Introduction:
- It is a technique specifically designed to limit the movement of an enyme.
- Immobilized enzymes refer to enzymes that have been physically or chemically bound or attached to a solid support or matrix, preventing them from freely diffusing in a solution.
- Immobilization is a widely used technique in biotechnology and various industrial processes due to the numerous advantages it offers over using free, soluble enzymes.
- Since 1960, the emergence of immobilizing enzymes has been an attractive topic
- The idea of enzymes’ immobilization was introduced by Nelson and Griffin in 1916 after they noticed that invertase can hydrolyse sucrose after being absorbed onto charcoal.
- Enzyme immobilization is a common practice, primarily to reduce the economic impact of enzyme costs on processes by allowing for multiple enzyme reuses. It also helps to reduce operating costs as the immobilization technique may change the behaviour of the enzyme, lowering costs for both the enzyme and the final product.
Benefits of immobilized enzyme:
- Increased stability: Immobilization can protect enzymes from harsh operating conditions, such as high temperatures, extremes of pH, and organic solvents, which could otherwise denature or inactivate the enzymes. This improved stability allows for longer enzyme lifetimes and more extended usage in industrial processes. Protection form degradation and deactivation.
- Enhanced reusability: Immobilized enzymes can be easily separated from the reaction mixture after use, making them simpler to recover, regenerate, and reuse. This reduces the cost of enzyme production and contributes to overall process efficiency.
- Improved product purity: Immobilization can aid in the separation of the enzyme from the reaction products and unreacted substrates, resulting in higher product yields and purities.
- Facilitated enzyme handling: Immobilized enzymes are often more manageable and convenient to handle than their soluble counterparts, especially in continuous flow processes.
Fig: Application of immobilised enzyme
Properties of carrier matrix for ideal immobilization:
Biocompatibility: The carrier matrix needs to be biocompatible in order to maintain the activity, stability, and conformation of the specific enzyme. It shouldn’t cause the enzyme to become denaturized or inactive.
Chemical Stability: The matrix should be chemically stable over a wide range of pH values and temperatures to ensure that it does not degrade or release harmful components into the reaction mixture.
Fig: Properties of carrier matrix for ideal immobilization of enzymes
Mechanical Stability: The carrier matrix should be mechanically stable in order to withstand physical pressures during handling, stirring, and other process conditions.
Non-Toxic: The carrier matrix should not be hazardous to cells, organisms, or humans, particularly if the immobilized enzyme will be used in biomedical or food-related applications.
Long-Term Stability: The carrier matrix should offer the immobilized enzyme with long-term stability, allowing for repeated cycles of use without significant loss of activity.
Economical: The carrier matrix should be cost-effective, particularly for large-scale applications.
Methods:
Covalent binding
- Covalent binding immobilizes enzymes by making strong bonds with support materials.
- Advantages include greater enzyme endurance, higher stereospecificity, and low enzyme leakage.
- Hydrophilic polysaccharide polymers and electrophilic groups are examples of common support materials.
- Covalent binding is possible with amino acid functional groups such as carboxylic, hydroxyl, amino, and sulfhydryl.
- Complex immobilization processes, probable enzyme denaturation, reduced enzyme loading capacity, and limited enzyme mobility are all disadvantages.
- Despite its limitations, covalent binding is preferred for its potential to improve enzyme stability and permit enzyme recovery and reuse.
Fig: Covalent binding: Derivation and Activation
Adsorption
This is the oldest and most basic strategy. In this form, enzyme adheres to the surface of the water-insoluble carrier matrix.
The binding is nonspecific, similar to electrostatic or hydrophobic affinity binding to a specific ligand.
The link between enzymes and the carrier matrix is typically strong, but it can be decreased by a variety of factors such as substrate addition, pH, or ionic strength.
The bonding in enzyme adsorption is performed through weak forces such as the hydrogen bond and the Vander Waal force.
Fig: Adsorption
Entrapment
Entrapment is a method of enzyme immobilization in which enzymes are physically trapped within a porous matrix or a gel-like material without generating strong covalent or chemical interactions.
Instead, the enzymes are trapped within the network of the support material, enabling substrates and products to diffuse in and out while keeping the enzymes isolated.
Fig: Entrapment
Encapsulation
It involves membrane confinement. In an aqueous solution, an enzyme is contained within the semipermeable membrane of a capsule.
This procedure permits the exchange of substrate and product, but not an enzyme. The efficiency of encapsulation depends on the stability of the enzyme.
The capsule’s matrix consists of a semi-permeable membrane, which may be polymeric, lipoid, non-ionic, etc.
Examples: It includes nitrocellulose, nylon semi-permeable matrix etc.
Fig: Encapsulation
Factors Affecting Enzyme Immobilization:
- Choice of Support Material
- Chemical Composition of the Support
- Enzyme Stability
- Immobilization Method
- pH and Temperature
- Cross-Linking Agents
kinetics of immobilized enzymes:
Enzyme kinetics aims to understand the quantitative aspects of enzyme-catalyzed reactions, which is essential for optimizing industrial processes, drug development, and gaining insights into biological mechanisms.
The kinetics of immobilized enzymes refers to the study of the enzymatic reactions that occur when enzymes are physically attached or immobilized onto a solid support or matrix, rather than being free in solution.
Partition Effects
Partition effects is related to the distribution of molecules between the immobilization matrix (solid support) and the surrounding solution. When an enzyme is immobilized on a solid support, it can interact with both the surface of the support and the solution in which it is immersed.
Fig: kinetics of immobilized enzymes
Due to the physio-chemical property, the polymer may attract or repel the substrate, product or inhibitor molecules, towards or away from it.
Due to this partitioning of molecules occurs between bulk solution and polymeric surface. This is called partition effect. It is represented by L.
If the polymer is positively charged. Then in case of negatively charged substrate, substrate is attracted towards polymers. Enzyme efficiency increased.
In case of positively charged substrate, it will be repelled by polymer. Enzyme efficiency is decreased.
Similarly, if the polymer is hydrophobic, it will attract hydrophobic molecules only and repel hydrophilic molecules and vice versa. So, we can conclude that during selection of immobilized enzyme, we must explore the properties of polymer also for the successful enzyme catalytic reaction.
Diffusion Effects
Diffusion effects are primarily concerned with the movement of substrates and products within the immobilization matrix. These effects can influence the rate at which reactants reach the enzyme’s active sites and the rate at which products are released.
For the successful catalysis of enzymatic reaction, substrate must diffuse towards the enzyme surface and product must diffuse away from the enzyme surface. Polymer also affect the diffusion of molecules i.e., substrate and products.
External diffusion- Transport of substrate towards the polymeric surface and products away from the polymeric surface into the bulk solution.
Internal diffusion- Transport of substrate and products within the pores of polymers to reach up to the surface of the internal diffusion.
Polymer must be selected in such a way so that it can allow the substrate and product to pass through it.
Applications:
Food Sector
- Alkaline Protease Immobilization: Immobilized alkaline protease onto mesoporous zeolite/silica, enabling the conversion of milk into cheese within a short 90-minute duration. The immobilized enzyme retained 74% of its catalytic activity after 16 storage days, compared to free proteases, which retained only 50% of their initial activity.
- Protease for Gluten Removal: Another application involved immobilizing protease by cross-linking with chitosan for eliminating gluten in beer. Immobilization significantly enhanced the enzyme’s effectiveness, reducing gluten content from 65 mg/kg to 15 mg/kg after 10 hours of treatment.
- Amylase Immobilization: Covalent immobilization of amylase with chitosan improved thermal stability by 35%, increased resistance to pH inactivation, and enhanced product yield during barley hydrolysis by 1.5-fold.
- Application in Processed Juice Production: Immobilized enzymes are extensively used in processed juice production. For instance, pectinase immobilized using poly (vinyl alcohol) effectively clarified apple juice, achieving an 80% reduction in turbidity after 3 cycles, with the enzyme being reusable up to 8 times while retaining 20% of its initial efficiency.
Detergent Industry
- Immobilized Protease on Wool: Vasconcelos et al. investigated the efficacy of covalently bonded immobilized protease with Eudragit on wool. The immobilized protease retained 76% of the original tensile strength, while samples with free enzyme retained only 37%. Furthermore, the immobilized enzyme did not damage the wool fibers.
- Immobilized Nanoenzyme: An immobilized protease in the form of a nanoenzyme retained a substantial catalytic efficacy for 12 cycles and maintained 63.6% activity after 1 hour at 60 °C, in contrast to free enzymes that lost their entire activity.
Textile Sector
- Immobilized Keratinase: Keratinase was immobilized using chitosan-β-cyclodextrin through cross-linking. The resulting conjugate exhibited high thermal stability (70 °C) and significantly improved storage stability, retaining up to 53.5% activity after 30 days, compared to the free enzyme.
- Immobilized Cellulases: Cellulases are widely used in textile processing for modifying cellulosic fibers. Immobilizing cellulases onto kaolin through covalent bonding/adsorption improved fabric quality, providing excellent pilling resistance, better tensile strength, and the ability to reuse cellulases for 3 successive cycles.
- Noncovalent Immobilization of Cellulases: Immobilized cellulase enzymes with Eudragit L-100 and Eudragit S-100 through noncovalent immobilization. The immobilized enzymes showed higher stability and preserved enzyme efficiency, with 51% and 42% retention of activity by the third cycle for Eudragit S-100 and Eudragit L-100, respectively. The treated fabrics were softer than control samples.
- Cellulase Immobilization on Calcium Alginate Starch: Cellulase enzymes were immobilized by adsorption on calcium alginate starch, resulting in minimal weight loss, lessening of tensile strength, and an improved index of whiteness.
- Immobilized Cellulase with Epoxy Resin: Immobilized cellulase with epoxy resin was effectively used in biopolishing of fabrics for 6 cycles without experiencing tensile strength loss.
- Chitosan Coating for Cellulase Immobilization: Chitosan was coated using poly (vinyl alcohol), and cellulases were immobilized on it, retaining 52% of the enzyme’s activity after 8 cycles.