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MGS0156 is a serine-dependent ⍺/β hydrolase originally identified from an environmental metagenomic analysis study in which various microbial genomes were sequenced for the identification of proteins with strong degradative activity against synthetic polyesters (Hajighasemi et al. 2016). MGS0156 has been shown to hydrolyze PLA into a mixture of lactic acid monomers, dimers, and higher oligomers as products, with a maximal activity between 35 and 40 °C (Hajighasemi et al. 2016). Our team was interested in MGS0156 due to its functionality in a lower temperature range, making it potentially useful in local composting facilities that often cannot maintain the sustained, high temperatures currently required for PLA degradation. Sequence and Features Status: 500 Content-type: text/html

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To characterize MGS0156, enzyme assays are performed with MGS0156-wt (wild type) and MGS0156-Nle (Norleucine) at different temperature to obtain Michaelis Menten parameters such as kcat, KM, and specificity constant. To prepare MGS0156-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 MGS0156 with 4-nitrophenyl octanoate (NPO). Assays were conducted with 1.5 µM of lipase in PBS with NPO concentration ranging from 0.0365 - 0.11 µM. Rate of change in absorbance was monitored at 410 nm for 1 min. A) Wild type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 25 ℃ B) Wild type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 60 ℃ C) MGS0156 chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25 ℃

Michaelis Menten parameters for MGS0156-wt and MGS0156-Nle when para-nitrophenyoctanoate (NPO) is used

MGS0156-wt
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 1.1x10^-3± 8.4x10^-4 1.6x10² ± 22.0 1.4x10⁵ ± 1.1x10⁵

25 5.3x10^-3 ± 4.7x10^-3 2.5x10² ± 48.0 4.8x10⁴ ± 4.3x10⁴

MGS0156-Nle
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
25 1.1x10^-3 ± 1.2x10^-3 2.1x10¹ ± 3.18 1.9x10⁴ ± 2.1x10⁴

Figure 2. Michaelis-Menten Esterase Activity Assay of MGS0156 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. Rate of change in absorbance was monitored at 410 nm for 1 min. A) Wild-type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 25 degrees C. B) Wild-type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 60 ℃. C) MGS0156 chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25℃

Michaelis Menten parameters for MGS0156-wt and MGS0156-Nle when para-nitrophenylbutyrate (pNOB) is used

MGS0156-wt
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
60 8.5x10^-3± 2.2x10^-2 2.0x10² ± 93.0 2.4x10⁴ ± 6.2x10⁴

25 1.0x10^-2 ± 6.6x10^-3 2.3x10² ± 38.0 2.2x10⁴ ± 1.5x10⁴

MGS0156-Nle
Temperature(℃) Km (µM) Kcat (s^-1) Specificity Constant (s^-1µM^-1)
25 2.2x10^-3 ± 1.7x10^-3 3.3x10¹ ± 4.5 1.5x10⁴ ± 1.2x10⁴

MGS1056-wt enzyme demonstrates better catalytic efficiency (kcat/km) when binding to NPO in comparison to pNOB, indicating a preference for NPO binding at 25℃. Enzyme-substrate binding exhibits a higher catalytic rate when the enzyme binds to 4-nitrophenyl octanoate (NPO) as opposed to 4-nitrophenyl butyrate (pNOB). It is hypothesized that this phenomenon can be attributed to the increased hydrophobicity and longer alkane chains present in NPO compared to pNOB. The hydrophobic binding of the substrate in the enzymatic active site contributes to enhanced enzyme-substrate binding affinity. The specificity of MGS1056-wt for NPO is 4.8x10⁴ s^-1µM^-1 at room temperature, while its specificity for pNOB is 2.2x10⁴ s^-1µM^-1. The heightened specificity of wild-type MGS1056 for NPO substrates relative to pNOB can be attributed to the larger size and increased hydrophobicity of the NPO substrate.

MGS1056-wt exhibits a higher Km value than variant MGS1056-Nle at 25℃, indicating that MGS1056-Nle demonstrates better substrate binding affinity than MGS1056 Nle. The Km value for MGS1056-Nle to NPO binding is approximately 4.82-fold smaller than MGS1056-wt. The Km value for MGS1056-Nle to pNOB binding is approximately 4.68-fold smaller than MGS1056-wt. Nevertheless, the catalytic efficiency of MGS1056-wt is still better than MGS1056-Nle due to higher Kcat in wild types. Our initial expectations were for MGS1056-Nle to exhibit improved catalytic efficiency and Km due to the substitution of Nle, which is less electronegative than the corresponding sulphur atom in Met, leading to increased hydrophobicity in the methylene group and favourable hydrophobic effects for enhanced binding and catalysis. Our experimental results indicate that although MGS1056-Nle exhibited better substrate binding affinity, MGS0156-wt maintained superior catalytic efficiency (kcat/km) compared to MGS1056-Nle.

Finally, wild-type enzyme activity is tested with varying temperatures. Results reveal that the wild-type MGS1056 enzyme, when acting on NPO, exhibits significantly higher catalytic efficiency at 60 ℃ compared to wild-type MGS1056 at 25 ℃ and both mutated forms of MGS1056 Nle at both room temperature and 60℃.

Reference
Hajighasemi, M., Nocek, B. P., Tchigvintsev, A., Brown, G., Flick, R., Xu, X., Cui, H., Hai, T., Joachimiak, A., Golyshin, P. N., Savchenko, A., Edwards, E. A., & Yakunin, A. F. (2016). Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases. Biomacromolecules, 17(6), 2027–2039. https://doi.org/10.1021/acs.biomac.6b00223