Making Water-Insoluble Enzymes — For bifunctional multifunctional agents used to crosslink enzymes, which molecular attribute is generally preferred?

Introduction:
Converting soluble enzymes into water-insoluble biocatalysts often relies on bifunctional crosslinkers that create covalent bridges between enzyme molecules or between enzyme and carrier. The molecular characteristics of the crosslinker influence penetration, reaction kinetics, and crosslink density.

Given Data / Assumptions:

• Goal: form stable, insoluble networks without excessive diffusion limitations.
• Crosslinkers possess at least two reactive groups (bifunctional).
• Reaction proceeds under conditions that preserve catalytic activity.

Concept / Approach:

Low-molecular-weight bifunctional reagents diffuse readily into enzyme matrices or onto crowded surfaces and efficiently bridge nearby reactive residues (amines, carboxyls, sulfhydryls). This produces uniform crosslinking without large steric hindrance. Classic examples include glutaraldehyde and short-chain carbodiimide-activated linkers. Excessively bulky or weakly reactive reagents may fail to create effective networks or can restrict access to active sites.

Step-by-Step Solution:

Verification / Alternative check:

Empirical protocols for CLEAs and carrier-bound enzymes commonly use low-MW bifunctional linkers to balance crosslinking speed and control over activity retention.

Why Other Options Are Wrong:

B and C introduce steric and functional disadvantages; D risks incomplete networks; E contradicts the premise by eliminating crosslinking capability.

Common Pitfalls:

Over-crosslinking can rigidify enzymes and reduce activity; optimize linker concentration and reaction time.

Final Answer:

Low molecular weight (e.g., glutaraldehyde-like) to diffuse and bridge reactive groups effectively