Immobilization of enzymes MCQs With Answer

Introduction: Immobilization of enzymes is a key topic in Bioprocess Engineering and Technology, crucial for M.Pharm students who aim to design robust biocatalytic processes. This blog presents focused multiple-choice questions to strengthen conceptual understanding and application of immobilization principles — methods (adsorption, entrapment, covalent binding, cross-linking, CLEAs), carrier selection, kinetics, mass transfer limitations, reactor design, and stability issues. Each question targets practical considerations such as enzyme orientation, multipoint attachment, spacer arms, and industrial reactor choices, preparing students for problem-solving in pharmaceutical bioprocesses. Use these MCQs to assess readiness for exams and to consolidate core concepts required for developing stable, reusable enzyme systems in drug manufacture.

Q1. What is the most accurate definition of enzyme immobilization?

• Physical or chemical confinement of an enzyme so that it retains catalytic activity and can be reused
• Mixing enzyme with substrate to form a transient complex
• Cloning and expressing enzyme genes in a host organism
• Covalent digestion of an enzyme into inactive peptides

Correct Answer: Physical or chemical confinement of an enzyme so that it retains catalytic activity and can be reused

Q2. Which primary advantage of immobilized enzymes is most relevant for industrial pharmaceutical processes?

• Elimination of the need for substrate purification
• Enhanced operational stability and ability to repeatedly use the biocatalyst
• Guaranteed increase in intrinsic catalytic rate (kcat)
• Complete prevention of product inhibition

Correct Answer: Enhanced operational stability and ability to repeatedly use the biocatalyst

Q3. Which immobilization method creates enzyme aggregates cross-linked without an external carrier?

• Physical adsorption onto activated carbon
• Entrapment in calcium alginate beads
• Cross-Linked Enzyme Aggregates (CLEAs)
• Covalent attachment to silica via silane coupling

Correct Answer: Cross-Linked Enzyme Aggregates (CLEAs)

Q4. Which support property most strongly determines internal diffusion limitations for substrates reaching immobilized enzymes?

• Surface hydrophobicity of the carrier
• Pore size distribution and porosity
• Color and optical transparency of the support
• Electrical conductivity of the carrier

Correct Answer: Pore size distribution and porosity

Q5. Which reagent is most commonly used for covalent cross-linking of enzymes and supports by forming Schiff bases?

• Calcium chloride
• Glutaraldehyde
• Sodium borohydride
• Polyethylene glycol

Correct Answer: Glutaraldehyde

Q6. How does immobilization typically affect the apparent Michaelis constant (Km) and maximal velocity (Vmax) compared to the free enzyme?

• Km decreases and Vmax increases due to enhanced substrate binding
• Km increases and Vmax decreases often because of mass transfer/resistance and steric effects
• Both Km and Vmax remain exactly the same under all conditions
• Km becomes zero and Vmax becomes infinite

Correct Answer: Km increases and Vmax decreases often because of mass transfer/resistance and steric effects

Q7. Which disadvantage is most associated with entrapment of enzymes in gels like alginate?

• Excessive covalent modification of active site residues
• High risk of enzyme proteolysis by gel components
• Strong mass transfer limitations leading to lowered apparent activity
• Immediate and irreversible enzyme denaturation upon entrapment

Correct Answer: Strong mass transfer limitations leading to lowered apparent activity

Q8. What is the main benefit of introducing a spacer arm between the carrier surface and the immobilized enzyme?

• Spacers permanently block substrate access to the active site
• Spacers reduce steric hindrance and improve substrate accessibility to the enzyme
• Spacers solely reduce enzyme stability under process conditions
• Spacers convert covalent binding into reversible adsorption

Correct Answer: Spacers reduce steric hindrance and improve substrate accessibility to the enzyme

Q9. Which immobilization technique generally results in the lowest enzyme leaching during operation?

• Physical adsorption onto hydrophobic supports
• Entrapment in highly porous beads without chemical bonds
• Covalent attachment to functionalized supports
• Simple mixing of enzyme with inert powder

Correct Answer: Covalent attachment to functionalized supports

Q10. For a high volumetric activity and no carrier-related dilution of activity, which approach is preferred?

• Using extremely large inert carriers to reduce enzyme proximity
• Cross-Linked Enzyme Aggregates (carrier-free CLEAs)
• Adsorbing tiny amounts of enzyme on glass slides
• Entrapment in very dilute hydrogel with low enzyme loading

Correct Answer: Cross-Linked Enzyme Aggregates (carrier-free CLEAs)

Q11. Which analytical technique is most appropriate to visualize the spatial distribution of enzyme within porous carriers?

• Nuclear Magnetic Resonance (NMR) spectroscopy
• Confocal Laser Scanning Microscopy with fluorescently labeled enzyme
• Gas chromatography of the immobilized matrix
• Simple UV absorbance of the supernatant only

Correct Answer: Confocal Laser Scanning Microscopy with fluorescently labeled enzyme

Q12. Multipoint covalent attachment of an enzyme to a support typically leads to which effect on enzyme properties?

• Increased conformational flexibility and reduced thermal stability
• Restricted mobility, increased rigidity and enhanced thermal stability
• Complete loss of all catalytic residues leading to inactivity
• Conversion of enzyme to a small molecule catalyst

Correct Answer: Restricted mobility, increased rigidity and enhanced thermal stability

Q13. Which reactor type is most commonly used for continuous industrial processes with immobilized enzymes?

• Packed-bed (fixed-bed) reactor
• Batch flask with free enzyme only
• Shake-flask static reactor
• Single-use disposable bag reactor for free enzyme

Correct Answer: Packed-bed (fixed-bed) reactor

Q14. What is the common cause of operational loss of activity in immobilized enzyme systems during repeated cycles?

• Complete chemical conversion of substrate to carrier material
• Leaching of loosely bound enzyme, mechanical shear, and gradual deactivation
• Spontaneous mutation of enzyme amino-acid sequence
• Permanent conversion of all substrates to inert compounds that poison the enzyme

Correct Answer: Leaching of loosely bound enzyme, mechanical shear, and gradual deactivation

Q15. Which support material is most appropriate when very high surface area and controlled pore size are required for enzyme immobilization?

• Controlled pore glass (CPG) or mesoporous silica
• Bulk polystyrene sheets with no porosity
• Solid metal discs without surface modification
• Transparent plastic film with no pores

Correct Answer: Controlled pore glass (CPG) or mesoporous silica

Q16. How is “activity recovery” defined after immobilization?

• The ratio of immobilized enzyme mass to support mass
• The percentage of initial free-enzyme catalytic activity that remains after immobilization
• The time taken for the immobilized enzyme to reach half its activity
• The absolute amount of protein bound per gram of carrier regardless of activity

Correct Answer: The percentage of initial free-enzyme catalytic activity that remains after immobilization

Q17. Which strategy is frequently used to achieve site-specific covalent immobilization of recombinant enzymes?

• Random adsorption on unmodified silica
• Use of engineered tags (e.g., His-tag, Cys-tag) and specific affinity or thiol-reactive chemistries
• Heating enzyme above its melting temperature to enhance binding
• Mixing enzyme with excess proteases to create new binding sites

Correct Answer: Use of engineered tags (e.g., His-tag, Cys-tag) and specific affinity or thiol-reactive chemistries

Q18. What is the most probable reason an immobilized enzyme shows a higher apparent Km than the free enzyme?

• The immobilized enzyme intrinsically has fewer active sites per molecule
• Diffusion limitations and reduced effective substrate concentration at the active site
• Complete denaturation of the enzyme tertiary structure
• Presence of enzyme inhibitors always formed during immobilization

Correct Answer: Diffusion limitations and reduced effective substrate concentration at the active site

Q19. Co-immobilization of multiple enzymes on the same carrier primarily benefits which phenomenon in multi-step biotransformations?

• Prevention of enzyme aggregation only
• Substrate channeling and improved intermediate transfer between enzymes
• Complete elimination of all mass transfer resistances
• Guaranteed identical pH optima for all enzymes involved

Correct Answer: Substrate channeling and improved intermediate transfer between enzymes

Q20. Which statement best describes Cross-Linked Enzyme Aggregates (CLEAs)?

• CLEAs require a solid inert carrier and are very dilute
• CLEAs are carrier-free, formed by precipitating enzymes followed by cross-linking to give high volumetric activity
• CLEAs are single-enzyme monolayers adsorbed on glass
• CLEAs are enzymes covalently attached to magnetic nanoparticles exclusively

Correct Answer: CLEAs are carrier-free, formed by precipitating enzymes followed by cross-linking to give high volumetric activity

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