Multienzyme Cascade Coimmobilization on ZIF-8-Coated Magnetic Nanoparticles for Efficient d-Allulose Synthesis

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Nanotechnology

Background:

• Efficient synthesis of d-allulose is crucial for its application as a low-calorie sweetener.
• Enzyme immobilization is a key strategy to improve enzyme stability and reusability for industrial processes.
• Developing robust carriers for multi-enzyme coimmobilization remains a challenge.

Purpose of the Study:

• To develop a multienzyme coimmobilization strategy on NZMNPs for efficient d-allulose synthesis.
• To enhance the stability and reusability of immobilized enzymes.
• To improve the overall catalytic efficiency of the d-allulose synthesis cascade.

Main Methods:

• Coimmobilization of d-allulose 3-epimerase (DPE), l-rhamnulose kinase (RhaB), and polyphosphate kinase (PPK) on NTA-functionalized ZIF-8-coated magnetic nanoparticles (NZMNPs).
• Optimization of immobilization conditions including enzyme-to-carrier ratio and time.
• Application of the PROSS strategy to improve the thermostability of RhaB.

Main Results:

• Achieved 93.7% immobilization efficiency and 107.1% activity retention.
• Immobilized DPE showed enhanced stability (60% activity after 360 min at 55 °C) and reusability (65.2% activity after 20 cycles).
• The coimmobilized system yielded 78.4% d-allulose conversion, with the S44K mutant improving cascade stability.

Conclusions:

• NZMNPs provide a robust and efficient platform for multienzyme coimmobilization.
• The developed strategy significantly enhances enzyme stability, reusability, and catalytic efficiency for d-allulose synthesis.
• This approach holds considerable potential for the industrial production of d-allulose.