Substrate affinity and spatial proximity synergistically guide multi-enzyme architecture rewiring for highly...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Carbohydrate Chemistry
• Biotechnology

Background:

• N-acetylchitosan oligosaccharides (NACOS) have broad applications in food, medicine, and agriculture.
• Synergistic action of lytic polysaccharide monooxygenases (LPMOs) and chitinases enhances NACOS production from crystalline chitin.
• Current enzyme combinations lack predictability and consistency in boosting NACOS yield.

Purpose of the Study:

• To develop a predictable multidimensional framework for designing LPMO-chitinase complexes.
• To guide the rational design of enzyme complexes for tunable enhancement of synergistic effects in chitin degradation.
• To improve the efficiency and yield of NACOS production.

Main Methods:

• Developed a framework based on substrate affinity of carbohydrate-binding modules (CBMs) and spatial proximity of catalytic domains.
• Designed and constructed an artificial fusion enzyme complex (CBM11) integrating LPMO (BcLPMO1_T3) and chitinase (BcChi2).
• Evaluated the catalytic activity, NACOS yield enhancement, and degradation rates on α-chitin and β-chitin substrates.

Main Results:

• The fusion CBM11 enzyme complex significantly increased NACOS yield by 6.33-fold on α-chitin and 20.16-fold on β-chitin, compared to chitinase alone.
• Achieved over 85% degradation rate of β-chitin using 3 μmol/L of the fusion CBM11.
• The fusion enzyme demonstrated good thermostability and tolerance to high hydrogen peroxide concentrations.

Conclusions:

• The developed framework enables predictable optimization of multi-enzyme synergistic systems for efficient chitin degradation.
• The artificial fusion enzyme complex shows significant potential for industrial applications in complex reaction systems.
• This strategy provides a valuable reference for developing biocatalysts for other insoluble polysaccharides.