Enzyme Activity: 1 Unit of enzyme activity is defined as the amount of enzyme required

to produce 1µM of glucose per minute per ml of enzyme extract under assay conditions.

Final volume of working standard = 3 ml

Standard graph:

Maltose standard graph

0.35
0.3
0.25
O.D at 540

0.2
0.15
0.1
Maltose
0.05
0
250 500 750 1000 1250
Conc. of Maltose microgram

250 µg ---- corresponds to 83.34 µg/ml

0.25 ml of enzyme source was taken and diluted up to 6.25 ml and OD was found but the
standard graph final volume is 3ml.

All readings (OD) have to be multiplied with (6.25/3) factor to get the final optical
density which can be compared with the standard graph.

For example: From Standard Graph: (Final volume) 3ml contains _______µg/ml of
Maltose ______ Optical Density.

But from the experiments: (Final Volume) 6.25 ml gives ______ Optical Density

Both can only be compared by multiplying with the factor (6.25/3).

0.25 ml of enzyme has given _______ concentration of Maltose but as per enzyme
activity definition was given for one ml of enzyme concentration so the final activity
calculated has to be multiplied with 4.

By considering all aspects:

Experiment 1: Optical Density for wheat bran – 0.410

For comparing this OD to the standard graph: 0.410 * (6.25/3) = 0.854

Using Standard Graph the concentration of Maltose:

By using Multiple Regressions:

Optical Density = 0.000232 (Maltose Concentration) + 0.008

Maltose Concentration (µg) = (Optical Density – 0.008)/0.000232

Using the above formula for our experimental value 0.854 the optical Density =

3646.55 µg of Maltose

Concentration of Maltose using Standard Graph (µg/ml) = 3646.55/3 = 1215.51 µg/ml

Concentration of glucose using standard graph = 1215.51 * (M. wt of Maltose/M. wt. of

glucose)

= 1215.51 * 2 = 2431.03 µg/ml

0.25 ml of enzyme was diluted to 6.25 ml final volume and the glucose concentration was
measured which has 2431.03 µg/ml of glucose

Enzyme concentration for 0.25 ml = 6.25 * 2431.03 / 0.25 = 60775.75 µg/ml

But as per enzyme activity we need 1 ml of enzyme:

Dilution factor = 4 * 6.25/.25 = 100

C(µ g/ml) * 1000

Unit of Enzyme Activtiy(µ M/ml-min) = * Dilution factor
Molecular weight of glucose* reaction time of enzyme with substrate

And that 1000 and Molecular wt of glucose are used for converting the concentration
g/ml into MOLAR.

U = 2431.03 * 1000 * 100 /(180 * 30) = 45019.07 µM/ml-min

Enzyme activity = 45019070.00 µM/l-min