1. Factors that affect cellobiase
Biofuel Lab
Factors that affect cellobiase

2. Cellobiase activity: Cellobiose glucose + glucose
(substrate) cellobiase (enzyme) (products)
Unfortunately, we can’t easily detect either the substrate or the products of this reaction.

3. Cellobiase also works on an artificial substrate:
p-Nitrophenyl glucopyranoside Glucose p-Nitrophenol
(substrate) cellobiase (enzyme) (products)
A strong base will denature the enzyme and stop the reaction.
p-Nitrophenol turns yellow in the presence of a base.
More yellow means more p-Nitrophenol.
More p-Nitrophenol means more of the substrate was broken apart.

4. Part 1: Rate of reaction
Set up 7 cuvettes of stop solution (strong base) – 2 of these are controls
Add enzyme solution to substrate solution and start timing
At intervals of 1, 2, 4, 6 and 8 minutes, transfer 500 uL of the enzyme/substrate mix to a stop solution cuvette
Record absorbance data on each cuvette

5. Level of yellow can be quantified with a spectrophotometer –
Likely hypothesis: As time increases, products of the reaction will increase.
Control minute min 4 min min min Control
no enzyme no enzyme
0 minutes min
Level of yellow can be quantified with a spectrophotometer –
more absorbance means more yellow, which means more product