Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome
| Autor: | Francesca Berini, Marco Pedroli, Kjell Morten Vårum, Loredano Pollegioni, Sara Sjöling, I. Presti, Fabrizio Beltrametti, Flavia Marinelli |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Antifungal Agents 030106 microbiology Chitin Bioengineering Biology medicine.disease_cause Applied Microbiology and Biotechnology Microbiology Rhizoctonia 03 medical and health sciences chemistry.chemical_compound Antifungal chitinase Functional metagenomics Heterologous expression Inclusion bodies Mode of action Protein purification Biotechnology Bioreactors Fusarium medicine Escherichia coli Glycosyl Lactic Acid Cloning Molecular Phylogeny Soil Microbiology Gene Library 2. Zero hunger chemistry.chemical_classification Research Chitinases Hydrogen-Ion Concentration Recombinant Proteins Fosmid Enzyme Hexosaminidases chemistry Chitinase biology.protein Metagenome Metagenomics Hydrophobic and Hydrophilic Interactions |
| Zdroj: | Microbial Cell Factories 16:16 |
| Popis: | Background Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases—was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. Results We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. Conclusions Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|