Biofuel Lab
Factors that affect cellobiase
Cellobiase activity:
Cellobiose
(substrate)
glucose
cellobiase (enzyme)
+ glucose
(products)
Unfortunately, we can’t easily detect either the substrate or the
products of this reaction.
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.
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
Likely hypothesis: As time increases, products of the reaction will increase.
Control
1 minute
2 min
4 min
6 min
8 min
Control
no enzyme
no enzyme
0 minutes
8 min
Level of yellow can be quantified with a spectrophotometer –
more absorbance means more yellow, which means more product