Dramatically increased pH and temperature stability of chymotrypsin using dual block polymer-based protein...

Area of Science:

• Bioconjugation Chemistry
• Polymer Science
• Enzyme Engineering

Background:

• Protein-polymer conjugates offer enhanced stability and tunable properties.
• Stimuli-responsive polymers can be used to control protein behavior.
• Atom Transfer Radical Polymerization (ATRP) is a versatile method for synthesizing block copolymers.

Purpose of the Study:

• To synthesize and characterize multimodal temperature-responsive chymotrypsin-poly(sulfobetaine methacrylamide)-block-poly(N-isopropylacrylamide) (CT-pSBAm-block-pNIPAm) conjugates.
• To investigate the temperature-dependent structural and functional properties of these bioconjugates.
• To evaluate the enhanced stability of the engineered enzyme under harsh conditions.

Main Methods:

• Polymer-Based Protein Engineering (PBPE) using aqueous ATRP.
• Synthesis of three different molecular weight CT-pSBAm-block-pNIPAm bioconjugates.
• Characterization of UCST and LCST phase transitions, substrate affinity, productivity, and stability.

Main Results:

• Synthesized CT-pSBAm-block-pNIPAm conjugates with dual temperature responsiveness (UCST and LCST).
• UCST transition was chain length dependent, while LCST was molecular weight independent.
• Conjugates demonstrated temperature-dependent substrate affinity and productivity from 0 to 40 °C.
• Enhanced stability against temperature, low pH, and protease degradation (active >8h at pH 1.0).

Conclusions:

• PBPE successfully created dual-zone, temperature-responsive shells around enzyme molecules.
• Engineered conjugates exhibit tunable properties and significantly improved stability.
• These findings open avenues for advanced enzyme applications in challenging environments.