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 (21μg/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,0L 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.

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome

Berini, Francesca;POLLEGIONI, LOREDANO;MARINELLI, FLAVIA
2017-01-01

Abstract

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 (21μg/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,0L 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.
2017
http://www.microbialcellfactories.com/home/
Antifungal chitinase; Functional metagenomics; Heterologous expression; Inclusion bodies; Mode of action; Protein purification; Biotechnology; Bioengineering; Applied Microbiology and Biotechnology
Berini, Francesca; Presti, Ilaria; Beltrametti, Fabrizio; Pedroli, Marco; Vårum, Kjell M.; Pollegioni, Loredano; Sjöling, Sara; Marinelli, Flavia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2061589
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