caa a22 4500 605504725 CHVBK 20210128100617.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6873-x doi (NATIONALLICENCE)springer-10.1007/s00253-015-6873-x Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase [Elektronische Daten] [Antonio Placido, Tran Hai, Manuel Ferrer, Tatyana Chernikova, Marco Distaso, Dale Armstrong, Alexander Yakunin, Stepan Toshchakov, Michail Yakimov, Ilya Kublanov, Olga Golyshina, Graziano Pesole, Luigi Ceci, Peter Golyshin] A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(l)-arabinopyranose with a high specific activity of about 2.7kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40min of incubation at 90°C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1. The Author(s), 2015 Vulcano Island nationallicence Fosmids nationallicence Metagenomic library nationallicence Screening nationallicence Hydrolase nationallicence Lipase nationallicence Esterase nationallicence Arabinopyranosidase nationallicence Placido Antonio Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut Hai Tran School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Ferrer Manuel Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain aut Chernikova Tatyana School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Distaso Marco School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Armstrong Dale School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Yakunin Alexander Department of Chemical Engineering and Applied Chemistry, University of Toronto, M5S 3E5, Toronto, Ontario, Canada aut Toshchakov Stepan Immanuel Kant Baltic Federal University, 236040, Kaliningrad, Russia aut Yakimov Michail Institute for Coastal Marine Environment CNR, 98122, Messina, Italy aut Kublanov Ilya S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, 117312, Moscow, Russia aut Golyshina Olga School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Pesole Graziano Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut Ceci Luigi Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut Golyshin Peter School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10031-10046 0175-7598 99:23<10031 2015 99 253 https://doi.org/10.1007/s00253-015-6873-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6873-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Placido Antonio Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut NATIONALLICENCE 700 1- Hai Tran School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 700 1- Ferrer Manuel Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Chernikova Tatyana School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 700 1- Distaso Marco School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 700 1- Armstrong Dale School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 700 1- Yakunin Alexander Department of Chemical Engineering and Applied Chemistry, University of Toronto, M5S 3E5, Toronto, Ontario, Canada aut NATIONALLICENCE 700 1- Toshchakov Stepan Immanuel Kant Baltic Federal University, 236040, Kaliningrad, Russia aut NATIONALLICENCE 700 1- Yakimov Michail Institute for Coastal Marine Environment CNR, 98122, Messina, Italy aut NATIONALLICENCE 700 1- Kublanov Ilya S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, 117312, Moscow, Russia aut NATIONALLICENCE 700 1- Golyshina Olga School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 700 1- Pesole Graziano Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut NATIONALLICENCE 700 1- Ceci Luigi Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy aut NATIONALLICENCE 700 1- Golyshin Peter School of Biological Sciences, Bangor University, LL57 2UW, Bangor, Gwynedd, UK aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10031-10046 0175-7598 99:23<10031 2015 99 253