Ordered Coimmobilization of Multimeric Enzyme Arrays with Enhanced Biocatalytic Cascade Performance

Abstract

Multiple enzyme coimmobilizations mimicking nature cascade enzymatic pathways have potential applications in diverse fields. We have developed a strategy for orderly coimmobilizing multienzymes by combining hierarchically self-assembled multimeric enzymes with specifically abundant polyhistidine tag affinity-mediated immobilization. Using this strategy, an ordered coimmobilization of the glycosyltransferase UGT51 mutant and sucrose synthase was constructed to realize the regeneration of costly sugar donor UDP-glucose that was used in the biosynthesis of the rare ginsenoside Rh2. The ordered coimmobilization array not only significantly boosted the immobilization and catalysis efficiency but also improved UDP-glucose regeneration, storage stability, and reusability compared to those of random coimmobilization and free enzyme-assembly systems. This study provides a great promise for fabricating enzyme arrays and highlights the synergistic benefits of nanocomplexes in enhancing biocatalytic cascade performance.

Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase

“…The SUS activity was determined in 250 μL reaction buffer containing the mixture contained 50 mM Tris–HCl pH 7.5, 10 mM UDP, and 500 mM sucrose, 10 μg protein ( Ali et al, 2021 ). After incubation for 1 h at 35°C, the reactions were terminated by heating at 95°C for 10 min.…”

“…Uridine diphosphate (UDP)-glycosyltransferases (UGTs) belongs to the largest glycosyltransferase family 1 and are capable of catalyzing glycosylation of various lipophilic molecules with the nucleotide UDP-sugars as the donor ( Schmid et al, 2016 ). Several UGTs have been exploited to alter the glycosylation patterns and sugar structures of natural products for developing potential therapeutic compounds ( Lairson et al, 2008 ; Xu and Kong, 2020 ; Ali et al, 2021 ). The conjugated glycosyl groups usually could alter water solubility, stability, and pharmacological properties of natural products ( Kren and Martínková, 2001 ).…”

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring “Above Room-Temperature” Bio-Catalytic Sustainability

“…In fact, due to multipoint covalent attachment, the sulfur-containing amino acid residue, the cysteine remains unchanged at higher temperatures; it enhances the activity and stability of immobilized papain . In doing so, it appreciably simplifies the designing of the bioreactors; − when individual catalysts in the selective compartments are sequentially retained by a chemical attachment to carry out the multi-enzyme reactions in cascade. , …”

Cry3Aa*SpyCatcher Fusion Crystals Produced in Bacteria as Scaffolds for Multienzyme Coimmobilization

“…To aid in bioconjugation, we chose to employ the SpyCatcher002/SpyTag002 system (SpyCat2/SpyTag2), a further optimized variant of the SpyCatcher/SpyTag covalent ligation technology developed by Mark Howarth. , The interaction of SpyCatcher002 with SpyTag002 results in the spontaneous formation of a covalent isopeptide bond. The SpyCatcher/SpyTag technology has been widely used for enzyme coimmobilization, and the benefits of this technology have been demonstrated. − For example, Wong et al have shown improvement of the pH stability and reusability of SpyTag proteins immobilized on a SpyCatcher-PHA synthase fusion displayed on the surface of polyhydroxyalkanoate particles. However, improvements in thermostability following SpyCatcher/SpyTag-mediated immobilization to date have been mostly limited .…”