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.
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 350 Mbp 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. 1360 kbp 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 2.7 kU/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 40 min 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.
Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy.
Antonio
A
Placido
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK. dr_tranhai@yahoo.de.
Tran
T
Hai
Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain.
Manuel
M
Ferrer
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
Tatyana N
TN
Chernikova
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
Marco
M
Distaso
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
Dale
D
Armstrong
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
Alexander F
AF
Yakunin
Immanuel Kant Baltic Federal University, 236040, Kaliningrad, Russia.
Stepan V
SV
Toshchakov
Institute for Coastal Marine Environment CNR, 98122, Messina, Italy.
Michail M
MM
Yakimov
S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, 117312, Moscow, Russia.
Ilya V
IV
Kublanov
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
Olga V
OV
Golyshina
Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy.
Graziano
G
Pesole
Institute of Biomembranes and Bioenergetics (CNR), Via Amendola 165/A, 70126, Bari, Italy.
Luigi R
LR
Ceci
School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
Peter N
PN
Golyshin
D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005820:Genetic Testing; D014644:Genetic Variation; D019015:Geologic Sediments / Q000382:microbiology*; D006867:Hydrolases / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D060086:Hydrothermal Vents; D062312:Islands; D007558:Italy; D054892:Metagenome; D013379:Substrate Specificity
D006867:Hydrolases
Arabinopyranosidase; Esterase; Fosmids; Hydrolase; Lipase; Metagenomic library; Screening; Vulcano Island