Enzyme-Directed Assembly of Antiparallel Cellulose II Nanocrystals: Unraveling the Mechanism Beyond Spontaneous Crystallization

Tomohiro Kuga; Naoki Sunagawa; Kei Kobayashi; Hirofumi Yamada; Tomoya Imai; Takayuki Uchihashi; Kiyohiko Igarashi

doi:10.1021/jacsau.5c00993

Enzyme-Directed Assembly of Antiparallel Cellulose II Nanocrystals: Unraveling the Mechanism Beyond Spontaneous Crystallization

Tomohiro Kuga
, Naoki Sunagawa
, Kei Kobayashi
, Hirofumi Yamada
, Tomoya Imai
, Takayuki Uchihashi
, Kiyohiko Igarashi^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Scientific › peer-review

Abstract

Humans have long utilized cellulose II, known as regenerated cellulose, for fibers like rayon and Cupra and films like cellophane. While cellulose I, found in nature, consists of parallel molecular chains, cellulose II is characterized by the stable arrangement of molecules in an antiparallel orientation. Enzymatic synthesis of cellulose in vitro also affords cellulose II with various morphologies, from monolayer lamellae crystals to gels, but its formation mechanism remains obscure. Here, we demonstrate that cellodextrin phosphorylase (CDP) catalyzes the synthesis and orchestrates the antiparallel self-assembly of cellulose II nanocrystals, exceeding the paradigm of spontaneous crystallization. High-resolution structural analysis reveals CDP’s key role in dictating crystal size and alignment, bridging the gap between enzymatic catalysis and biodirected material architecture. Our research unveils a unique protein-templated assembly process for advanced cellulose materials, paving the way for enzyme-guided construction of next-generation functional nanostructures.

Original language	English
Pages (from-to)	154-165
Number of pages	12
Journal	JACS Au
Volume	6
Issue number	1
DOIs	https://doi.org/10.1021/jacsau.5c00993
Publication status	Published - 26 Jan 2026
MoE publication type	A1 Journal article-refereed

Funding

We thank Prof. Motomitsu Kitaoka of Niigata University for providing pET28a vectors harboringcdpgene andblspgene. We thank Dr. Satoshi Kimura of the University of Tokyo for his cooperation in X-ray diffraction, Prof. Tsuguyuki Saito of the University of Tokyo and Dr. Kazuho Daicho of Kanazawa University for solid-state13C NMR, and Dr. Hiroaki Kominami for technical help during FM-AFM observations. We thank Prof. Masahide Kikkawa and Yoichi Sakamaki of the University of Tokyo for their help in data collection with cryo-EM. The electron diffraction experiment was done by the Analysis and Development System for Advanced Materials (ADAM) facility at Research Institute for Sustainable Humanosphere, Kyoto University. This work was supported by the ”Development of recycling system for bio-organic materials in space” program, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Coordination Funds for Promoting AeroSpace Utilization, Japan; Grant Number JPJ000959. This work was also supported by JSPS KAKENHI (Grant no. 22J12566 to T.K. Grant no. 19K15884 to N.S.) and by a Grant-in-Aid for Innovative Areas from MEXT (Grant no. 18H05494 to K.I.), the Academy of Finland through research grant SA-FOSSOK [Decision No. 309384]. This research was partially supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP21am0101001 (support number: 2869). K.I. thanks the Finnish Funding Agency for Innovation for the support of the Finland Distinguished Professor Program “Advanced approaches for enzymatic biomass utilization and modification (BioAD)”. This work was supported by the ”Development of recycling system for bio-organic materials in space” program, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Coordination Funds for Promoting AeroSpace Utilization, Japan; Grant Number JPJ000959. This work was also supported by JSPS KAKENHI (Grant no. 22J12566 to T.K. Grant no. 19K15884 to N.S.) and by a Grant-in-Aid for Innovative Areas from MEXT (Grant no. 18H05494 to K.I.), the Academy of Finland through research grant SA-FOSSOK [Decision No. 309384]. This research was partially supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP21am0101001 (support number: 2869). K.I. thanks the Finnish Funding Agency for Innovation for the support of the Finland Distinguished Professor Program “Advanced approaches for enzymatic biomass utilization and modification (BioAD)”.

Keywords

cellulose
glycoside hydrolase
in vitrosynthesis
phosphorylase
self-assembly

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10.1021/jacsau.5c00993

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title = "Enzyme-Directed Assembly of Antiparallel Cellulose II Nanocrystals: Unraveling the Mechanism Beyond Spontaneous Crystallization",

abstract = "Humans have long utilized cellulose II, known as regenerated cellulose, for fibers like rayon and Cupra and films like cellophane. While cellulose I, found in nature, consists of parallel molecular chains, cellulose II is characterized by the stable arrangement of molecules in an antiparallel orientation. Enzymatic synthesis of cellulose in vitro also affords cellulose II with various morphologies, from monolayer lamellae crystals to gels, but its formation mechanism remains obscure. Here, we demonstrate that cellodextrin phosphorylase (CDP) catalyzes the synthesis and orchestrates the antiparallel self-assembly of cellulose II nanocrystals, exceeding the paradigm of spontaneous crystallization. High-resolution structural analysis reveals CDP{\textquoteright}s key role in dictating crystal size and alignment, bridging the gap between enzymatic catalysis and biodirected material architecture. Our research unveils a unique protein-templated assembly process for advanced cellulose materials, paving the way for enzyme-guided construction of next-generation functional nanostructures.",

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AU - Igarashi, Kiyohiko

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N2 - Humans have long utilized cellulose II, known as regenerated cellulose, for fibers like rayon and Cupra and films like cellophane. While cellulose I, found in nature, consists of parallel molecular chains, cellulose II is characterized by the stable arrangement of molecules in an antiparallel orientation. Enzymatic synthesis of cellulose in vitro also affords cellulose II with various morphologies, from monolayer lamellae crystals to gels, but its formation mechanism remains obscure. Here, we demonstrate that cellodextrin phosphorylase (CDP) catalyzes the synthesis and orchestrates the antiparallel self-assembly of cellulose II nanocrystals, exceeding the paradigm of spontaneous crystallization. High-resolution structural analysis reveals CDP’s key role in dictating crystal size and alignment, bridging the gap between enzymatic catalysis and biodirected material architecture. Our research unveils a unique protein-templated assembly process for advanced cellulose materials, paving the way for enzyme-guided construction of next-generation functional nanostructures.

AB - Humans have long utilized cellulose II, known as regenerated cellulose, for fibers like rayon and Cupra and films like cellophane. While cellulose I, found in nature, consists of parallel molecular chains, cellulose II is characterized by the stable arrangement of molecules in an antiparallel orientation. Enzymatic synthesis of cellulose in vitro also affords cellulose II with various morphologies, from monolayer lamellae crystals to gels, but its formation mechanism remains obscure. Here, we demonstrate that cellodextrin phosphorylase (CDP) catalyzes the synthesis and orchestrates the antiparallel self-assembly of cellulose II nanocrystals, exceeding the paradigm of spontaneous crystallization. High-resolution structural analysis reveals CDP’s key role in dictating crystal size and alignment, bridging the gap between enzymatic catalysis and biodirected material architecture. Our research unveils a unique protein-templated assembly process for advanced cellulose materials, paving the way for enzyme-guided construction of next-generation functional nanostructures.

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Enzyme-Directed Assembly of Antiparallel Cellulose II Nanocrystals: Unraveling the Mechanism Beyond Spontaneous Crystallization

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Keywords

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