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General PropertiesActive Site TheoryDenaturationFactors Affecting Enzyme Action | Bioprocessing | Mandatory Activities

Enzymes  

Proteins that function as biological catalysts are called enzymes.

Enzymes speed up specific metabolic reactions.

Low contamination, low temperature and fast metabolism are only possible with enzymes.

Metabolism is fast, with the product made to a high degree of purity.


General Properties  

  • Catalysts
  • Protein
  • Specific
  • Reversible — can catalyse the reaction in both directions
  • Denatured by high temperature and change in pH
  • Rate of action affected by temperature and pH

Protein Nature of Enzymes

  • Composed of C, H, O and N. Sulphur (S) may also be present.
  • One or more polypetide chains - large number of linked amino acids.
  • Formed by the ribosomes – translation of mRNA during protein synthesis.
  • Denatured by high temperature and unfavourable pH.

Folded Shape of Enzymes

  • The polypeptide chains are folded into a particular three-dimensional shape.
  • The correct folded shape is essential for enzyme action.
  • The shape gives the enzyme special domains that function as active sites.
  • The compatible substrate molecules bind to the active site.
  • Different enzymes have a differently shaped active site.

Roles of Enzymes in Plants and Animals
Enzymes catalyse all metabolic reactions.

  • Lower the activation energy – the energy input needed to bring about the reaction.
  • Regulate the thousands of different metabolic reactions in a cell and in the organism.
  • The activity of a cell is determined by which enzymes are active in the cell at that time.
  • Cell activity is altered by removing specific enzymes and/or synthesising new enzymes.


Active Site Theory  

“Lock and Key Hypothesis and Induced Fit”

  • The enzyme’s active site has a shape closely complementary to the substrate The substrate locks into the active site of the enzyme.
  • The active site alters its shape holding the substrate more tightly and straining it.
  • An enzyme-substrate complex is formed.
  • The substrate undergoes a chemical change – a new substance, product, is formed.
  • The product is released from the active site.
  • The free unaltered active site is ready to receive fresh substrate.

Textbook Diagram: Enzyme Action Sequence.

Native Enzyme: an enzyme that can function normally because its active site has the correct shape.

Denatured Enzyme: an enzyme that cannot operate because the shape of its active site is altered and so the substrate cannot combine with it – change in shape resulting in loss of biological function.

Renatured Enzyme: the denatured enzyme has recovered it shape and function when the temperature and/or pH are again favourable.


Denaturation  

Heat is a form of energy. The addition of heat can cause a change in the three-dimensional shape of a protein.

The new shape results in a change in the chemical properties of the protein.

The protein is said to be denatured if the shape change causes it to lose its normal biological activity. Denaturation is not usually reversible.

Some denatured proteins do renature when their normal environmental conditions are restored.


Factors Affecting Enzyme Action  

Enzyme action occurs when the enzyme and substrate collide.

During the collision the substrate slots into the active site of the enzyme.

Collisions happen because of the rapid random movement of molecules in liquids.

(i) Temperature

Textbook Graph: Temperature-Enzyme Graph

  • at 0°C enzyme action is low because the movement of molecules is low
  • the collision frequency between enzyme and substrate is therefore low
  • increasing the temperature speed up the movement of molecules
  • collision frequency increases raising the collision frequency
  • therefore enzyme action increases
  • maximum enzyme action at 40°C - maximum collision frequency between native enzymes and substrates
  • enzyme action decreases above 40°C because the enzymes are denaturing
  • when all the enzymes are denatured enzyme action stops

(ii) pH

Textbook Graph: pH-Enzyme Graph

  • enzyme action is greatest within a narrow range of pH, because
  • all the enzymes are in their native state
  • increased acidity or alkalinity decreases the ability of the substrates to bind to the active site
  • and so enzyme action decreases
  • a major pH change denatures the enzymes so enzyme action stops


Optimum Enzyme Activity
Enzymes function best within a narrow range of temperature and pH.

Human intracellular enzymes work best at 37°C and pH 7.


Bioprocessing  

Bioprocessing is the use of biological materials (organisms, cells, organelles, enzymes) to carry out manufacturing or treatment prodedures of commercial or scientific interest.

Examples of Bioprocessing with Enzymes:

  • Glucose Isomerase: production of fructose from glucose.
  • Sucrase: production of glucose and fructose from sucrose.

Immobilised enzymes are not free in solution – for example they cam be held in a bead of soft permeable gel or coat the internal surface of a porous solid.

Teztbook Diagram: Bioreactor Setup.

Bioprocessing Procedure

  • Bioprocessing with immobilised enzymes is carried out in a bioreactor.
  • The gel beads, with the immobilised enzymes, are held in suspension in the nutrient medium.
  • The bioreactor is sterile – micro-organisms would have a major negative impact.
  • Temperature, pH, substrate and product concentration and waste level are checked constantly.
  • The product can be produced by continuous flow or batch processing.

Advantages of Immobilised Enzymes

  • Easier purification of the product as the separation of the enzyme beads is not a problem.
  • Easy to recover and recycle the enzymes – more economical process.
  • The enzymes remain functional for much longer as it is a gentler process.


Mandatory Activities  

To Determine the Effect of pH on the Rate of Enzyme Action.

Textbook Diagram: show set up of the apparatus.

  • Substrate: starch. Enzyme: amylase.
  • Use the same volume of the same substrate and enzyme solutions.
  • Temperatrue at 37°C: heated water bath and thermomenter.
  • Different pH values: use buffer solutions – pH 3, pH 5, pH 7, pH 9, pH 11.
  • Experiment: starch + buffer + amylase.
  • Control: starch + buffer + water.
  • Each minute test a small sample of experiment and control for starch using iodine.
  • Control Results: no starch break down as blue-black is the constant result.
  • Experiment Results: record the time at which each first produced a yellow-brown result.
  • Yellow-brown means starch is not present, therefore starch breakdown occurred.
  • Calculate rate of enzyme activity: Rate = 1 ???ime
  • Repeat many times to verify the results.
  • Graph the results – pH on x-axis.


To Determine the Effect of Temperature on the Rate of Enzyme Action

  • Substrate: starch. Enzyme: amylase.
  • Use the same volume of the same substrate and enzyme solutions.
  • Suitable constant pH: pH 8 – use a buffer solution.
  • Different Temperatures: 0°C – use ice bath, 20°C – room temperature, use a heated water bath and thermometer for temperatures greater than room temperature (30°C, 40°C, 50°C….)
  • Experiment: starch + buffer + amylase.>
  • Control: starch + buffer + water.
  • Each minute test a small sample of experiment and control for starch using iodine.
  • Control Results: no starch break down as blue-black is the constant result.
  • Experiment Results: record the time at which each first produced a yellow-brown result.
  • Yellow-brown means starch is not present, therefore starch breakdown occurred.
  • Calculate rate of enzyme activity: Rate = 1 ???time
  • Repeat many times to verify the results.
  • Graph the results – temperature on x-axis.


Investigate the Effect of Heat Denaturation on the Activity of an Enzyme

  • Substrate: starch. Enzyme: amylase.
  • Use the same volume of the same substrate and enzyme solutions.
  • Suitable constant pH: pH 8 – use a buffer solution.
  • Temperatures: 37°C – human body temperature.
  • Control: starch + buffer + native amylase.
  • Experiment: starch + buffer + boiled saliva (helded at 100°C for 10 minutes).
  • Each minute test a small sample of experiment and control for starch using iodine.
  • Experiment: no starch break down as blue-black is the constant result.
  • Control: the yellow-brown colour indicates starch breakdown.
  • Native Amylase: starch breakdown. Denatured Amylase: no breakdown of starch
  • Conclusion: heat denaturation of the enzyme results in the loss of its catalytic activity.
  • Repeat many times to verify the results.


Prepare an Enzyme Immobilisation and Examine its Application

Preparation of Immobilised Enzyme

  • Prepare a solution of calcium chloride.
  • Mix sodium alginate and amylase solutions.
  • Draw the mixture into a syringe.
  • Gently squeeze drops of the mixture into the calcium chloride solution.
  • Allow the beads of immobilised amylase to harden in the calcium chloride solution.
  • Collect the beads in a strainer and wash with distilled water.


Examination of the Application of Immobilised Enzyme

  • Control Jar: amylase solution mixed with a starch solution.
  • Experiment Jar: immobilised enzyme beads in the same volume of the starch solution.
  • Temperature: 20°C – room temperature.
  • Swirl both jars equally.
  • Every minute test a sample from each for starch using iodine.
  • Record the time to achieve a yellow-brown colour – starch breakdown completed.
  • Now test for reducing sugar using Benedict’s Reagent – brick-red colour forms.
  • Note that it is much easier to remove the immobilised enzyme than the free enzyme from the product solution and the immobilised enzyme beads can be easily reused.
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