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Production and char...
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Berini, FrancescaPolitecnico di Milano and University of Insubria, Varese, Italy
(författare)
Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome
- Artikel/kapitelEngelska2017
Förlag, utgivningsår, omfång ...
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2017-01-31
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BioMed Central,2017
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printrdacarrier
Nummerbeteckningar
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LIBRIS-ID:oai:DiVA.org:sh-31935
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https://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-31935URI
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https://doi.org/10.1186/s12934-017-0634-8DOI
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Språk:engelska
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Sammanfattning på:engelska
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Ämneskategori:ref swepub-contenttype
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Ämneskategori:art swepub-publicationtype
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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.
Ämnesord och genrebeteckningar
Biuppslag (personer, institutioner, konferenser, titlar ...)
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Presti, IlariaPolitecnico di Milano and University of Insubria, Varese, Italy / Chemo Biosynthesis, Corana, Pavia, Italy
(författare)
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Beltrametti, FabrizioActygea, Gerenzano, Varese, Italy
(författare)
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Pedroli, Marco
(författare)
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Vårum, Kjell MNorwegian University of Science and Technology, Trondheim, Norway
(författare)
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Pollegioni, LoredanoPolitecnico di Milano and University of Insubria, Varese, Italy
(författare)
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Sjöling, SaraSödertörns högskola,Miljövetenskap(Swepub:sh)SHSASG
(författare)
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Marinelli, FlaviaPolitecnico di Milano and University of Insubria, Varese, Italy
(författare)
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Politecnico di Milano and University of Insubria, Varese, ItalyPolitecnico di Milano and University of Insubria, Varese, Italy / Chemo Biosynthesis, Corana, Pavia, Italy
(creator_code:org_t)
Sammanhörande titlar
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Ingår i:Microbial Cell Factories: BioMed Central16:11475-2859
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