Name:

Km

Category:

Activity

Property:

Michaelis Constant (Km)

Units:

mM (milliMolar)

Measurement Technique:

Enzymatic Hydrolysis

Buffers:

50 mM sodium citrate–sodium phosphate

Temperature:

30 °C

pH:

6.0

Protein Concentration:

None

Wavelength:

None

Ionic Strength:

None

Details:

The initial rates for the hydrolysis of the p-nitrophenyl β-glycosides (NPβglc, p-nitrophenyl β-glucoside; NPβgal, p-nitrophenyl β-galactoside; NPβfuc, p-nitrophenyl β-fucoside) and cellobiose were determined at 30 °C using substrates prepared in 50 mM sodium citrate–sodium phosphate buffer at pH 6. The hydrolysis of the p-nitrophenyl β-glycoside substrates was detected by following the production of p-nitrophenolate, whereas hydrolysis of cellobiose was detected by the glucose production. The production of two glucoses from one cellobiose was taken into account in the calculations. At least ten substrate concentrations, bracketing the Km values, were used for the determination of the enzyme kinetic parameters (Km and kcat). Rate and substrate concentration data were fitted to the Michaelis-Menten equation using the Origin 2017 software, version b9.4.0.220 (Origin Lab, Northampton, MA, USA).