Protocol

Highlights:
Using a
colorimetric
substrate to
track reaction
rate

Cellobiose and glucose are colorless when

dissolved
Use of the artificial substrate p-nitrophenyl
glucopyranoside allows the reaction to be
tracked by monitoring the appearance of
yellow color

cellobiose

p-nitrophenyl glucopyranoside

Cellobiase
breakdown of pnitrophenyl
glucopyranoside

p-nitrophenyl glucopyranoside + H2O

glucose

p-nitrophenol

Basic
conditions

Clear

Yellow

How can this

enzymatic
reaction be
easily
quantified?

Basic solution (STOP SOLUTION):

- will develop color of any p-nitrophenol
present
- will stop the reaction
Each reaction time point can be directly
compared to a standard of known
concentration of p-nitrophenol
The amount of yellow color in the
reaction solution can be quantified by
measuring the absorbance at 410 nm
using a spectrophotometer.

Biofuel Enzyme
Kit
Procedure
Overview

Prepare and run reactions

Example of
Standards'
Absorbance
Readings

Standard
S1

Amount of
p-nitrophenol
(nmol)
0

Absorbance
410 nm
0

S2

12.5

0.2

S3

25

0.4

S4

50

0.8

S5

100

1.6

Qualitative
Determination
of Amount of
Product
Formed

Visually compare the color of the reaction

time points E1-E5 and the controls Start
and End against the standards of known
amount
Plot the amount of p-nitrophenol formed
at each time point to generate a reaction
curve

Quantitative
Determination of
p-nitrophenol
Amount
Read Samples
Analyze Results

Read the absorbance at 410 nm for each

standard and generate a standard curve
Determine the amount of product for each
reaction time point using the standard curve

Quantitative
Determination
of p-nitrophenol
Amount

Calculating
initial reaction
rate with and
without an
enzyme
present

Initial reaction rate =

Amount of p-nitrophenol
produced (nmol)
Time (min)

Initial reaction rate =

50 nmol - 0 nmol
4 min - 0 min

= 12.5 nmol/min

Conditions
affecting
reaction rate

pH
Temperature
Substrate Concentration
Enzyme Concentration

Effects of pH
Prepare and
run reactions

Calculating
initial
reaction rate
at different
pH values

Initial reaction rate =

Amount of p-nitrophenol
produced (nmol)
Time (min)

This is the amount of p-nitrophenol produced in

2 minutes

Further
activities
included in
the kit

Effect of temperature on the reaction

rate
Effect of substrate concentration on
the reaction rate
Effect of enzyme concentration on the
reaction rate
Ability of a mushroom extract to
catalyze the breakdown of the
substrate