Enzymes in Action: Cellulase
- Biochemical processes in the cell are controlled by the nature and arrangement of internal membranes, the presence of specific enzymes, and environmental factors
- Enzymes have specific functions, which can be affected by factors including temperature, pH, the presence of inhibitors, and the concentrations of reactants
Photosynthesis produces glucose which plants channel into energy storage (as starch) or deploy as structural polymer (cellulose). Although evolution has equipped many organisms with some form of amylase to digest starch, very few, apart from some micro-organisms, possess an enzyme that can digest cellulose. As a result, cellulose remains indigestible to most animals. Even herbivores that need to digest cellulose to survive are unable to produce the enzyme themselves. They rely on symbiotic bacteria in their gut to provide cellulase. Apart from being necessary for digestion by herbivores, cellulase is becoming commercially important for the role it could play in producing biofuels via the fermentation of sugars.
In this experiment, you will study the effect of cellulase on a water soluble derivative of cellulose called methyl cellulose. Whereas cellulose is not soluble in water, methyl cellulose is a water soluble derivative that can be hydrolysed by cellulase. In this experiment, a viscous solution of methyl cellulase is treated with cellulase and the reduction in viscosity can be used as a measure of the extent of reaction.
PREPARATION - BY LAB TECHNICIAN
Methyl Cellulose Solution 3%
- To make a methyl cellulose solution, place 1.5g of methyl cellulose powder in a 200mL beaker.
- Measure 50mL distilled water into a measuring cylinder.
- Pour 20mL of the water into a small beaker and heat to boiling then remove from heat and wait 2 minutes.
- Using a glass rod, stir small aliquots of the hot water into the methyl cellulose powder to form a stiff paste that is free of lumps. You will use 10-15mL of the hot water to accomplish this.
- Pour any remaining hot water back into the residual cold water.
- Add the cold water to the paste in small aliquots with stirring until it has been completely mixed.
- Add a stir bar to the mixture and stir on a magnetic stirrer for 60-90 minutes. As the viscosity rises you will need to adjust the stirrer speed to keep it moving.
- Remove from the stirrer, cover and allow to sit overnight to homogenise.
METHOD - STUDENT PRACITCAL
- Add 20mL water to one beaker of methyl cellulose solution. This will be your control.
- Add 20mL of 1.0% cellulase solution to the other beaker of methyl cellulose solution.
- Stir the solutions on magnetic stirrers until both are homogeneous. The time will depend on the stirrer being used. In any case, stir both solutions for the same time, even if one becomes evenly blended before the other.
- Note the significant difference in viscosity. Hydrolysis of the cellulose backbone by the cellulase reduces the molecular weight of the methyl cellulose polymer and hence lowers its viscosity.
Extension work: Measure of Viscosity
- To obtain a numerical measure of viscosity, reduce the amount of liquid added to the methyl cellulose solution from 20mL to 1.0mL. That is, add 1.0mL of water to the 50mL volume of 3% methyl cellulose that is the control and add 1.0mL of 1.0% cellulase solution to the test beaker. Note the start time and stir each beaker on a magnetic stirrer.
- Set up two 10mL syringe barrels (plunger removed) vertically on a retort stand with 50mL beakers below the outlets. Block the outlets. (A plug of “blue tack” is suitable if you do not have syringe caps).
- After 2.5 minutes of stirring, pour 10mL of each mix into a syringe barrel then return the two main mixtures to their respective stirrers.
- When ready, remove the outlet plugs from the syringes simultaneously.
- Measure how long it takes for the contents to pass through the syringe barrels.
- For the test mixture, pour a second volume of 10mL into the syringe barrel after a total of 5 minutes of stirring.
- Repeat after stirring for 10 minutes, 15 minutes and 20 minutes.
- Plot the data with viscosity (i.e. seconds to pass through the syringe barrel) on the vertical axis and treatment time (i.e. stirring time in minutes) on the horizontal axis.
- If the data appear too compressed, repeat the experiment but reduce the amount of liquid added in step 1 above to just 0.5mL.
Extension work: Temperature effects on cellulase stability
- Study the effect of temperature on the stability of cellulase. Enzymes are denatured by exposure to heat
- Set up a series of six beakers, each containing 50mL of 3% methyl cellulose.
- Set up a beaker of 1.0% cellulase (150mL) on a hot plate with a thermometer inserted.
- As a control, add 20mL of water to the first beaker of methyl cellulose and stir until homogeneous.
- Turn on the hot plate and withdraw 20mL of cellulase solution when the temperature reaches 50°C. Add the cellulase to the methyl cellulose and stir until homogeneous. Note whether the viscosity is lower than the control or about the same.
- Repeat when the temperature reaches 60°C, 70°C, 80°C and 90°C.
- Note the temperature at which the cellulase is denatured (ceases to be effective at hydrolysing methyl cellulose).
Methyl cellulose solutions should be prepared at least 24 hours in advance. You can make them up to a week before planned use. Because of its high viscosity, a solution of methyl cellulose is difficult to pour. To solve this issue, prepare the volume you need in each experiment beaker (50mL), rather than making a single large batch and trying to pour it into smaller volumes.
- 40 min
- Methyl Cellulose 3%
- Cellulase 1% (v.v)
- Distilled water
- Transfer Pipettes
- Magnetic Stirrers
- Beakers (200ml)
- Measuring cylinder
- 10ml syringe x 2
- Safety Gloves Required
- Safety Goggles Required
- Exercise caution handling lab equipment and chemicals
- Avoid any direct contact with the solution and wash hands thoroughly.
- Keep chemicals away from open flames