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Production and char...
Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome
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- Berini, Francesca (author)
- Politecnico di Milano and University of Insubria, Varese, Italy
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- Presti, Ilaria (author)
- Politecnico di Milano and University of Insubria, Varese, Italy / Chemo Biosynthesis, Corana, Pavia, Italy
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- Beltrametti, Fabrizio (author)
- Actygea, Gerenzano, Varese, Italy
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Pedroli, Marco (author)
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- Vårum, Kjell M (author)
- Norwegian University of Science and Technology, Trondheim, Norway
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- Pollegioni, Loredano (author)
- Politecnico di Milano and University of Insubria, Varese, Italy
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- Sjöling, Sara (author)
- Södertörns högskola,Miljövetenskap
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- Marinelli, Flavia (author)
- Politecnico di Milano and University of Insubria, Varese, Italy
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(creator_code:org_t)
- 2017-01-31
- 2017
- English.
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In: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 16:1
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https://doi.org/10.1...
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http://www.ncbi.nlm....
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Abstract
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- 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 Ca(2+)-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.
Subject headings
- NATURVETENSKAP -- Geovetenskap och miljövetenskap -- Miljövetenskap (hsv//swe)
- NATURAL SCIENCES -- Earth and Related Environmental Sciences -- Environmental Sciences (hsv//eng)
Keyword
- Environmental Studies
- Miljövetenskapliga studier
Publication and Content Type
- ref (subject category)
- art (subject category)
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