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Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome
Politecnico di Milano and University of Insubria, Varese, Italy.
Politecnico di Milano and University of Insubria, Varese, Italy / Chemo Biosynthesis, Corana, Pavia, Italy.
Actygea, Gerenzano, Varese, Italy.
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2017 (Engelska)Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 16, nr 1, artikel-id 16Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
2017. Vol. 16, nr 1, artikel-id 16
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Miljövetenskap
Forskningsämne
Miljövetenskapliga studier
Identifikatorer
URN: urn:nbn:se:sh:diva-31935DOI: 10.1186/s12934-017-0634-8ISI: 000397718400001PubMedID: 28137256Scopus ID: 2-s2.0-85011032503OAI: oai:DiVA.org:sh-31935DiVA, id: diva2:1071282
Forskningsfinansiär
EU, FP7, Sjunde ramprogrammet, 222625Tillgänglig från: 2017-02-03 Skapad: 2017-02-03 Senast uppdaterad: 2017-11-29Bibliografiskt granskad

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