Enzyme activity exercise - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Eliasson, A, E Boles, B Johansson, M Österberg, J M Thevelein, I Spencer-Martins, H Juhnke, and B Hahn-Hägerdal. 2000. “Xylulose Fermentation by Mutant and Wild-Type Strains of Zygosaccharomyces and Saccharomyces Cerevisiae.” Appl. Microbiol. Biotechnol. 53 (4): 376–82. https://doi.org/10.1007/s002530051629.

The XK reaction was coupled with the pyruvate kinase reaction as follows:

xylulose + ATP ⟶ xylulose 5-phosphate + ADP ADP + phosphoenolpyruvate ⟶ pyruvate + ATP pyruvate + NADH ⟶ lactate + NAD�

The reaction mixture contained the following: 50 mM TRIS/HCl bufferr (pH 7.5), 2.0 mM MgCl2, 2.0 mM ATP, 0.2 mM phosphoenolpyruvate, 0.2 mM NADH, 10 U pyruvate kinase (E.C. 2.7.1.40), 10 U lactate dehydrogenase (E.C. 1.1.1.27) and 8.5 mM xylulose.

Consumption of NADH was measured spectrophotometrically at 340 nm.

The molar absorption coefficient (extinction coefficient) of NADH at 340 nm (ε340) is 6220 M-1 cm-1, whereas that of NAD+ at the same wavelength can be assumed to be 0.

U / mg of protein

One unit of enzyme activity is defined as 1 µmol of substrate converted per min.

Beer-Lambert Law

The Beer-Lambert Law describes the linear relationship between the absorbance of a solution and the concentration of the absorbing species in the solution.

Formula

The equation is given by:

A = ε * c * l

where:

  • A = Absorbance (no units)
  • ε = Molar absorptivity or extinction coefficient (L·mol⁻¹·cm⁻¹)
  • c = Concentration of the solution (mol·L⁻¹)
  • l = Path length through the sample (cm)

Explanation

  • Absorbance (A) is the amount of light absorbed by the solution.
  • Molar absorptivity (ε) is a constant that depends on the nature of the absorbing species and the wavelength of light.
  • Concentration (c) is the amount of the absorbing species per unit volume.
  • Path length (l) is the distance that light travels through the solution.

The Beer-Lambert Law is often used in spectrophotometry to determine the concentration of a solute in a solution by measuring the absorbance.

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