10^-4µM for the TTL-Nle. However, the catalytic activity of Nle TTL decreased from 6.3 s^-1 for wild-type to 5.3 s^-1 for TTL-Nle. The specificity constant increases significantly in TTL-Nle. In comparison, the assays at 25°C the catalytic efficiency decreased. Although 25°C is much lower than the optimal temperature of TTL, resulting in smaller values for catalytic activity and enzyme specificity for both the wild type and modified TTL-Nle.

The results from the pNOB assays at 60 °C suggest a small decrease in catalytic efficiency after the incorporation of norleucine. However, at 25°C the catalytic efficiency was shown to improve in the TTL-Nle, from 19.0 to 31.0 s^-1µM^-1. Note the substrate binding affinity decreased from 2.0x10^-1 µM to 4.2x10^-2 µM, although the catalytic activity was still quite low in both the wildtype and modified TTL which is consistent with assays being done below TTL’s optimal temperature. The results from the pNOB assays showed lower activity across all conditions, likely due to its shorter and less hydrophobic chain.

Overall, the norleucine incorporation assays at 60 °C showed an overall improvement of enzymatic activity. This is consistent with the increased hydrophobicity of TTL-Nle likely improved the interactions with the substrate. The results from the assays at 25°C decreased catalytic efficiency due to the increased substrate binding affinity (Km) value. Outside the optimal temperature range, the norleucine modification did not appear to improve.

Referece:
Haernvall, K., Fladischer, P., Schoeffmann, H., Zitzenbacher, S., Pavkov-Keller, T., Gruber, K., Schick, M., Yamamoto, M., Kuenkel, A., Ribitsch, D., Guebitz, G. M., & Wiltschi, B. (2022). Residue-specific incorporation of the non-canonical amino acid norleucine improves lipase activity on synthetic polyesters. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.769830