When using whole cells as biocatalysts for intracellular enzymes, which pretreatment is commonly required to allow substrates and products to pass through the cell boundary efficiently?

Introduction:
Whole-cell biocatalysis can provide cofactor regeneration, enzyme stability, and multi-step pathway coupling. However, when the target enzymes are intracellular, mass transfer across the cell envelope becomes the rate-limiting step. Controlled permeabilization is a widely used solution to enhance transport while maintaining catalytic competence.

Given Data / Assumptions:

• Intracellular enzymes are desired for a bioconversion.
• Cell integrity is initially intact, limiting diffusion.
• We seek a method that increases permeability without catastrophic loss of activity.

Concept / Approach:
Permeabilization employs solvents (for example, toluene traces), surfactants, ionic treatments, or mild physical methods to create controlled pores. This reduces transport resistance for substrates, intermediates, and products while preserving the metabolic machinery required for cofactor recycling (for example, NADH/NAD+).

Step-by-Step Solution:

Verification / Alternative check:
Compare reaction rates before and after permeabilization; increased initial rate and higher final conversion indicate improved mass transfer. Leakage assays (for example, protein release) guide the window between under- and over-treatment.

Why Other Options Are Wrong:

• Lyophilize: Freeze-drying may increase brittleness but often inactivates sensitive enzymes or eliminates cofactor regeneration.
• Heat-kill: Destroys metabolic activity and denatures many enzymes; not suitable when active catalysis is required.
• Denature: By definition abolishes enzyme activity, defeating the purpose.

Common Pitfalls:
Over-permeabilization causing enzyme leakage or cell lysis, solvent residues inhibiting enzymes, and ignoring mass transfer at the pellet scale. Always validate with small-scale experiments before scale-up.

Final Answer:
permeabilize