Biofuel Lab

Factors that affect cellobiase

Cellobiase activity:

Cellobiose glucose + glucose(substrate) cellobiase (enzyme) (products)

Unfortunately, we can’t easily detect either the substrate or the products of this reaction.

p-Nitrophenyl glucopyranoside Glucose + p-Nitrophenol(substrate) cellobiase (enzyme) (products)

Cellobiase also works on an artificial substrate:

•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

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

Likely hypothesis: As time increases, products of the reaction will increase.