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Functional Characterization and Low-Resolution Structure of an Endoglucanase Cel45A from the Filamentous Fungus Neurospora crassa OR74A: Thermostable Enzyme with High Activity Toward Lichenan and β-Glucan

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Abstract

Biomass is the most abundant and short-term renewable natural resource on Earth whose recalcitrance toward enzymatic degradation represents significant challenge for a number of biotechnological applications. The not so abundant but critically necessary class of GH45 endoglucanases constitutes an essential component of tailored industrial enzyme cocktails because they randomly and internally cleave cellulose molecules. Moreover, GH45 glucanases are core constituents of major-brand detergent formulations as well as enzymatic aid components in the cotton processing industry, clipping unwanted cellulosic fibers from cotton (cellulosic)-based tissues. Here we report on a recombinant high-yield Neurospora crassa OR74A NcCel45A production system, a single-band GH45 endoglucanase purification, and a complete enzyme functional characterization. NcCel45A is a bimodular endoglucanase showing maximum activity at pH 6.0 and 60 °C, while most active against lichenan and β-glucans and lesser active toward filter paper, carboxymethylcellulose, and phosphoric acid-swollen cellulose. Gluco-oligosaccharide degradation fingerprinting experiments suggest cellopentaose as the minimal length substrate and ThermalFluor studies indicate that NcCel45A displays excellent stability at elevated temperatures up to 70 °C and pHs ranging from 5 to 9. Remarkably, we show that NcCel45A is uniquely resistant to a wide-range of organic solvents and small-angle X-ray scattering show a monkey-wrench molecular shape structure in solution, which indicates, unlike to other known cellulases, a non-fully extended conformation, thus conferring solvent protection. These NcCel45A unique enzymatic properties maybe key for specific industrial applications such as cotton fiber processing and detergent formulations.

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Abbreviations

CBM:

Carbohydrate-binding module

CMC:

Carboxymethyl cellulose

DAM:

Dummy atom models

DNS:

Dinitrosalycylic acid

D max :

Maximum particle size

EDTA:

Ethylenediaminetetraacetic acid

GH:

Glycoside hydrolase

HCCA:

Alpha-cyano-4-hydroxy cinnamic acid

PASC:

Phosphoric acid-swollen cellulose

PDB:

Protein Data Bank

R g :

Radius of gyration

pNPG:

p-Nitrophenyl-β-d-glucoside

Rpm:

Rotations per minute

SAXS:

Small-angle X-ray scattering

T m :

Melting temperature

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Acknowledgments

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) via grants 2008/56255-9, 2009/54035-4, 2009/11536-3, 2010/08680-2, 11/20505-4 and 09/11536-3; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) via grants 490022/2009-0, and 301981/2011-6; and the University of São Paulo (USP) via NAP de Bioenergia & Sustentabilidade (NAPBS) e NAP de Instrumentação (CIEA_MNB). We would like to thank Livia Manzine, Alexandre Antoniazzi, Maria Auxiliadora Morim Santos and Mariana Ortiz de Godoy for technical support and the Brazilian Synchrotron Light Laboratory (LNLS, Campinas) for access to the SAXS beam line.

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  1. Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, São Carlos, SP, 13566-590, Brazil

    Marco Antonio Seiki Kadowaki, Cesar Moises Camilo, Amanda Bernardes Muniz & Igor Polikarpov

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  1. Marco Antonio Seiki Kadowaki
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Correspondence to Igor Polikarpov.

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Kadowaki, M.A.S., Camilo, C.M., Muniz, A.B. et al. Functional Characterization and Low-Resolution Structure of an Endoglucanase Cel45A from the Filamentous Fungus Neurospora crassa OR74A: Thermostable Enzyme with High Activity Toward Lichenan and β-Glucan. Mol Biotechnol 57, 574–588 (2015). https://doi.org/10.1007/s12033-015-9851-8

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