Table 1 Representative techniques for enzyme activation and stabilization in vitro
System | Preferred Enzyme Characteristic(s) | Activation | Stabilization | Operating Conditions | Concern(s) | Key Reference(s) |
|---|---|---|---|---|---|---|
Condensate | ||||||
Charged Polymer | Charged surface-exposed regions | 〇 | Aqueous solution; low ionic strength recommended | Susceptible to condensate fusion or solidification, potentially affecting enzyme activity | ||
Peptide Tag | Suitable for tag fusion (e.g., via exposed N- or C-terminal regions) | 〇 | Aqueous solution | Requires recombinant expression | ||
Enzyme engineering | ||||||
Rational Design | Available structural insights to enable precise modification | 〇 | 〇 | Aqueous solution | Requires detailed structural information | [2] |
Directed Evolution | Suitable for high-throughput screening and selection | 〇 | 〇 | Aqueous solution | Labor-intensive and time-consuming | |
Immobilization | ||||||
Covalent Binding | Surface-exposed functional groups (e.g., amino, carboxyl, or thiol) | 〇 | Aqueous solution | Irreversible binding may limit recovery and reuse | ||
Physical Adsorption | Hydrophobic regions or charged residues for surface attachment | 〇 | Aqueous solution; low ionic strength recommended | Weak binding may lead to enzyme leaching into the bulk solution | ||
Entrapment | Tolerant to diffusion limitations or require protection from harsh conditions | 〇 | Aqueous solution; low ionic strength recommended | Limited substrate diffusion may reduce efficiency | ||
Micellar system | ||||||
Polymer Micelles | Retention of activity in micellar environments that provide hydrophobic/hydrophilic compartmentalization | 〇 | Aqueous solution; low ionic strength recommended | Risk of enzyme denaturation during micelle formation | [53] | |
Polyion Complex Micelles | Surface-exposed charged regions | 〇 | Aqueous solution; low ionic strength recommended | Similar to condensates; however, the membrane may limit substrate influx | ||
