Purification of enzymes MCQs With Answer

Purification of enzymes MCQs With Answer is designed for M.Pharm students studying Advanced Pharmaceutical Biotechnology. This quiz collection focuses on theoretical concepts and practical strategies used to isolate and characterize enzymes for research and therapeutic applications. Questions cover classical and modern purification techniques — centrifugation, precipitation, membrane concentration, chromatographic separations (ion exchange, affinity, size exclusion, hydrophobic interaction), analytical methods, yield and fold calculations, stabilization, refolding, and quality control. Each MCQ aims to test understanding of principles, experimental design choices, troubleshooting, and interpretation of results that are critical for efficient enzyme purification in pharmaceutical development and bioprocessing.

Q1. What does an increase in specific activity during a purification process indicate?

• Loss of enzyme activity due to denaturation
• Enrichment of the target enzyme relative to total protein
• Increase in total protein concentration
• Improved substrate conversion by all proteins present

Correct Answer: Enrichment of the target enzyme relative to total protein

Q2. Which statement correctly distinguishes differential centrifugation from density gradient centrifugation?

• Differential centrifugation separates particles solely by buoyant density while gradients separate by size
• Differential centrifugation uses increasing speeds to pellet components by size/mass; density gradients separate components by buoyant density at equilibrium
• Both techniques are identical and interchangeable for purifying soluble enzymes
• Density gradient centrifugation cannot resolve organelles and is only for small molecules

Correct Answer: Differential centrifugation uses increasing speeds to pellet components by size/mass; density gradients separate components by buoyant density at equilibrium

Q3. What is the principal mechanism by which ammonium sulfate precipitation concentrates proteins?

• Specific binding of ammonium sulfate to the active site of enzymes
• Salting-in effect increasing protein solubility at high ionic strength
• Salting-out effect decreasing protein solubility by competing for water molecules
• Covalent modification of surface residues to promote aggregation

Correct Answer: Salting-out effect decreasing protein solubility by competing for water molecules

Q4. During purification, dialysis is primarily used to:

• Concentrate dilute protein solutions by removing water
• Remove small solutes such as salts or denaturants while retaining macromolecules
• Break disulfide bonds to denature the protein
• Fragment large proteins into peptides

Correct Answer: Remove small solutes such as salts or denaturants while retaining macromolecules

Q5. Which parameter is most critical when selecting an ultrafiltration membrane for enzyme concentration?

• Membrane color
• Nominal molecular weight cut-off (MWCO) relative to enzyme size
• Electrical conductivity of the membrane material
• Presence of immobilized ligand on the membrane

Correct Answer: Nominal molecular weight cut-off (MWCO) relative to enzyme size

Q6. Ion-exchange chromatography separates proteins primarily based on:

• Molecular weight differences under denaturing conditions
• Hydrophobic patches on the protein surface
• Net surface charge of the protein at the working pH
• Specific affinity for metal ions irrespective of charge

Correct Answer: Net surface charge of the protein at the working pH

Q7. Which of the following is a correct advantage of affinity chromatography for enzyme purification?

• It separates proteins exclusively by size without ligands
• It provides high selectivity based on reversible, specific binding to a ligand
• It requires no optimization of binding or elution conditions
• It always yields fully active enzyme without further polishing steps

Correct Answer: It provides high selectivity based on reversible, specific binding to a ligand

Q8. In size-exclusion chromatography (SEC) performed under native conditions, which molecules elute first?

• Small molecules that penetrate the pores deeply
• Molecules with the highest isoelectric point
• The largest molecules that are excluded from the pores
• Molecules with the strongest affinity for the matrix

Correct Answer: The largest molecules that are excluded from the pores

Q9. Which analytical technique is most appropriate to determine the molecular weight of a denatured enzyme subunit?

• Native PAGE followed by activity staining
• SDS-PAGE with molecular weight standards
• Ion-exchange chromatography retention time
• Hydrophobic interaction chromatography elution salt profile

Correct Answer: SDS-PAGE with molecular weight standards

Q10. Isoelectric focusing separates proteins by:

• Affinity to hydrophobic matrices in high salt
• Charge differences by migration to the position where pH equals their pI
• Molecular size in a denaturing gel
• Binding to immobilized metal ions

Correct Answer: Charge differences by migration to the position where pH equals their pI

Q11. Hydrophobic interaction chromatography (HIC) typically requires which condition to promote binding of proteins to the matrix?

• Low ionic strength buffers to expose hydrophobic regions
• High salt concentration to strengthen hydrophobic interactions
• Strong chaotropic agents to denature proteins
• Presence of imidazole to compete for binding

Correct Answer: High salt concentration to strengthen hydrophobic interactions

Q12. How is purification fold calculated during an enzyme purification?

• Final total protein divided by initial total protein
• Initial specific activity divided by final specific activity
• Final specific activity divided by initial specific activity
• Final total activity divided by total protein

Correct Answer: Final specific activity divided by initial specific activity

Q13. Percent yield (recovery) of an enzyme purification step is calculated as:

• (Final total activity / Initial total activity) × 100
• (Final specific activity / Initial specific activity) × 100
• (Initial total protein / Final total protein) × 100
• (Final concentration / Initial concentration) × 100

Correct Answer: (Final total activity / Initial total activity) × 100

Q14. Which additives are commonly used to stabilize enzymes during purification and storage?

• Glycerol, BSA, low concentrations of reducing agent, and appropriate buffer salts
• SDS, high urea, and strong oxidizing agents
• High concentrations of free metal ions that catalyze oxidation
• Concentrated HCl to lower pH drastically

Correct Answer: Glycerol, BSA, low concentrations of reducing agent, and appropriate buffer salts

Q15. Which protease inhibitors would you include in an extraction buffer to broadly protect a target enzyme from degradation?

• PMSF (serine protease inhibitor), EDTA (metalloprotease inhibitor), and pepstatin (aspartic protease inhibitor)
• Imidazole, high salt, and urea
• Sodium dodecyl sulfate, mercuric chloride, and perchloric acid
• Only protease-free water without any inhibitors

Correct Answer: PMSF (serine protease inhibitor), EDTA (metalloprotease inhibitor), and pepstatin (aspartic protease inhibitor)

Q16. What is a standard strategy to refold a recombinant enzyme recovered from inclusion bodies?

• Rapid dilution of denatured protein into refolding buffer with appropriate redox agents and gradual removal of denaturant by dialysis
• Heating the inclusion bodies to 95 °C to accelerate folding
• Treating with proteases to generate smaller fragments that refold independently
• Adding strong organic solvents such as methanol to promote correct disulfide bond formation

Correct Answer: Rapid dilution of denatured protein into refolding buffer with appropriate redox agents and gradual removal of denaturant by dialysis

Q17. Which characteristic best distinguishes FPLC from typical analytical HPLC when purifying enzymes?

• FPLC operates at low pressure with aqueous, protein-friendly buffers, whereas HPLC often uses high pressure and organic solvents
• FPLC uses only organic solvents while HPLC uses only water
• FPLC is suitable for small molecules while HPLC is only for proteins
• There is no practical difference; they are the same technique

Correct Answer: FPLC operates at low pressure with aqueous, protein-friendly buffers, whereas HPLC often uses high pressure and organic solvents

Q18. What is a common elution method for His-tagged recombinant enzymes bound to Ni-NTA affinity resin?

• Increasing concentration of imidazole to competitively displace the His-tag
• Raising the temperature to 100 °C to denature and release bound protein
• Adding SDS to disrupt hydrophobic interactions
• Dialysis against pure water without imidazole

Correct Answer: Increasing concentration of imidazole to competitively displace the His-tag

Q19. Which approach is effective for removal of endotoxin (lipopolysaccharide) contamination from purified enzyme preparations?

• Phase separation with Triton X-114, ion-exchange chromatography, or polymyxin B affinity
• Heating to 80 °C for prolonged periods to inactivate endotoxin
• High concentrations of MgCl2 to precipitate endotoxin- enzyme complexes
• Adding bacterial lysate to neutralize endotoxin

Correct Answer: Phase separation with Triton X-114, ion-exchange chromatography, or polymyxin B affinity

Q20. Which combination of methods provides both identity and purity assessment of a purified enzyme preparation?

• SDS-PAGE with densitometry for purity plus mass spectrometry for identity
• Only measuring buffer conductivity
• Observation of solution color and viscosity alone
• Counting colonies on LB agar plates

Correct Answer: SDS-PAGE with densitometry for purity plus mass spectrometry for identity

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