Immobilized cutinases: Preparation, solvent tolerance and thermal stability

Area of Science:

• Biocatalysis
• Enzyme immobilization
• Organic media transformations

Background:

• Enzyme selectivity and substrate specificity studies in organic media require active immobilized enzymes.
• Characterizing enzyme use conditions is a critical prerequisite for such studies.

Purpose of the Study:

• To develop active immobilized cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using physical immobilization.
• To characterize their use conditions, including solvent tolerance and thermal stability.

Main Methods:

• Physical immobilization of AoC, HiC, and TtC onto Lewatit VP OC 1600 via hydrophobic interactions.
• Optimization of immobilization conditions (loading ratio, buffers, temperature, time, mixing speed).
• Characterization of enzyme activity, solvent tolerance, thermal stability, and kinetic stability in organic media.

Main Results:

• Immobilization yields exceeded 98% for all three cutinases under optimized conditions.
• HiC demonstrated the highest tolerance to solvents of increased polarity.
• TtC exhibited the highest thermal stability, retaining activity up to 80°C in a bulk reaction system and showing superior kinetic stability at 80°C.
• All immobilized cutinases retained >64% activity at 90°C in nonane and >75% residual activity at 70°C over 3 hours.

Conclusions:

• Active immobilized cutinases with high yields and stability were successfully developed.
• The characterized properties of these immobilized enzymes, particularly HiC and TtC, make them suitable catalysts for selective transformations in organic media.
• These findings establish a foundation for further in-depth evaluation of these biocatalysts.