Investigating Enzymes and Industrial Uses

Factors affecting enzyme activity

Enzymes are proteins with a specific 3D shape. The substrate fits the active site (induced fit), a reaction happens and the products leave. The enzyme is not used up. Anything that changes how often substrate and active site meet, or changes the shape of the active site, changes the rate of reaction.

The rate is measured as the amount of product formed (or substrate used up) per unit time:

Enzymes work by lowering the activation energy of a reaction, the energy needed to start it. In the induced fit model, the active site changes shape slightly as the substrate binds. This strains the substrate's bonds, so the reaction starts more easily and much faster.

Four factors you must know: temperature, pH, substrate concentration and enzyme concentration.

Temperature

• As temperature rises, molecules move faster. Collisions between enzyme and substrate are more frequent and more energetic, so the rate increases.

• At the optimum temperature the rate is highest (about 37 °C for human enzymes).

• Above the optimum, the bonds that hold the 3D shape break. The active site changes shape and the substrate no longer fits. The enzyme is denatured. This is usually permanent.

• Cold does not denature an enzyme. At low temperatures the enzyme is just slow.

pH

Each enzyme has an optimum pH, the pH at which its rate is highest. Pepsin in the stomach works best at about pH 2, amylase in saliva at about pH 7 and trypsin in the small intestine at about pH 8. Extreme pH changes the charges on the amino acids, disrupts the bonds that maintain the shape, and denatures the enzyme.

Substrate and enzyme concentration

• More substrate: the rate rises steeply at first because more active sites are occupied. Then it levels off, since all active sites are in use (the enzyme is saturated). Only adding more enzyme can raise the rate further.

• More enzyme (with plenty of substrate): the rate increases in proportion, because there are more active sites.

Reading rate data

In the exam you are often given a table or graph and asked to calculate a rate, find the optimum and explain the shape.

Investigating enzymes in the lab

You must carry out investigations of factors affecting enzyme activity. Typical set-ups:

• Catalase (liver or potato extract) with hydrogen peroxide: measure the height of foam or the volume of oxygen collected.

• Amylase with starch: take samples at intervals and test with iodine solution. Blue-black means starch is still present, and the time to reach no colour change gives a measure of rate.

Variables and controls

• Independent variable: the one you change (for example pH).

• Dependent variable: the one you measure (for example foam height).

• Controlled variables: everything else, kept the same (volumes, concentrations, temperature, time).

Getting a range of values

• Temperature: water baths set at, for example, 10, 20, 30, 40, 50, 60 °C. Let the tubes reach the temperature before mixing.

• pH: use buffer solutions at different pH values (for example pH 3, 5, 7, 9, 11).

• Substrate concentration: dilute a stock solution to give a series of concentrations.

The control

A control shows that the effect is due to the enzyme. Use a boiled (denatured) extract in place of the active enzyme. If no reaction happens, the enzyme was responsible.

Reliability and precision

• Repeat each value at least three times and calculate a mean (more reliable).

• Use accurate measuring equipment (a syringe or a graduated pipette) for more precise results.

• Use a timer rather than estimating, and remove anomalous results.

Inhibitors and substrate concentration

An inhibitor is a substance that reduces enzyme activity.

• A competitive inhibitor has a shape similar to the substrate. It binds to the active site and blocks the substrate. If you add more substrate, it outcompetes the inhibitor, so the rate can recover. The graph reaches the same maximum rate, but only at a higher substrate concentration.

• A non-competitive inhibitor binds elsewhere on the enzyme and changes its shape, so the active site no longer works. Adding more substrate does not overcome it.

Industrial uses and immobilised enzymes

Enzymes work at mild temperatures and pH, they are specific and they save energy. So they are used in many industries:

Immobilised enzymes

An immobilised enzyme is fixed to, or trapped in, an inert insoluble material so that it stays in one place while the substrate flows past.

Common methods:

• Entrapment in alginate beads (jelly-like beads of calcium alginate).

• Adsorption onto a surface, such as glass beads.

• Covalent bonding to a support.

• Membrane retention behind a partially permeable membrane.

In a column, the substrate (for example milk) is poured in at the top, flows slowly through the beads, and the product (for example lactose-free milk) leaves at the bottom.

Advantages and disadvantages

Advantages

• The enzyme can be reused, which lowers costs.

• The product is not contaminated with enzyme, so there is no need for an extra purification step.

• Allows a continuous process rather than batches.

• The enzyme is often more stable. Attached to the support (by bonds or trapped in the bead), its 3D shape is held in place, so heat or a change in pH is less likely to unfold it. It tolerates changes in temperature and pH better.

Disadvantages

• Setting up costs more (the beads, supports and equipment), although the reuse can repay this over time.

• The substrate must diffuse into the beads, so the reaction can be slower than with free enzyme.

• Some activity may be lost when the enzyme is attached, since the active site can be distorted.

Back to the start: the dairy reuses its lactase because it is immobilised in beads in a column. To test what makes an enzyme work best, change one variable at a time, control the rest, include a boiled-enzyme control and repeat.

Summary

• Rate of reaction = amount of product ÷ time. It depends on temperature, pH, enzyme concentration and substrate concentration.

• Above the optimum temperature or outside the optimum pH, enzymes are denatured: the shape of the active site changes. Cold only slows them down.

• Substrate concentration: the rate rises, then levels off when the enzyme is saturated. More enzyme raises the maximum rate.

• Competitive inhibitors block the active site and can be overcome with more substrate. Non-competitive inhibitors cannot.

• Investigations: identify the independent, dependent and controlled variables, use buffers or water baths, a boiled-enzyme control and repeats.

• Immobilised enzymes (for example lactase in alginate beads) can be reused, give a pure product, allow continuous processes and are more stable, but cost more to set up and may react more slowly.