title: Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.
abstract: Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8-70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.
authors.affiliation: Department of Microbiology, School of Life Sciences Weihenstephan, Technische Universität München , Freising-Weihenstephan , Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Leis
authors.affiliation: Department of Microbiology, School of Life Sciences Weihenstephan, Technische Universität München , Freising-Weihenstephan , Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Heinze
authors.affiliation: Department of Microbiology, School of Life Sciences Weihenstephan, Technische Universität München , Freising-Weihenstephan , Germany.
authors.forename: Angel
authors.initials: A
authors.lastname: Angelov
authors.affiliation: Department of Microbiology, School of Life Sciences Weihenstephan, Technische Universität München , Freising-Weihenstephan , Germany.
authors.forename: Vu Thuy Trang
authors.initials: VT
authors.lastname: Pham
authors.affiliation: Göttingen Genomics Laboratory, Department of Genomic and Applied Microbiology, Georg-August University Göttingen , Göttingen , Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Thürmer
authors.affiliation: Laboratoire de Microbiologie des Environnements Extrêmes-UMR 6197 (CNRS-Ifremer-UBO), Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale , Plouzané , France.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Jebbar
authors.affiliation: School of Biological Sciences, Bangor University , Bangor , UK.
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
authors.affiliation: Fakultät für Mathematik, Informatik und Naturwissenschaften Biologie, Biozentrum Klein Flottbek, Universität Hamburg , Hamburg , Germany.
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Streit
authors.affiliation: Göttingen Genomics Laboratory, Department of Genomic and Applied Microbiology, Georg-August University Göttingen , Göttingen , Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Daniel
authors.affiliation: Department of Microbiology, School of Life Sciences Weihenstephan, Technische Universität München , Freising-Weihenstephan , Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liebl
keywords: archaeal endoglucanase; enzymatic characterization; extreme thermostable protein; functional screenings