High performance crystalline nanocellulose using an ancestral endoglucanase

Borja Alonso-Lerma,Leire Barandiaran,Rainer Hillenbrand,Raquel Olmos-Juste,Iban Amenabar,Lorena Ugarte,Antonio Reifs,Raul Perez-Jimenez,Nerea Barruetabeña,Arantxa Eceiza,Izaskun Larraza

doi:10.1038/s43246-020-00055-5

Borja Alonso-Lerma, Leire Barandiaran + Show 9 more

Open Access

https://doi.org/10.1038/s43246-020-00055-5

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Abstract

Improving the efficiency of enzymes towards decomposing substrates has been one of the central goals in the biotechnology industry. However, the modification of enzymes for upgrading natural materials to high-value performant materials is largely unexplored. Here, we demonstrate that the ancestral form of a Cel5A bacterial endoglucanase, unlike its modern descendant from Bacillus subtilis, was able to generate cellulose nanocrystals (EnCNC) chemically pure, maintaining native cellulose structure and displaying higher thermal stability and crystallinity than standard CNC obtained by acidic treatment. We demonstrate that EnCNC alone is a suitable matrix to grow cells in 2D and 3D cultures. Importantly, EnCNC accepts well graphene derivatives to fabricate conductive hybrids inks forming a stable flat surface where cells also attach and proliferate. Our results demonstrate that EnCNC has physicochemical properties unattainable with standard CNC, making it a unique material ideal as a matrix for the design of biocompatible advanced materials for tissue engineering and other applications.

Highlights

Improving the efficiency of enzymes towards decomposing substrates has been one of the central goals in the biotechnology industry
We compared the yield to that of the modern descendant from B. subtilis, used as query sequence for the ancestral reconstruction with its own carbohydratebinding module (CBM), the difference between ancestral endoglucanase (ANC EG) and B. subtilis EG can be only attributed to the catalytic domain
We report that the ancestral form of an endoglucanase Cel5A is able to upgrade cellulose to a nanomaterial suitable for high-performance applications, EG alone can produce pure nanocellulose (EnCNC)

Summary

Introduction

Improving the efficiency of enzymes towards decomposing substrates has been one of the central goals in the biotechnology industry. We demonstrate that the ancestral form of a Cel5A bacterial endoglucanase, unlike its modern descendant from Bacillus subtilis, was able to generate cellulose nanocrystals (EnCNC) chemically pure, maintaining native cellulose structure and displaying higher thermal stability and crystallinity than standard CNC obtained by acidic treatment. A less investigated aspect in biotechnology is how to modify enzymes in order to upgrade or transform substrates into highvalue performants to be used as platform materials[7] Such development would expand the range of applicability of enzymes, making them useful for downgrading and upgrading new biomaterials derived from enzymatic transformations. CNC production is mainly achieved through chemical treatments using sulfuric acid that swells cellulose amorphous regions keeping the crystalline part This process leaves sulfate groups attached to the CNCs surface affecting their physicochemical properties[13]. We present EnCNC as a potential platform for bioinks, biosensors, and other biocompatible advanced materials

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