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

Abstract

BackgroundThrough 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.ResultsWe 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.ConclusionsExpression 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0634-8) contains supplementary material, which is available to authorized users.

Antimicrobial and Anti-Proliferative Effects of Skin Mucus Derived from Dasyatis pastinaca (Linnaeus, 1758)

“…Elevated levels of chitinases have been reported in a variety of diseases including infections, chronic inflammation, and degenerative disorders [ 39 , 40 ]. Recent studies reported that CHIT1 play a role as antifungal and antibacterial activity [ 41 , 42 , 43 , 44 , 45 ]. We also demonstrated an enzymatic activity of CHIT1 in FSM that can partially explain its anti-microbial activity.…”

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

“…Strain engineering to increase monoterpene precursor production has recently led to significant improvements in geraniol titers. (Jiang et al, 2017; Zhao et al, 2017). The G8H-mediated ω-hydroxylation of geraniol at C-8 was described as a major pathway bottleneck in S. cerevisiae due to poor activity of the P450 (Brown et al, 2015).…”

Serratia marcescenssecretes proteases and chitinases with larvicidal activity againstAnopheles dirus

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