Construction of Bienzyme Self-Assembling Clusters Based on SpyCatcher/SpyTag for the Efficient Biosynthesis of
Yuanyuan Wu1, Xinyu Zhang1, Yuyan Xiao1
1College of Food and Biological Engineering, Hefei University of Technology, Hefei, Anhui 230601, China.
Abstract:
The development of spatially organized enzyme systems with enhanced substrate channeling is critical for biocatalytic applications in food and agricultural biotechnology. Here, we constructed bienzyme self-assembling clusters (BESCs) using SpyCatcher/SpyTag technology to integrate prokaryotic dextransucrase (YG) and eukaryotic dextranase (padex). Through the modular engineering of eight enzyme chimeras, six covalently linked BESCs were generated, with YGST-SCpadex exhibiting superior performance. This optimized assembly reduced lag time by 59% and produced low-molecular-weight dextran (3500 Da) with enhanced uniformity (D̵ = 1.28), features desirable for food-grade applications. Mechanistic analysis confirmed that precise spatial organization enabled efficient intramolecular substrate channeling, improving cascade catalysis while maintaining operational stability. This modular strategy provides a versatile framework for engineering tailored enzyme complexes, offering transformative potential for sustainable biomanufacturing of polysaccharide-based ingredients in the food and agriculture sectors.
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