Prior to kinetic studies, experiments were conducted to determine enzyme concentrations, dNTP concentrations, and times for which product accumulation was linear as a function of time.
Crude cellulase concentration yielding the highest reducing sugar concentration from the previous study determined the enzyme concentration to be added to the substrate in each flask.
These include using three points to determine the enzyme concentration ([I]=0, 0.5[E]0, and [E]0), using a narrow dilution series with only two or three points to determine the asymptote at high inhibitor concentration, and avoiding fixing [E]0 to a constant value in the fitting if at all possible.
As the Michaelis-Menten plots were linear up to 0.25 mM, it was only possible to determine enzyme velocities at this concentration (k values for these substrates were 5.9±0.6 min−1 and 5.3±0.6 min−1, respectively).
The absorbance of the reference was subtracted from the reaction absorbance, prior to determining the enzyme concentration.
Optimal conditions for the structured lipid containing omega-3 to omega-6 fatty acids in the ratio of 1 1 were determined to be; enzyme concentration 3.75% (w/w), temperature 37.5 °C, incubation time 30.81 h and ratio of free fatty acid concentrate from linseed oil to groundnut oil 1.16 (w/w).
The initial rate was determined with constant enzyme concentration, varying substrate concentrations and constant cosubstrate concentration.
It was interesting to find that the decrease of the scattering intensity was not accompanied by a decrease of the average size of the PCL nanoparticles, indicating that the enzyme, Lipase Pseudomonas (PS), "eats" the PCL nanoparticles one-by-one, so that the biodegradation rate is determined mainly by the enzyme concentration.
The minimal concentration of PF-bgl for the complete conversion of ginsenoside R1 to APPT was determined by varying the enzyme concentration from 0.156 to 10 µg ml−1 with 1.0 mg ml−1 R1 and 4.0 mg ml−1 DT-bgl for 6 h.
The Vmax values were converted to kcat using the total enzyme concentration determined from a Bradford assay (kcat = Vmax/ Etot).
To establish the apparent KM (K1/2) for each substrate, rate constants (kobs) were determined at varying enzyme concentrations, and plots of kobs vs [E] were fit to the equation, kobs = kst[E]/(K1/2 + [E]).
