Extraction of enzymes MCQs With Answer

Introduction: Extraction of enzymes is a critical unit operation in Advanced Pharmaceutical Biotechnology that bridges upstream production and downstream purification. For M.Pharm students, understanding enzyme extraction involves not just isolating proteins from biomass but preserving catalytic activity, maximizing yield, and minimizing contaminants. This blog focuses on core principles: selection of cell disruption techniques, choice of buffers and stabilizers, precipitation and membrane separations, and strategies to prevent denaturation and proteolysis. Emphasis is placed on process parameters (pH, temperature, ionic strength), scale-up considerations, and analytical measures such as specific activity and recovery. These MCQs are designed to test conceptual depth and practical decision-making for enzyme extraction in pharmaceutical contexts.

Q1. What is the primary objective of the enzyme extraction step in downstream processing?

• To convert enzymes into inactive forms for easier handling
• To isolate the enzyme in an active, soluble form with high specific activity and minimal contaminants
• To crystallize enzymes for structural studies
• To change enzyme specificity through chemical modification

Correct Answer: To isolate the enzyme in an active, soluble form with high specific activity and minimal contaminants

Q2. Which of the following cell disruption methods is most suitable for scale-up of bacterial intracellular enzyme extraction while minimizing heat generation?

• Sonication (probe ultrasonic)
• High-pressure homogenization
• Bead milling with small laboratory beads
• Freeze–thaw cycling

Correct Answer: High-pressure homogenization

Q3. For maintaining enzyme stability during extraction, which buffer property is most critical to control?

• Buffer color
• pH close to the enzyme’s optimum and within a range that minimizes denaturation
• Buffer volatility
• Buffer osmolarity alone without considering pH

Correct Answer: pH close to the enzyme’s optimum and within a range that minimizes denaturation

Q4. Ammonium sulfate precipitation is commonly used for enzyme recovery. What principle underlies this technique?

• Hydrophobic interaction chromatography
• Salting-in effect increasing solubility at high salt
• Salting-out by reducing protein solubility due to competition for water molecules
• Size exclusion based on molecular sieving

Correct Answer: Salting-out by reducing protein solubility due to competition for water molecules

Q5. Aqueous two-phase systems (ATPS) are attractive for enzyme extraction because they:

• Require organic solvents that denature enzymes
• Allow simultaneous concentration and partial purification in a gentle, biocompatible environment
• Are only applicable to small molecules, not proteins
• Always yield crystalline enzyme products

Correct Answer: Allow simultaneous concentration and partial purification in a gentle, biocompatible environment

Q6. Which stabilizer is commonly added during extraction to inhibit proteolysis and protect enzyme activity?

• Sodium dodecyl sulfate (SDS)
• Protease inhibitors such as PMSF or EDTA, depending on protease class
• High concentrations of ethanol
• Strong oxidizing agents

Correct Answer: Protease inhibitors such as PMSF or EDTA, depending on protease class

Q7. In membrane-based concentration of enzymes, which membrane property is most important to select for retaining the target enzyme?

• Color of membrane
• Molecular weight cut-off (MWCO) relative to the enzyme’s molecular weight
• Electrical conductivity of membrane
• Membrane opacity

Correct Answer: Molecular weight cut-off (MWCO) relative to the enzyme’s molecular weight

Q8. Why is low temperature commonly maintained during extraction of labile enzymes?

• To increase enzymatic activity artificially during extraction
• To minimize thermal denaturation and reduce protease activity
• To facilitate organic solvent evaporation
• To promote microbial growth that stabilizes enzymes

Correct Answer: To minimize thermal denaturation and reduce protease activity

Q9. Which chromatographic technique is most appropriate for capturing enzymes based primarily on specific binding interactions (e.g., affinity tags)?

• Size-exclusion chromatography
• Ion-exchange chromatography
• Affinity chromatography
• Normal-phase chromatography

Correct Answer: Affinity chromatography

Q10. Specific activity is an important measure during extraction. What does it represent?

• The absolute mass of protein recovered per liter
• The ratio of enzyme activity units to total protein amount, indicating purity
• The total number of enzyme molecules in solution
• The recovery yield expressed as percent volume

Correct Answer: The ratio of enzyme activity units to total protein amount, indicating purity

Q11. Which additive is often used to prevent oxidation of sensitive enzymes during extraction?

• Hydrogen peroxide
• Dithiothreitol (DTT) or reduced glutathione as reducing agents
• Strong acids to lower pH drastically
• Organic peroxides

Correct Answer: Dithiothreitol (DTT) or reduced glutathione as reducing agents

Q12. During extraction from plant tissues rich in polyphenols, which strategy helps prevent enzyme inactivation?

• Add polyphenol oxidase to the buffer
• Include polyvinylpolypyrrolidone (PVPP) or activated charcoal to bind polyphenols
• Increase temperature to denature polyphenols
• Omit buffer and extract in pure water

Correct Answer: Include polyvinylpolypyrrolidone (PVPP) or activated charcoal to bind polyphenols

Q13. Which parameter best describes the efficiency of enzyme recovery after an extraction step?

• Specific gravity of the extract
• Percent recovery or yield (activity recovered relative to starting material)
• Optical density at 600 nm
• Viscosity of the extract

Correct Answer: Percent recovery or yield (activity recovered relative to starting material)

Q14. When extracting an intracellular enzyme, what is the main advantage of using gentle non-ionic detergents at low concentrations?

• They completely denature proteins to facilitate purification
• They solubilize membranes to release enzymes while preserving native structure
• They act as strong oxidizing agents
• They remove all lipids permanently

Correct Answer: They solubilize membranes to release enzymes while preserving native structure

Q15. Which analytical technique is most appropriate for monitoring enzyme integrity and detecting degradation during extraction?

• Thin-layer chromatography of small metabolites
• SDS-PAGE to assess polypeptide size distribution and detect proteolysis
• pH meter alone
• Simple refractometry

Correct Answer: SDS-PAGE to assess polypeptide size distribution and detect proteolysis

Q16. What is a key limitation of using organic solvent extraction for enzyme recovery?

• Solvents always increase enzyme activity
• Many organic solvents denature proteins and inactivate enzymes
• Solvents selectively remove only nucleic acids
• Organic solvents are cost-free and entirely safe

Correct Answer: Many organic solvents denature proteins and inactivate enzymes

Q17. During ultrafiltration concentration, what phenomenon can lead to flux decline and reduced process efficiency?

• Increased transmembrane pressure improving flux indefinitely
• Membrane fouling and concentration polarization at the membrane surface
• Complete transparency of the solution
• Constant viscosity regardless of concentration

Correct Answer: Membrane fouling and concentration polarization at the membrane surface

Q18. Which approach is recommended to minimize loss of enzyme activity during large-scale homogenization?

• Operate at elevated temperatures to reduce viscosity
• Use short processing times, control temperature, and add stabilizers to buffer
• Omit buffering agents entirely
• Prolong homogenization until all cells are visibly destroyed regardless of heat

Correct Answer: Use short processing times, control temperature, and add stabilizers to buffer

Q19. If an enzyme has an isoelectric point (pI) of 5.0, at which buffer pH would ion-exchange chromatography using an anion exchanger be most effective?

• pH 3.0, where the enzyme is strongly positively charged
• pH 7.0, where the enzyme carries net negative charge and binds to an anion exchanger
• pH 5.0 exactly, where the enzyme has no net charge
• pH 2.0, to protonate all residues

Correct Answer: pH 7.0, where the enzyme carries net negative charge and binds to an anion exchanger

Q20. Lyophilization (freeze-drying) is often used as a final step for enzyme preparations because it:

• Always increases enzymatic activity relative to liquid form
• Removes water under low temperature and pressure to stabilize enzyme for long-term storage when proper excipients are used
• Denatures all proteins making them insoluble
• Is a rapid, heat-intensive dehydration method

Correct Answer: Removes water under low temperature and pressure to stabilize enzyme for long-term storage when proper excipients are used

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