title: Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample.
abstract: Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851 Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.
authors.affiliation: Department of Plant Biology and Biotechnology, Westphalian Wilhelm's University Münster, Schlossplatz 8, 48143 Münster, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Stöveken
authors.affiliation: Department of Plant Biology and Biotechnology, Westphalian Wilhelm's University Münster, Schlossplatz 8, 48143 Münster, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.affiliation: evocatal GmbH, Alfred-Nobel-Strasse 10, 40789 Monheim am Rhein, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Kolkenbrock
authors.affiliation: Center for Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Zakrzewski
authors.affiliation: Center for Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Wibberg
authors.affiliation: Center for Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: F G
authors.initials: FG
authors.lastname: Eikmeyer
authors.affiliation: Center for Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Pühler
authors.affiliation: Center for Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schlüter
authors.affiliation: Department of Plant Biology and Biotechnology, Westphalian Wilhelm's University Münster, Schlossplatz 8, 48143 Münster, Germany. Electronic address: moersch@uni-muenster.de.
authors.forename: B M
authors.initials: BM
authors.lastname: Moerschbacher
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D050260:Carbohydrate Metabolism; D002686:Chitin; D002688:Chitinases / Q000235:genetics*; D048271:Chitosan; D054892:Metagenome / Q000235:genetics*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002686:Chitin; D048271:Chitosan; D002688:Chitinases
keywords: Chitin/chitosan modifying enzymes; Chitinase; Dot assay; Next generation metagenomic sequencing; Soil