Part:BBa_K4949002

MGS0156

MGS0156 is a serine dependent ⍺/β hydrolase originally identified from an environmental metagenomic analysis study to look for enzymes to degrade PLA

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 380
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 982
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 82
  Illegal NgoMIV site found at 253
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 766
  

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     1.6² ± 22.0               1.4x10⁵ ± 1.1x10⁵

25                                            5.3x10^-3 ± 4.7x10^-3    2.5² ± 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℃