Immobilising enzymes
Immobilised enzymes are enzymes that have been fixed in some way to an inert, stationary, and insoluble material to prevent them from diffusing freely in a solution. This process is crucial for various industrial, medical, and food technology applications.
Method of Immobilisation (using Alginate Beads): A common method involves trapping enzymes within alginate beads. The process typically involves:
Preparing a solution of sodium alginate (a polysaccharide).
Stirring the chosen enzyme solution into the sodium alginate solution.
Filling a syringe with this alginate-enzyme mixture and dripping it into a beaker of calcium chloride solution.
The sodium alginate and calcium chloride react instantly to form jelly, turning each droplet into a small bead. These insoluble pellets can then be separated and washed with distilled water to harden.
Once immobilised, these beads can be gently packed into a column, and the substrate liquid can be allowed to trickle steadily over them. The enzymes within the beads then catalyse the reaction, converting the substrate into product, which can be collected from the bottom of the column.
Advantages of Using Immobilised Enzymes: There are several significant advantages to using immobilised enzymes compared to using enzymes free in solution:
Reusability: The enzyme preparation can be re-used multiple times, which is efficient and cost-effective, as enzymes can be expensive.
Product Purity: The product is obtained enzyme-free. This means there is no need for further processing or filtering to separate the enzyme from the product, reducing purification costs.
Increased Stability/Tolerance: Immobilised enzymes tend to be more stable and long-lasting, exhibiting a greater tolerance to changes in temperature and pH compared to enzymes in solution. This is partly because their molecules are held firmly in shape by the inert matrix (e.g., alginate), which prevents them from denaturing as easily. Additionally, parts of the molecules embedded in the beads may not be fully exposed to temperature or pH changes.
Disadvantages of Immobilisation: While highly advantageous, immobilisation does have some drawbacks:
Cost of Preparation: The creation of stable, hardened pellets is an added expense, which can reflect in the cost of the industrial product.
Potential for Detachment: If the enzyme becomes detached from the matrix, it will appear in the product as a contaminant, potentially unnoticed.
Altered Activity: The immobilisation mechanism chosen must not alter the enzyme's shape or catalytic ability. Sometimes, the active site may be distorted by immobilisation.
Diffusion Limitations: The substrate needs to pass through the matrix to reach the enzyme, and some product may be retained within the matrix.
Applications and Examples: Immobilised enzymes are used in various practical applications:
Lactose-free milk production: The enzyme lactase can be immobilised to hydrolyse lactose in milk into glucose and galactose, making it suitable for lactose-intolerant individuals.
Biosensors: Immobilised enzymes, such as glucose oxidase, are used in biosensors and dipsticks for measuring glucose concentration in blood or urine. In these devices, glucose is oxidized by the enzyme, producing hydrogen peroxide, which then causes a color change or an electrical signal, indicating glucose levels.
Industrial processes: They are widely used in processes like fructose syrup manufacture (using glucose isomerase), and in detergents (using proteases).
Medical treatments: Examples include the use of adenosine deaminase (AD) for severe combined immunodeficiency disease (SCID), where the enzyme may be administered to patients or gene therapy involves genetically modifying stem cells with the normal AD gene.
Overall, immobilised enzymes represent a significant advancement in biotechnology, offering enhanced control, reusability, and stability, making them highly valuable for numerous applications.
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