Fabrication of a 3D nano-bienzyme cascade reactor based on a Dps protein scaffold for chiral amine synthesis

Abstract

Based on the self-assembling properties of the SpyCatcher/SpyTag system and the structural advantages of Dps protein, this study successfully constructed a three-dimensional nano-enzyme cascade reactor (3DNECR) through the covalent self-assembly of SpyTag-ADH and SpyCatcher-Dps-ATA117 fusion proteins. The 3DNECR exhibited significantly enhanced catalytic efficiency compared to the two-dimensional control, attributed to optimized spatial organization promoting substrate channeling. The reactor exhibited remarkable storage, pH, and thermal stability. It maintained over 80% activity after 9 days of storage, showed superior pH tolerance across pH 8–10, and remained stable in the temperature range of 4–40 ℃. Molecular docking confirmed strong interfacial binding (− 17.3 kcal/mol) between assembly components and favorable substrate binding (− 7.4 kcal/mol) within the active site. Furthermore, the 3DNECR was applied to the asymmetric synthesis of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine (R-BPA) in an oil-water biphasic system. Under optimized conditions, the 3DNECR consistently achieved high yields (99.9%) and excellent enantioselectivity (99.9%). The 3DNECR maintained a relative enzyme activity as high as 92% even after six cycles of reuse. This integrated platform showcases substantial potential for efficient and sustainable biocatalytic applications.

Graphical abstract

Keywords

Scaffold / Multi-enzyme complex / Cascade reactor / Preparation / Chiral amines

References

[1] Aer L, Jiang QF, Zhong LLet al. . Optimization of polyethylene terephthalate biodegradation using a self-assembled multi-enzyme cascade strategy. J Hazard Mater. 2024, 476: 11.

[2] Cai XL, Huang YT, Zhu CZ. Immobilized multi-enzyme/nanozyme biomimetic cascade catalysis for biosensing applications. Adv Healthc Mater. 2025, 14(8): 16.

[3] Che SJ, Konno H, Makabe K. SpyTag peptide with alkoxyl aspartic acids for pH-dependent activation of the SpyCatcher/Tag system. Bioconjug Chem. 2024, 355616-622.

[4] Dang MH, Dong JT, Wu CJ, Hu XC, Ren LJ. Efficient synthesis of nicotinamide mononucleotide by a novel multi-enzyme cascade system based on DNA scaffolding. Food Biosci. 2025, 71: 10.

[5] Duarte M, Carvalho AL, Ferreira MCet al. . Tripartite binding mode of cohesin-dockerin complexes from Ruminococcus flavefaciens involving naturally truncated dockerins. J Biol Chem. 2025, 301716.

[6] Fegade SL. Questionable green ionic liquid: comment on extractive desulfurization of liquid fuels by energy efficient green thiazolium based ionic liquids. Ind Eng Chem Res. 2015, 5472259-2259.

[7] Fegade SL (2024a) Assessment of greenness of catalytic deoxygenation of crop oil for green diesel production. Clean Circ Bioecon 8(c):100091

[8] Fegade SL. Green hydrocarbons and fuels from municipal polymer waste co-fed with natural gas using a batch catalytic slurry process. Green Technol Sustain. 2024, 23100099.

[9] Fegade SL. Red chemistry: principles and applications. Next Sustain. 2024, 4: 100048.

[10] Gentili S, Miglioli F, Borghesani Vet al. . Exploiting SpyTag/SpyCatcher technology to design new artificial catalytic copper proteins. ChemBioChem. 2025, 261411.

[11] Hosseini B, Ashraf M, Kitchen P et al (2025) Engineering protein-peptide interfaces via combinatorial mutagenesis and mass photometric screening. Biomolecules 15(8). https://doi.org/10.3390/biom15081183

[12] Jongkind E, Domenech J, Govers Aet al. . Reductive aminases discovered from bacteria to synthesize chiral amines. New Biotech. 2025, 85175-175.

[13] Lai CF, Shim WY, Li JLet al. . Biocatalytic conversion of CO₂ to value-added chemicals using hybrid protein scaffolds. Chem Eng J. 2025, 520: 15.

[14] Li NN, Han Y, Huang WQet al. . Hierarchical encapsulation of enzymes with multishell metal-organic frameworks for sensitive detection of α-amylase activity in complex fermentation samples. Int J Biol Macromol. 2025, 30510.

[15] Lim S, Kim J, Kim Y, Xu DW, Clark DS. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem Commun. 2020, 56364950-4953.

[16] Lin P, Dinh H, Morita Y, Nakata E, Morii T. Dynamic assembly of cascade enzymes by the shape transformation of a DNA scaffold. Adv Funct Mater. 2023, 331510.

[17] Lu Y, Wang JM, Xu HBet al. . Efficient synthesis of key chiral intermediate in painkillers (r)-1- 3,5-bis(trifluoromethyl)phenyl Ethanamine by bienzyme cascade system with r-ω-transaminase and alcohol dehydrogenase functions. Molecules. 2022, 272117.

[18] Lu Y, Wang JM, Cheng PP et al (2023) Efficient biosynthesis of (R)-1-[3,5-bis(trifluoromethyl)phenyl] Ethanamine with self-assembled dual-enzyme clusters. Process Biochem 134:151–164

[19] Luo MX, Zhang M, Chi CB, Chen G. Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni²⁺. Int J Biol Macromol. 2024, 261: 10.

[20] Ma CL, Wang YH, Guo K, Wu RR, Zhu ZG. Enzymatic electrosynthesis system based on multi-enzyme catalysis or coupled with microbial transformation. Catal Sci Technol. 2025, 1551390-1405.

[21] Patil PD, Gargate N, Tiwari MS, Nadar SS. Compartmentalization of multi-enzymes within designable metal-organic frameworks (MOF): A review of systematic approaches. Int J Biol Macromol. 2025, 328: 19.

[22] Peng F, Chen QS, Zong MH, Lou WY (2021) Sequential co-immobilization of multienzyme nanodevices based on spycatcher and spytag for robust biocatalysis. Mol Catal 510. https://doi.org/10.1016/j.mcat.2021.111673

[23] Röder J, Fischer R, Commandeur U. Engineering potato virus x particles for a covalent protein based attachment of enzymes. Small. 2017, 134812.

[24] Schmid-Dannert C, López-Gallego F. Advances and opportunities for the design of self-sufficient and spatially organized cell-free biocatalytic systems. Curr Opin Chem Biol. 2019, 4997-104.

[25] Si M, Xu Q, Jiang L, Huang H. SpyTag/SpyCatcher cyclization enhances the thermostability of firefly luciferase. PLoS ONE. 2016, 11916.

[26] Veron BS, Lethcoe K, Ryan RO (2025) Immobilized nanodisks for study of ligand binding interactions. Biochim Et Biophys Acta-Biomembr 1867(8). https://doi.org/10.1016/j.bbamem.2025.184459

[27] Wang JM, Lu Y, Cheng PPet al. . Construction of bi-enzyme self-assembly clusters based on Spycatcher/Spytag for the efficient biosynthesis of (R)-Ethyl 2-hydroxy-4-phenylbutyrate. Biomolecules. 2023, 13124.

[28] Wang Z, Gao GS, Gao YD, Yang LC. Application of Imine reductase in bioactive chiral amine synthesis. Org Process Res Dev. 2024, 2883035-3054.

[29] Yan GB, Li X, Yu XL, Zhai C, Li WQ, Ma LX. Argonaute-driven programmable multi-enzyme complex assembly on ribosomal RNA scaffolds. Int J Biol Macromol. 2025, 311: 10.

[30] Zhong XR, Ma YJ, Zhang XZet al. . More efficient enzymatic cascade reactions by spatially confining enzymes via the SpyTag/SpyCatcher technology. Mol Catal. 2022, 5217.

[31] Zhu WW, Fang T, Zhang WJet al. . A ROS scavenging protein nanocage for in vitro and in vivo antioxidant treatment. Nanoscale. 2021, 1384634-4643.

Funding

Jiangxi University of Chinese Medicine Doctoral Research Start-up Fund(2004/5252301021)

2024 Key R&D Project of Jiangxi Province(20243BCC31008)

National Major Scientific Research Cultivation Project of Jiangxi University of Traditional Chinese Medicine(2023ZDPY0011)

RIGHTS & PERMISSIONS

The Author(s)